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STUDIES ON THE AETIOPATHOGENESIS
OF TYPE 1 AUTOIMMUNE DIABETES
By
Hayder A. Al-Domi
Doctor of Philosophy
University of Western Sydney
2006
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“For by doubting we are led to question and by questioning
we arrive at the truth” Peter Abelard (1099-1142)
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Statement of Authentication
The work presented in this thesis is, to the best of the researcher’s knowledge and
belief, original except as acknowledged in the text. I hereby declare that I have not
submitted this material either in whole or in part, for a degree at this or any other
institution.
Hayder A. Al-Domi ………………………………….
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Ethical Approval
The Human Research Ethics Committee of the University of Western Sydney as well
as the authorities of all Jordanian participating centres (The National Centre for
Diabetes, Endocrinology and Genetics, Princess Rahmah Children’s Hospital, and Al-
Zahrawi Medical Laboratory) approved all studies described in this thesis. All
subjects, or their parents when appropriate, gave written informed consent, or verbal
witnessed consent when appropriate, for the studies undertaken.
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Acknowledgments
The present work was carried out at the University of Western Sydney over the years
2001-2005. In this tempestuous and self-isolating journey to the prize at the other end,
I have been fortunate to have the generous contribution of many individuals. I would
like first to express my deepest and warmest gratitude to my supervisors Dr. Mark R.
Jones and Dr Jim Bergan for their valuable intellectual advice and guidance into the
scientific work and for their everlasting patience over these years. Their
encouragement and unrelenting attitude towards scientific work has carried my fate
over minor and major problems. It has been a privilege to work under their guidance.
They created an opportunity to generate, test, and learn. I think myself lucky to have
had two such devoted mentors and friends to assist me in this arduous task putting in
concert the bits and pieces from the puzzle of diabetes.
I specially thank the hopeful families and their young children who have had the time,
fate, and endurance to participate in this study.
I wish to express my gratitude to Prof. Wajih Owais, President, Jordan University of
Science, and Technology (JUST), Jordan, Prof. Kamil Ajlouni, President, and Prof.
Mohamed Al Khatib, Vice president, The National Centre for Diabetes,
Endocrinology and Genetics, Jordan. Thanks also go to Prof. Nizar Abou-Harfeil,
Research Lab, Department of Biotechnology, and Genetic Engineering, JUST, Jordan
as well as to Mr. Mustafa Telfah, Head, Al-Zahrawi Medical Laboratory, Jordan, and
to the Administration of Princess Rahmah Children’s Hospital, Jordan for creating the
possibilities for identifying the participants, sample collection, and analysis within
their institutions. I am also grateful to all research assistants for their skilful
assistance.
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I owe my sincere gratitude to my best friend Mrs Gillian Wilkins, School
Administrative Coordinator, who has made me, as an overseas student, become
acquainted with the local Australian cultural background. Her relentless passion and
encouragement were precious and gave me boundless vigour to embark on this study.
I wish also to thank our research lab technical staff members for their highly valued
technical assistance. Thanks also go to Mrs Helen Farrell, Student Support Services,
who, on short notice, has skilfully revised the language of this thesis.
My warmest thanks go to the memory of Dad. Thanks also go to Mama for her
support and endless patience throughout my never-ending years of studying and
émigré as well as to my supportive brothers, Mohammed, Zohair and Ahmed to my
loving sisters, Azizah, Nijah and Inshrah. Nephews, nieces who never lost faith in me
and to my dear friend Dr. Saleh Ashourfat for his consistent support.
Finally, I love to thank the dearest, my soulmate, Narcisa -Sarah. You have made me
to remember every single day that life is worth living for. You have provided refuge
in the time of need. To emerge without your confidence and endless working days I
would never have begun and certainly not finished.
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Table of Contents
TABLE OF CONTENTS .............................................................................................................................. I
LIST OF TABLES........................................................................................................................................V
LIST OF FIGURES.................................................................................................................................VIII
LIST OF ABBREVIATIONS.................................................................................................................... XI
LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS ............................................ XII
CONFERENCE PRESENTATIONS ....................................................................................................XIII
ABSTRACT ............................................................................................................................................. XIV
FORWARD.............................................................................................................................................XVII
RATIONALE OF THE STUDY ......................................................................................................... XVIII
OBJECTIVES OF THE STUDY ........................................................................................................... XIX
SCOPE OF THE THESIS ...................................................................................................................... XXI
MIND MAP I: THE NATURAL HISTORY OF TYPE 1 AUTOIMMUNE DIABETES................XXII
MIND MAP II: PROTEIN STUDIES ................................................................................................ XXIII
OVERVIEW OF THE THESIS .......................................................................................................... XXIV
1 REVIEW OF THE LITERATURE................................................................................................... 1
1.1 INTRODUCTION............................................................................................................................. 1
1.2 AUTOIMMUNE DISEASES: GENERAL BACKGROUND ...................................................................... 3
1.2.1 Type 1 autoimmune diabetes .................................................................................................. 4
1.2.1.1 Definition.............................................................................................................................................4
1.2.1.2 A conflicting nomenclature ................................................................................................................5
1.2.1.3 Phases...................................................................................................................................................6
1.3 FACTS AND FIGURES..................................................................................................................... 7
1.4 THE AETIOPATHOGENESIS OF AUTOIMMUNE DIABETES .............................................................. 11
1.4.1 Who is more prone to have the disease?............................................................................... 11
1.4.2 Autoantigens cross-reactivity ............................................................................................... 14
1.4.3 Invasion of autoreactive lymphocytes................................................................................... 15
1.4.4 Circulating autoantibodies: Early markers .......................................................................... 17
1.4.5 ABO and Rh blood groups: Signs of protective effect .......................................................... 21
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1.4.6 Maturity and architecture of gut system............................................................................... 24
1.4.6.1 Uptake of macromolecules ...............................................................................................................26
1.4.6.2 Breastfeeding or bottle-feeding........................................................................................................27
1.5 NON-GENETIC DETERMINANTS OF THE DISEASE ......................................................................... 31
1.5.1 Implication of viral infections............................................................................................... 32
1.5.2 Casein: A renowned diabetogen ........................................................................................... 33
1.5.3 Bovine serum albumin: A strong candidate.......................................................................... 37
1.5.3.1 Humoral immunity ............................................................................................................................38
1.5.3.2 Cellular immunity .............................................................................................................................40
1.5.3.3 Secretory immune system.................................................................................................................41
1.5.4 Bovine insulin: The contender counterpart .......................................................................... 42
1.6 PROTEINS MODIFICATION: ENHANCED IMMUNOGENICITY .......................................................... 48
1.7 THERMAL PROCESSING: ALTERED IMMUNOGENICITY ................................................................ 50
1.8 PREVENTION TRIALS: PROS AND CONS ....................................................................................... 53
1.9 TO SUM UP.................................................................................................................................. 57
2 TYPE 1 AUTOIMMUNE DIABETES: IMPLICATIONS OF WORLDWIDE IMPORTED
MILK AND MILK PROTEIN CONSUMPTION................................................................................... 58
2.1 ABSTRACT.................................................................................................................................. 58
2.2 INTRODUCTION........................................................................................................................... 60
2.3 RESEARCH DESIGN AND METHODS............................................................................................. 61
2.3.1 Statistical analysis ................................................................................................................ 62
2.4 RESULTS .................................................................................................................................... 63
2.5 DISCUSSION ............................................................................................................................... 72
2.6 CONCLUSION.............................................................................................................................. 75
3 BASELINE DIETARY PROFILE OF YOUNG JORDANIAN CHILDREN WITH
DIABETES: BREASTFEEDING AND EARLY INFANT FEEDING PRACTICES.......................... 76
3.1 ABSTRACT.................................................................................................................................. 76
3.2 INTRODUCTION........................................................................................................................... 78
3.3 RESEARCH DESIGN AND METHODOLOGY .................................................................................... 79
3.3.1 The population sample.......................................................................................................... 79
3.3.2 Survey tool ............................................................................................................................ 80
3.3.3 Exclusion criteria ................................................................................................................. 80
3.3.4 Estimation of validity and reliability .................................................................................... 81
3.3.5 Estimation of glycosylated haemoglobin (HbA1c)................................................................ 81
3.3.6 Statistical analysis ................................................................................................................ 81
3.4 RESULTS .................................................................................................................................... 82
3.4.1 General nutritional status and sociodemographic characteristics....................................... 82
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3.4.2 General medical history related to diabetes mellitus ........................................................... 86
3.4.3 Breastfeeding and early infant feeding practices ................................................................. 88
3.4.4 Risk of having the disease..................................................................................................... 93
3.5 DISCUSSION ............................................................................................................................... 94
3.6 CONCLUSION.............................................................................................................................. 99
4 PROPORTION OF ABO AND RH BLOOD SYSTEMS IN YOUNG JORDANIANS WITH
DIABETES: A SIGN OF PROTECTIVE EFFECT ............................................................................. 100
4.1 ABSTRACT................................................................................................................................ 100
4.2 INTRODUCTION......................................................................................................................... 101
4.3 RESEARCH DESIGN AND METHODOLOGY .................................................................................. 102
4.3.1 The population sample........................................................................................................ 102
4.3.2 Determination of ABO and Rh blood groups...................................................................... 103
4.3.3 Statistical analysis .............................................................................................................. 103
4.4 RESULTS .................................................................................................................................. 104
4.5 DISCUSSION ............................................................................................................................. 106
4.6 CONCLUSION............................................................................................................................ 108
5 EVIDENCE ON THE HETEROGENEITY OF NATIVE, MODIFIED AND HEAT
PROCESSED MILK PROTEINS: A SPECTRAL PROFILE............................................................. 109
5.1 ABSTRACT................................................................................................................................ 109
5.2 INTRODUCTION......................................................................................................................... 111
5.3 METHODS................................................................................................................................. 112
5.3.1 Milk samples ....................................................................................................................... 112
5.3.2 Reconstitution, separation, and modification of milk protein solutions ............................. 113
5.3.3 Water bath heat processing ................................................................................................ 113
5.3.4 Hot plate heat processing ................................................................................................... 114
5.3.5 Absorbance spectrum.......................................................................................................... 114
5.3.6 Reversed-phase high-pressure liquid chromatography (RV-HPLC) .................................. 115
5.4 RESULTS .................................................................................................................................. 116
5.4.1 Fat and precipitant yield .................................................................................................... 116
5.4.2 Identification standard curve.............................................................................................. 117
5.5 MODIFICATION AND HEAT PROCESSING.................................................................................... 122
5.5.1 Modification of bovine serum albumin with bovine insulin ................................................ 122
5.5.2 Heat processing of native and modified bovine serum albumin with bovine insulin .......... 125
5.5.3 Water bath versus hot plate heat treatment ........................................................................ 128
5.6 DISCUSSION ............................................................................................................................. 130
5.7 CONCLUSION............................................................................................................................ 134
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6 A NEWLY DEVELOPED SALIVA-BASED ELISA TO DETERMINE IMMUNE RESPONSE
TO MILK PROTEINS IN YOUNG DIABETIC JORDANIANS: LACK OF ASSOCIATION FOR
INFANTILE FEEDING PRACTICES................................................................................................... 135
6.1 ABSTRACT................................................................................................................................ 135
6.2 INTRODUCTION......................................................................................................................... 137
6.3 RESEARCH DESIGN AND METHODOLOGY .................................................................................. 138
6.3.1 The population sample........................................................................................................ 138
6.3.2 Breastfeeding and early infant feeding practices ............................................................... 139
6.3.3 Blood samples collection .................................................................................................... 139
6.3.4 Saliva samples collection.................................................................................................... 139
6.3.5 C - reactive protein (CRP).................................................................................................. 140
6.3.6 Preparation of the testing antigens..................................................................................... 140
6.3.7 Enzyme linked immunosorbent assay (ELISA) ................................................................... 141
6.3.8 Statistical analysis .............................................................................................................. 142
6.4 RESULTS .................................................................................................................................. 143
6.5 DISCUSSION ............................................................................................................................. 148
6.6 CONCLUSION............................................................................................................................ 158
7 FUTURE PROSPECTS.................................................................................................................. 159
7.1 MIND MAP III: ASPECTS OF FUTURE STUDIES........................................................................... 163
8 CONCLUSION ............................................................................................................................... 164
9 REFERENCES................................................................................................................................ 167
10 APPENDICES................................................................................................................................. 206
10.1 PPENDIX A: INFORMATION SHEET, CONSENT, AND SURVEY (ENGLISH)..................................... 207
10.1.1 Participant information sheet (English)......................................................................... 207
10.1.2 Consent to participate in research (English) ................................................................. 213
10.1.3 Survey form (English) .................................................................................................... 216
10.2 APPENDIX B: INFORMATION SHEET, CONSENT, AND SURVEY (ARABIC) ................................... 223
10.2.1 Participant information sheet (Arabic).......................................................................... 223
10.2.2 Consent to participate in research (Arabic) .................................................................. 229
������ Survey form (Arabic)...................................................................................................... 231
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List of Tables
TABLE �2-1 WORLDWIDE MEAN (PER 100,000) AND RELATIVE INCREASE IN OCCURRENCE OF TYPE 1
‘AUTOIMMUNE’ DIABETES AMONG CHILDREN AGED 14 YEARS OR LESS MODIFIED FROM THE ONKAMO
STUDY (1999). RELATIVE INCREASE IN THE DISEASE OCCURRENCE IN PERU (LIMA), AUSTRALIA
(WEST), AND ISRAEL (YEMENITE JEWS) IS ASSUMED TO REPRESENT THAT OF THE WHOLE POPULATIONS
OF THESE COUNTRIES. .......................................................................................................................... 65
TABLE �2-2 WORLDWIDE MEAN IMPORTED MILK EXCLUDING BUTTER (IMEB) (TRILLION METRIC TONNES).
MEAN IMEB FOR EACH INDIVIDUAL COUNTRY MODIFIED FROM FOOD AND AGRICULTURE
ORGANISATION OF THE UNITED NATIONS (2004) AND CALCULATED FOR THE PERIOD MATCHING THAT
OF THE ONKAMO STUDY (1999). THE POPULATIONS ARRANGED IN DESCENDING ORDER IN
ACCORDANCE WITH THE RELATIVE INCREASE IN THE DISEASE OCCURRENCE PER YEAR. RELATIVE
INCREASE IN THE DISEASE OCCURRENCE IN PERU (LIMA), AUSTRALIA (WEST), AND ISRAEL (YEMENITE
JEWS) ASSUMED TO REPRESENT THAT OF THE WHOLE POPULATIONS OF THESE COUNTRIES. ALL DATA
WERE STATISTICALLY SIGNIFICANT AT P<0.05. ................................................................................... 66
TABLE �2-3 WORLDWIDE MEAN RELATIVE CHANGE IN MEAN IMPORTED MILK EXCLUDING BUTTER (IMEB)
(TRILLION TON) MODIFIED FROM FOOD AND AGRICULTURE ORGANISATION OF THE UNITED NATIONS
(2004) AND CALCULATED FOR THE PERIOD MATCHING THAT OF THE ONKAMO STUDY (1999). THE
POPULATIONS ARRANGED IN DESCENDING ORDER IN ACCORDANCE WITH THE RELATIVE INCREASE IN
THE DISEASE OCCURRENCE PER YEAR. RELATIVE INCREASE IN THE DISEASE OCCURRENCE IN PERU
(LIMA), AUSTRALIA (WEST), AND ISRAEL (YEMENITE JEWS) ASSUMED TO REPRESENT THAT OF WHOLE
POPULATIONS OF THESE COUNTRIES. DATA WERE CONSIDERED STATISTICALLY SIGNIFICANT AT P<0.05.
............................................................................................................................................................ 67
TABLE �2-4 WORLDWIDE MEAN MILK PROTEIN CONSUMPTION (MPC) (G/CAPITA/DAY). MEAN MPC FOR EACH
INDIVIDUAL COUNTRY MODIFIED FROM FOOD AND AGRICULTURE ORGANISATION OF THE UNITED
NATIONS (2004) AND CALCULATED FOR THE PERIOD MATCHING THAT OF THE ONKAMO STUDY (1999).
THE POPULATIONS ARE ARRANGED IN DESCENDING ORDER IN ACCORDANCE WITH THE RELATIVE
INCREASE IN THE DISEASE OCCURRENCE PER YEAR. RELATIVE INCREASE IN THE DISEASE OCCURRENCE
IN PERU (LIMA), AUSTRALIA (WEST), AND ISRAEL (YEMENITE JEWS) IS ASSUMED TO REPRESENT THAT
OF THE WHOLE POPULATIONS OF THESE COUNTRIES. ALL DATA WERE STATISTICALLY SIGNIFICANT AT
P<0.05. ................................................................................................................................................ 69
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TABLE �2-5 WORLDWIDE RELATIVE CHANGE IN MEAN MILK PROTEIN CONSUMPTION (MPC) (G/CAPITA/DAY).
MEAN MPC FOR EACH INDIVIDUAL COUNTRY MODIFIED FROM FOOD AND AGRICULTURE
ORGANISATION OF THE UNITED NATIONS (2004) AND CALCULATED FOR THE PERIOD MATCHING THAT
OF THE ONKAMO STUDY (1999). THE POPULATIONS ARE ARRANGED IN DESCENDING ORDER IN
ACCORDANCE WITH THE RELATIVE INCREASE IN THE DISEASE OCCURRENCE PER YEAR. RELATIVE
INCREASE IN THE DISEASE OCCURRENCE IN PERU (LIMA), AUSTRALIA (WEST), AND ISRAEL (YEMENITE
JEWS) ASSUMED TO REPRESENT THAT OF THE WHOLE POPULATIONS OF THESE COUNTRIES. DATA WERE
STATISTICALLY SIGNIFICANT AT P<0.05. ............................................................................................. 70
TABLE �3-1 SUMMARY OF THE SOCIODEMOGRAPHIC CHARACTERISTICS OF DIABETIC AND NON-DIABETIC
JORDANIAN CHILDREN AGED 14 YEARS OR LESS. UPPER LIMIT NORMAL (ULN) = 6.1%. THE RESULTS
PRESENTED AS MEAN ± STANDARD DEVIATION AND AS PERCENTAGE WITHIN EACH GROUP. DATA WERE
CONSIDERED STATISTICALLY SIGNIFICANT AT P<0.05. ........................................................................ 84
TABLE �3-2 SUMMARY OF THE SOCIODEMOGRAPHIC CHARACTERISTICS OF THE PARENTS OF DIABETIC AND
NON-DIABETIC JORDANIAN CHILDREN AGED 14 YEARS OR LESS. THE RESULTS PRESENTED AS MEAN ±
STANDARD DEVIATION AND PERCENTAGE WITHIN EACH GROUP. DATA WERE CONSIDERED
STATISTICALLY SIGNIFICANT AT P<0.05. ............................................................................................. 85
TABLE �3-3 FAMILY HISTORY OF PARTICIPATING DIABETIC AND NON-DIABETIC YOUNG JORDANIAN CHILDREN
AGED 14 YEARS OR LESS. THE RESULTS PRESENTED AS PERCENTAGE WITHIN EACH GROUP. DATA WERE
CONSIDERED STATISTICALLY SIGNIFICANT AT P<0.05. ........................................................................ 87
TABLE �3-4 BREASTFEEDING AND EARLY INFANT FEEDING PRACTICES IN YOUNG JORDANIAN DIABETIC AND
NON-DIABETIC CHILDREN AGED 14 YEARS OR LESS. RESULTS PRESENTED AS PERCENTAGE WITHIN EACH
GROUP AND MEAN ± STANDARD DEVIATION. DATA WERE CONSIDERED STATISTICALLY SIGNIFICANT AT
P<0.05. ................................................................................................................................................ 90
TABLE �4-1 COMPARISON BETWEEN THE ABO AND RH (D) BLOOD GROUPS OF YOUNG MALE AND FEMALE
DIABETIC AND NON-DIABETIC CHILDREN. THE RESULTS PRESENTED AS PERCENTAGE WITHIN EACH
GROUP. DATA WERE CONSIDERED STATISTICALLY SIGNIFICANT AT P<0.05....................................... 105
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TABLE �5-1 SPECTRAL OF RAW WHOLE POOLED BOVINE MILK (RWPBM), PASTEURISED AND HOMOGENISED
FRESH BOVINE MILK (PHFBM), BOVINE-BASED ADULT CANNED FORMULAE (ADULT H, J AND D),
CANNED WHEY DOMINANT BOVINE-BASED STARTER INFANT FORMULAE (ISFS AND ISFK), AND
CANNED ENZYMATICALLY HYDROLYSED WHEY PROTEIN INFANT FORMULA (ISFN). MILK SOLUTIONS
SEPARATED BY PRECOOLING TO 4ºC/20MIN AND CENTRIFUGED AT 25,000 G/60MIN/4ºC; 70MM
DIAMETER ROTOR. ABSORBANCE OBTAINED AT �MAX: 270NM AND 275NM ((�MAX FOR WAVELENGTH
AT WHICH THE MAXIMUM ABSORBANCE OBTAINED) AND �280NM. ................................................... 120
TABLE �5-2 THE EFFECT OF INCUBATION AND CONCENTRATION ON THE ABSORBANCE MAXIMUM OF BOVINE
INSULIN (BI, 1MG/ML), BOVINE SERUM ALBUMIN (BSA, 1MG/ML) AND BSA MODIFIED WITH
ALTERNATIVE DIFFERENT CONCENTRATIONS AND EQUAL VOLUMES OF BI (MG/ML) UNDER
PHYSIOLOGICAL CONDITIONS (37ºC/24H/25RPM, PH 7.4) AND UNDER ROOM TEMPERATURE (RT:
17ºC/24H/25RPM, PH 7.4). ABSORBANCE OBTAINED AT �MAX: 275NM ((�MAX FOR WAVELENGTH AT
WHICH THE MAXIMUM ABSORBANCE OBTAINED) AND AT �280NM. ................................................... 123
TABLE �6-1 MULTIVARIATE ANALYSIS OF TESTS BETWEEN-SUBJECTS; EFFECTS FOR NATIVE BOVINE SERUM
ALBUMIN (BSA) AND MODIFIED AND HEAT PROCESSED BSA WITH BOVINE INSULIN (MBI-BSA) ON
SERUM IGG AND SECRETORY IGA (SIGA) TITRE LEVELS IN BOTH DIABETIC AND NON-DIABETIC
JORDANIAN CHILDREN AGED 14 YEARS OR LESS. DATA WERE CONSIDERED STATISTICALLY
SIGNIFICANT AT P<0.05. .................................................................................................................... 148
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List of Figures
FIGURE �2-1 WORLDWIDE TRENDS (1961-2001) IN MILK PROTEIN CONSUMPTION (G/CAPITA/DAY). DATA
MODIFIED FROM FOOD AND AGRICULTURE ORGANISATION OF THE UNITED NATIONS (2004)............. 71
FIGURE �3-1 PERCENTILE CURVES FOR BODY WEIGHT, HEIGHT AND BODY MASS INDEX OF DIABETIC CHILDREN
(DC) AND NON-DIABETIC (NDC) JORDANIAN CHILDREN AGED 14 YEARS OR LESS. THE RESULTS
PRESENTED AS PERCENTAGE WITHIN EACH GROUP. DATA WERE CONSIDERED STATISTICALLY
SIGNIFICANT AT P<0.05. ...................................................................................................................... 86
FIGURE �3-2 PROPORTION OF JORDANIAN DIABETIC CHILDREN (DC) AND NON-DIABETIC CHILDREN (NDC)
AGED 14 YEARS OR LESS WHO HAD BREASTFEEDING (BRF), BOTTLE-FEEDING (BOF) AND EXPOSED TO
SOLID FOODS SUPPLEMENTS AT THREE MONTHS (M) OF AGE OR MORE. THE RESULTS PRESENTED AS
PERCENTAGE WITHIN EACH AGE GROUP. DATA WERE CONSIDERED STATISTICALLY SIGNIFICANT AT
P<0.05. NO BRF (P=0.079), BRF >3 MONTHS (P=0.672), NO BOF (P=0.041), BOF >3 MONTHS
(P=0.672) AND SOLID S >3 MONTHS (P=0.000). ................................................................................... 91
FIGURE �3-3 HOME HEAT PROCESSING PROCEDURES THAT PARENTS USED TO PREPARE THEIR INFANTS LIQUID
BOTTLE MILK FEEDS BOTH DIABETIC CHILDREN (DC) AND NON-DIABETIC (NDC) JORDANIAN
CHILDREN. THE RESULTS PRESENTED AS PERCENTAGE WITHIN EACH GROUP. DATA WERE
STATISTICALLY INSIGNIFICANT (P<0.907). .......................................................................................... 92
FIGURE �4-1 DISTRIBUTION (%) OF THE ABO AND RH (D) BLOOD GROUPS OF YOUNG DIABETIC CHILDREN
(DC) AND NON-DIABETIC CHILDREN (NDC). THE RESULTS PRESENTED AS PERCENTAGE WITHIN EACH
GROUP. ABO BLOOD GROUPS AND RH (D) POSITIVE OF BOTH DC AND NDC WERE STATISTICALLY
SIGNIFICANT (P=0.002 AND P�0.001 RESPECTIVELY). ....................................................................... 104
FIGURE �5-1 SPECTRAL PROFILE OF HUMAN BREAST MILK (HBM). MILK SOLUTIONS SEPARATED BY
PRECOOLING TO 4ºC/20MIN AND CENTRIFUGED AT 15,000 ROUND PER MINUTE/60MIN/4ºC;70MM
DIAMETER ROTOR. ABSORBANCE OBTAINED AT �MAX: 270NM (�MAX FOR WAVELENGTH AT WHICH
THE MAXIMUM ABSORBANCE OBTAINED) AND �280NM..................................................................... 118
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FIGURE �5-2 SPECTRAL PROFILE OF BOVINE SERUM ALBUMIN (BSA) AND BOVINE INSULIN (BI). ABSORBANCE
OBTAINED AT �MAX: 275NM ((�MAX FOR WAVELENGTH AT WHICH THE MAXIMUM ABSORBANCE
OBTAINED) AND �280NM. .................................................................................................................. 119
FIGURE �5-3 COMPARISON BETWEEN THE CHROMATOGRAPHIC PROFILES OF UNTREATED HUMAN BREAST MILK
(HBM), RAW WHOLE POOLED BOVINE MILK (RWPBM), PASTEURISED AND HOMOGENISED FRESH
BOVINE MILK (PHFBM), ADULT FULL CREAM (ADULT D), ADULT SKIM (ADULT H) AND INFANT
STARTER FORMULAE (ISF)K, ISFS AND ISFN. COLUMN (C18) ISOCRATIC MOBILE PHASE SOLVENTS
(100% ACETONITRILE AND 0.1% TRIFLUOROACETIC ACID IN DOUBLE-DEIONISED WATER). FLOW RATE
1ML/MIN. DETECTION AT 220NM. ...................................................................................................... 121
FIGURE �5-4 CHROMATOGRAPHIC PROFILE OF BOVINE INSULIN (BI, 1MG/ML), BOVINE SERUM ALBUMIN (BSA,
1MG/ML) AND BSA MODIFIED WITH ALTERNATIVE CONCENTRATIONS AND EQUAL VOLUMES OF BI
(MG/ML) UNDER PHYSIOLOGICAL CONDITIONS (PC: 37ºC/24H/25RPM, PH 7.4) AND UNDER ROOM
TEMPERATURE (RT: 17 ºC/24H/25RPM, PH 7.4). COLUMN (C18), ISOCRATIC MOBILE PHASE SOLVENTS
(100% ACETONITRILE AND 0.1% TRIFLUOROACETIC ACID IN DOUBLE-DEIONISED WATER). FLOW RATE
1ML/MIN. DETECTION AT 220NM. ...................................................................................................... 124
FIGURE �5-5 PERCENT CHANGE IN THE EFFECT OF WATER BATH HEAT PROCESSING AT VARIOUS
TEMPERATURES AND EXPOSURE TIME ON THE ABSORBANCE MAXIMUM OF BOVINE SERUM ALBUMIN
(BSA) AND BSA MODIFIED WITH BOVINE INSULIN (MBI-BSA) UNDER PHYSIOLOGICAL CONDITIONS (37
ºC/24H/25 RPM). THE PERCENT CHANGE IN THE ABSORBANCE MAXIMUM OF HEAT PROCESSED BSA AND
MBI-BSA CALCULATED IN REFERENCE TO THAT OF UNTREATED BSA AND UNTREATED MBI-BSA
RESPECTIVELY. ABSORBANCE DETERMINED AT �280NM....................................................................... 126
FIGURE �5-6 EFFECT OF WATER BATH HEAT PROCESSING AT VARIOUS TEMPERATURES AND EXPOSURE TIME ON
THE CHROMATOGRAPHIC PROFILES OF BOVINE SERUM ALBUMIN (BSA), AND BSA MODIFIED WITH
BOVINE INSULIN (MBI-BSA) UNDER PHYSIOLOGICAL CONDITIONS (37 ºC/24H/25 RPM). COLUMN C18,
ISOCRATIC MOBILE PHASE SOLVENTS (100% ACETONITRILE AND 0.1% TRIFLUOROACETIC ACID IN
DOUBLE-DEIONISED WATER). FLOW RATE 1ML/MIN. DETECTION AT 220NM...................................... 127
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FIGURE �5-7 EFFECT OF WATER BATH HEAT PROCESSING (WB) AT (40-100 ºC FOR 15 MIN) AND HOT PLATE
HEAT PROCESSING (HP) AT (63 ºC FOR 15 AND 30 MIN) PROCEDURES ON THE PERCENT CHANGE IN THE
ABSORBANCE MAXIMUM OF BOVINE SERUM ALBUMIN (BSA), RAW WHOLE POOLED BOVINE MILK
(RWPBM), CANNED WHEY DOMINANT BOVINE-BASED STARTER INFANT FORMULAE (ISFS), AND
CANNED ENZYMATICALLY HYDROLYSED WHEY PROTEIN INFANT FORMULA (ISFN). THE PERCENT
CHANGE IN ABSORBANCE CALCULATED IN REFERENCE TO THAT OF NOT HEAT PROCESSED MILK
PROTEIN. ABSORBANCE DETERMINED AT �280NM................................................................................ 129
FIGURE �6-1 PERCENT CHANGE (*100) IN SERUM IGG AND SECRETORY IGA (SIGA) TITRE LEVELS OF
DIABETIC CHILDREN PRODUCED TO MODIFIED AND HEAT PROCESSED BOVINE SERUM ALBUMIN (BSA)
WITH BOVINE INSULIN (MBI-BSA) AT DIFFERENT TEMPERATURES AND EXPOSURE DURATIONS
(70C/5MIN, 60ºC/15MIN, 60ºC/5MIN, 50ºC/5MIN, 40ºC/10MIN AND UNTREATED MBSA-BI). RELATIVE
CHANGE CALCULATED IN REFERENCE TO THAT PRODUCED AGAINST UNTREATED BSA. PERCENT
CHANGE CALCULATED IN REFERENCE TO IGG AND SIGA ANTIBODY TITRE LEVELS PRODUCED AGAINST
UNTREATED BSA............................................................................................................................... 144
FIGURE �6-2 COMPARISON BETWEEN THE SAMPLE PAIRED TEST MEAN EFFECT THAT NATIVE BOVINE SERUM
ALBUMIN (BSA) AND MODIFIED AND HEAT PROCESSED BSA WITH BOVINE INSULIN (MBI-BSA) HAVE
ON BOTH LOGARITHMIC (LOG) SERUM IGG AND SECRETORY IGA (SIGA) ANTIBODY TITRE LEVELS IN
BOTH DIABETIC AND NON-DIABETIC JORDANIAN CHILDREN AGED 14 YEARS OR LESS. MEAN EFFECT
WAS STATISTICALLY SIGNIFICANT AT P<0.05 FOR ALL HEAT AND MODIFIED BSA AND BI. ............... 146
xi
List of Abbreviations
ABBOS a peptide fragment of 13 amino acids of bovine serum albumin ADA American Diabetes Association ALA alpha-lactalbumin AU absorbance unit BB-rat bio-breeding rat BI bovine insulin BLG beta-lactoglobulin BMP bovine milk proteins BOF bottle-feeding BRF breastfeeding BSA bovine serum albumin CI confidence interval DERI the Diabetes Epidemiology Research International Group DIAMOND the World Health Organisation Project for Childhood Diabetes DC diabetic children DIPP Type 1 Diabetes Prediction and Prevention Study DPT-1 the Diabetes Prevention Trial GAD glutamic acid decarboxylase ELISA enzyme-linked immunosorbent assay ENDIT the European Nicotinamide Diabetes Intervention Study HbA1c haemoglobin A1c HLA human leukocyte antigen HBM human breast milk IAA insulin autoantibodies IA-2 islet antigen 2 IA-2A antibodies to islet antigen 2 ICA islet cell antibodies IDDM insulin-dependent diabetes mellitus Ig immunoglobulin ISF infant starter formula mBI-BSA modified BSA with BI MHC major histocompatibility complex MPs milk protein solutions NDC non-diabetic children NOD mouse non-obese diabetic mouse OD optical density PHFBM pasteurised homogenised fresh bovine milk RWPBM raw whole pooled bovine milk SD standard deviation TRIGR the Trial to Reduce IDDM in the Genetically at Risk WHO World Health Organisation
xii
List of publications and conference presentations
The following publications (under review) have been derived from this thesis:
• Al-Domi, H., Bergan, J., Jones, M. R. (2005). Type 1 autoimmune diabetes:
Implications of worldwide imported milk and milk protein consumption. Human
Ecology. In review.
• Al-Domi, H., Bergan, J., Jones, M. R., Ajlouni, K. (2005). Baseline dietary profile
of young Jordanian children with diabetes: Breastfeeding and early infant feeding
practices. Diabetologia. In review.
• Al-Domi, H., Jones, M. R., Bergan, J. (2005). Proportion of ABO and Rh blood
systems in young Jordanians with diabetes: A sign of protective effect. Nutrition
and Dietetics. In review.
• Al-Domi, H., Jones, M. R., Bergan, J. (2005). Evidence on the heterogeneity of
native, modified and heat processed milk proteins: A spectral profile. Australian
Journal of dairy Technology. In review.
• Al-Domi, H., Jones, M. R., Bergan, J., Owais, W. (2005). A newly developed
saliva-based ELISA to determine immune response to milk proteins in young
diabetic Jordanians: Lack of association for infantile feeding practices. European
Journal of Clinical Nutrition. In review.
xiii
Conference presentations
• Al-Domi H., Jones, R., and Bergan, J. Possible diabetogenicity of modified and
denatured bovine milk proteins. In: Second annual College of Science,
Technology and Environment Innovation Conference. Sydney, Australia:
University of Western Sydney; 7-8 June 2005.
• Al-Domi, H., Jones M. R., and Bergan J. (2004). Spectrophotometric
characteristics of human breast milk, and native and heat processed bovine milk-
based formula. Biological Sciences and Society: Services and Obstacles, Jordan,
Yarmouk University. PP 13.
• Al-Domi H., Jones, M. R., and Bergan, J. Autoimmune diabetes versus bovine
milk proteins antigenicity: A puzzling link. In: First Annual College of Science,
Technology, and Environment Innovation Conference. Sydney, Australia:
University of Western Sydney. 9-11 June 2004.
• Al-Domi H., Jones, M. R., and Bergan, J. Aetiology of autoimmune diabetes In:
Postgraduate Annual Conference. College of Science, Technology and
Environment, University of Western Sydney, NSW, Australia, June 2003. (Won
the first prize in the Conference Competition).
• Invited lecture: Type 1 autoimmune diabetes: Controversy and future
perspectives. Agriculture Faculty summer seminars programme, Department of
Nutrition and Food Technology, Jordan University of Science and Technology,
Jordan, November 21, 2005.
xiv
Abstract
Type 1 autoimmune diabetes mellitus (T1ADM) is a serious worldwide health problem.
A minimum of three million individuals worldwide have the disease for the majority
onset occurs during childhood. The disease is widely considered as a complex, chronic,
metabolic, slowly progressive, polygenic, inflammatory organ-specific autoimmune-
mediated disorder induced by activated autoreactive T cells. The natural history of the
disease involves four phases of variable duration and is characterised by genetically
determined susceptibility, occurrence of autoimmune markers, and selective aggressive
destruction of insulin-producing pancreatic beta cells.
There is a strong assertion that every case of every disease has both genetic and
environmental causes. Nevertheless, the exact aetiopathogenesis of autoimmune diabetes
is not yet fully understood, what is evident however, is that autoimmune diabetes is a
multifactorial disorder featured by a complex interplay between genetic and non-genetic
(dietary and non-dietary) environmental factors which trigger the destruction of insulin
producing pancreatic beta cells. Studies on the association between early exposure to
foreign environmental triggering factors and the development of the disease remain
discrepant and widely controversial.
Worldwide geographic variation in the disease occurrence among young children was
significantly associated with global variations in mean and relative changes in both total
milk imports and the amount of milk proteins consumed. This was influenced by the
socioeconomic status of populations. Evidence on the diversity of milk protein solutions
as well as the effect of modification and thermal processing on these proteins was
established by the identification of changes in their spectral profiles and chromatographic
profiles.
xv
Studies on type 1 autoimmune diabetes in Jordanian children with diabetes are very
limited. Baseline dietary profile, breastfeeding and early infant feeding practices,
susceptibility relationship with ABO and Rh (D) blood system, and secretory and
humoral immune responses to native, modified and thermally processed bovine serum
albumin with bovine insulin were investigated in 50 young Jordanian diabetic children
aged 14 years or less and 50 age and sex matched controls.
Breastfeeding in Jordan is common and is usually extended for a minimum of one year
for the majority of children. In this study, diabetic and non-diabetic children were
breastfed for a period of time with a higher proportion of diabetic children received no
bottle-feeding in their infancy. Disease diagnosis age was approximately at six years of
age, and was mainly in winter with female predominance.
Dietary compliance in diabetic children was very poor with signs of undernutrition in
both groups. Better nutritional counselling and standardisation of national dietary
guidelines as well as establishing a national diabetes registry may improve their
nutritional status. Blood group O+ was predominant in diabetic children and a higher
proportion of NDC had Rh (D) negative blood groups with gender variation.
Although bovine milk proteins are among the most investigated possible diabetogens,
few studies have examined the modification and thermal effect on its immunogenicity.
Changes in the mucosal architecture as well as interactions between different dietary
(native, modified, and thermally processed), and non-dietary (normal flora and viral)
factors coexisting in the gut environment may either boost or suppress the autoimmune
process. Concomitant investigation of these factors may constitute a new ground for
identifying possible primary environmental factors involved in triggering the pancreatic
autoimmune process.
xvi
The newly developed saliva-based ELISA is non-invasive, rapid, and convenient method
and constitutes a useful proxy of immune response studies in young diabetic children.
The heat processing of bovine proteins at various temperatures as well as exposure times
has caused substantial change in the secretory and humoral immune responses with
significant variations. Immune responses were neither limited to diabetic patients nor
associated with the breastfeeding or early infant feeding practices with the higher
proportion of diabetic children receiving no bottle-feeding in their early infancy. This
may reflect an unspecific defect of the immune system in young Jordanian children.
Considerable variations between low and high-risk populations require further
investigation.
Enhancing our understanding of the biological history of the preclinical stage of the
disease, achieving ample insights into disease immunopathogenesis, and identification of
persons for prevention trials could lead to more efficient early diagnosis prior to the
manifestation of the disease. Identifying the nature and timing of environmental factors
as well as genetic factors that may predispose to the initiation of the disease should
continue. Eventually, the benefits to individuals and the world society will be enormous
and that should provide the momentum to continue clinical studies on therapeutic
intervention.
xvii
Forward
Type 1 diabetes mellitus remains a serious, costly, and rising worldwide health problem
resulting in substantial morbidity and mortality. Although the majority of people with
type 1 diabetes have autoimmune aetiology, the aetiopathogenesis of autoimmune
diabetes as a form of diabetes for immune-mediated diabetes remains unknown.
Understanding the aetiopathogenesis of autoimmune diabetes requires setting down the
nature of the triggering factors, target autoantigens and the effector mechanisms. The
natural history of the disease involves four phases of variable duration. In addition to
genetic susceptibility linkage to or protection from the disease, environmental factors are
widely implicated in initiating the pancreatic autoimmune process. Nonetheless, given the
complexity of the aetiology of autoimmune diabetes and that multiple islet molecules are
the target of autoimmune process, it is apparent that a number of various mechanisms of
beta cell destruction are operative in type 1 autoimmune diabetes.
Short-term breastfeeding and early introduction of bovine milk-based infant formula may
predispose young genetically susceptible children to progressive signs of beta cell
destruction. Although dietary proteins, particularly bovine milk proteins are among the
most investigated putative non-genetic determinants implicated in the disease
aetiopathogenesis, the timing, nature, and characteristics of environmental diabetogenic
factors remain unknown and no causal links have been established unequivocally.
Therefore, it is important to embark on identifying changes that persevere up to the
initiation of the destruction of insulin secreting pancreatic beta cells, not only to minimise
or stop the progression of the ongoing process, but also to prevent the destruction
process.
xviii
Rationale of the study
It is well recognised that every disease has both genetic and environmental causes. There
are indications that environmental factors may play a crucial role over a limited period of
time in genetically susceptible individuals to initiate the pancreatic insulin producing
beta-cells autoimmune process. The nature of triggering factors, target autoantigens, and
the effector mechanisms are not clearly understood. No causal links between
environmental triggering factors and the pancreatic autoimmune process have yet been
identified unequivocally.
Although the occurrence of autoimmune diabetes in Jordan is increasing by
approximately one percent annually, studies on diabetes in young Jordanian children
remain limited. Therefore, the rationale of this study hinges upon establishing a base-line
dietary profile, and determining possible humoral and secretory immune response to
native and heat processed bovine serum albumin modified with bovine insulin in young
Jordanian children with diabetes.
To minimise the increasing occurrence of the disease through proposing safe and
inexpensive preventative interventions of the disease, this study is directed at improving
the understanding of the aetiopathogenesis of the disease by participating in identifying
potential primary environmental triggering factors. Obviously, the health care system is a
vital element in every society to maintain people’s health of the highest standard.
xix
Objectives of the study
The objectives of the present study were:
• to study possible associations between both worldwide mean and relative change
in both imported milk excluding butter and milk protein consumption, and both
mean and global increase in the disease occurrence;
• to examine the diversity of native bovine milk proteins and the effect of
modification and heat processing on those proteins by studying changes in their
spectral and chromatographic profiles;
• to establish a baseline dietary profile of young diabetic and non-diabetic
Jordanian children aged 14 years or less in terms of breastfeeding and early infant
feeding practices;
• to determine serum IgG antibody titre levels produced to native bovine serum
albumin as well as to native and heat processed modified bovine serum albumin
with bovine insulin in diabetic Jordanians aged 14 years or less;
• to develop an a new non-invasive enzyme linked immunosorbent assay (ELISA)
for determination of saliva IgA antibody titre levels produced to both native
bovine serum albumin, and native and heat processed modified bovine serum
albumin with bovine insulin compared to serum IgG antibody titre levels in
diabetic Jordanians aged 14 years or less; and
xx
• to establish a ‘susceptibility relationship’ between ABO and Rh blood systems
and the development of type 1 autoimmune diabetes mellitus in young diabetic
Jordanian children.
xxi
Scope of the thesis
The initiation and progression to type 1 autoimmune diabetes in genetically susceptible
individuals involve a number of mechanisms of beta cell destruction. The interruption of
multiple mechanisms, rather than one single pathway, leading to the destruction of beta
cell can be the key method to prevent type 1 autoimmune diabetes. The scope of the
present study is exemplified in the following two Mind Maps. The first Mind Map shows
the main processes implicated in the natural history of type 1 autoimmune diabetes, and
the second Mind Map illustrates the design of the protein studies.
xxii
Mind map I: The natural history of type 1 autoimmune diabetes
xxiii
Mind map II: Protein studies
xxiv
Overview of the thesis
This thesis contains seven chapters compiled in a manuscript format. Chapter 1 provides
a historical scientific background and a framework needed to understand the
aetiopathogenesis of the disease. Chapter 2 studies possible association between
worldwide mean and relative change in both imported milk and milk protein
consumption, and mean and global increase in the disease occurrence. Chapter 3 embarks
on establishing baseline data on the dietary profile of Jordanian children with diabetes
aged 14 years or younger in terms of breastfeeding and early infant feeding practices.
Chapter 4 investigates susceptibility association for the proportion of ABO and Rh (D)
blood systems in young Jordanians with diabetes. Chapter 5 aims at examining the
heterogeneity of native, modified and heat processed bovine milk proteins by studying
changes in their spectral and chromatographic profiles compared to that of human breast
milk. Chapter 6 focuses on developing a new non-invasive saliva-based enzyme linked
immunosorbent assay to determine the secretory immune response as well as humoral
immune response to native, modified and heat processed bovine milk diabetogens in
young Jordanian diabetic children with regard to their early feeding practices. Chapter 7
suggests the main aspects of future studies. Chapter 8 concludes the work.
1
1 Review of the literature
Diabetogenicity of bovine milk proteins: A puzzling link
1.1 Introduction
Autoimmune diseases are serious and costly disorders. They include an organ and a non-
organ-specific diseases (Roitt et al. 2001; Ghaffar 2004). In addition to thyroiditis,
Graves disease, Addison disease and polyglandular syndromes, type 1 diabetes mellitus is
an autoimmune endocrine disease (Baker 1997). It is a worldwide life-threatening serious
and complex lifelong health condition, which results in substantial morbidity and
mortality despite great advances in disease control and treatment interventions (Rewers
and Klingensmith 1997). It ranks third among the most widespread of severe chronic
childhood diseases.
Nearly half of the world’s 200,000 individuals annually diagnosed with type 1 diabetes
are children (International Diabetes Federation 2003). The disease occurrence increases
with age in different paediatric age groups (Karvonen et al. 2000), affecting 0.5-1% of
the general population throughout its lifetime, and is marked by obvious variation
(Rewers and Klingensmith 1997; Onkamo et al. 1999). Geographic variation in risk
genotypes for T1ADM (Kukko et al. 2004) and 350-fold worldwide variation in the
disease occurrence rate among and within major ethnic and racial groups appear to follow
a global ethnic and racial distribution (Karvonen et al. 2000). This indicates an important
interplay between genetic susceptibility and environmental factors in the initiation and
development of the disease.
2
Type 1 diabetes, an immune-mediated complex chronic metabolic disorder of multiple
unknown aetiology is more dangerous than other forms of diabetes and is a direct life
threat to persons with the disease (Bogardus and Lillioja 1992; Bach 1994; Williams
1999). Even with good care, diabetic patients have an increased risk of long-term
complications which are directly associated with duration of the diabetic state (Akerblom
et al. 1997; Williams 1999). The natural history of autoimmune diabetes involves four
phases of variable durations (Rewers and Klingensmith 1997). It is strongly asserted that
every case of diabetes has both genetic and environmental causes (Rothman 2002).
There are indications that the destruction of insulin producing pancreatic beta cells may
result from a series of unfavourable interactions between environmental factors over a
limited period in genetically susceptible individuals. Having the knowledge that no causal
links between any of the putative environmental factors and the initiation of the
autoimmune process of the pancreatic beta cell have yet been identified unequivocally; it
is apparent that a range of mechanisms of pancreatic beta cell destruction are operative in
type 1 autoimmune diabetes (Kawasaki et al. 2004). A better definition of the nature of
the environmental triggering factors, the target autoantigens and the effector mechanisms
required for the autoimmune process will broaden our understanding of the disease
aetiopathogenesis (Bach 1994).
It was therefore pivotal to embark on identifying the changes that lead to the initiation of
insulin secreting pancreatic beta cell (-cells) destruction, to not only minimise or stop its
progression, but also to prevent the initiation of beta-cell destruction. The present review
attempts to examine main controversial issues linked to the role of bovine milk proteins
(BMP) in provoking the pancreatic autoimmune process in children, to explain how
evident the relationship is, and what remains to be done to control it.
3
1.2 Autoimmune diseases: General background
Differentiation between self and non-self determinants enables the body to establish a
must self-tolerance, a specific immunological non-reactivity to an antigen resulting from
a previous exposure to the same antigen, mechanisms. The breakdown of these
mechanisms triggers the initiation of an immune response against self-components. In
autoimmune diseases, products of the immune system, antibodies and effector T cells,
can cause damage to the self (Roitt et al. 2001; Ghaffar 2004).
Autoimmune diseases can be classified according to the organ or tissue involved into an
organ-specific or a non-organ-specific disease. The immune response in the former
category is directed against certain antigen(s) associated with the damaged target organ,
for example Hashimoto’s thyroiditis, primary myxoedema, pernicious anaemia,
Addison’s disease, and type 1 autoimmune diabetes. The immune response in the latter
category is directed against antigens not associated with the target organ, for example
ulcerative colitis, rheumatoid arthritis, anti-phospholipids syndrome, discoid and
systemic lupus erythematosus (Roitt et al. 2001; Ghaffar 2004).
Human and animal studies have demonstrated the importance of genetic susceptibility in
the development of the autoimmune disease particularly human leucocyte antigen (HLA)
genotypes (B, B27, DR2, DR3, DR4, DR5) (Ghaffar 2004). Although the precise
aetiopathogenesis of the autoimmune diseases remains unknown, it has been suggested
that a number of factors are involved in the aetiology of the autoimmune disease
including sequestered antigen, a void of autoreactive T cell clones, loss of suppressor
cells, and cross reactive antigens (Roitt et al. 2001; Ghaffar 2004).
4
1.2.1 Type 1 autoimmune diabetes
1.2.1.1 Definition
The concept of type 1 diabetes as an immune-mediated disease developed rapidly in the
mid-1970s (Gale 2001). Based on the disease aetiological heterogeneity and given that
not all persons with T1ADM are children at onset and not all are insulin-dependent or
having an immune-mediated form of the disease, the American Expert Committee on the
Diagnosis and Classification of Diabetes Mellitus has recommended a new aetiological
diagnostic criterion.
The first category of this four-category classification represents type 1 (insulin-
dependent) diabetes, which is further divided into two subgroups: “type 1A” diabetes for
immune-mediated diabetes, and ‘type IB’ for non-immune diabetes with severe insulin
deficiency. The second category represents type 2 (non-insulin-dependent) diabetes ‘type
2 diabetes’. This category includes idiopathic types of diabetes with insulin resistance
and without severe insulin deficiency or significant loss of cells. The third category
stands for gestational diabetes. Finally, a category for individuals with specific genetic
syndromes (The Expert Committee on the Diagnosis and Classification of Diabetes
Mellitus 2002) such as Maturity Onset Diabetes of Youth-MODY (Winter et al. 1987).
5
The aetiology of autoimmune diabetes as a form of diabetes for immune-mediated
diabetes is unknown. What is known however, is that the disease is a degenerative,
multifactorial, complex, chronic, metabolic, slowly progressive, polygenic, organ-
specific, inflammatory, autoimmune-mediated disorder induced by activated autoreactive
T cells (Van Vliet et al. 1989; Roep et al. 1990; Thorsby and Ronningen 1992; Bach
1994; Davies et al. 1994; Neophytou et al. 1996; Wilder 1998; Roep et al. 1999; Mordes
et al. 2004; Ogasawara et al. 2004; von Boehmer 2004; Nakayama et al. 2005), and
characterised by genetically determined susceptibility (Heward and Gough 1997; Rich
and Concannon 2002).
The occurrence of autoimmune markers (Kimpimaki et al. 2001), selective and
aggressive destruction of insulin-producing pancreatic -cells (Winer et al. 2003), the
reported progress to glucose intolerance and chronic hyperglycaemia, and significant
decrease in insulin receptors leads to insulin resistance, or “exceptionally” it happens due
to the presence of receptor antibodies (McCarty et al. 1996).
1.2.1.2 A conflicting nomenclature
The massive amount of data generated worldwide on diabetes is described in many
amorphous and often contradictory ways. To avoid the inevitable overlap between the
published work of various research groups as well as to avoid arriving at conflicting
nomenclatures, for example type 1 diabetes, type 1 (insulin-dependent) diabetes mellitus
and juvenile diabetes. The term type 1 autoimmune diabetes mellitus (T1ADM) will be
used throughout the present study to denote other popular ways of naming the disease,
(The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus 2002).
6
1.2.1.3 Phases
It is well established that the natural history of type 1 autoimmune diabetes involves four
phases (Rewers and Klingensmith 1997). Firstly, the pancreatic -cell pre-clinical slowly
progressive prodromal phase characterised by selective destruction of insulin-producing
pancreatic islet -cells asymptomatically until around 90% of the -cells are destroyed
through T-cell infiltrate accumulating around the islet cells and subsequently inside these
cells leading to invasive ‘insulitis’ (Saukkonen et al. 1994; Levy-Marchal et al. 1995b;
Simone and Eisenbarth 1996; Akerblom et al. 1997; Imagawa et al. 2000; Winer et al.
2003).
The first stage exists for up to 13 years, but it may be concluded in a few months
(Plotinick and Hinderson 2001), even in utero, during which autoimmune and metabolic
changes can be detected (Leslie and Elliot 1994; Knip 1997). Secondly, the clinical
diabetes onset phase. Thirdly, the transient remission phase, and finally, the established
diabetes phase (Rewers and Klingensmith 1997; Rewers et al. 2005). The presentation of
clinical diabetes at variable ages may reflect various disease progression rates rather than
different periods of exposure to possible critical environmental events (Otonkoski et al.
2000).
7
1.3 Facts and figures
Type 1 autoimmune diabetes has a worldwide distribution with substantial variation
(Karvonen et al. 2000). Approximately 124 million individuals worldwide have diabetes,
a minimum of three million have T1ADM, and the majority are children (International
Diabetes Federation 2003). The global increase in the disease occurrence seems to affect
both at low (Hungary) and at high (Sweden) risk populations (Soltesz et al. 1994)
throughout the world and marked by obvious variations (Karvonen et al. 2000). The
relative increase is predominantly higher in populations with lower occurrence (Bingley
and Gale 1989; Handelsman and Jackson 1999; Onkamo et al. 1999). A Finnish
occurrence trend analysis (1965-1996) study showed an absolute 0.67 annual increase in
the disease occurrence (Tuomilehto et al. 1999), the overall increase in the disease
occurrence calculated from pooled data based on 37 studies compiled in 27 countries
from 1960 to 1996 was three percent per year (95% CI: 2.6; 3.3, P=0.0001) (Onkamo et
al. 1999).
The occurrence of T1ADM will continue to increase above 30/100,000/year in several
other populations (Onkamo et al. 1999). The increase in the occurrence of the disease in
Finland (1987-1996) at diagnosis was the highest in children aged one to four years
(Tuomilehto et al. 1999); while, the worldwide age-specific disease occurrence rate has
increased with age, the highest was among 10-14 years-old children (P<0.0001)
(Karvonen et al. 2000). Whether that trend in disease occurrence indicates a change in the
age at disease onset instead of a definite increase in prevalence requires further
investigation (Onkamo et al. 1999).
8
Globally, the Diabetes Mondiale (DiabMond) Study Project (Karvonen et al. 2000) has
demonstrated a more than 350-fold variation in age adjusted disease occurrence rates; it
has ranged from 0.1/10,000/year in Zuny, China to 36.8/10,000/year in Finland. The vast
majority of Asian populations have a very low age-adjusted disease occurrence rate
(<1/100,000/year), whereas an intermediate disease occurrence rate was observed in
North African populations (5-9.9/100,000/year).
In Europe, the occurrence rate has ranged from 5.7/100,000/year in Latvia;
19.9/100,000/year in Norway; and 26.9/100,000/year in Sweden to approximately
37/100,000/year in both Sardinia and Finland. While the populations of North America
have a high disease occurrence rate (10-19.99/100,000/year), the occurrence rate among
South American populations has ranged from less than one to 9.99/100,000/year. The
occurrence rate of the disease in the majority of Central America and West Indies
populations has ranged from one to 9.9/100,000/year, whereas in Oceania, the age-
adjusted occurrence rate has ranged from 14.5/100,00/year in New South Wales,
Australia to 21.9/100,000/year in Canterbury, New Zealand (Karvonen et al. 2000).
Type 1 autoimmune diabetes is prevalent in the Middle East and estimates indicate that a
minimum of 100,000 individuals have the disease (Amos et al. 1997a). The estimated
annual disease occurrence was approximately 6000 with some 3000 being children below
15 years. In Jordan (1997), approximately 5200 Jordanians had the disease with 700
being children below 15 years old (Green 1997). The disease occurrence in Jordanian
children aged 0-14 years is among the lowest in the region and has remained relatively
unchanged.
9
Although the occurrence of the disease in Jordan has slightly increased from 2.8 per
100,000 in 1992 to 3.6 per 100,000/year in 1996 (Ajlouni et al. 1999), it remains among
the lowest in its region and matches that of the Arab population who live in neighbouring
Israel (2.9/100,000/year) (Shamis et al. 1997). Marked increase in the disease occurrence
of T1ADM is occurring among the Jewish population with an exceptional increase in
Yemenite Jews (18.5/100,000/year) compared to the overall occurrence rate in the Jewish
population (5.7/100,000/year) (Shamis et al. 1997). The disease occurrence has also
increased substantially in Kuwaitis. It has increased from 3.95/100,000/year (1980-1981)
(Taha et al. 1983) to 14.4/100,000/year (1992-1993) (Shaltout et al. 1995).
An obvious variation in the disease occurrence rate exists between the northern and
southern hemispheres with a positive polar equator gradient. No country below the
equator has an occurrence more than (15/100,000/year) (Karvonen et al. 1993). The
highest disease occurrence was observed in Caucasian people, and the lowest was
observed in Asia and South America. Except in Nordic countries where certain
exceptions from the overall disease occurrence pattern exist; Finland and Sardinia have
the highest disease occurrence rates and are characterised with a unique genetic
background, dissimilar environments, and are all geographically far from each other
(Karvonen et al. 2000). European countries have shown the largest difference in the
occurrence rates of the disease (Karvonen et al. 1993; Thorsdottir et al. 2000).
Considerable variation was also observed between the four countries around the Baltic
sea (Padaiga et al. 1997). Although the disease occurrence rate within-country vary in
Nordic countries, United Kingdom, America (Reunanen et al. 1988), Italy, New Zealand
and China, dissimilarity within populations was not often reported. Possible
underestimation of the occurrence of the disease in populations (i.e. Asia) with low
disease occurrence necessitate careful interpretation of these worldwide variations in the
occurrence rate of the disease (Karvonen et al. 2000).
10
The increase in the disease occurrence is not restricted to a particular ethnic group
(Onkamo et al. 1999). Worldwide geographic variation in disease occurrence rates
indicates that the distribution of ethnic groups and the degree of difference in genetic
susceptibility exists between different races. For example, Blacks and Mongoloids have a
lower disease occurrence compared to that of Caucasian populations (Reunanen et al.
1988). While European populations have the highest disease occurrence rate, it has been
reported that tropical and nontropical populations have a relatively high disease
occurrence rate marked with a relatively wider risk gradient among non-European ethnic
groups (Karvonen et al. 2000).
Furthermore, it has been demonstrated that gender variations exist between populations.
Populations with low disease occurrence rate have a female predominance (Rewers et al.
1988), whereas populations with high disease occurrence rate have a male predominance
(Padaiga et al. 1997). In addition, the occurrence rate of diabetes follows an inverse
association with mean annual temperatures. Seasonal variation of the disease occurrence
in Europe follows a sinusoidal model in both sexes and all childhood age groups, with a
peak in winter (Levy-Marchal et al. 1995a). Worldwide variation in the occurrence rates
of T1ADM among and within major ethnic and racial groups in different populations
signifies an important interplay between genetic susceptibility and environmental factors
in the initiation and development of the disease (Karvonen et al. 2000).
11
1.4 The aetiopathogenesis of autoimmune diabetes
1.4.1 Who is more prone to have the disease?
The clustering of the disease in certain families as well as twin studies have demonstrated
that genetic background plays a significant role in the occurrence of T1ADM (Heward
and Gough 1997). A genome-wide linkage screen for human T1ADM susceptibility
genes has identified more than 18 genomic regions that may harbour susceptibilities
linkage for the disease. The polygenic inheritance nature of the disease reflects a genetic
heterogeneity of T1ADM in diverse ethnicities (Davies et al. 1994).
Depending on the HLA DQ genotype in humans, the risk of developing T1ADM varies
by a minimum of a thousand fold (Nakayama et al. 2005). Human leucocyte antigen
genes class II region encoded within the major histocompatibility complex (MHC) on the
short arm of chromosome 6p21 (referred to as IDDM1) comprises the major locus
conferring disease susceptibility. An association with the insulin gene region on
chromosome 11p15 (referred to as IDDM2) has also been proposed (Davies et al. 1994;
Tosi et al. 1994). In addition to HLA gene region, it has been demonstrated that other
genes on different chromosomes are implicated in susceptibility to have T1ADM. For
instance, vitamin D receptor gene polymorphism (Motohashi et al. 2003) and
susceptibility locus on chromosome 8q24 (Sale et al. 2002) have been implicated in
susceptibility to T1ADM. Therefore, they may play a role in predicting the disease in
individuals at high-risk.
12
The MHC class II includes at least three loci: HLA-DP, DQ and DR alleles, which
comprise the major locus conferring disease susceptibility (Tosi et al. 1994). It appears
that while more than a half of the genetic susceptibility to T1ADM is related to the HLA
gene, the remaining portion is non-HLA associated. Ninety percent of patients with
autoimmune diabetes have both or either DR3 and DR4 alleles (Reece et al. 1991).
Although the association of particular combinations of DQ (A1 and B1) genes among
Caucasoid, Black and Orientals has indicated that the greatest susceptibility is in the HLA
DQ molecules (Thorsby and Ronningen 1992), a complete HLA DQ genotype should be
determined to estimate the maximum risk for developing the disease (Tosi et al. 1994).
Sequence analysis of polymorphic heterodimeric HLA-DQ gene products has suggested
that variation may also account for changes in the risk of developing T1ADM. For
example, while an amino acid variation at position 57 on the DQ beta chain and non-
aspartic acid was associated with a significant increase in risk of developing the disease,
those having an aspartic acid residue in the same position were related to disease
resistance. The presence of non-aspartic DQB1 and arginine DQA1 alleles has exhibited
the highest risk of developing the disease. On the other hand, heterozygosis at DQB1
position 57 or DQA1 position 52 was adequate to negate the significance for association
with developing T1ADM (Tosi et al. 1994).
13
Individuals who carry the HLA-DQB1*02/0302 risk genotype have a high increased
genetic risk of developing T1ADM; whereas individuals who carry the HLA-
DQB1*0302/x (x = other than *02, *0301, or * 0602) have a moderate risk (Kimpimaki
et al. 2002). A highly significant geographic variation in the risk HLA-DQ genotypes
associated with T1ADM in five different populations with low, moderate, or high-risk
HLA-DQ genotypes may explain the 30 fold hypothesised variation in the occurrence of
autoimmune diabetes across ethnic groups as well as across populations (Dorman et al.
1990; Jacobs et al. 1992; Ilonen et al. 2000; Kukko et al. 2005). Correlating these
variations with various HLA-DQ alleles particular function generate appealing insights
into the potential mechanisms triggering off T1ADM (Moustakas and Papadopoulos
2002).
Emerging findings on gene and cell replacement therapy may constitute a novel approach
for the treatment of T1ADM. It has recently been demonstrated that delivering a
pancreatic and duodenal homeobox gene 1 (PDX-1), a known active gene in the
development of insulin-producing pancreatic beta cells, to both foetal progenitor liver
cells (Zalzman et al. 2003) and adult human, non-insulin producing, liver cells (Sapir et
al. 2005) has induced a comprehensive developmental shift of these cells into functional
insulin-secreting cells. High blood glucose levels of diabetic immunodeficient mice,
recipients of the genetically engineered liver cells, were regulated for a prolonged period
of time (Zalzman et al. 2003; Sapir et al. 2005).
Although the mechanisms of disease inheritance remain controversial (Reece et al. 1991),
progress in the ability to identify persons at high risk has led to an ongoing worldwide
intensive and extensive research. Consequently, Type 1 Diabetes Genetics Consortium
(T1DGC) has been established to orchestrate the ongoing international endeavour to
identify genes that designate an individual’s risk of type 1 diabetes (T1DGC 2003).
14
1.4.2 Autoantigens cross-reactivity
In view of host immune responses, it is increasingly evident that cellular response to
autoantigens cross-reactivity may take a more significant part in disease
aetiopathogenesis than was formerly anticipated. Molecular mimicry between host and
foreign environmental factors are to a certain extent a frequent episode, and most likely
take place much more frequently than once indicated (Visvanathan and Zabriskie 2000).
A significant body of evidence indicates that an invading foreign protein has an amino
acid homology comparable to that in a host might initiate a breaking tolerance to self-
antigens. Therefore, a viral, bacterial, or food peptide similar enough to a human peptide
can cause an autoimmune reaction. For example, bovine serum albumin (BSA) and islet
cell proteins ICA 69/p69; -lactoglobulin (BLG) and retinolbinding protein; -casein and
GLUT-2 glucose transporters; conjoint autoantigenicity of proinsulin (amino acids 24-36)
and GAD65 (amino acid 506-518) (Rudy et al. 1995; Narendran et al. 2003) have been
anticipated to provide a molecular base to explain the initiation of cross-reactive immune
responses directed not only at the invading foreign protein, but also toward self-proteins
(Cavallo et al. 1996; Vaarala et al. 1998; Atkinson and Eisenbarth 2001). However, to
accurately institute the relevance of molecular mimicry in naturally occurring human
disease is, yet the most complicated phase (Oldstone 2000).
15
1.4.3 Invasion of autoreactive lymphocytes
Increased immunity to BMP is not a disease-specific immunity; it reflects associated
HLA haplotypes genetic susceptibility to increased immunity to dietary proteins that is
associated with the HLA-A1-B8-DR3-DQ2 (A1*0501, B1*0201) haplotype (Harrison
and Honeyman 1999). The exact mechanism by which the HLA-DQ molecules and other
class II molecules are directly involved in the initiation of -cell destruction remains
unclear. Nonetheless, it seems likely that -cell destruction involves antigens peptide
fragments presentation by the class II gene encode heterodimeric proteins expressed on
the surface of antigen-presenting cells such as dendritic cells, lymphocytes,
monocytes/macrophages to CD+ T-helper cells (Thorsby and Ronningen 1992;
Moustakas and Papadopoulos 2002; von Boehmer 2004).
The detection of particular -cell derived peptides by autoreactive CD8+ T helper cells
may well be a key episode in initiating the autoimmune process of the disease (Thorsby
and Ronningen 1992; Ogasawara et al. 2004; von Boehmer 2004). Activated islet-
specific T cell clones have been found to transfer diabetes to non-obese diabetic (NOD)
mice demonstrating that activation of autoreactive T cell is vital for the initiation of
autoimmune diabetes (Peterson et al. 1994; Wucherpfenning and Strominger 1995;
Peterson and Haskins 1996). It has been found also that the mutation of proinsulin
transgene (residue 16 on the insulin B chain was replaced by alanine) has abrogated the
T-cell stimulation of the major insulin autoreactive NOD T-cell clones(Abiru et al. 2000).
Vitamin D compounds may suppress the activation process by binding to vitamin D
receptor (Motohashi et al. 2003), nevertheless the activation process might be triggered
by a number of mechanisms such as molecular mimicry, viral or bacterial superantigens;
release of autoantigens during inflammation; and bystander activation (Fujinami 2001).
16
Innate immune Natural killer (NK) cells express neither T-cell nor B-cell antigen
receptors; most surface antigens detectable on NK cells by monoclonal antibodies are
shared with T cells (Roitt et al. 2001). The balance between signals transmitted by
activating receptors regulates the function of the NK cells (Cerwenka and Lanier 2001).
The identification of receptors of NK cells has enhanced our understanding of the role of
NK cells in immune responses (Yokoyama and Plougastel 2003). Studies on NOD mice
have demonstrated that CD8+ T infiltrating the pancreas express NKG2D, an activating
receptor on CD8+ T cells and NK cells; antibodies blocking NKG2D during the
prediabetic stage can completely prevent the disease progression through impairing the
expansion and function of autoreactive CD8+ T cells (Ogasawara et al. 2004). It has been
also found that the suppressor CD25+ and CD4+ regulatory T cells can suppress
autoimmune disease in vivo (Tarbell et al. 2004).
A small number of CD25+ and CD4+ regulatory T cells can reverse diabetes after the
onset of disease in animals (Tang et al. 2004). This may harness a new approach for
autoimmune diabetes cellular immunotherapy (Tang et al. 2004; Tarbell et al. 2004).
Better understanding of the mechanisms policing the migration and subsequent invasion
of autoreactive lymphocytes and other leukocytes from the blood into the pancreas
causing insulitis and beta cell destruction will offer a better understanding of the
aetiopathogenesis of autoimmune diabetes (Yang et al. 1996).
17
1.4.4 Circulating autoantibodies: Early markers
Type 1 autoimmune diabetes is characterised by the early presence of autoantibodies
against islet -cell derived proteins (Narendran et al. 2003). Cytoplasmic islet cell
antibodies (ICAs), a heterogenous group of islet autoantibodies (Eisenbarth et al. 1992;
Zanone et al. 2003), are deemed to a great extent as a significant humoral marker for the
prediction of a -cells autoimmune process, an early indicator of T1ADM (Bingley et al.
1994). In addition, a wide range of circulating autoantibodies to islet autoantigens are
anticipated as possible pancreatic autoimmunity markers of both human and NOD mouse.
The main autoantigens implicated in the disease aetiology include insulin and proinsulin
(Palmer et al. 1983; Ziegler et al. 1989; Narendran et al. 2003; Nakayama et al. 2005),
glutamic acid decarboxylase 65 (GAD65) (Baekkeskov et al. 1990; Tisch et al. 1993;
Tisch et al. 2001; Zanone et al. 2003), tyrosine phosphate related enzyme protein IA-2
(ICA512/IA-2A) (Lan et al. 1994; Kawasaki et al. 1996; Wasmeier and Hutton 1996;
Zanone et al. 2003) and heat shock protein 65 (Elias et al. 1990; Elias et al. 1991; Birk et
al. 1996). Other alternatives include increased levels of local nitric oxide, oxygen
radicals, and certain cytokines (Kolb et al. 1995).
In view of the number of autoantibodies positivity, siblings of children newly diagnosed
with T1ADM can be categorised into four stages: “no prediabetes”; “early prediabetes”,
“advanced prediabetes” and “late prediabetes” for no autoantibodies, one, two and more
than three autoantibodies respectively (Mrena et al. 1999). It has been demonstrated that
insulin autoantibodies (IAA) are the first to appear in diabetic children less than three
years of age (Yu et al. 2000) whereas IA-2A antibodies are the most specific predictor,
islet cell autoantibodies (ICA) and IAA are the most sensitive predictors for T1ADM
(Kukko et al. 2005).
18
While GAD65 autoantibodies and IA-2A autoantibodies were considered as the main
markers for the prediction of T1ADM (Zanone et al. 2003), the presence of
autoantibodies for all possible autoantigens, ICA, IAA, GAD65 and IA-2A was
considered as a stronger indicator of a late sign of an in-progress -cells autoimmune
process (Kimpimaki et al. 2001). Nevertheless, GAD65, and IA-2A had the highest
predictive value for T1ADM (Kukko et al. 2005).
Increased islet autoantibody levels among infants most likely exhibit a sign of de novo
production of autoantibodies associated with T1ADM(Ziegler et al. 1999; Hamalainen et
al. 2000). The detection of de novo produced islet autoantibodies prior to one year of age
is vital as offspring have an extremely high risk of developing the disease. Children born
to mothers with T1ADM have maternally acquired antibodies that continue for nine
months at least after birth; antibodies to GAD65 were nearly transient maternally
acquired at nine months of age, whereas IAA were often non-maternally acquired at nine
months of age (Naserke et al. 2001).
Foetal exposure of the offspring of mothers with diabetes to islet autoantibodies may be
protective to potential islet autoimmunity and, subsequently diabetes (Koczwara et al.
2004). Although most cord blood autoantibodies seem to be maternally acquired, the
presence of autoantibodies in infants cord blood has no predictive value of future
development of islet -cell autoimmunity (Stanley et al. 2004).
19
Replace testing for ICAs, as a primary screening test, by testing for either IA-2A/GAD65
or IAAs/GAD65 if combined with second line tests for ICAs and/or IAAs was found to
be more sensitive. Therefore, the sample of population required will be reduced to half of
that needed if tested for ICAs only (Bingley et al. 1999). The increased levels of
antibodies to ICAs, insulin, GAD65, and IA-2A identified more frequently at two years
of age with no particular order in the occurrence of positive autoantibodies tested have
indicated that the pancreatic autoimmune process starts very early in life among
genetically susceptible children. As such, more attention to this age-group may be
necessary in screening stages (Roll et al. 1996).
It has been found that of six children tested positive for all four-islet autoantibodies, three
children developed clinical diabetes before five years of age. These findings demonstrate
an increased risk for a probable future development of the disease among genetically
susceptible children, and therefore, exhibit a high predictive value (Roll et al. 1996). A
Finnish prospective study has proposed that while ICAs seem to be more specific, IAAs
are characterised by high sensitivity, early emergence, and high occurrence of transit
antibody positivity for the screening process among children genetically susceptible to
autoimmune diabetes during the first two years of age (Komulainen et al. 1999; Paronen
et al. 2000b; Kimpimaki et al. 2002). Autoantibodies positivity to either IA-2A or
GAD65 denotes a highly sensitive marker for -cell autoimmunity in the paediatric age
group. The perseverance of IA-2A confers a sign for residual function of -cell, hence
providing clinical and predictive significance to these autoantibodies (Zanone et al.
2003).
20
A study on dizygotic and monozygotic non-diabetic twins and non-twins compared to
diabetic patient siblings and controls has indicated that only 5.9% of the controls
(P<0.0001), 10.7% of the non-twin siblings (P<0.0001) and 20% of the dizygotic twin
siblings (P<0.05) compared to 41.5% of the monozygotic twin siblings exhibited ICAs,
IAA, GAD65, and IA-2A. Monozygotic twin siblings have shown multiple
autoantibodies positivity more frequently than the dizygotic twin siblings. Survival
analysis demonstrated that monozygotic twin siblings with HLA-DQ8/DQ2 genotype
were more likely to express positive autoantibodies than monozygotic twin siblings
without this genotype (64.2%; 95% CI 32.5-96, and 23.5% CI 7-40, respectively) at ten
years discordance (P<0.05). These findings suggest a possible predominance of a genetic
susceptibility in islet -cell autoimmune process (Redondo et al. 1999).
21
1.4.5 ABO and Rh blood groups: Signs of protective effect
A specific complex of antigens present on red blood cells genetically determines blood
group characteristics. Immunological characteristics determine and classify the
differentiation of blood by type (Watkins and Morgan 1959; Morgan and Watkins 2000).
A wide range of blood group systems is identified. The ABO blood groups system, first
introduced in 1901, is the most used blood group system. Hitherto, more than 25 major
blood group systems, a minimum of 270 red cell phenotypes and more than six hundred
red cell membrane antigens are recognised (Green 1989; Garratty et al. 2000; Hughes-
Jones and Gardner 2002).
Studies have demonstrated the implication of different blood group phenotypes in disease
pathogeneses. The ABO and Rhesus [Rh (D)] blood groups have been implicated in
increased susceptibility to certain diseases (Blackwell 1989), for instance Helicobacter
pylori and the increased risk of having peptic ulcer (Alkout et al. 1997; Alkout et al.
2000), haemolytic uremic syndrome and Escherichia coli (Blackwell et al. 2002),
elevated serum antibody titre levels to vibrio cholera (Swerdlow et al. 1994), carcinomas
(Su et al. 2001) and infertility in women (Lurie et al. 1998).
22
The ABO blood group system is the most important and most common human
alloantigen system in blood transfusion (Yamamoto et al. 1992). The genetic role in the
determination of ABO and Rh (D) blood groups (Green 1989; Garratty et al. 2000;
Hughes-Jones and Gardner 2002) exhibited extensive variation in the distribution of
ABO and Rh (D)D blood groups in different populations (May and du Toit 1989; Wagner
et al. 1995; Falusi et al. 2000; Chiaroni et al. 2004). The main ABO alleles that create
familiar A, B, AB and O blood groups exhibit broad sequence heterogeneity and
extensive sequence variation in both the coding and non-coding region of the gene, given
that a minimum of 70 ABO alleles was reported (Yip 2002).
An increased risk of developing type 1 autoimmune diabetes due to maternal-child blood
group incompatibility was established in Swedish diabetic children aged 14 years or less
(Dahlquist and Kallen 1992). Studies on type 2 NIDDM (non-insulin-dependent diabetes
mellitus) showed inconsistency in the distribution pattern of ABO and Rh (D) blood
groups. While the distribution of B blood group was elevated among Pakistanis who have
type 2 NIDDM (Qureshi and Bhatti 2003), the proportion of A, AB and Rh (D) positive
blood groups were increased among Indians who have the disease (Sidhu et al. 1988),
whereas no significant difference was established in Iranians with diabetes (Karamizadeh
and Amirhakimi 1996). These differences in the frequency of ABO and Rh blood groups
indicate racial and ethnic variations in persons indigenous to various parts of the world
(May and du Toit 1989; Wagner et al. 1995; Falusi et al. 2000; Chiaroni et al. 2004).
Furthermore, a significant relationship was found for the prevalence of fast acetylator
phenotype in diabetic adults having blood group B, given fast acetylator prevalence was
significantly higher in diabetic children than adults and non-diabetics (Pontiroli et al.
1984). While no associations were found for MN, KIDD and Lewis blood group systems
in both diabetic adults and diabetic children grouped according to insulin dose
administered, it was statistically significant for the ABO blood group in patients
receiving higher insulin doses (Kanazawa et al. 1983).
23
Numerous studies have challenged the importance of ABO blood group in the occurrence
of various diseases. For instance, the increased binding of Helicobacter pylori to
epithelial cells and increased risk of peptic ulcer (Alkout et al. 1997; Alkout et al. 2000),
duodenal and gastric ulcers (Mentis et al. 1991), haemolytic uremic syndrome caused by
Escherichia coli O157 (Blackwell et al. 2002), diarrhoea and heat-labile enterotoxin-
producing Escherichia coli (Black et al.) and elevated serum antibody titre levels to
vibrio cholera (Swerdlow et al. 1994) are associated with a higher proportion of people
of O blood group phenotype.
The ABO histo-blood system includes three antigens (ABH). Unlike people of A, B and
AB groups who can convert the H antigen into A or B antigens, people with O blood
group having guanine (258 residue) deleted in the O gene, produce an inactive protein
incapable of converting the H antigen (Yamamoto F 1990). The prevalence of
Helicobacter pylori in non-secretor people with duodenal ulcer was significantly higher
than that of non-secretor people who had gastric ulcer (Mentis et al. 1991). An
association between secretor status and the susceptibility to develop type 1 autoimmune
diabetes with elevated proportion of non-secretors among them was also demonstrated
(Blackwell et al. 1987).
Given the heterogeneity in the glycoconjugates expression associated with different
histo-blood group ABO (Yamamoto F 1990; Yamamoto et al. 1992; Yamamoto et al.
1993), it was established that the secretor gene shows signs of a protective effect
particularly in immunocompromised people (Blackwell 1989). Studying the secretor
status of humans with type 1 autoimmune diabetes and viral infections particularly
rotaviruses (Honeyman et al. 2000) and enteroviruses (Hiltunen et al. 1997; Salminen et
al. 2004), raises some further important questions with regard to the aetiopathogenesis of
the disease (Blackwell 1989).
24
1.4.6 Maturity and architecture of gut system
Few studies have been carried out on the gut mucosa of patients with T1ADM,
nonetheless the role of the gut immune system has been implicated in the
aetiopathogenesis of the disease (Westerholm-Ormio et al. 2003). Gut mucosa constitutes
the first immunoregulatory barrier (Harrison and Honeyman 1999). Not fully matured gut
mucosa is most probably temporarily permeable to intact BMP during the first months of
age (Savilahti et al. 1988; Kuitunen et al. 1994).
Mononuclear cell mass accumulates in the islets of Langerhans and obliterated pancreatic
-cells. T-cell lines isolated from the islets of children newly diagnosed T1ADM have
demonstrated a strong adherence to the endothelium of both the diabetic pancreas and to
the mucosal lymphoid tissue. Cell lines of the pancreatic or control show no adherence to
the vessels of healthy pancreas suggesting that lymphocytes derived from the mucosal
lymphoid tissue may be involved in developing the disease (Hanninen et al. 1993).
Breast milk has growth factors and cytokines, for instance insulin (Arsenault and Menard
1984; Buts et al. 1997a; Marandi et al. 2001; Shamir et al. 2001) among other
immunomodulatry factors which enhance the functional maturation of intestinal mucosal
tissues (Harrison and Honeyman 1999). Studies on artificially reared rat models have
demonstrated that bovine milk-based formulae is a primary factor provoking intestinal
overgrowth and precocious maturation of particular intestinal functions (Dvorak et al.
2000) particularly leading to structural and enzymatic alterations (Yeh 1983).
25
Human breast milk contains a higher concentration of insulin compared to that of bovine
milk (60.23 ± 41.05 and 16.32 ± 5.98 U/ml) (Shehadeh et al. 2001). No matter how
insulin is administered (i.e., systemically or orally), it can interact with intestinal mucosa
producing significant physiological consequences on gastrointestinal tract growth
(Shamir et al. 2001). However, insulin available naturally in milk is most likely to be
much more active on the gut than free orally administered insulin (Buts et al. 1997b).
Changes in dietary ingredients, microbial flora (Sharma et al. 1995b; Sharma et al.
1995a), bacterial metabolites (Fontaine et al. 1996), and enteric viral infections (Hiltunen
et al. 1997; Honeyman et al. 2000; Salminen et al. 2004) as well as interaction between
these factors may significantly change the mucosal architecture, the mucosal adaptation
(Sharma and Schumacher 1995), the epithelial cell production (Clark et al. 1981), and the
regional number of the enteroendocrine cells (Sharma and Schumacher 1996). This may
increase the gut permeability to possible foreign, dietary, and non-dietary antigens. The
interaction of the gut immune system with oral antigens may either boost or suppress the
autoimmune process that depends on the functional status of the gut immune tissue (Scott
et al. 1996a; Harrison and Honeyman 1999).
It is implicated that intraepithelial T lymphocytes of the small intestine have the ability to
mature and differentiate independently either with the presence or absence of the thymus;
therefore, the intestinal-associated lymphoid tissue could mediate the effect of
diabetogenic dietary factors (Lefrancois and Puddington 1995). Therefore, the
stabilisation of the mucosal barrier may play an important role in the maintenance of the
gut health (Kleessen et al. 2003).
26
Diabetes-prone BioBreeding (BBdp) rats exposed to oral diabetogenic diets and
immunomodulators were able to regulate the expression of T1ADM in connection with
the changes in the gut local cytokine balance (Scott et al. 2002), resolving whether
impaired mucosal architecture as well as the functionality of the immune system
predisposes to the development of type 1 diabetes or not will enhance our understanding
on the aetiopathogenesis of autoimmune diabetes in humans (Harrison and Honeyman
1999).
1.4.6.1 Uptake of macromolecules
While it has been suggested that elevated antibodies in humans may reflect de-novo
production of autoantibodies associated with T1ADM (Hamalainen et al. 2000), the
detection of circulating antibodies to BSA in young rabbits born to immunised mothers
compared to those born to unimmunised mothers has given evidence on passive
transplacental immunization in animals (Kleinman et al. 1983). Less intact BSA and
more fragments of BSA were recovered from orally immunised rats than from
unimmunised rats which demonstrates the role immunisation had on enhancing the
degradation of antigen (Pang et al. 1981).
Colostrum is essential to the growth and function of the intestine particularly just after
birth as it provides immunological defence. It has been demonstrated that it stimulates
both intestinal endocytotic and enzymatic capability, and therefore failure to administer
colostrum on time may affect the mediation of intestinal macromolecules transmission as
well as the activity of disaccharidases (Westrom et al. 1985; Jensen et al. 2001).
27
In vitro and in vivo animal studies have shown that very small amounts of intact and
polypeptide fragments of BSA may transfer across the intestine mucosa into the systemic
circulation of mature animals (Bloch et al. 1988). Animal studies have further
demonstrated the presence of immunoreactive BSA, molecular characteristics
approximated that of native BSA and porcine albumin in the circulation of young piglets
was enhanced during the early invasive stage of viral gastroenteritis (Bloch et al. 1979;
Keljo et al. 1987; Egberts et al. 1991). An increase in intestine permeability following
viral enteritis seems to take place only in Peyer’s patch-free jejunum segments and
therefore future immunological implications have to take the specialised function of
Peyer’s patch regions of the small intestine into consideration (Keljo et al. 1985).
1.4.6.2 Breastfeeding or bottle-feeding
The hypothesis of an inverse correlation between the protective effect that breastfeeding
may have on the development of T1ADM and the implication of the early introduction of
BMP at early age with the disease aetiopathogenesis has undergone worldwide extensive
investigation. Evidence on the protective effect that breastfeeding may have on the
development of the disease and the avoidance of BMP in early infancy has come from
retrospective studies (Mayer et al. 1988; Glatthaar et al. 1988,; Blom et al. 1991; Virtanen
et al. 1991; Dahlquist et al. 1992; Virtanen et al. 1992; Verge et al. 1994; Gimeno and de
Souza 1997; Kimpimaki et al. 2001). The only prospective clinical dietary intervention
trial on humans has recently suggested a possible role that dietary intervention in infancy
may have on the initiation of pancreatic autoimmunity in genetically susceptible infants
(Akerblom et al. 1999; Akerblom et al. 2005).
28
Whole population ecological studies have indicated a positive relationship between the
exposure to BMP and the development of T1ADM in 12 countries and further have
demonstrated in another 18 countries that populations having the lowest occurrence of
breastfeeding at three months of age experience the highest occurrence of the disease
(Scott 1990). These findings were supported by a previous study of 12 different countries
(Dahl-Jorgensen et al. 1991) as well as in nine regions within Italy (Fava et al. 1994).
Studies have indicated that independent of duration of breastfeeding, young age at
introducing BMP is the most vital risk factor for the development of autoimmune
diabetes (Kostraba et al. 1993; Virtanen et al. 1993; Verge et al. 1994); exposing infants
before eight days of age to dietary proteins other than breastfeeding can be a risk factor
for the development of the disease (OR 2.29; 95% CI 1.37–3.83) (Gimeno and de Souza
1997). A population-based-control study has ascertained that while increased risk of
developing T1ADM was associated with early introduction of bovine milk products
before three months of age, exclusive breastfeeding for more than three months of age
was associated with lower risk (OR 0.66; 95% CI 0.45-0.97). Increased risk was
associated with high dietary intake of BMP in the 12-month period preceding the
appearance of the disease symptoms later in childhood (OR 1.84; 95%CI 1.12-3.00)
(Verge et al. 1994).
29
Retrospective human studies were not able to demonstrate the protective effect of the
duration of breastfeeding and the delay in the introduction of BMP may have on the
development of the disease (Fort et al. 1986; Siemiatycki et al. 1989; Bodington et al.
1994; Norris et al. 1996; Meloni et al. 1997; Esfarjani et al. 2001). Meta-analysis studies
were able to establish a weak relation between the protective effect of breastfeeding and
age at introducing BMP (Gerstein 1994; Norris and Scott 1995). A critical analysis of the
clinical literature (available up to 1994) that links breastfeeding and early consumption of
BMP to the development of T1ADM has concluded that diabetic patients in case control
studies were more likely breastfed for less than 3 months of age (OR 1.43; 95% CI 1.15–
1.77), and exposed to bovine milk before the age of 4 months (OR 1.63; 95% CI 1.22–
2.17 (Gerstein 1994). This was confirmed by data from a summary of 17 case-control
studies (Norris and Scott 1995) which demonstrated that early exposure to breast-milk
substitutes (OR 1.38; 95% CI 1.18–1.61) and bovine milk-based substitutes (OR 1.61;
95% CI 1.31–1.98) before three months of age have a moderate effect on the risk of the
development of autoimmune diabetes.
No significant difference has been found for the proportion of genetically susceptible
children and autoantibodies associated with exposure to BMP. Children with
autoantibodies were breastfed for a relatively longer period of time than their controls
(Norris et al. 1996). Regardless of the HLA genotypes (Hummel et al. 2000), the
Australian AudDiab (Couper et al. 1999) and the German BABYDIAB (Hummel et al.
2000) study groups were not able to demonstrate that breastfeeding may have a protective
effect on the development of the disease. Although Sardinia has one of the highest
disease occurrence rates in Europe, and the population is at a high risk of developing the
disease, a case-control study has argued that even the mainstream proportion of children
with diabetes were breastfed. The risk of developing the disease amongst those who were
not breastfed was insignificant (Meloni et al. 1997). A Swedish and Norwegian time-
series study (1940-1980) has ascertained an inverse relationship between the duration of
breastfeeding and the development of T1ADM (Borch-Johnsen et al. 1984).
30
Similarly, a cross-sectional study screening children from families with a history of
T1ADM for beta-cell autoimmunity markers (IAA, GAD, and IA-2) has found no
significant differences for the relation to exposure to BMP between children who have
autoantibodies and their controls. Children with autoantibodies were breastfed for a
longer duration than the controls (Norris et al. 1996). These findings were supported by a
prospective study which has indicated that even in high-risk children (Relative risk: 0.91-
1.09) no potential relation has been established between the duration of breastfeeding or
exposure to BMP and the initiation of beta-cell autoimmune process (Couper et al. 1999).
31
1.5 Non-genetic determinants of the disease
The data indicate that while there is an important genetic component to T1ADM,
(Heward and Gough 1997; Rich and Concannon 2002), different putative environmental
factors may also play a major role in development of the disease. Twin studies (Verge et
al. 1995; Petersen et al. 1997; Redondo et al. 1999; Redondo et al. 2001), migrant studies
(Bodansky et al. 1992; Shamis et al. 1997), worldwide geographic variations (Karvonen
et al. 2000; Atkinson and Eisenbarth 2001), interethnic and racial differences (Shamis et
al. 1997; Karvonen et al. 2000), geographic variation in the risk genotypes for diabetes
(Kukko et al. 2004), and disease occurrence transitory tendency have indicated that
environmental factors operating in genetically susceptible individuals may play a crucial
role over a limited period of time in the disease aetiopathogenesis (Elliot et al. 1996;
Knip 1997).
Epidemiological studies have indicated a strong aetiological role of non-genetic risk
determinates for T1ADM without discriminating between low (Hungarian) and high
(Swedish) at risk populations (Soltesz et al. 1994). Certain viral infections (Yoon et al.
1979) and several dietary factors, particularly BMP, wheat and soy proteins are among
the most putative environmental triggering agents in the initiation of a pancreatic -cell
destruction process (Akerblom and Knip 1998; Norris et al. 2003; Ziegler et al. 2003).
32
In addition, toxins, particularly n-nitroso compounds, vaccine administration,
psychological stress (Atkinson and Eisenbarth 2001), age group, seasonal variation
(Levy-Marchal et al. 1995b), geographic variation (Levy-Marchal et al. 2001), sex,
maternal blood incompatibility, infectious diseases, high growth rates (Dahlquist 1994),
social status, foetal and perinatal events (Soltesz et al. 1994), and overweight (Libman et
al. 2003) are all non-genetic factors implicated in the aetiology of the disease.
Furthermore, it has been indicated that vitamin D deficiency has a potential role in the
aetiopathogenesis of autoimmune diabetes (Stene et al. 2000; Hypponen et al. 2001;
Norris 2001) and that autoimmune process targeting nervous system structures
surrounding insulin producing islet beta cells appear to be an integral early part of the
development of the disease (Winer et al. 2003).
1.5.1 Implication of viral infections
Exemplifying a model for intrauterine virus-induced diabetes, congenital rubella
syndrome is the only viral infection evidently linked to the disease (Ginsberg-Fellner et
al. 1984). Nonetheless, since there is no evidence of increased frequency for humoral beta
cell destruction in patients with congenital rubella syndrome, it has been proposed that
the development of T1ADM in response to this infection may be triggered by a
mechanism other than autoimmune mechanisms (Menser et al. 1974; Viskari et al. 2003).
Although most of the evidence on the role of viral infections in the aetiopathogenesis of
T1ADM in genetically susceptible children has come from epidemiological studies
(Dahlquist 1991), a few prospective studies have shown a significant and specific
potential association between both rotavirus (Honeyman et al. 2000) and enteroviruses
(Hiltunen et al. 1997; Salminen et al. 2003; Salminen et al. 2004), and the initiation of the
pancreatic autoimmune process.
33
Although there is a tendency for higher enterovirus infections among enterovirus positive
participants than controls, it provides no evidence to support the view that enteroviral
infection frequency is a possible risk factor for pancreatic autoimmunity among
genetically at risk children. Further investigation is required to determine whether
continuous or recurring viral infections occur more commonly among individuals with
pancreatic autoimmunity or not (Graves et al. 2003).
1.5.2 Casein: A renowned diabetogen
Although 2.5 fold increased risk of developing autoimmune diabetes is associated with
early exposure to solid foods, the evaluation of infants’ exposure to other foods in their
diet has not been studied as extensively as BMP (Rewers and Norris 1996). Studies on
BBdp rats (Elliot and Martin 1984; Scott et al. 1989; Hoofar et al. 1991; Schatz and
Maclaren 1996) have showed subsequently that diabetes occurrence is highest in animals
fed mainly on plant protein-based diet, mainly wheat and soy proteins.
It remains unknown how wheat proteins increase disease expression in genetically
susceptible animals. Scott’s group has described and partially characterized food
diabetogens in wheat protein and soy protein, which might be more antigenic than BMP.
These food antigens are linked to MHC class I expression on cells, reduced islet area,
-cell mass and a preponderance of Th1 cytokines in pancreas (Scott 2003). In the period
prior to the development of significant pancreas inflammation in BBdp rats, a number of
diet-modifiable changes have taken place in gastrointestinal tract inflammatory mediators
and permeability (Hurley 1993; Scott 2003).
34
Nordic countries have a similar genetic background as well as similar breastfeeding
practices. Although Icelanders are amongst the highest in the world-consuming bovine
milk per capita (International Dairy Federation 1993; Elliot et al. 1999), the occurrence of
autoimmune diabetes in Iceland is less than 50% of that in other Nordic countries. No
significant difference has been found between exclusively breast-feed diabetic children
and newly diagnosed diabetic children who received bovine milk-based formula and the
control group. The frequency and extent of breastfeeding were comparable. Lower
T1ADM occurrence might be explained by the significantly lower levels of A1 and B -
casein variants of Icelandic cow milk compared to that in other Scandinavian countries
(Thorsdottir et al. 2000).
Similarly, Elliot found that all analysed Icelandic milk samples have lower -casein
fraction A1 and B -casein than other milk samples from Scandinavia. Per capita A1 and B
-casein and autoimmune diabetes occurrence are significantly correlated (Elliot et al.
1999). Unlike other Scandinavian cattle breeding, Icelandic cattle have been isolated for
about 1100 years leading to less frequent gene coding for A1 and B -casein (Lien et al.
1999).
Studies on NOD mice have demonstrated that whole casein diabetogenicity is produced
from -casein A1 A1 cows; nonetheless, casein produced from -casein A2 A2 -casein
cows did not. This effect may be due to the presence of a bioactive -Casomorphin-7
peptide (BCM-7) that has opioid characteristics including a dramatic inhibitory effect on
immune cell function in both susceptible human and animals. BCM-7 is released from the
-casein variant (A1 and B) with a histidine at position 67. However, it cannot be released
from of the -casein variant with a proline at position 67 (Elliot 1989; Elliot et al. 1997;
Elliot et al. 1999).
35
Unlike -casein A1 60-67, bovine -casein A2, and human -casein contain a proline
amino acid at a corresponding position in their sequence. It is likely due to this proline
peptide that an equivalent to BCM-7 cannot be formed upon digestion of human -casein
(Elliot et al. 1997). Specific proliferation of T lymphocytes with bovine casein in persons
with T1ADM may account for the response of pancreatic -cells cellular and humoral
immune response to -casein (Cavallo et al. 1996). This explains the specificity of the
connection between the occurrence of diabetes and the consumption of a number of -
casein variants (Elliot et al. 1999). However, casein hydrolysate-based bovine milk
formula was found to be highly effective in preventing autoimmune diabetes in NOD
mice (Karges et al. 1997).
Human preventive studies have shown that the elimination of dietary gluten for six
months does not decrease autoantibody titres associated with autoimmune diabetes
although it may have an advantageous outcome on the protection of –cells in diabetic
patients (Hummel et al. 2002; Pastore et al. 2003). Whether casein hydrolysate-based
bovine milk formula is effective in preventing T1ADM in animals or not is still
controversial. While an animal prevention study has found that casein hydrolysate-based
bovine milk formula was highly effective in preventing autoimmune diabetes in NOD
mice (Karges et al. 1997), another animal study on BBdp rats has concluded that protein
free-based diets do not protect rats from developing diabetes (P<0.05). Rats fed on free
amino acid-based diet showed a slight delay of developing the disease. Nevertheless, that
concurred with a reduction in weight gain compared with rats fed on standard diet
(Simonson et al. 2002).
36
A leading prospective study of T1ADM at high-risk population (BABYDIAB) has found
that feeding newborn children with gluten-containing foods prior to three months of age
was accompanied with a significant increase in islet autoantibodies risk (Adjusted hazard
ratio 4.0; 95% CI 1.4-11.5; P=0.01) compared to those exclusively breast-fed until three
months of age. The duration of exclusive breastfeeding or shortened total breastfeeding
did not increase the risk of developing islet autoantibodies significantly (Ziegler et al.
2003).
The American Diabetes Autoimmune Study in the Young (DAISY), established in 1994
to investigate newborns with and without a family history of type 1 DM, has concluded
that apart from the type of cereals introduced, the risk of islet autoimmunity was higher in
infants aged zero to three months and those aged seven months when first given cereals
than those aged between four to six months. This has pointed to a possible window of the
introduction of dietary cereals in early infancy where primary exposure may add to the
risk of developing islet autoimmunity (Norris et al. 2003). Nevertheless, the German
BABYDIAB prospective study of high-risk population established in 1989, and the
DAISY studies were not able to offer adequate proof to propose that feeding an infant on
cereals triggers diabetes. The link between early infant diet and the development of
autoantibodies produced against the pancreatic -islet cells remains controversial
(Atkinson and Gale 2003).
37
1.5.3 Bovine serum albumin: A strong candidate
A number of factors have prompted researchers to investigate bovine milk proteins
particularly bovine serum albumin and bovine insulin as a putative environmental factors
implicated in the destruction of the pancreatic beta cell. These factors include: first, the
exposure of children to bovine-based formulae as their first foreign dietary supplement;
second, extreme increase in the disease occurrence observed among children under 15
years old at diagnosis (Onkamo et al. 1999; Gale 2002); and finally, increased antibody
titre levels produced against BMP in diabetic children compared to that produced in the
controls have placed BMP particularly BSA under intensive and extensive investigation
(Karjalainen et al. 1992b; Saukkonen et al. 1994; Levy-Marchal et al. 1995a).
Albumin is the most abundant protein in the blood circulatory system. Bovine serum
albumin constitutes only one per cent of whole bovine milk (Carter and Ho 1994;
National Health and Medical Research Council 2003). The predominantly alpha-helical
polypeptide globin structure of BSA (Carter et al. 1989) consists of 607 amino acids
(66430.3 Daltons) and seventeen disulphide bridges which form nine loops creating three
domains (Feldhoff and Peters 1975; McLachlan and Walker 1977; Carter et al. 1989;
Hirayama et al. 1990; Carter and Ho 1994). Bovine serum albumin amino acid sequence
of peptide (1-24) has a high degree of similarity with that of human serum albumin. They
only differ at four amino acid sites (2 [Threonine-Alanine], 6 [Isoleucine-Valine], 18
[Histidine-Asparagine] and 21 [Glycine-Alanine] respectively) (Bradshaw and Peters
1969).
38
1.5.3.1 Humoral immunity
Humoral immunity has been widely implicated in the initiation of the pancreatic -cells
autoimmune process. Studies have suggested that BMP may constitute putative triggering
factors of the autoimmune response, attributing to antibody production and succeeding
the destruction of -cells in newly diagnosed children with the disease (Karjalainen et al.
1992a; Savilahti et al. 1993; Saukkonen et al. 1994; Levy-Marchal et al. 1995a;
Saukkonen et al. 1995; Ahmed et al. 1997). The most direct evidence on humoral
immunity to BMP has come from a Finnish family retrospective study, which has
demonstrated that IgG antibodies to BSA, but not other BMP, were elevated in all newly
diagnosed children. Most of the antibodies were age dependent and specific to the
ABBOS epitope (pre-BSA position 153-169 peptide).
Since ABBOS shares an epitope with p69 –cell surface protein may induce a cross-
reactive immune response, ABBOS peptide has been implicated as a possible triggering
factor of the autoimmune process (Karjalainen et al. 1992a). Studies have demonstrated
that while IgA antibodies produced against BSA were elevated, IgM antibodies to BSA
were not (Karjalainen et al. 1992a; Levy-Marchal et al. 1995a; Saukkonen et al. 1995;
Saukkonen et al. 1998a). It has been observed that bovine milk protein hydrolysates are
competitively inhibiting ABBOS binding to antibody. Therefore, to warrant that
hydrolysed infant formulas are free of cross-reactive ABBOS antibody binding sites, a
significant variation in residual reactive sites among hydrolysed BMP requires specific
identification (van Beresteijn and Meijer 1996).
39
While elevation in anti-BLG IgG antibodies was widely demonstrated (Savilahti et al.
1988; Savilahti et al. 1993; Saukkonen et al. 1995; Ahmed et al. 1997; Saukkonen et al.
1998a), IgA antibodies to BLG were considered independently associated with the risk of
developing the disease (Dahlquist et al. 1992; Saukkonen et al. 1995). Similarly, IgA
antibodies to whole BMP in recently diagnosed young diabetic children were high
(Savilahti et al. 1988; Dahlquist et al. 1992; Savilahti et al. 1993; Saukkonen et al. 1998a)
and were also considered as an independent disease risk determinant. A few studies were
not able to establish associations for anti-BSA antibodies in recently diagnosed children
(Ivarsson et al. 1995; Ahmed et al. 1997).
Antibodies to BSA in HLA-DR3 recently diagnosed children were higher than that in
both HLA-DR3 non-diabetic children and non-HLA-DR3 diabetics (Krokowski et al.
1995). Identical diabetic sibling pairs for HLA-DQB1* (0201, 0302, 0602/03) alleles
associated with the risk or protection from diabetes have shown elevated antibodies to
BMP than their HLA-DQB1 matched sibling control group which was an independent
risk factor regardless of feeding practices (Kostraba et al. 1993; Saukkonen et al. 1998b).
Nevertheless, a French study was not able to establish a significant association between
antibodies to BSA and HLA-DQB genotype (Levy-Marchal et al. 1995a).
40
1.5.3.2 Cellular immunity
Cellular immune mechanisms are implicated as the main mediator of the pancreatic -
cells autoimmunity process. A dichotomous relation has been established between
autoantibodies circulating to glutamic acid decarboxylase and peripheral-blood T-cell
proliferation in at-risk relatives of persons with diabetes (Harrison et al. 1993). Mean
stimulation index of proliferation of T-cell to -casein, but not to BSA or BMPS is
significantly higher in newly diagnosed children with diabetes than in non-diabetic
children or patients with autoimmune thyroid disease (Cavallo et al. 1996).
Cellular responsiveness of peripheral-blood mononuclear cells was not observed to BSA
or common food antigens. Enhanced cellular response with BLG in newly diagnosed
diabetic children was significant and was not associated with HLA-DQB1* TIAD risk
allele (Vaarala et al. 1996). No cellular response was observed in HLA and/or age
matched at-high risk controls, but peripheral lymphocytes T-cell produced from newly
diagnosed diabetic children were significantly responsive to insulin B chain 9-23 amino
acid residue (Alleva et al. 2000).
A few studies have not been able to demonstrate either cellular response (Atkinson et al.
1993) or humoral response to BSA or ABBOS peptide (Atkinson et al. 1993; Ivarsson et
al. 1995). Elevated cellular and humoral immune responses to BSA among patients with
other autoimmune diseases (chronic autoimmune thyroiditis, rheumatoid arthritis, and
systemic lupus erythematosus) and at the risk of developing diabetes may defer its
possible role as a diabetogenic factor (Atkinson et al. 1993).
41
One study has indicated that cellular immunity and humoral immunity to a particular
food antigen is not limited to diabetic persons. Cellular responsiveness to � and -casein
found higher in both diabetic and non-diabetic children had HLA-DR3 alleles, with no
difference in the cellular response in either groups (Sarugeri et al. 1999). Lack of
relationship between antibodies to BSA and ICA may indicate that humoral immunity to
BSA may be a sign of an unspecific and a broad defect in the course of immunologic
tolerance (Atkinson et al. 1993; Luhder et al. 1994; Ivarsson et al. 1995).
1.5.3.3 Secretory immune system
Secretory IgA is one dominant characteristic humoral factor of the local immune system
in the body secretions. Saliva, for instance may act as a first-line of defence against local
infections as well as prevent access of foreign antigenic factors to the immune system
(Roitt et al. 2001). The levels of sIgA, following the development and maturation of the
salivary glands, has a parabolic rapport with age (Smith et al. 1987; Ben-Aryeh et al.
1990; Kugler et al. 1992; Percival et al. 1997). Thus, sIgA has been widely used as a
biomarker in biobehavioural studies (Ben-Aryeh et al. 1984; Worthman et al. 1990;
Hertsgaard et al. 1992; Shirtcliff et al. 2000; Shirtcliff et al. 2001), determination of
toxins and pollutants (Gilfrich et al. 1981; Bauer et al. 1983), and as a diagnostic tool
(Behets et al. 1991; Ciclitira and Ellis 1991; Kozlowski and Jackson 1992).
42
The synthesis of sIgA is directly associated with foreign factors, given a high sIgA
synthesis rate and a short half-life, and associated directly to the activation of T and B
cell activation as well (Smith et al. 1987; Paul 1993). Although, antibody immune
responses at various mucosal effector sites are antigen type and dosage dependent, the
whole mucosal immune system faraway from the sensitisation site can be engaged by the
sensitisation of a specific site of the system that may well present a thorough view of the
performance of the entire mucosal immune system (Externest et al. 2000). A salivary-
based ELISA has been used in the determination of antibodies produced against gliadin
in patients with celiac autoimmune disease (Ciclitira and Ellis 1991; Fasano et al. 2003).
Given that salivary samples collection is a non-invasive, stress-free, simple method, and
can be easily collected and stored (James-Ellison et al. 1997) it may, therefore constitute
an alternative system to detect humoral immunity in diabetic children.
1.5.4 Bovine insulin: The contender counterpart
There is a growing evidence indicating that insulin and its precursor proinsulin
particularly the 9-23 immunodominant residue peptide of the B chain are widely
anticipated as potential autoantigens capable of initiating the destruction of insulin
secreting pancreatic beta cells sanctioning other pancreatic proteins to become the
primary target leading to the disease (Wegmann et al. 1994; Abiru et al. 2000; Alleva et
al. 2000; Wong et al. 2002; Kent et al. 2005; Nakayama et al. 2005). It has been also
anticipated that insulin becomes a target ensuing the initiation of the autoimmune process
by another autoantigen (Atkinson et al. 1986; Srikanta et al. 1986; Vaarala et al. 1998;
Komulainen et al. 1999; Alleva et al. 2000; Kimpimaki et al. 2002; Juvenile Diabetes
Research Foundation International 2005b).
43
It has been recently demonstrated that breeding a knockout of the insulin 1 gene and a
knockout of the insulin 2 gene into NOD mice has prevented most progression to diabetes
in the latter and has accelerated the development of the disease and the development of
the insulin autoantibodies in the former (Nakayama et al. 2004; Nakayama et al. 2005),
however insulin knockouts were not able to prevent all pancreatic autoimmunity, but
were able to prevent islet-specific autoimmunity.
Neither was deletion of the GAD65 gene able to affect the disease occurrence in NOD
mice indicating that GAD65 expression is not a primary autoantigen for the development
of the disease (Kash et al. 1999). These findings indicate that insulin and proinsulin
molecules have a target sequence of the pancreatic autoimmune process that bolsters the
hypothesis that, if primary autoantigens do exist for specific autoimmune process,
proinsulin is a primary autoantigen of the NOD mouse. This constitutes a novel ground
for possible disease prevention by the application of deletion therapy (Nakayama et al.
2005).
The intensity of autoimmune T-cell mediated response triggered by a certain autoantigen
is proportional to the level of that antigen expressed in the target tissue. It has been
suggested that islet-specific T-cell mediated immune response candidate autoantigens in
persons with autoimmune diabetes are important in the destruction of the pancreatic beta-
cells (Van Vliet et al. 1989; Roep et al. 1990; Neophytou et al. 1996; Roep et al. 1999).
44
In humans, the expression of insulin is adequate to target a tissue to T-cell mediated
pathology. Cellular immunity to proinsulin, peptide (amino acids 24-36) was virtually
limited entirely to individuals at risk of developing the disease. Newly diagnosed children
with diabetes developed a substantial cellular immune response to 9-23 immunodominant
amino acid region of the insulin B chain (Alleva et al. 2000). Recent findings support the
hypothesis that insulin may be a primary autoantigen. It has been demonstrated that a
high level of T-cell clonal expansion was observed in pancreatic lymph nodes from
diabetic patients with HLA-DR allele that was not observed in controls. Clonally
expanded CD4+ clones recognised specifically the insulin A chain 1-15 amino acid
residue.
A prospective cohort study has demonstrated that cellular and humoral immune response
to bovine insulin at three months of age was higher in infants exposed to bovine milk-
based formula than that in infants exclusively breastfed. At three months of age, infants
exposed to bovine milk-based formula showed higher anti-bovine insulin IgG antibodies
levels than in infants who received casein hydrolysate-based formula, but there was no
difference in cellular response among both groups (Paronen et al. 2000b). Early exposure
of infants to bovine milk proteins resulted in decreased levels of antibodies produced to
bovine insulin at 18 months of age. Although dietary antigens present in human breast
milk may have a role in early tolerance induction resulting in immunisation, that role
remains intriguing and requires further investigation (Paronen et al. 2000a).
45
A human workshop on T cell has pointed out that populations diversity and inability to
differentiate between non-diabetic controls and newly diagnosed patients, and difficulties
in identifying and preparing candidate autoantigens; as well as interlaboratory variation
and availability of suitable assays for standardised use may explain conflicting findings
and problems associate with autoreactive T cell assays in T1ADM (Roep et al. 1999). It
remains difficult to identify which antigens serve as targets for beta-cell destruction in
humans with autoimmune diabetes and whether they are the same as those involved in
NOD mice (Wegmann and Eisenbarth 2000).
Insulin is a 5,808 Daltons (Harfenist and Craig 1952; Sanger 1958), 51 amino acids, dual-
chain hormone; chain A (21 amino acids) and B chain (30 amino acids). Insulin is the
main product of the pancreas islet -cells and is not produced in other tissues in
hormonally significant quantities (Wegmann and Eisenbarth 2000). Also, it is necessary
for the intestinal growth and maturation (Shamir et al. 2001) as well as for the control of
several cellular metabolic courses (Buts et al. 1997a).
Human breast milk contains a stable (5 ng/ml) concentration of immunoreactive insulin
(Harrison and Honeyman 1999), whereas bovine milk contains (327 ng/ml) in the first
milking. Variation in bovine milk insulin is considerable, particularly during the first
period of lactation; bovine milk insulin concentration fell to approximately 50% within
the first day postpartum and it reaches a stable concentration on day seven postpartum
(46 ng/ml; about 14% of its first milking concentration). Bovine milk insulin
concentration is almost 100 times higher than that of serum insulin, which suggests a
specific mechanism of transferring insulin from serum to milk (Aranda et al. 1991).
Immunisation to insulin arises primarily from exposure to bovine insulin and that may
play a key role in the pancreatic autoimmune process (Vaarala et al. 1999; Paronen et al.
2000b).
46
Although human, bovine and porcine insulin share high degree of sequence homology,
they differ at certain amino acid residues. Human insulin differs only at one B30
(Threonine) amino acid residue from that of porcine insulin (Alanine), whereas human
insulin differs at three residues from that of bovine insulin (A8 [Threonine replaced with
Alanine], A10 [Isoleucine replaced with Valine] and B30 [Threonine replaced with
Alanine], respectively). It has been demonstrated that two-fold increase in the exposed
area of the bovine insulin compared to that of the porcine insulin analog was
accompanied with a significant reduction of the hydrophilic area on the same insulin
hexamer surfaces, given changes in amino acid residues (Yip et al. 1998).
The absence of prominent differences between human and porcine insulin of similar
purity in terms of their antigenicity (Larkins et al. 1986) is likely due to the closer
structure of porcine insulin to that of human insulin (Little et al. 1977). The discernible
variation between bovine insulin, human insulin and porcine insulin indicates that the
physical characteristics of insulin rather than the variation in the amino acid sequence
may determine its immunogenicity (Kurtz et al. 1985). A comprehensive literature review
available up to 2002 aiming at assessing the effect of different insulin species has
indicated that antibodies produced against insulin did not show relevant dissimilarities
between most of the investigated purified porcine insulin and semisynthetic human
insulin although the methodological quality of most studies was poor (Richter and Neises
2004). Nonetheless, it has been demonstrated that conventional bovine insulin contains
immunogenic amounts of proinsulin to humans when administered to individuals with
T1ADM and is more immunogenic than purified pork insulin (Kawazu et al. 1979; Kurtz
et al. 1980).
47
Bovine insulin is a small protein of 5,808 Daltons (Harfenist and Craig 1952; Sanger
1958) and cannot elicit an antibodies immune response by itself; this requires coupling to
a carrier protein such as BSA (Harlow and Lane 1988) of 66430.3 Daltons (Carter and
Ho 1994). Modification of both BSA (Teale and Benjamin 1976) and bovine insulin
(Speth and Lee 1984) with various binding molecular as well as heat processing have a
substantial effect on their immunogenic response (Hanson and Mansson 1961;
Karjalainen et al. 1992a; Alting et al. 1997). These modifications either alter regions of
the immunogen to present better sites for T-cell binding or expose new epitopes for B-
cell binding (Harlow and Lane 1988).
The application of the suitable heating procedure may play a role in determining the
diabetogenicity degree of certain dietary constituents (Strand 1994). Studies on the
thermo lability of insulin and its role in the diabetogenicity of insulin remain scarce.
Nonetheless, its widely accepted that for insulin to maintain its viability, insulin-
dependent diabetic patients are encouraged not to store insulin vials under extreme
temperatures (<2 or > 30 ºC) (American Diabetes Association 2002).
Furthermore, insulin suspensions exposed to temperatures more than 25 ºC for extended
periods may become difficult to homogenise, whereas exposing insulin to 50 ºC or more
leads to the coagulation of the insulin suspensions (Brange 1987). Studies on the thermo
lability of insulin and its role in altering the immunogenicity of the insulin molecule
remain scarce. It is, therefore, pivotal to examine the effect of both modification and
thermal processing on the diabetogenicity of insulin, given that canned infant milk
formulae undergo a wide range of both large and home scale heat processing (Pisecky
1997).
48
1.6 Proteins modification: Enhanced immunogenicity
Albumin is the most copious protein of the blood circulatory system. Bovine serum
albumin constitutes only one per cent of whole bovine milk (Carter and Ho 1994;
National Health and Medical Research Council 2003). The predominantly alpha-helical
polypeptide globin structure of BSA (Carter et al. 1989) consists of 607 amino acids
(66430.3 Daltons) and seventeen disulphide bridges which form nine loops creating three
domains (Feldhoff and Peters 1975; McLachlan and Walker 1977; Carter et al. 1989;
Hirayama et al. 1990; Carter and Ho 1994). Bovine serum albumin amino acid sequence
of peptide (1-24) has a high degree of similarity with that of human serum albumin. They
only differ at four amino acid residues (2 [Threonine-Alanine], 6 [Isoleucine-Valine], 18
[Histidine-Asparagine] and 21 [Glycine-Alanine] respectively) (Bradshaw and Peters
1969).
Native BSA binds heterogeneously with detergents involving many sites of varying
affinity; given native BSA has four distinct binding sites for detergents. The acceptor
activity of BSA with detergents occurs without conformational changes and results in
major denaturation (Nozaki et al. 1974). The modification of arginine at its amine
terminal first enhances the acceptor activity of BSA significantly (Leibowitz and Soffer
1971). Glycation reaction with BSA involves reversible and irreversible formation of
modified BSA which occurs mainly on arginine residues (Lo et al. 1994). The increased
levels of advanced glycation end products are implicated in diabetes vascular
complication (Thornalley et al. 2003).
49
Restricted antibodies population to isolated different antigenically active BSA fragments
representing BSA on both domains and subdomains showed that some regions of the
molecule refold more rapidly than other domains. However, the entire polypeptide chain
was not necessary for the reformation of native BSA structure indicating a possible
interdomain influence. It has been demonstrated that when the antigenic sites of modified
BSA with methoxypolyethylene glycol were destroyed, modified BSA did not induce any
immune responses compared to a strong native albumin immune response (Teale and
Benjamin 1976).
Specific protein phosphatases may have a certain binding site or sites for porcine insulin
and that modified insulin with phosphatases may enhance catalytic activity. Porcine
insulin was capable, in vitro, of activating and binding to purified rabbit skeletal muscle
phosphatase. It was temperature, time, and concentration dependent and was not
mimicked with either insulin A chain or insulin B chain or BSA. The activation
phenomena may be prevented by insulin anti sera (Speth and Lee 1984). The kinetics of
tryptic hydrolysis of the arginyl and lysyl bonds at bovine insulin B chain (B22, and B29)
have demonstrated that the zinc-free bovine insulin B chain folded into the secondary and
tertiary characteristic of the bovine insulin. The basic residues of insulin become more
unavailable to tryptic hydrolysis than the matching residues in the oxidised chain (Wang
and Carpenter 1969). Substantial interactions with other ingredients in food systems may
take place, resulting in modified behaviour of the proteins (Kinsella and Whitehead 1989)
and therefore may play a crucial role in our understanding toward the disease
aetiopathogenesis.
50
1.7 Thermal processing: Altered immunogenicity
Human breast milk is the perfect initial food to meet the rapid growth infants experience
during their first year. If certain health or personal conditions in either mother or infant
prevent breastfeeding, bovine milk-based infant formula is the formula of choice (Wold
and Adlerberth 2000; Oddy 2002). Mature human milk differs considerably in
composition compared to that of other species. It particularly contains protein and sodium
chloride at very low concentrations and high concentrations of lactose and
oligosaccharides (National Health and Medical Research Council 2003).
In spite of all efforts to duplicate human milk, it nevertheless remains challenging. While
emphasising “closeness to breast milk”, a report has warned against continuous
manufacturers’ violation of the international code on the marketing of human breast milk
substitutes (Mayor 2004). Requirements to assimilate human milk high lactose content
have made infants formulas as one of the most difficult-to-dry foods. Modern infant
formulas require both large scale and home food processing that can bring about extreme
alteration in the chemical composition of food products (Rechcigl 1982).
Bovine milk is a complex polydispersion system. Individual components of milk have
various impacts on its physical properties. Therefore, the variation in the composition of
the milk system has a significant impact on its properties and thus on the properties of the
final product (Pisecky 1997). Bovine milk contains more than 25 separate proteins, 80%
of them are heterogeneous casein occurring as a micellar complex and whey proteins
form the remaining (20%) of the milk protein components (Whitney 1988). Heat
processing is the most commonly used in canned bovine milk-based foods processing
technologies and is attributing to some undesirable possibly severe quality defects in
bovine milk, a heat sensitive liquid, including the denaturation of its proteins (Pisecky
1997).
51
The effect of heat depends among other factors on pH (Law and Leaver 2000), heating
temperature, presence or absence of co-denaturation associate, and industrial thermal pre-
treatment (Bertrand-Harb et al. 2002). Therefore, it is critical to identify possible
conformational changes of milk proteins resulting from heat processes (Relkin 1996).
Bovine milk proteins are implicated in causing allergenic reactions in infants (Goldman
et al. 1963; Kletter et al. 1971b; May et al. 1982; Bahna and Gandhi 1983) as well as in
inducing a specific immune response in humans (Bahna and Gandhi 1983; Karjalainen et
al. 1992b; Saukkonen et al. 1994; Levy-Marchal et al. 1995a). The immunogenicity of
antigens can be often substantially altered by slight changes in the structure of antigen;
many molecules can be made more immunogenic by heat denaturation, given that protein
antigens aggregate caused by heat processing are usually more immunogenic.
This treatment can change the structure of many compounds, particularly proteins, and
expose new epitopes. Even small epitope structural changes can prevent antigen
recognition and can prevent antibodies differentiating between conformations of protein
antigens. These modifications either alter regions of the immunogen to present better
sites for T-cell binding or expose new epitopes for -cell binding (Harlow and Lane
1988; Harlow and Lane 1999).
Immune electrophoretic studies on bovine colostrum and other milk products showed that
BSA (a highly folded tertiary conformation of 608 amino acid hydrophilic whey protein
that renders protection to casein) (Feldhoff and Peters 1975; Hirayama et al. 1990;
Pisecky 1997) is destroyed at 70 to 80ºC for fifteen minutes. Beta-casein, a 209 amino
acids molecule exists in an open structure, and beta-lactoglobulin are very stable. They
retain their total solubility and antigenicity even under extreme heat treatment for
extended periods of time (120˚C/15 min and 100˚C/15min, respectively) (Hanson and
Mansson 1961).
52
Radial immunodiffusion assay for BSA has found that bovine-based infant formulae,
ultra-high temperature (UHT) milk, ultrapasteurised milk, and milk boiled at elevation (<
6400 feet) were free of BSA. Heat processing at various temperatures and exposure time
(84, 85 and 86ºC/>4 min, 89ºC/>3 min, 90 ºC/90 Seconds and 94ºC/60 Seconds) and
have rendered milk free of BSA (Strand 1994). Thermal processing of canned infant
formulae as well as liquid formulae at different temperatures and exposure time has
rendered milk free of BSA (Monte et al. 1994; Strand 1994).
The physical nature of the antigen affects its immunogenicity (Saukkonen et al. 1994).
Serum antibody titre levels produced against pasteurised bovine milk were the highest
compared to antibodies produced against heat processed bovine milk proteins (May et al.
1982). Antibodies to BSA are common in the general population, given the detection of
antibodies associated with diabetes is technically demanding (Levy-Marchal et al.
1995a). Although bovine serum albumin has many epitopes capable of inducing a broad
range of high and low affinity general antibodies population not only to conformational
epitopes, but also to denatured BSA it can be also recognised by the general antibodies
population (Karjalainen et al. 1992b; Savilahti et al. 1993; Saukkonen et al. 1994).
Heat processed milk contains active (Saperstein and Anderson 1962), but less antigenic
BLG and alpha-lactalbumin (ALA) in both liquid and powder form and results in less
frequent reactions than pasteurised milk (Crawford 1960). Thermal processing of
enzymatic hydrolysates of the whey protein at 90ºC/10 minutes has reduced its
antigenicity significantly (Boza et al. 1994). Individuals sensitive to BSA may tolerate
heat processed bovine milk proteins; tolerance was reduced for individuals responsive to
other protein fractions (Crawford 1960). Animal studies (Kilshaw et al. 1982; Heppell et
al. 1984) have shown that unlike pasteurised whey proteins or skimmed milk, antibodies
produced to extremely heat treated whey proteins (100 or 115ºC for 30 minutes) were
very low or undetected. If detected antibodies were residual casein specific that indicates
an extensive denaturation of these proteins.
53
1.8 Prevention trials: Pros and cons
Most of the evidence on the diabetogenicity of the dietary proteins particularly bovine
milk proteins has come from retrospective studies, yet these findings remain widely
discrepant and controversial. Discrepancy in these findings in both human and animal
retrospective studies on autoimmune diabetes indicates variations in the diabetogenicity
of milk proteins. In addition, this discrepancy is a result of a number of factors. They
include: (1) the actual exposure to diabetogenic factors in certain populations but not in
others (Norris et al. 2003), (2) timing of exposure to foreign environmental factors
(Verge et al. 1994; Kimpimaki et al. 2001) (3) study design, (4) analysis methods
(Karjalainen et al. 1992b), and (4) differences between populations examined (Esfarjani
et al. 2001).
The secondary prevention trials of diabetes are models to obtain large cohorts for reliable
evaluation of the secondary prevention. The European Nicotinamide Diabetes
Intervention Trial (ENDIT), a multicentre five-years randomised double-blind placebo-
controlled intervention trial of nicotinamide, has concluded that nicotinamide was
ineffective at preventing or delaying the clinical onset of T1ADM in individuals with a
first-degree family history of the disease (Gale et al. 2004).
Similarly, the American Diabetes Prevention Trial-type 1 (DPT-1), a double-blind,
placebo-controlled clinical trial, has concluded that the administration of oral insulin was
ineffective in delaying or preventing T1ADM in first and second-degree non-diabetic
relatives at risk for diabetes (Skyler et al. 2005). Findings of secondary prevention trials
on the role of administration of either oral or intranasal insulin may have on the
destruction of the pancreatic beta cells were contradictory.
54
The Nasal Insulin in Prevention of Type 1 Diabetes Trial, a randomised controlled
clinical trial under the umbrella of the Finnish Diabetes Prediction and Prevention
(DIPP), has indicated that a decreased response of the first-phase insulin can be an initial
phenomenon in the course of prediabetes in young children (Kupila et al. 2001; Keskinen
et al. 2002; Juvenile Diabetes Research Foundation International 2005a) and the
Melbourne Nasal Insulin, a crossover randomised controlled clinical trial, has suggested
that intranasal insulin does not accelerate the loss of beta-cell function in individuals at
risk for T1ADM and induces immune changes consistent with mucosal tolerance to
insulin (Harrison et al. 2004). Both the Italian IMDIAB study group (Pozzilli et al. 2000)
and the French Diabetes Insulin Oral Group (Chaillous et al. 2000), multicentre
randomised controlled clinical trials, have demonstrated that the administration of oral
insulin initiated at the clinical onset of T1ADM is not able to prevent the deterioration of
the pancreatic beta-cells.
Reviews of the designs and inclusion criteria of the secondary prevention trials of
diabetes have indicated that there were pros and cons to the initiation of each of these
studies (Knip and Akerblom 1998; Rosenbloom et al. 2000; Schatz 2002). Therefore,
there are lessons to be learned as negative outcome in these studies is reported (Pozzilli
2002). The establishment of a number of coordinated secondary prevention intervention
trials remain an appropriate response to this life-long overwhelming disease (Gale et al.
2004; Harrison et al. 2004).
55
Primary prevention trials aiming at warding off the disease from occurring in high-risk
populations prior to any detectable indications of prediabetes islet -cell autoimmunity
take place constitutes a main challenge to the ongoing research efforts. Two human
primary prevention trials have been established: the Trial to Reduce IDDM in the
Genetically at Risk (TRIGR) and the Type 1 Diabetes TrialNet. The former is the first
dietary intervention primary prevention study warranted to prove or disprove whether
avoidance of bovine milk-based formula has an effect on the development of T1ADM in
children.
This trial has shown that substituting BMP with hydrolysed casein-based infants formula
over the first six to eight months of age might protect genetically susceptible children
from developing pancreatic -cell autoimmunity (Akerblom et al. 1999; Akerblom et al.
2005). The latter is a multicentre study group established recently to conduct natural
history, preventive research and study intervention therapies for people with newly
diagnosed diabetes (Type 1 Diabetes TrialNet n.d.).
The Triggers and Environmental Determinants in Diabetes of the Young (TEDDY), a
consortium of six international centres, has been established to examine, confirm and
extend the findings of the ongoing and the future prospective cohorts. This consortium is
to coordinate the ongoing international efforts to identify infectious agents, dietary
factors, or other environmental factors that contribute to the risk of T1ADM in
individuals with genetic susceptibility (National Institute of Health 2003).
56
Enhancing the understanding of the biological history of the preclinical stage of T1ADM
will achieve ample insights into disease immunopathogenesis. The identification of
individuals for prevention trials may lead directly to more efficient early diagnosis prior
to the manifestation of the disease. Nevertheless, the ability to predict the disease without
a capability of primary prevention raises a number of ethical issues including induced
stress, changes in the lifestyle and insurability (Schatz et al. 2000). Lack of effective
intervention remains the single most significant barrier against population wide scale
trials (Schatz et al. 2002).
Given that large-scale trials screening stages and prevention procedures are based on the
assumption of eventually positive findings, the possibility to fail to prevent the disease
constitute a threat, anxiety, uncertainty, conflict and depression to individuals at high risk
of developing the disease and to their families. Therefore, families participating in these
trials require psychological consultation and support at every stage of the study (Weber
and Roth 1997; Roth 2001). Further investigations into the underlying mechanisms of the
disease are essential prior to undertaking additional intervention trials (Atkinson and Gale
2003).
Overcoming the inherited ethical issues accompanying the likelihood of instigating the
awareness of risk in healthy persons and possible false positivity requires minimising
controversy surrounding the prerequisites of any disease prevention trials (Rosenbloom et
al. 2000). Integration of behavioural research (Johnson 2001) as well as more
fundamental research are justified prior to effective and safe prevention trials commence
(Dahlquist 1999). Curtailing the duration of time-consuming clinical trials is essential to
set up surrogate markers of clinical T1ADM and is one approach that will ease the initial
assessment of potential intervention modalities and selection of the most promising
therapies for full-scale testing with overt disease as the endpoint (Knip and Akerblom
1998).
57
1.9 To sum up
Human studies on T1ADM (Kimpimaki et al. 2001; Norris et al. 2003) and human
studies on celiac disease (Ivarsson et al. 2002) as well as animal studies (Elliot and
Martin 1984; Elliot et al. 1988; Johnston and Monte 2000; Scott et al. 2002) have
concluded that the risk of developing the disease was reduced if the diabetogenic dietary
agent, such as BMPs and cereals was introduced while the newborn was still breastfed
independent of the age of exposure. These findings indicate a possible diabetogenicity of
certain early infant foods, but it should not be misinterpreted by parents and the public
(Atkinson and Gale 2003).
Compliance with the current six months infant exclusive breastfeeding recommendations
of the World Health Organisation should be maintained (World Health Organization
2002). The need for complementary nourishments afterwards in combination with
continuous breastfeeding up to two or three years of age or beyond should also be
recognised (National Health and Medical Research Council 2003; World Health
Organization and United Nations Children's Fund 2003). Success in the ongoing research
on preventing the occurrence of the disease in animals and the exciting findings about its
causes are imperative for diagnosis, treatment, and minimising or even preventing the
onset of the disease in humans.
58
2 Type 1 autoimmune diabetes: Implications of worldwide
imported milk and milk protein consumption
2.1 Abstract
Objective: Claims of adverse health effects from milk ingredients are on the increase.
The increasing incidence of type 1 diabetes mellitus due to pancreatic autoimmunity has
been related to the source and amount of milk proteins consumption (MPC). This study
examined the possible association between imported milk excluding butter (IMEB) and
MPC and the occurrence of the disease.
Design and methods: Data for type 1 diabetes mean incidence and percent increase in
the disease incidence per year in children aged 14 years or less were taken from the
Onkamo study (1999). Data for IMEB and MPC were obtained for each individual
country from FAO food balance sheets. An average percent relative change in IM and
MPC for each country was calculated for the period matching that of the Onkamo study.
Correlations between both mean and relative increase in the disease incidence, and both
mean and average percent relative change in IMEB; and mean and average percent
relative change in MPC were computed. A linear regression model was used to address
the strength of that relationship.
Results: The data fit a linear regression model. Correlation between disease mean
incidence and both relative change in IMEB and mean MPC was r = 0.648 (P = 0.05),
and r = 0.595 (p = 0.05), respectively. A significant inverse association (r = -0.657, P =
0.05) was established between the increase in diabetes incidence and mean MPC.
59
Conclusion: Worldwide changes in both imported milk proteins and milk protein
consumption may have a possible link to mean disease incidence as well as the global
increase in type 1 diabetes mellitus.
60
2.2 Introduction
Type 1 autoimmune diabetes affects approximately 0.5-1% of the general population
throughout its lifetime, and is marked by obvious geographic, ethnic and racial variation
(Rewers and Klingensmith 1997; Onkamo et al. 1999; Karvonen et al. 2000). This
indicates an important interplay between genetic susceptibility and environmental factors
in the initiation and development of the disease.
All Nordic countries (Norway, Denmark, Sweden, and Finland) have a similar genetic
background and relatively similar breastfeeding practices (International Dairy Federation
1993; Freysteinsson and Sigurdsson 1996; Elliot et al. 1999; Thorsdottir et al. 2000;
Erkkola et al. 2005), nevertheless the occurrence of type 1 autoimmune diabetes
(T1ADM) varies considerably in these countries. Whereas the relative increase in the
disease occurrence is relatively similar, the occurrence rate of the disease in Iceland is
50% lower than that in other Nordic countries (Onkamo et al. 1999).
The hypothesis of an inverse correlation between the protective effect breastfeeding may
have on the development of T1ADM and the implication of the early introduction of
bovine milk proteins at early age with the disease aetiopathogenesis has undergone
worldwide extensive investigation (Borch-Johnsen et al. 1984; Gerstein 1994; Verge et
al. 1994; Norris and Scott 1995; Gimeno and de Souza 1997; Meloni et al. 1997; Couper
et al. 1999; Hummel et al. 2000; Thorsdottir et al. 2000; Akerblom et al. 2005).
61
Animal and human studies were able to establish a significant association between
differences in the amount of bovine milk protein consumed, which may govern the
intestinal transmission of macromolecules, and the occurrence of the disease (Westrom et
al. 1985; Scott 1990; Dahl-Jorgensen et al. 1991). Studies in Iceland were able to
demonstrate that only beta-casein variants may be correlated with lower disease
occurrence in Iceland (Elliot et al. 1999; Thorsdottir et al. 2000; Birgisdottir et al. 2002).
If bovine milk proteins are diabetogenic, the controversy and discrepancy of the findings
of these studies may indicate variations in diabetogenicity of bovine milk proteins. The
purpose of the present study was to investigate, retrospectively, possible associations for
worldwide mean and relative change in both imported milk excluding butter (IMEB) and
milk protein consumption (MPC) per capita per day, and both mean and global increase
in the disease occurrence.
2.3 Research design and Methods
Data for the T1ADM mean occurrence, and percent increase in disease occurrence per
year in children aged 14 years or less were taken from the Onkamo study (Onkamo et al.
1999), which has a strict inclusion criteria. Data for IMEB (trillion metric tonnes), and
MPC gram (per capita per day) were obtained from FAO food balance sheets (Food and
Agriculture Organisation of the United Nations 2004).
62
FAO food balance sheets do not provide specific data on a specific region or an ethnic
group in a specific country. Therefore, we assumed that available FAO balance sheets
data on mean and percent change in both IMEB and MPC for West Australia, Lima
Peruvians, and Yemenite Israelites represent that of Australian, Peruvian, and Israeli
populations. Countries with more than one region included in the Onkamo study were
excluded from the present study.
Twelve countries were included to represent the global variation in both the mean and the
increase in disease occurrence. Mean of both IMEB excluding butter and MPC per capita
per day for each individual country as well as average percent relative change in IMEB
and MPC for each country were calculated for the period matching that of the Onkamo
study, given that the reference year was the study starting year of each study included in
the Onkamo study.
2.3.1 Statistical analysis
Assuming that errors were normally distributed and that data fit a linear regression
model, correlations between both mean and relative increase in disease occurrence; and
both mean and average percent relative change in IMEB as well as mean and average
percent relative change in milk protein consumption MPC were computed by the
bivariate correlations procedure. The One-Sample T-Test was applied to determine
significant differences between means by using the Statistical Package for Social
Sciences (SPSS). Data were considered statistically significant at P<0.05.
63
2.4 Results
There was a significant and strong positive correlation between mean disease occurrence;
and both relative change in IMEB and mean MPC (r = 0.648, P = 0.05, and r = 0.595, p =
0.05 respectively). Although not significant, there was an inverse association between
mean disease occurrence and both mean IMEB and the relative change in MPC (r = -
0.214, P = 0.05, and r = -0.313, P = 0.05, respectively).
For relative increase in occurrence of T1ADM in children aged 14 years or less, there
was a strong significant inverse correlation with mean MPC (r = -0.657, P = 0.050). An
inverse and not significant correlation was established with relative change in IMEB
excluding butter (r = -0.226, P = 0.050) and a positive not significant correlation with
relative change in MPC (r = 0.279, P = 0.050).
Mean and relative increase in the disease occurrence is shown in Table 2-1. It shows that
while Iceland and Finland have the same relative increase in the disease occurrence,
Finland has the highest disease occurrence. Imports of milk excluding butter into France
was the highest among all countries (2.63 trillion ton, 95% CI 2.16; 3.11, P = 0.000)
(Table 2-2) with relatively low variations in IMEB (0.03%) (Table 2-3), whereas imports
of milk into Iceland was the lowest (0.001 trillion metric tonnes, 95% CI -2.4; -1.9, P
=0.000) and relative change in IMEB was the lowest (0.02%, 95% CI -0.17; 0.22, P
0.809).
64
Although imported milk excluding butter into Finland was relatively low (0.047 trillion
metric tonnes, 95% CI 0.35; 0.53, P = 0.000), relative change in IMEB in Finland was
higher than any other country (6.26%, 95% CI 5.05; 7.42, P = 0.0000). This was higher
than that of worldwide relative change in IMEB by more than 5-fold. Relative change in
IMEB in Hungary was 0.044 trillion metric tonnes (- 0.60; 95% CI -0.81; -0.46, P =
0.0000).
65
Table 2-1 Worldwide mean (per 100,000) and relative increase in occurrence of type 1
‘autoimmune’ diabetes among children aged 14 years or less modified from the Onkamo
study (1999). Relative increase in the disease occurrence in Peru (Lima), Australia (West),
and Israel (Yemenite Jews) is assumed to represent that of the whole populations of these
countries.
Country Study period Mean disease
occurrence
% Increase
in disease
occurrence
Hungary 1978-87 6.1 8.5
Peru: Lima 1985-94 0.5 7.7
Australia: West 1985-92 14.9 6.3
Libya 1981-90 8.7 6.3
France 1988-95 8 3.9
Israel: Yemenite 1965-93 5 3.2
Norway 1973-82 20.8 3.2
Austria 1979-93 7.8 2.7
Finland 1965-96 30.3 2.3
Iceland 1970-88 9 2.3
Sweden 1978-92 24.9 1.2
Malta 1980-96 14.7 0.5
Global 3
66
Table 2-2 Worldwide mean imported milk excluding butter (IMEB) (Trillion metric
tonnes). Mean IMEB for each individual country modified from Food and Agriculture
Organisation of the United Nations (2004) and calculated for the period matching that of
the Onkamo study (1999). The populations arranged in descending order in accordance
with the relative increase in the disease occurrence per year. Relative increase in the disease
occurrence in Peru (Lima), Australia (West), and Israel (Yemenite Jews) assumed to
represent that of the whole populations of these countries. All data were statistically
significant at P<0.05.
Country Mean imported milk excluding
butter (trillion metric tonnes) P-Value
Hungary 0.044 0.002
Peru 0.314 0.000
Australia 0.214 0.000
Libya 0.298 0.000
France 2.626 0.000
Israel 0.095 0.000
Norway 0.028 0.005
Austria 0.277 0.000
Finland 0.047 0.000
Iceland 0.001 0.000
Sweden 0.196 0.000
Malta 0.050 0.000
Global 0.229 0.000
67
Table 2-3 Worldwide mean relative change in mean imported milk excluding butter
(IMEB) (Trillion ton) modified from Food and Agriculture Organisation of the United
Nations (2004) and calculated for the period matching that of the Onkamo study (1999).
The populations arranged in descending order in accordance with the relative increase in
the disease occurrence per year. Relative increase in the disease occurrence in Peru (Lima),
Australia (West), and Israel (Yemenite Jews) assumed to represent that of whole
populations of these countries. Data were considered statistically significant at P<0.05.
Country Mean relative change in imported
milk excluding butter P-Value
Hungary -0.06 0.000
Peru 0.03 0.418
Australia 0.01 0.602
Libya 0.02 0.050
France 0.03 0.038
Israel -0.02 0.001
Norway 0.32 0.012
Austria 0.02 0.144
Finland 6.26 0.000
Iceland 0.02 0.809
Sweden 0.00 0.653
Malta 0.01 0.010
Global 1.15 0.000
Bold indicates statistical insignificant at P<0.05.
68
Mean MPC is shown in Table 2-4. The table shows that Icelanders consume more milk
proteins (34 g/day/capita, 95% CI 30.9; 33.6, P = 0.000) than that of the Finnish (27
g/day/capita, 95% CI 24.8; 26.0, P = 0.000), whereas the relative change in MPC was
declining in both populations (-6.4%, 95% CI -12.5; -8.7 and -8.3%, 95% CI -12.4; -4.9,
P = 0.000, respectively) (Table 2-5). In Hungary, MPC was 14.6 g/day/capita (95% CI
6.4; 10.5, P = 0.0000) whereas relative change in MPC was 5-fold higher than that of the
global change in MPC (22.8%, 95% CI -4.3; 33.0, P 0.115). This trend was also observed
in Israel. Milk protein consumption in Malta and Sweden MPC was among the highest
among all studied countries (17.10; 95% CI 16.10; 17.10, 29.36; 95% CI 27.57; 28.75, P
= 0.0000, respectively).
69
Table 2-4 Worldwide mean milk protein consumption (MPC) (g/capita/day). Mean MPC
for each individual country modified from Food and Agriculture Organisation of the United
Nations (2004) and calculated for the period matching that of the Onkamo study (1999).
The populations are arranged in descending order in accordance with the relative increase
in the disease occurrence per year. Relative increase in the disease occurrence in Peru
(Lima), Australia (West), and Israel (Yemenite Jews) is assumed to represent that of the
whole populations of these countries. All data were statistically significant at P<0.05.
Country Mean milk protein consumption
(g/day/capita) P-Value
Hungary 14.6 0.000
Peru 4.6 0.000
Australia 21.6 0.000
Libya 10.7 0.000
France 26 0.000
Israel 17.4 0.000
Norway 26.8 0.000
Austria 21.7 0.000
Finland 27.7 0.000
Iceland 34.6 0.000
Sweden 29.4 0.000
Malta 17.1 0.000
Global 22.2 0.000
70
Table 2-5 Worldwide relative change in mean milk protein consumption (MPC)
(g/capita/day). Mean MPC for each individual country modified from Food and Agriculture
Organisation of the United Nations (2004) and calculated for the period matching that of
the Onkamo study (1999). The populations are arranged in descending order in accordance
with the relative increase in the disease occurrence per year. Relative increase in the disease
occurrence in Peru (Lima), Australia (West), and Israel (Yemenite Jews) assumed to
represent that of the whole populations of these countries. Data were statistically significant
at P<0.05.
Country Relative change in mean milk
protein consumption P-Value
Hungary 22.8 0.115
Peru -1.9 0.096
Australia 4.4 0.240
Libya -1.1 0.001
France 0.33 0.020
Israel 27.4 0.000
Norway 12 0.011
Austria 6.4 0.002
Finland -8.3 0.000
Iceland -6.4 0.000
Sweden 3.6 0.031
Malta -3 0.024
Global 4.6 0.177
Bold indicates statistical insignificant at P<0.05.
71
Positive trend in MPC with a marked variation among populations is shown in Figure 2-
1. Globally, milk protein consumption has increased from 6.7 g/day/cap/day (1961) to 7.2
g /day/cap/day (2001). Milk protein consumption in Finland and Iceland has declined
from 31.3 and 37.4 g/capita/day, (1961) to 27.7 and 24.2 g/capita/day (2001)
respectively, while consumption has doubled in Libya, and increased by 1.5-fold in
Hungary.
Figure 2-1 Worldwide trends (1961-2001) in milk protein consumption (g/capita/day). Data
modified from Food and Agriculture Organisation of the United Nations (2004).
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72
2.5 Discussion
All Nordic countries (Norway, Denmark, Sweden, and Finland) have a similar genetic
background and relatively similar high breastfeeding practices (International Dairy
Federation 1993; Freysteinsson and Sigurdsson 1996; Elliot et al. 1999; Thorsdottir et al.
2000; Erkkola et al. 2005). In Iceland, the occurrence of diabetes is 50% lower than that
in other Nordic countries, and approximately one third lower than that in Finland,
whereas the relative increase in the disease occurrence among children aged 14 years or
less in Iceland and Finland was the same (Onkamo et al. 1999).
Icelanders are amongst the highest in the world in the consumption of bovine milk and
the results presented here indicated that although the milk protein consumption in both
countries has declined in 2000 compared to that in 1960s, both Icelanders and Finnish
populations consume a relatively similar amount of milk proteins. This may explain the
similarity in the low relative increase in the disease occurrence in both populations
(Onkamo et al. 1999). On the other hand relative change in IMEB in Finland was higher
than that of Iceland by a minimum of 300-fold (Table 2-3) which may again explain wide
variation in the mean disease occurrence in both populations (International Dairy
Federation 1993; Elliot et al. 1999).
It has been demonstrated that the amount of protein available in the intestine of piglets
govern the intestinal transmission of macromolecules in the newborn preclousre piglet
(Westrom et al. 1985). In addition a significant relationship was established between the
consumption of milk proteins and the occurrence of disease (Scott 1990). This was
marked with geographic variation (Dahl-Jorgensen et al. 1991). No significant
differences were found between exclusively breastfed Icelandic children with diabetes
and newly diagnosed diabetic children who received bovine milk-based formula and the
non-diabetic children.
73
The frequency and extent of breastfeeding were comparable. While low disease
occurrence among Icelandic children was not correlated with either total protein
consumption (Elliot et al. 1999; Thorsdottir et al. 2000) or consumption of bovine serum
albumin (Birgisdottir et al. 2002), it may be explained by significant lower levels of A1
and B beta-casein variants of Icelandic bovine milk compared to that in other
Scandinavian countries (Thorsdottir et al. 2000). Similarly, Elliot (1999) has found that
all analysed Icelandic milk samples have lower -casein fraction A1 and B -casein than
other milk samples from Scandinavia. Per capita A1 and B -casein and diabetes
occurrence are significantly correlated (Elliot et al. 1999).
Unlike Scandinavian cattle breeds, Icelandic cattle have been isolated for about 1100
years leading to less frequent gene coding for A1 and B -casein (Lien et al. 1999). This is
dissimilar to -casein A1 60-67, bovine -casein A2 and human -casein that contain a
proline amino acid at a corresponding position in their sequence. It is likely due to this
that a proline peptide equivalent to BCM-7 cannot be formed upon digestion of human -
casein (Elliot et al. 1997). Furthermore, specific proliferation of T lymphocytes with
bovine casein in those with T1ADM may account for the response of pancreatic -cells
cellular and humoral immune response to -casein (Cavallo et al. 1996). This explains the
specificity of the connection between the occurrence of diabetes and the consumption of
a number of -casein variants (Elliot et al. 1999). However, casein hydrolysate-based
bovine milk formula was found to be highly effective in preventing autoimmune diabetes
in NOD mice (Karges et al. 1997). Early reports on humans also indicate a protective
effect of milk hydrolysates in humans as well (Akerblom et al. 2005).
74
People in developing countries are exposed to certain features of the affluent Westernised
lifestyle including foods that find their way into their markets. Urbanisation is often
accompanied by a shift in the availability and accessibility of foodstuffs. They often
displace local and traditional food items and food habits resulting in a possible negative
impact on health status (Williams 1999). Marked increase in the disease occurrence of
T1ADM is occurring among the Jewish population with an exceptional increase among
Yemenite Jews (18.5/100,000) compared to the overall occurrence rate among the Jewish
population (5.7/100,000), and (2.9/100,000) among the Arab populations (Shamis et al.
1997).
Yemenite Jews have immigrated from voluntary isolated communities in underdeveloped
countries (Shamis et al. 1997) to Israel which is a high-income Westernised country
(Gross national income [GNI]: US$ 16,000/capita, 2002, Atlas method) (The World Bank
Group 2004). Furthermore, it has been demonstrated that Yemenite Jews are genetically
distinctive from other Israeli ethnic groups (Weintrob et al. 2001). The sharp increase in
the disease occurrence among them may be attributed to changes in affluent economic
status and Westernised lifestyle as a consequence of immigration (Shamis et al. 1997).
Worldwide economic disparities remain entrenched among and within different countries
(Sen and Bonita 2000). Economic status under which populations live are among other
conditions in determining the risks to which populations are exposed (World Health
Organization Western Pacific Region 2002). The Kuwaiti population is approximately
half that of Jordan, and has a high-income developing population (GNI per capita 16,340
US$, 2002) similar to that in the industrialised countries. In Kuwait, the mean and
relative change in IMEB were higher by 2-fold, and 3-fold, respectively than that of
Jordan, a severely-indebted lower-middle-income country (GNI per capita: US$ 1,760,
2002) (The World Bank Group 2004).
75
Milk protein consumption in Kuwait (14.51 ± 2.75 g/capita/day) was not only twice that
of Jordanians, but also similar to that of affluent populations with substantial relative
changes (36.49 ± 25.3). In Jordan MPC has increased from 3.6 g/capita/day in 1961 to
6.9 g/capita/day in 2001 (Beaudry 2004). Interestingly, while the disease occurrence
among Kuwaitis has risen substantially from 3.95/100,000, (980-1981) (Taha et al. 1983)
to 14.4/100,000 (1992-1993) (Shaltout et al. 1995), the disease occurrence in Jordanian
children aged 14 years or less remained relatively unchanged. It has increased from
2.8/100,000 in 1992 to 3.6/100,000 in 1996 (Ajlouni et al. 1999). It remains among the
lowest in its region and matches that of the Arab population who live in neighbouring
Israel. Arab-Israelites for instance are voluntarily isolated and live in poor communities
similar to that the Yemenite Jews encountered prior to immigrating to Israel. Arab-
Israelites continue to live in rural areas maintaining more traditional lifestyles that may
explain the low disease occurrence among them (Shamis et al. 1997).
2.6 Conclusion
The results presented here agree with previous findings indicating a strong correlation
between differences in milk consumption and the occurrence of autoimmune diabetes
(Westrom et al. 1985; Scott 1990; Dahl-Jorgensen et al. 1991). It does seem reasonable to
suggest that geographic variation in the disease occurrence among children aged 14 years
or younger was associated with a worldwide variation in both mean and relative change
in imported and consumed milk protein. Whether this variation is an indication of
variation in the diabetogenicity of bovine milk proteins requires further longitudinal
animal and human trials.
76
3 Baseline dietary profile of young Jordanian children with
diabetes: Breastfeeding and early infant feeding practices
3.1 Abstract
Objective: A study was conducted to establish a dietary profile for diabetic and non-
diabetic Jordanian children aged 14 year or younger by investigating the occurrence of
breastfeeding and early infant feeding practices
Design and methods: Fourteen male ad 36 female diabetic children (DC), and 50 (22
male and 28 female) unrelated aged matched non-diabetic children (NDC) were
identified by the National Centre for Diabetes, Endocrinology and Genetics, Jordan. All
children were 14 years old or less, and children with known autoimmune disease were
excluded. A constructed three-part questionnaire was developed. The percent HbA1c in
blood was determined.
Results: Mean age at which children were diagnosed with diabetes was seven year with
the majority being diagnosed in winter. No significant difference was found for body
mass index. Signs of undernutrition were observed in both groups, whereas signs of
obesity were seen in NDC. Dietary compliance in DC was poor (mean HbA1c 9.32%).
Extended breastfeeding was equally prevalent in both groups to age 15 months. Sixty two
percent of DC and 92% of NDC had bottle-feeding after 3 months of age, 8% of the NDC
had no bottle-feeding. None of participants in both groups had solid foods before 3
months of age.
77
Conclusion: No significant association has been established for neither breastfeeding nor
early infant feeding practices between both DC and NDC Jordanian children aged 14
years or less. Children in both groups were breastfed for at least some period of time
with higher proportion of diabetic children receiving no bottle feeding in their infancy.
Provisions for dietary compliance and nutritional counselling are necessary for young
Jordanian children.
78
3.2 Introduction
In Jordan, the percentage of population aged below 15 years constitutes 37.8% of the
overall four and a half million Jordanians (Department of Statistics 2004). In terms of
health outcomes and access, however underutilised, inefficient and expensive, Jordan has
one of the best performing health sectors in its region (Obermeyer and Potter 1991;
Partners for Health Reform Plus 1997). Estimates indicate that more than 100,000
persons in the Middle East are prevalent with type 1 autoimmune diabetes (T1ADM)
(Amos et al. 1997b). The estimated annual occurrence is around 6000; of these some
3000 are children below the age of 15 years.
In 1997, around 5,200 Jordanians had the disease; of them some 700 were children below
15 years old (Green 1997). The disease occurrence among Jordanian aged 14 years or
less has increased from 2.8 per 100,000 children in 1992 to 3.6 per 100,000 children in
1996 (Ajlouni et al. 1999), nonetheless it still has among the lowest occurrence rates in
the region (Shaltout et al. 1995; Kadiki et al. 1996; Soliman et al. 1996; Kulaylat and
Narchi 2000). There is a dearth of information on both the epidemiology and the dietary
profile of people with type 1 autoimmune diabetes in Jordan. The purpose of the present
study was to embark on establishing baseline data on the dietary profile of Jordanian
children with diabetes aged 14 years or younger in terms of their breastfeeding and early
infant feeding practices.
79
3.3 Research design and methodology
3.3.1 The population sample
The cohort of this random case control prospective study consisted of 50 young Jordanian
diabetic children (DC) who were identified during routine consultation visits to the
outpatient’s diabetic clinic of The National Centre for Diabetes, Endocrinology, and
Genetics (NCDEG), Jordan. Fifty non-diabetic Jordanian children were drawn from the
general population and identified by Princess Rahmah Children’s Hospital (PRCH), and
Al-Zahrawi Medical Laboratory, Jordan. The majority of participants in both groups live
in Amman and Irbid, which account for more than a half of the Jordan population. Only
children aged 14 years or less with T1ADM were included.
Diagnosis with diabetes was confirmed by the medical history of the diabetic children,
including the presence of clinical symptoms and the need for insulin therapy. Participants
were unrelated to those with diabetes or to each other and were age and gender matched
with that of the participants of the control group. Ethical approval was granted by the
Human Research Ethics Committee of the University of Western Sydney as well as by
the authorities of all Jordanian participating institutions. All subjects, or their parents
when appropriate, gave either written informed consent or a witnessed verbal consent
when appropriate.
80
3.3.2 Survey tool
A constructed questionnaire (Appendix 10.1.3 and 10.2.3) was developed and ethically
approved by all participating institutions. All subjects, or their parents when appropriate,
gave either written informed consent (Appendix 10.1.2, and 10.2.2) or a witnessed verbal
consent when appropriate. Participants and their parents received an information sheet
explaining the research general background (Appendix 10.1.1, and 10.2.1). The
questionnaire consisted of three main parts: the first part evaluates general nutritional
status and sociodemographic characteristics, the second part examines general medical
history related to diabetes mellitus, and the third assesses breastfeeding and early infant
feeding practices prior to and after developing the disease.
3.3.3 Exclusion criteria
Children with any known form of rheumatic fever, rheumatoid arthritis, multiple
sclerosis, and system lupus erythematosus at the time of surveying were not included in
the study. These diseases may have affected the initiation of pancreatic autoimmune
diabetes. To prevent genetic bias no more than one member of a nuclear family was
included in either diabetic nor non-diabetic group.
81
3.3.4 Estimation of validity and reliability
A panel of paediatricians, nurses, university academics, parents, and day-care centres
carers have reviewed the questionnaire to evaluate its content validity. After undertaking
a pilot study and re-standardising the survey tool to meet the Jordanian cultural
background the internal consistency reliability was estimated. Coefficient � was more
than 81.2, which indicates that the test reliability must be high (Allen and Yen 1979).
3.3.5 Estimation of glycosylated haemoglobin (HbA1c)
The levels of HbA1c of diabetic children are routinely estimated during diabetic children
routine visit to NCDEG. The percent HbA1c in human whole blood was determined using
automated ion-exchange high performance liquid chromatography (Bio-Rad D-10®, Bio-
Rad Labs, Inc, USA). Haemoglobin A1c was measured within the normal range less than
6.05%. The upper limit of normal (ULN) equals 6.1% (The Diabetes Control and
Complications Trial Research Group 1993).
3.3.6 Statistical analysis
All data were coded, computer entered and analysed using the Statistical Package for
Social Sciences (SPSS). Crude analysis comparing diabetic and control group was
performed using parametric [independent T sample test for equality of means and
analysis of variance (ANOVA)], and nonparametric [Chi-square (x2)] statistic tests as
appropriate. Under the assumption of normally distributed errors, the data fit a linear
regression model.
82
Correlations between variables were computed by the bivariate correlations procedure.
Phi and Cramer’s phi was calculated to measure the degree of association between
variables derived from a table’s x2 value. Based on values of a set of predictor variables,
logistic regression model was used to estimate odds ratios (OR) with a 95% confidence
interval (CI) for each of the independent variables in the model. Data were considered
statistically significant at P<0.05.
3.4 Results
3.4.1 General nutritional status and sociodemographic characteristics
Table 3-1 and Table 3-2 summarise the mean, standard deviation, percentages and
statistical significance for the following variables: (1) age, weight, height, body mass
index (BMI), birth order of the participants, and (2) age, education and occupation of the
parents. There were no statistical differences between DC and NDC. Diabetic female
children numbered more than males (72 % and 28%, respectively). While the majority of
diabetic males aged four to ten years, females were ten to fourteen years (43%, and 61%
respectively, P = 0.034, phi = 0.36). No significant relationship was found for the age
groups of the NDC within the diabetic children group (P = 0.246).
No statistical significant difference was found for the mean body mass index (BMI) in
both DC and NDC. Percentile curves for body weight, height and body mass index of DC
and NDC ranged between the 5th and 95th percentiles for mean age, respectively (P =
0.323). Mean BMI percentiles curves of the diabetic males and females were above the
(25th and 50th percentile for mean age, respectively. P = 0.064), that was above the 75th
percentile for both sexes in the NDC (P = 0.739).
83
Approximately 20% of both DC and NDC were below the fifth percentile curve. A
minimum of 10 percent of children in both groups were overweight, and six percent of
the NDC were obese (Figure 3-1). Mean haemoglobin A1c levels of DC was 9.32 (±
Standard deviation [SD] 2.25, P = 0000), given a reference point of less than 6.05%;
upper limit of normal (ULN) = 6.1%) (The Diabetes Control and Complications Trial
Research Group 1993). The levels of mean haemoglobin A1c among individual levels
have ranged from 6.2 to 14.7 %, only one diabetic child has a normal level.
Wide variation was observed for the occupation of the parents of participants in both
groups. Fathers were mainly government employees, whereas mothers were often
unemployed (i.e. homemakers). Variation for the education of the parents was also
observed. Proportions of mothers in both groups who have had undergraduate and
postgraduate degrees were higher than that of fathers (Table 3-2).
84
Table 3-1 Summary of the sociodemographic characteristics of diabetic and non-diabetic
Jordanian children aged 14 years or less. Upper limit normal (ULN) = 6.1%. The results
presented as mean ± standard deviation and as percentage within each group. Data were
considered statistically significant at P<0.05.
Variable Diabetic group
(n = 50)
Control
group
(n = 50)
P-Value
Gender (Male: Female) 14:36 22:28 0.096
Mean 9.1 ± 3.67 8.80 ± 3.69 0.684
0 - �4 12% 14%
>4 - �10 36% 21%
Child age (year)
>10 �14 26% 22%
0.010
Child weight (kg) 31.48 ± 14.73 32.12 ± 13.10 0.819
Child height (m) 1.32 ± 0.18 1.31 ± 0.16 0.753
Birth weight (kg) 2.96 ± 0.7 3.00 ± 0.20 0.698
Child body mass index (kg/m2) 16.90 ± 4.18 17.77 ± 4.54 0.323
Glycosylated haemoglobin A1c (%) 9.32 ± 2.25 6.0 (ULN)º 0.000
Birth order 2.64 ± 1.79 2.90 ± 1.92 0.486
85
Table 3-2 Summary of the sociodemographic characteristics of the parents of diabetic and
non-diabetic Jordanian children aged 14 years or less. The results presented as mean ±
standard deviation and percentage within each group. Data were considered statistically
significant at P<0.05.
Variable Diabetic group
(n = 50)
Control
group
(n = 50)
P-Value
Mother age (year) 35.43 ± 6.50 33.94 ± 7.78 0.306
Father age (year) 41.27 ± 9.15 41.80 ± 8.54 0.768
�12 years 44 % 44 % Mother
education >12 years 56 % 56 %
0.230
�12 years 58 % 68 % Father
education >12 years 42 % 32 %
0.300
Unemployed 72 % 58 %
Labour 12 % 12 %
Government employee 14 % 24 %
Mother
occupation
Private professionals 2 % 6 %
0.381
Unemployed 4 % 4 %
Labour 24 % 8 %
Government employee 58 % 80 %
Father
Occupation
Private professionals 14 % 8 %
0.139
86
Figure 3-1 Percentile curves for body weight, height and body mass index of diabetic
children (DC) and non-diabetic (NDC) Jordanian children aged 14 years or less. The results
presented as percentage within each group. Data were considered statistically significant at
P<0.05.
0
10
20
30
40
50
60
% DC & NDC
5th 10th 75th 85th 95th
Percentiles
Diabetic Non-diabetic
3.4.2 General medical history related to diabetes mellitus
Median age at which children were diagnosed with diabetes was seven years
(interquartile range (IQR): 2.9 to 10.0 years), mean age was 6.6 years (SD ± 3.8). Male
and female DC were diagnosed at 5.4 and 7.11 years (SD ± 4.47 and ± 3.55 respectively,
p = 0.163). Table 3-3 shows a statistically significant relationship between DC and NDC
was established for both having a family member with diabetes (P � 0.001, phi = 0.50).),
and the type of diabetes they had (P = 0.000, phi = 0.50). No significant relationships
were established for either the total number of relatives who had diabetes or the nature of
that relationship.
87
Table 3-3 Family history of participating diabetic and non-diabetic young Jordanian
children aged 14 years or less. The results presented as percentage within each group. Data
were considered statistically significant at P<0.05.
Variable Patients
group
(n = 50)
Control
group
(n = 50)
P-Value
Number of participants having
a family member with diabetes
68% 18% P=0.000
One person 60.6 % 100 %
Two persons 18.2 % 0 %
Three persons 12.1 % 0 %
Number of family members
with diabetes the participants
have
> three persons 9.1 % 0 %
P=0.162
Sisters
and brothers
24 % 11 %
Parents 19 % 22 %
Relationship
to the family members
Grandparents 57 % 66 %
P� 1.000
Type 1
autoimmune
diabetes
40 % 0 % Type of diabetes family
relatives had
Type 2
(non-insulin-
dependent)
60 % 100 %
P=0.000
Mothers having gestational diabetes 4.1 % 0 P= 0.149
88
The findings presented here also demonstrated that approximately two thirds of DC were
diagnosed in winter (January, September, October, November, and December). More
than two thirds of the parents of the interviewees have indicated that prior to clinical
onset of the diabetes, their children had suffered undiagnosed viral infections described
as “colds, influenza, and gastric problems” that was accompanied with severe feverish
signs. Approximately 38 percent of them were diagnosed at a routine hospital or primary
health care centre.
Two thirds of the parents were educated on the signs and symptoms of the disease that
was significantly affected by mothers education (P = 0.008). None of the DC participated
in this study have any autoimmune disease, 12 percent of them had other chronic diseases
(partial deafness, dwarfism, brucellosis, meningitis and hypothyroidism). One diabetic
child had food allergy to fish and none of the participants had any chronic gastrointestinal
diseases.
3.4.3 Breastfeeding and early infant feeding practices
Table 3-4 and Figure 3-2 exemplify that except for three diabetic children (P = 0.079), all
other DC and NDC were breastfed for at least some period of time (P = 0.466) and also
shows that however the proportion of the DC exclusively breastfed for less than three
months was higher than that in the NDC (22% and 6% respectively). There was no
significant relationship between the DC and NDC for either the duration of exclusive
breastfeeding (BRF) for three months or less (94% and 100% respectively, P = 0.466) or
for the duration of sustained BRF (72% and 94%, P = 0.672 respectively).
89
The duration of BRF for less than three months of life was significantly correlated with
the age and the occupation of the mothers of the participants (r = - 0.570 and 0.616, P
<0.05, respectively). The mean duration of sustained breastfeeding in the diabetic and
non-diabetic children was 15.94 ± 13.09 and 15.06 ± 4.89 (P = 0.672), respectively. This
was correlated with the birth order of the participant (r = 0.237, P <0.05).
Diabetic children who had no bottle-feeding (BOF) at any age were significantly different
from the NDC who had no bottle-feeding at any age (P= 0.041). Nonetheless, there was
no significant relationship between both groups in neither introducing BOF at age
younger than three months (P = 0.130) nor in the duration of continued BOF (P = 0.672).
None of the NDC had any solid foods prior to three months of age. A statistically
significant relationship between DC and NDC was found for the introduction of
supplementary solid foods at the age of nine months or less (P =0.000, Cramers’phi =
0.436). Wheat was the most supplementary food introduced to NDC and rice was the
most common supplementary food DC had (P = 0.020, Cramer’s phi= 0.279). The
introduction of solid foods after nine months of age was significantly correlated with the
gender of the participants (r = 0.841, P <0.01).
Bovine milk-based formulae are widely used in nurturing both DC and NDC (P = 0.295).
Neither DC nor NDC had received soymilk. Few parents had switched between bovine-
milk based formula and fresh bovine milk as the child develops diabetes (P = 0.295).
Figure 3-3 illustrates that a third of the parents of DC and NDC prepared their children’s
liquid bottle-milk feeds by dissolving canned infant milk powder in lukewarm water and
then boiling together. This was not significantly related with those who prepared it by
mixing canned infant milk formula with preboiled water (P = 0.907).
90
Table 3-4 Breastfeeding and early infant feeding practices in young Jordanian diabetic and
non-diabetic children aged 14 years or less. Results presented as percentage within each
group and mean ± standard deviation. Data were considered statistically significant at
P<0.05.
Variable Patients
group
(n = 50)
Control group
(n = 50)
P-Value
No breast feeding 6% 0 P =0.079
<1 week 4% 0
>2 weeks – 1 month 4% 0
Exclusive Breastfeeding
>1 month – 3 months 14% 6%
P =0.466
Continued breastfeeding For >3 months
(72%) 15.94 ± 13.09
(94%) 15.06 ± 4.89
P = 0.672
No bottle-feeding 8% 0 P = 0.041
<1 week 16% 0
>1 – 2 weeks 2 %
>2 weeks – 1 month 8 % 8%
>1 – 3 months 4 % 0
P = 0.130
Initiation Of bottle-feeding
>3 months (62%) 13.09 ± 7.18
(92%) 11.17 ± 3.36
P = 0.672
Bovine milk-based 90% 98% Type formula introduced Fresh bovine milk 2% 2%
P = 0.295
3 months 16% 0%
3-6 months 30% 66%
6-9 months 46% 26%
P =0.000
Introduction of supplementary solid foods
> 9 months (8%) 14.25 ± 4.50
(8%) 10.25 ± 0.96
P = 0.132
Wheat 62% 84%
Rice 30% 16%
Solid foods introduced
Others 8% 0%
P = 0.020
91
Figure 3-2 Proportion of Jordanian diabetic children (DC) and non-diabetic children (NDC)
aged 14 years or less who had breastfeeding (BRF), bottle-feeding (BOF) and exposed to
solid foods supplements at three months (m) of age or more. The results presented as
percentage within each age group. Data were considered statistically significant at P<0.05.
No BRF (P=0.079), BRF >3 months (P=0.672), no BOF (p=0.041), BOF >3 months (p=0.672)
and solid s >3 months (P=0.000).
0
10
20
30
40
50
60
70
80
90
100
% DC & NDC
No BRF BRF>3m No BOF BOF>3m Solids >3m
Diabetic
Non-diabetic
92
Figure 3-3 Home heat processing procedures that parents used to prepare their infants
liquid bottle milk feeds both diabetic children (DC) and non-diabetic (NDC) Jordanian
children. The results presented as percentage within each group. Data were statistically
insignificant (P<0.907).
0
10
20
30
40
50
60
70
% DC & NDC
Milk powder boiled with water Milk powder mixed with
preboiled water
Diabetic Non-diabetic
93
3.4.4 Risk of having the disease
The logistic regression analysis yielded that BRF and BOF at least for some period was
associated with a smaller risk of developing diabetes (OR 0.560; 95% CI 0.199; 1.571,
P=0.432). Introducing BOF both prior to and after three months of age to children
breastfed at least for some period of time was associated with a relatively greater risk of
having the disease (OR 1.56; 95% CI 0.792; 3.067 P=0.091, and OR 0.926; 95% CI
0.845; 1.041, P=0.342 respectively). This was similar when BOF was initiated at an age
less than three months to children breastfed for less than three months (OR 1.423; 95%
CI 0.915; 2.212, p=0.780). The risk was relatively lower if BOF was introduced to
children breastfed for more than three months of age (OR 0.999; 95% CI 0.995; 1.003,
P=0.278).
Regardless of the duration of BRF, introduction of supplementary foods was associated
with nearly the same risk of having the disease. Introduction of wheat or rice to children
breastfed for both less and more than three months was associated with relatively the
same risk (OR 0.998; 95% CI 0.697; 1.430, P=0.320 and OR 0.956; 95% CI 0.914;
0.999, P=0.430). Nonetheless, the risk was higher with the age at which BOF was
introduced, particularly at an age less than three months compared to introduction at more
than three months of age (OR 1.362; 95% CI 0.769; 2.413, P=0.850 and 0.896; 95% CI
0.825; 0.973, P=.462). The way by which parents prepared bottle-milk feeds and
introducing BOF prior to three months of age was associated with a greater risk of having
the disease (OR 1.884; 95% CI 0.838; 4.239, P=0.820). The risk was lower when
introduced at more than three months of age (OR 0.962; 95% CI 0.915; 1.012, P=0.294).
94
3.5 Discussion
The first and only study on the epidemiology of type 1 autoimmune diabetes was in 1999
(Ajlouni et al. 1999). This study, to the best of our knowledge, constitutes the first to
establish a primary baseline data on the dietary profile of Jordanian children with
diabetes aged 14 years or less by studying their breastfeeding and early infant feeding
practices. Unlike type 2 (non-insulin-dependent) diabetes, type 1 autoimmune diabetes, at
the present time, does not constitute a health problem in Jordan (Ajlouni et al. 1998a).
Jordan faces critical challenges regarding high population growth rates that will double
the population in 24 years (Department of Statistics 2004). It is projected that the number
of children with diabetes will increase 10-fold by 2028, given the current disease
occurrence rate and if the crude death rate remains unchanged.
The structure of Jordan population is complex and shaped by both historical and cultural
factors (Nabulsi et al. 1997). Jordan is a severely-indebted lower-middle-income country
(GNI per capita 1760, 2002) (The World Bank Group 2004). More than 70% of the
Jordanian population are urban dwellers with an average 5.7 persons per household. More
than 37% are children below 15 years of age (Department of Statistics 2004). The Jordan
population is endogamic involving inbreeding subunits with a wide range of genetic
diversity (Nabulsi 1995; Nabulsi et al. 1997).
Jordan is a north of the equator Asian country, at low risk population and fits the
worldwide geographic distribution model of the disease (Karvonen et al. 1993). It has one
of the lowest disease rates. The age-specific disease occurrence rate was the highest
among Jordanian children aged 10-14 years and the lowest was among children less than
four years of age (5.5 and 1.3 per 100,000 respectively) without gender differences
(Ajlouni et al. 1999).
95
In the early years of the twenty first century, six percent of the general Jordanian
population were undernourished. Five percent of children aged five years are
underweight. Eight percent of infants were born with low-birth weight (United Nations
Development Programme 2003; UNICEF 2004). The distribution of anaemic children,
haemoglobin less than seven, has risen from 0.13% in 2003 to 0.22% in 2004. For
anaemic children of 10 to <11 haemoglobin the distribution has risen from 24.7% in 2003
to 25.6% in 2004 (Ministry of Health Jordan 2005) with signs of protein-energy
malnutrition among infants (Hijazi 1974).
A study on Jordanian children has shown that the height-for-age values fluctuated
between the 5th and 10th percentiles for both males and females. The body weight-for-
age was just above the 25th percentile for males, whereas it fluctuated between the (25th
and 50th percentiles) for females. Body mass index of male children were just above the
50th percentile and for females it varied between the 50th and 75th percentiles until 13.5
years of age (Hasan et al. 2001). Data on BMI were compared with the American
percentile curves of body mass index (Hammer et al. 1991) given the unavailability of
standardised national BMI percentile curves and the implications of ethnic differences
(Hosseini et al. 1999; Denney-Wilson et al. 2003; Karasalilhoglu et al. 2003).
Jordan provides free healthcare and nutrition programmes for children, including free
insulin injections instituted by The Monarch of Jordan, King Abdullah II in 2000 (Jordan
Times 1999). The World Food Program-Assisted Project provides school children with
approximately 690 calories per day (Hijazi and Adbulatif 1986). In addition, the School
Nutrition Program (SNP) also provides a daily fortified mid-morning snack containing
essential vitamins for approximately 54,000 school children to schools in remote
underprivileged areas (Jordan Times 2004).
96
In addition to specialised medical services rendered by the National Centre for Diabetes,
Endocrinology and Genetics, a state-of-art centre was established in 1996 (The National
Center for Diabetes 2005). The Jordan River Foundation (JRF), a leading non-
government organization, chaired by Her Majesty the Queen has recently launched a
three-year strategic plan focusing on the Jordan River Children’s Program as one of the
Queen Rania Centre for the Family and Child major agendas on child safety and early
childhood development issues (Al-Abdullah 2004).
Obesity seems to affect more than half of Jordanian youths above 25 years (Ajlouni et al.
1998b), while females have a propensity to develop obesity after puberty (Hasan et al.
2001). Obesity and rapid linear growth are considered as risk factors for T1ADM in
children as reported in most industrialised countries (Hypponen et al. 2000). The
occurrence of childhood type 2 diabetes is now prevailing among children as well (Drake
et al. 2002). The increased occurrence of childhood obesity is of concern and requires
preventative measures (Gahagan et al. 2003). Early identification of childhood obesity is
a key factor in preventing obesity and its health related complications in later adulthood
(Leong and Wilding 1999; Howdle and Wilkin 2001; Al-Domi et al. 2005).
Average HbA1c levels more than two percent above the upper limit of normal (6.1%)
indicate considerable risks for microvascular complications (The Diabetes Control and
Complications Trial Research Group 1993; Davidson et al. 1999). The level of HbA1c
was elevated well above the upper normal limit in 98% of the DC Jordanian children and
was above the 10% level in one third of them. Haemoglobin A1c levels indicate how
blood glucose is handled in the long-term rather than one instance of time (Davidson
2001).
97
Poor dietary habits and sedentary lifestyle as well as poor dietary counselling are most
likely the main reasons why diabetics are not achieving the recommended HbA1C levels.
Variation in physician expectations and the availability of licensed dieticians and
nutrition counselling practitioners may contribute significantly to dietary compliance. In
Jordan, nutrition and nutrition counselling are not recognised as medical professions
rather nutrition and dietetics practitioners are often graduates of either food sciences or
general public health with major in human nutrition disciplines. Furthermore, the patient
load for each nutrition practitioner is burdensome. An interdisciplinary and collaborative
team of which the person with diabetes is the main focus is the most suitable team to
undertake (Al-Domi et al. 2005).
The rate of breastfeeding in the developing countries was 52% in 2001 (UNICEF 2001).
In Jordan, the percentage of infants exclusively breastfed was 67% and 23% (0-4 and less
than 6 months, respectively) and 70% of children six to nine months of age were
breastfed with complementary food, whilst at 20 to 23 months, 12% were still BRF. A
minimum of 90% of all mothers initiate some BRF and 27% of them initiate BRF within
the first hour after birth (UNICEF 2004; Ministry of Health Jordan 2005). The reduction
of BOF and improved BRF practices may save an estimated 1.5 million children each
year. However, it was reported that only 16 countries, Jordan among them, have achieved
full compliance with the code of baby formula marketing (UNICEF 1997). Jordan has
adopted the Baby-Friendly Hospital Initiative in 1993 with three existing baby-friendly
facilities (UNICEF 2005).
98
The hypothesis of an inverse correlation between the protective effect breastfeeding may
have on the development of T1ADM, the implication of the early introduction of BMP at
early age with the disease aetiopathogenesis has undergone worldwide extensive
investigation, and it remains widely controversial. Meta analysis of nearly thirty
retrospective studies have failed to establish a strong relation between the protective
effect of breastfeeding and age when introducing BMP (Gerstein 1994; Norris and Scott
1995). The only prospective clinical dietary intervention trial on humans has recently
suggested a possible role dietary intervention may have on the initiation of pancreatic
autoimmunity in genetically susceptible infants (Akerblom et al. 1999; Akerblom et al.
2005).
While a Sardinian case-control study has demonstrated that the risk of developing the
disease in Sardinian children who were not BRF was insignificant (Meloni et al. 1997), a
Swedish and Norwegian time-series (1940-1980) study has ascertained an inverse
relationship between the duration of breastfeeding and the development of T1ADM
(Borch-Johnsen et al. 1984). On the other hand, studies have indicated that independent
of the duration of BRF, young age at introducing BMP is the most vital risk factor (Verge
et al. 1994; Gimeno and de Souza 1997). Regardless of the HLA genotypes, two
prospective studies, the Australian AudDiab study (Couper et al. 1999) and the German
BABYDIAB study (Hummel et al. 2000), were not able to establish that breastfeeding
may have a protective effect on the development of the disease. These findings remain
controversial and discrepant with strong indications. It indicates that beta cell destruction
is a complex process, which is affected by inert-individual variations as well as variation
between low and high-risk populations.
99
3.6 Conclusion
Regardless of their group, there was no significant relationship between DC and NDC for
the duration of either BRF, or in the instigation of BOF or the introduction of
supplementary foods. Breastfeeding in Jordan is common and is usually extended for a
minimum of one year for the majority of children. A higher proportion of diabetic
children received no bottle-feeding in their infancy. Dietary compliance in Jordanian
children with diabetes was poor with signs of undernutrition in both groups as well as
signs of obesity in controls. In Jordan, for better nutrition compliance there does seem a
need for better dietary monitoring and professional dietary counselling programmes for
both diabetic and non-diabetic children as well as establishing a national diabetes registry
and developing national dietary guidelines.
100
4 Proportion of ABO and Rh blood systems in young
Jordanians with diabetes: A sign of protective effect
4.1 Abstract
Objective: A study was conducted to examine whether ABO and Rh (D) blood groups
are associated with having type 1 autoimmune diabetes in young Jordanian children.
Design and methods: diabetic children (DC) and their unrelated aged matched non-
diabetic children (NDC) were identified. All children were 14 years old or less. The ABO
and Rh (D) phenotypes of the participants were determine by slide method at room
temperature. The specifications of the manufacturer were maintained. Positive and
negative controls were tested in parallel with each batch of tests.
Results: Statistically significant relationships were found for ABO blood group of both
DC and NDC (P = 0.002). The most frequent blood group in DC and NDC was O+
blood group (54% and 38% respectively) followed by A+ blood group (30% and 22%,
respectively) and B+ blood group (16% and 8%, respectively). None of the DC had AB+,
A- or O- blood group. All DC were Rh (D) positive, whereas 80% of NDC were Rh (D)
positive (P � 0.001), 8% of NDC of Rh (D) negative had B blood group.
Conclusion: Blood group O+ was associated with a greater risk of developing diabetes.
Whether certain blood group may confer a protective effect from diabetes or not requires
further investigation on the secretor status of humans with type 1 autoimmune diabetes
and viral infections.
101
4.2 Introduction
A specific complex of antigens present on red blood cells genetically determines blood
groups characteristics. Immunological characteristics determine and classify the
differentiation of blood by type (Watkins and Morgan 1959; Morgan and Watkins 2000).
Although there are more than 25 major blood group systems, a minimum of 270 red cell
phenotypes and more than six hundred red cell membrane antigens are recognised (Green
1989; Garratty et al. 2000; Hughes-Jones and Gardner 2002), the ABO blood groups
system, first introduced in 1901, remains the most used system.
Studies have demonstrated the implication of different blood group phenotypes in disease
pathogeneses. The ABO and Rhesus [Rh (D)] blood groups have been implicated in
increased susceptibility to certain diseases (Blackwell 1989) for instance, Helicobacter
pylori and the increased risk of peptic ulcer (Alkout et al. 1997; Alkout et al. 2000),
haemolytic uremic syndrome and Escherichia coli (Blackwell et al. 2002), elevated
serum antibody titre levels to vibrio cholera (Swerdlow et al. 1994), carcinomas (Su et al.
2001) and infertility in women (Lurie et al. 1998).
102
Studies on maternal-child blood group incompatibility (Dahlquist and Kallen 1992), type
2 (non-insulin-dependent diabetes) (Sidhu et al. 1988; Qureshi and Bhatti 2003) and
T1ADM (Kanazawa et al. 1983; Pontiroli et al. 1984) have demonstrated associations for
the distribution of the ABO blood groups in diabetics different to the general population.
Differences in the frequency of ABO and Rh blood groups indicate racial and ethnic
variations in individuals indigenous to various parts of the world (May and du Toit 1989;
Wagner et al. 1995; Falusi et al. 2000; Chiaroni et al. 2004). The worldwide paucity of
studies on type 1 autoimmune diabetes particularly in young Jordanian children has
encouraged the researcher to study the frequency of ABO and Rh (D) blood groups
among them.
4.3 Research design and methodology
4.3.1 The population sample
The cohort of this study consisted of 50 young Jordanian diabetic children (DC) (36
females and 14 males) identified by The National Centre for Diabetes, Endocrinology
and Genetics (NCDEG), Jordan, and 50 (28 females and 22 males) unrelated, age and sex
matched non-diabetic children (NDC) were identified by Princess Rahmah Children’s
Hospital (PRCH)) and Al-Zahrawi Medical Laboratory, Jordan. Ethical approval was
granted by the Human Research Ethics Committee of the University of Western Sydney
as well as by the authorities of all Jordanian participating institutions. All subjects, or
their parents when appropriate, gave either written informed consent or a witnessed
verbal consent when appropriate.
103
4.3.2 Determination of ABO and Rh blood groups
A licensed phlebotomist drew (5ml) venous blood samples. Specimens were tested
immediately following collection. Anti-A, anti-B and anti-D monoclonal murine IgM
blood grouping reagents obtained from Bioscot®, (Serologicals Ltd, UK) were used to
determine the ABO and Rh (D) phenotypes of the participants by slide method at room
temperature. Manufacturer’s specifications were maintained and positive and negative
controls were tested in parallel with each batch of tests.
4.3.3 Statistical analysis
All data were coded, computer entered and analysed using the Statistical Package for
Social Sciences (SPSS). Nonparametric [Chi-square (x2)] statistic tests used as
appropriate. Phi and Cramer’s phi were calculated to measure the degree of association
between variables derived from a table’s x2 value. Based on values of a set of predictor
variables, the logistic regression model was used to estimate odds ratios (OR) with 95%
confidence interval (CI) for each of the independent variables in the model. Data were
considered statistically significant at P<0.05.
104
4.4 Results
Figure 4-1 shows statistically significant relationships found for ABO blood groups of
both DC and NDC (P = 0.002, Cramer’s phi = 0.44). The most frequent blood group in
DC and NDC was O+ blood group (54% and 38% respectively) followed by A+ blood
group (30% and 22%, respectively) and B+ blood group (16% and 8%, respectively).
None of the DC had AB+, A- or O- blood group. All DC were Rh (D) positive, whereas
80% of NDC were Rh (D) positive (P � 0.001, phi = 0.33), 8% of NDC of Rh (D)
negative have B blood group.
Figure 4-1 Distribution (%) of the ABO and Rh (D) blood groups of young diabetic children
(DC) and non-diabetic children (NDC). The results presented as percentage within each
group. ABO blood groups and Rh (D) positive of both DC and NDC were statistically
significant (P=0.002 and P�0.001 respectively).
0
10
20
30
40
50
60
(%)
Proportion of
DC & NDC
A +A +A +A + A -A -A -A - B +B +B +B + AB +AB +AB +AB + O +O +O +O + O -O -O -O -
ABO and Rh (D) blood groups
DC
NDC
105
Table 4-1 shows the gender variation of ABO and Rh (D) blood groups among
participants with regard to their gender. A significant relationship was found for the ABO
blood groups between diabetic and non-diabetic females. No significant relationship was
found for ABO or Rh (D) blood groups between diabetic and non-diabetic males and
diabetic and non-diabetic females.
Table 4-1 Comparison between the ABO and Rh (D) blood groups of young male and
female diabetic and non-diabetic children. The results presented as percentage within each
group. Data were considered statistically significant at P<0.05.
A+ A- B+ AB+ O+ O- P-Value
Diabetic Males
(n = 14)
28.5% 0% 14.5% 0% 57.0% 0.0%
Non-diabetic Males
(n = 22)
23% 9.0% 9.0% 9.0% 41.0% 9.0%
0.464
Diabetic Females
(n = 36)
30.5% 0.0% 16.5% 0.0% 53.0% 0.0%
Non-diabetic Females
(n = 28)
21.5% 10.7% 7.0% 14.1% %36 10.7%
0.008
The logistic regression analysis has shown that ABO and Rh (D) blood groups were
associated with a greater risk of developing diabetes (OR 1.022; 95% CI 0.884; 1.183).
Blood groups were also associated with relatively low risk of developing the disease with
regard to the gender (OR 0.978; 95% CI 0.909; 1.054).
106
4.5 Discussion
The present study, to the best of the researcher’s knowledge, constitutes the first data on
ABO and Rh (D) blood groups of diabetic Jordanian children aged 14 years or less. The
findings of the present study constitute baseline data for both diabetic and non-diabetic
Jordanian children aged 14 years or less, given the unavailability of reliable records for
the distribution of the blood groups in the general population. The ABO blood groups
system is the most important and the most common human alloantigen system in blood
transfusion (Yamamoto et al. 1992).
Genetic role in the determination of ABO and Rh (D) blood groups system (Green 1989;
Garratty et al. 2000; Hughes-Jones and Gardner 2002) exhibited extensive variation in
the distribution of ABO and Rh (D) blood groups in different populations (May and du
Toit 1989; Wagner et al. 1995; Falusi et al. 2000; Chiaroni et al. 2004). The main ABO
alleles that create familiar A, B, AB and O blood groups exhibit broad sequence
heterogeneity and extensive sequence variation in both the coding and non-coding region
of the gene, given that a minimum of 70 ABO alleles are reported (Yip 2002).
Studies on maternal-child blood group incompatibility (Dahlquist and Kallen 1992), type
2 non-insulin-dependent diabetes (Sidhu et al. 1988; Qureshi and Bhatti 2003) and type 1
insulin-dependent diabetes (Kanazawa et al. 1983; Pontiroli et al. 1984; Karamizadeh and
Amirhakimi 1996) have demonstrated associations for the distribution of the ABO blood
groups in diabetics different to the general population. An increased risk of developing
type 1 autoimmune diabetes due to maternal-child blood group incompatibility was
established in Swedish diabetic children aged 14 years or less (Dahlquist and Kallen
1992).
107
Studies on type 2 diabetes mellitus showed inconsistency in the distribution pattern of
ABO and Rh (D) blood groups. While the distribution of B blood group was elevated
among Pakistanis (Qureshi and Bhatti 2003), the proportion of A, AB and Rh(D) positive
blood groups were increased among Indians with the disease (Sidhu et al. 1988), whereas
no significant difference was established in Iranians with diabetes (Karamizadeh and
Amirhakimi 1996). These differences in the frequency of ABO and Rh blood groups
indicate racial and ethnic variations in persons indigenous to various parts of the world
(May and du Toit 1989; Wagner et al. 1995; Falusi et al. 2000; Chiaroni et al. 2004).
Significant relationships were found for the prevalence of fast acetylator phenotype in
diabetic adults who had blood group B, given fast acetylator prevalence was significantly
higher in diabetic children than adults and non-diabetics (Pontiroli et al. 1984). While, no
associations were found for MN, KIDD and Lewis blood group systems in both diabetic
adults and diabetic children grouped according to insulin dose administered, it was
statistically significant for the ABO blood groups in patients receiving higher insulin
doses (Kanazawa et al. 1983).
Numerous studies have challenged the importance of ABO blood groups. For instance,
the increased binding of Helicobacter pylori to epithelial cells and increased risk of
peptic ulcer (Alkout et al. 1997; Alkout et al. 2000), duodenal and gastric ulcers (Mentis
et al. 1991), haemolytic uremic syndrome caused by Escherichia coli O157 (Blackwell et
al. 2002), diarrhoea and heat labile enterotoxin producing Escherichia coli (Black et al.)
and elevated serum antibody titre levels to vibrio cholera (Swerdlow et al. 1994) are
associated with a higher proportion of people of O blood group phenotype.
108
The ABO histo-blood system includes three antigens (ABH). Unlike people of A, B and
AB groups can convert the H antigen into A or B antigens, people of O blood group
having guanine (258) deleted in the O gene produce an inactive protein incapable of
converting the H antigen (Yamamoto F 1990). The prevalence of Helicobacter pylori in
non-secretor people with duodenal ulcer was significantly higher than that of non-
secretor people who had gastric ulcer (Mentis et al. 1991).
The majority of diabetic children who participated in this study were diagnosed in winter
and the majority have encountered a viral infection preceding the clinical onset of
autoimmune diabetes. An association between secretor status and the susceptibility to
develop type 1 autoimmune diabetes with elevated proportion of non-secretors among
them was also demonstrated (Blackwell et al. 1987). Given the heterogeneity in the
expression of glycoconjugates associated with different histo-blood group ABO
(Yamamoto F 1990; Yamamoto et al. 1992; Yamamoto et al. 1993), it was established
that secretor gene shows signs of protective effect particularly in immunocompromised
people (Blackwell 1989).
4.6 Conclusion
Blood group O+ is significantly predominant in young Jordanian children with diabetes
and is associated with a greater risk of developing diabetes. A higher proportion of NDC
had Rh (D) negative blood groups with gender variation. Whether certain blood groups
confer a protective effect from diabetes requires further investigations on the secretor
status of humans with type 1 autoimmune diabetes.
109
5 Evidence on the heterogeneity of native, modified and
heat processed milk proteins: A spectral profile
5.1 Abstract
Objective: Bovine milk proteins have been widely implicated in the development of type
1 autoimmune diabetes in early infancy. Solubilized proteins have distinctive absorption
properties which can be quantitated independently devoid of extensive procedures.
Design and methods: Human breast milk, bovine-based liquid, and reconstituted adult
and infant bovine milk-based formulas were cooled (4 ºC/ 20 min) and separated at
15,000 rpm for one hour at 4 ºC. to replicate home and industrial heat processes; milk
proteins were heat processed by both water bath heat treatment (40-100 ºC/5-60 min)
and/or hot plate heat treatment (63 ºC/15-30min). Absorbance spectra of native and
treated milk proteins were screened in the ultraviolet region, and the pH was recorded at
25 ºC.
Results: A unique identification standard curve was established for each of the tested
proteins. The linear range of the assay for human-breast milk, bovine serum albumin and
the rest of the tested proteins was 20-100mg/ml, 1mg/ml, and 10mg/mL respectively. The
absorption maxima of human-breast milk proteins, BSA, and the rest of the tested milk
proteins fall into three maxima in the ultraviolet region (�270nm, �275, nm, and �270-
�275nm nm; respectively). Changes in the absorption maxima of the tested milk proteins
subjected to various heat treatments were detectable at temperatures as low as 40 ºC for
30min, and were concentration dependent. Human breast-milk has a neutral pH (7.2),
whereas the vast majority of the tested bovine milk proteins were slightly acidic.
110
Conclusion: The findings of the present study have ascertained the heterogeneity of both
human breast milk and bovine milk protein solutions. Different native, modified and heat
processed milk protein solutions have exhibited a distinctive spectral profile with various
intensities. The present assay was rapid, sensitive, and reproducible.
111
5.2 Introduction
Although bovine milk proteins have undergone an intensive and extensive investigation
as a putative environmental factors implicated in the destruction of pancreatic beta cell,
no causal links have been established and findings remain widely controversial and
discrepant. Milk proteins are widely heterogeneous (Goff and Hill 1993) and that
discrepancy in the findings indicates variations in diabetogenicity of milk proteins
(Thorsdottir et al. 2000).
Heat processing is most commonly used in canned bovine milk-based foods processing
technologies and is attributing to some undesirable and possibly severe quality defects to
milk products including the denaturation of its protein (Pisecky 1997), which depends
among other factors on heating temperature (Bertrand-Harb et al. 2002). Heat processing
often causes protein antigens to aggregate, which affect both the immunogenicity
(Harlow and Lane 1988) as well as physical nature of the proteins (Saperstein and
Anderson 1962).
Complex proteins have distinctive absorbance properties and can be quantitated
independently devoid of extensive purification procedures (Kaplan and Pesce 1996). The
measurement of the light absorbance of substances is ordinary to many basic scientific
and clinical research applications. It is one of the most widely used analytical techniques
in determining the concentration of almost any molecule or class of molecules, studying
the biochemical properties of various molecules (Lowry et al. 1951; Bradford 1976;
Smith et al. 1985; Stoscheck 1990; Kaplan and Naito 1996) and studying the degree of
denaturation of milk proteins (Garcia-Hernandez et al. 1998).
112
The spectral characteristics of milk and milk products influence their appearance. This
has provided the bases for many rapid indirect methods of analysis (Goff and Hill 1993;
Goff 2004). The purpose of the present study was to verify the diversity of milk proteins
as well as to identify changes that modification and thermal processing may cause to milk
proteins by studying changes in their spectral and chromatographic profiles.
5.3 Methods
5.3.1 Milk samples
Human breast milk (HBM) donated from a volunteer mother was collected over 24 hours
and kept at (4ºC), raw whole pooled bovine milk (RWPBM) was obtained from the
University of Western Sydney Dairy Farm and commercial whole pasteurised and
homogenised fresh bovine milk (PHFBM) was obtained from local markets.
In addition, six canned bovine-based milk formulae were analysed. Three adult formulae
(not suitable for infants). (Adult H (skim generic, Australia), Adult J (skim; Australia)
and Adult D (full cream; Australia) were analysed. Two canned whey dominant bovine-
based starter infant formulae (ISFK; New Zealand and ISFS; USA) and one canned
enzymatically hydrolysed whey protein infant formula (ISFN; Germany), and bovine
serum albumin (BSA) (Sigma, USA) and bovine insulin (BI) (Sigma-Aldrich, Australia)
were also analysed. pH was recorded at 25ºC.
113
5.3.2 Reconstitution, separation, and modification of milk protein solutions
Milk powders, BSA and BI were reconstituted in double deionised ultra-filtered water
(0.18 µm) at room temperature (24ºC). The separation process of whole milk solutions
was standardised; the best separation of HBM, and liquid and reconstituted bovine milk
protein solutions (BMPs) was achieved by precooling milk solutions to 4ºC for 20
minutes followed by centrifugation at 25,000 g for 60 minutes at 4 ºC (70 mm diameter
rotor, Beckman J2-HS).
Supernatant protein layers were gently aspirated, sterile-filtered through a 0.22 µm filter
(Millipore, UK) and stored frozen at -70ºC. The wet weight of fat and precipitants was
recorded. One milliter of BSA (1mg/ml) was modified with an equal volume of BI
(1mg/ml) and then 0.2 – 0.8 mg/ml of BSA was modified with one minus 0.2 to 0.8
mg/ml BI alternatively. The modification of BSA with BI was strictly carried out under
aseptic conditions under room temperature (RT) (17ºC/24h, pH 7.4) and under
physiological conditions (37ºC/24h), both were shaken continuously at 25 round per
minute (rpm).
5.3.3 Water bath heat processing
Sterile-filtered BMPs, native BSA and modified BSA with BI (m-BI-BSA) were
subjected to various water bath heat treatment at temperatures ranging from 40 to100ºC
and held for five to sixty minute. Treatments were carried in duplicates and strictly under
the same the conditions. Samples of milk protein solutions (10mg/ml) were placed in
heat-resistance thin-wall test tubes (Pyrex 18 x 150mm).
114
Sealed test tubes were submerged in a preheated water bath at a constant temperature at
different preset times. One minute was allowed to warm the sample to the required
temperature. Heat processed BMPs were cooled immediately to zero ºC by submerging
them in ice cubes mixed with water and table salt for 15 minutes. Cooled samples were
stored frozen at -70ºC for 24 hours prior to analysis.
5.3.4 Hot plate heat processing
To reach a constant temperature of 63ºC, Sterile-filtered BMPs (10mg/ml) were
preheated in sealed thin-walled heat-resistant beakers (Pyrex 80 x 100 mm) at 100ºC and
held for 30 seconds. Preheated samples duplicates were transferred immediately to a
preheated hot plate yielding a constant temperature of 63ºC and held for 15 and 30
minutes. Heat processed MPs cooled immediately as described above and stored at -70ºC
for 24 hours before analysis.
5.3.5 Absorbance spectrum
The absorbance maximum of HBM, and native and heat processed BMPs was measured
in the ultraviolet region of the spectrum using a dual beam Multiskan® Spectrum
spectrophotometer (Thermo Electron Corp., SkanIt software research edition). It has both
the sample and the reference beams, which enable scanning of the sample spectrum with
simultaneous substraction of the blank spectrum.
115
Technically Multiskan® Spectrum provides a zero to four absorbance unit (AU) read-out-
range; 0-3 AU, ± 2% linearity; 0-2 AU, ± 0.005 AU accuracy ; precision CV < 1%, 0-2
AU, CV< 2%, 2-3 AU precision; an optical bandpass of 2nm (± 1nm wavelength
accuracy) and a stray light of <0.02% at 230nm.
To avoid non-specific absorbance from buffers or other chemical moieties, only Sterile-
filtered deionised water was used. Prior to scanning, standard matched semi-micro
cuvettes (0.5 ml) were shaken at (30rpm); scanning the absorbance spectrum was strictly
carried out under the same conditions at 25ºC at five nanometre wavelength interval first
in the 200 to 100nm wavelength range. The absorbance maximum was confirmed by
rescanning the samples in the ultraviolet region at one-nanometre wavelength interval.
The absorbance at �max (max = wavelength at which the maximum absorbance peak
obtained) and absorbance at �280nm were recorded. Samples with more than 0.005 AU
variations were rejected.
5.3.6 Reversed-phase high-pressure liquid chromatography (RV-HPLC)
All chromatographic separations were performed on a Shimadzu LC (Class VP software)
system, incorporating LC10Ai solvent delivery unit, online DGU-14A degasser, CT0-
10A column oven, SPP-M10A Diode array detector, CDD-6A conductivity detector, and
SCL-10A system control. A silica-based C18 column, 5u, 250 X 4.6 mm (3354, Alltima,
Alltech Associates, Inc.) was employed for separation employing an isocratic elution at
solvent flow rate of 1.0 ml/min with HPLC-grade 100% acetonitrile (Labscan Asia Co.,
Ltd., Thailand) and 0.1% trifluoroacetic acid in double-deionised water (Pierce Chemical,
USA).
116
Fifty microliter of eluant was filtered through 0.22m filter and the effluent was
monitored at 220nm. The column was washed with the mobile phase solvents until the
absorbance at 220nm returned to the base line. High reproducibility of the retention time
was obtained run to run. Due to the complexity in comparing the various chromatographs
of tested milk protein solutions and the limited scope of the present study,
chromatographs were used mainly for comparisons and verification of changes in the
spectral profile of native, heat processed and modified milk BMPs.
5.4 Results
5.4.1 Fat and precipitant yield
Human breast milk and ISFS have almost a neutral pH (7.3 and 7.2, 25 ºC, respectively).
Slight acidity was identified in RWPBM, PHFBM, ISFN and ISFK, and adult formulae
pH (mean ± standard deviation (STD) 6.7 ± 0.1 and 6.4 ± 0.1; 25 ºC, respectively). The
precipitants yield (percent wet weight) of HBM was the lowest among all analysed BMPs
(0.15%) and the fat yield was relatively low (2.6%), whereas adult formulae have the
highest precipitants yield (18.3% ± 0.01) and a trace of fat (0.04% ± 0.001). On the other
hand, the fat and precipitants yield of ISF was 4.1% ± 0.01 and 1.7% ± 1.4*10-05,
respectively, and the fat yield of RWPBM and PHFBM was 5.5% and 6.2%, respectively
and precipitants yield was 4.6% and 3.6%, respectively.
117
5.4.2 Identification standard curve
Human and BMPs have a relatively similar spectral profile in 250 to 310nm spectral
region with variations in the intensity of the absorbance peak. A concentration dependent
linear range identification standard curve was established for HBM and all tested BMPs.
At a higher concentration range, the relationship was nonlinear. Figure 5-1 shows that
while the concentration range required to establish a linear range for HBM was 100-fold
higher than that required to BSA as well as BI (Fig 5-2). This was 10-fold higher than
that required by RWPBM, PHFBM, adult and infant bovine-based canned formulae
(Table 5-1).
Table 5-1 shows that while hypo allergic enzymatically hydrolysed whey protein infant
formula (ISFN) yielded the highest absorbance maximum among all tested BMPs and
HBM (�270: 1.902 and �280:1.758) this was followed by adult skimmed formulae (Adult H
and J), the absorbance maximum of whole pasteurised and homogenised bovine milk was
the lowest (�275: 0.264 and �280: 0.258). It has been also demonstrated that while HBM
and infant starter formulae have yielded its maximum absorbance at 270nm, adult and
whole fresh and pasteurised bovine milk protein solutions have exhibited a maximum
absorbance at �275nm. Distinctive chromatographic profiles of HBM and all tested BMPs
are shown in figure 5-3.
118
Figure 5-1 Spectral profile of human breast milk (HBM). Milk solutions separated by
precooling to 4ºC/20min and centrifuged at 25,000 g/60min/4ºC;70mm diameter rotor.
Absorbance obtained at �max: 270nm (�max for wavelength at which the maximum
absorbance obtained) and �280nm.
0
0.2
0.4
0.6
0.8
1
1.2
20 40 60 80 100
[mg/ml]
Ab
so
rban
ce
HBM � 270 nm HBM � 280 nm
119
Figure 5-2 Spectral analysis of native bovine serum albumin (BSA) and bovine insulin (BI).
Absorbance obtained at �max: 275nm ((�max for wavelength at which the maximum
absorbance obtained) and �280nm.
0.2
0.4
0.6
0.8
1.0BI �275 nm BI �280 nm BSA �275 nm BSA �280 nm
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Absorbance
[mg/ml]
Milk proteins
120
Table 5-1 Spectral of raw whole pooled bovine milk (RWPBM), pasteurised and
homogenised fresh bovine milk (PHFBM), bovine-based adult canned formulae (Adult H, J
and D), canned whey dominant bovine-based starter infant formulae (ISFS and ISFK), and
canned enzymatically hydrolysed whey protein infant formula (ISFN). Milk solutions
separated by precooling to 4ºC/20min and centrifuged at 25,000 g/60min/4ºC; 70mm
diameter rotor. Absorbance obtained at �max: 270nm and 275nm ((�max for wavelength at
which the maximum absorbance obtained) and �280nm.
Milk product Wavelength
(nm)
Concentration range(mg/ml)
2.0 4.0 6.0 8.0 10.0
Fresh �275 0.036 0.092 0.1405 0.202 0.269
RWPBM
�280 0.035 0.090 0.137 0.198 0.263
Pasteurised �275 0.038 0.089 0.130 0.187 0.264
PHFBM
�280 0.037 0.087 0.128 0.183 0.258
�275 0.247 0.510 0.755 1.044 1.337 Adult H
Skimmed �280 0.245 0.508 0.750 1.036 1.327
�275 0.211 0.431 0.662 0.890 1.138 Adult J
Skimmed �280 0.209 0.426 0.655 0.880 1.127
�275 0.115 0.236 0.355 0.480 0.608
Bovine-
based adult
formulae
Adult D
Full cream �280 0.107 0.220 0.331 0.448 0.568
�270 0.179 0.370 0.562 0.757 0.959 ISFS
�280 0.173 0.358 0.543 0.731 0.926
�270 0.369 0.749 1.142 1.517 1.902 ISFN
�280 0.34 0.69 1.052 1.399 1.757
�270 0.203 0.415 0.638 0.863 1.085
Bovine-
based
infant
formulae
ISFK
�280 0.198 0.404 0.621 0.841 1.057
121
Figure 5-3 Comparison between the chromatographic profiles of untreated human breast
milk (HBM), raw whole pooled bovine milk (RWPBM), pasteurised and homogenised fresh
bovine milk (PHFBM), adult full cream (Adult D), adult skim (Adult H) and infant starter
formulae (ISF)K, ISFS and ISFN. Column (C18) isocratic mobile phase solvents (100%
acetonitrile and 0.1% trifluoroacetic acid in double-deionised water). Flow rate 1ml/min.
Detection at 220nm.
Fig 5-5-1 HBM
Fig 5-5-2 RWPBM
Fig 5-5-3 PHFBM
Fig 5-5-4 Adult D
Fig 5-5-5 Adult D
Fig 5-5-6 ISFS
Fig 5-5-7 ISFN
Fig 5-5-8 ISFK
122
5.5 Modification and heat processing
5.5.1 Modification of bovine serum albumin with bovine insulin
Concentration dependent changes in the spectral profile as well as the chromatographic
profile of modified BSA with BI are shown in Table 5-2 and Figure 5-4, respectively.
Unlike the incubation of equal volumes and concentrations of BSA (1mg/ml) modified
with BI (1mg/ml) at room temperature (17ºC/24h/25rpm, pH 7.4), the incubation of mBI-
BSA under physiological conditions (37ºC/24h/25rpm, pH 7.4) has yielded the highest
absorbance peak, which was also higher than that of BSA modified with BI at
physiological conditions with different alternative concentrations.
123
Table 5-2 The effect of incubation and concentration on the absorbance maximum of bovine
insulin (BI, 1mg/ml), bovine serum albumin (BSA, 1mg/ml) and BSA modified with
alternative different concentrations and equal volumes of BI (mg/ml) under physiological
conditions (37ºC/24h/25rpm, pH 7.4) and under room temperature (RT: 17ºC/24h/25rpm,
pH 7.4). Absorbance obtained at �max: 275nm ((�max for wavelength at which the
maximum absorbance obtained) and at �280nm.
� 275nm � 280nm
Bovine serum albumin 0.73 0.74
Bovine insulin 1.05 0.94
BSA (0.2 mg/ml) / BI (0.8 mg/ml) 0.48 0.44
BSA (0.4 mg/ml / BI (0.6 mg/ml) 0.46 0.43
BSA (0.6 mg/ml) / BI (0.4 mg/ml) 0.46 0.44
0.8BSA (0.8 mg/ml) / BI (0.2 mg/ml) 0.56 0.54
BSA (1.0 mg/ml) / BI (1.0 mg/ml)
PC: 37 ºC/24h/25rpm
0.95 0.89
BSA (1.0 mg/ml) / BI (1.0 mg/ml)
(RT:17 ºC/24h/25rpm
0.56 0.56
124
Figure 5-4 Chromatographic profile of bovine insulin (BI, 1mg/ml), bovine serum albumin
(BSA, 1mg/ml) and BSA modified with alternative concentrations and equal volumes of BI
(mg/ml) under physiological conditions (PC: 37ºC/24h/25rpm, pH 7.4) and under room
temperature (RT: 17 ºC/24h/25rpm, pH 7.4). Column (C18), isocratic mobile phase solvents
(100% acetonitrile and 0.1% trifluoroacetic acid in double-deionised water). Flow rate
1ml/min. Detection at 220nm.
Fig 5-6-1 BI
Fig 5-6-2 BSA
Fig 5-6-3 BSA (1)/BI(1)
RT: 17Cº/24h/25rpm
Fig 5-6-4 BSA (1)/BI(1)
PC:: 37Cº/24h/25rpm
Fig 5-6-5 BSA (0.2)/BI (0.8)
Fig 5-6-6 BSA (0.4)/BI (0.6)
Fig 5-6-7 BSA (0.6)/BI (0.4)
Fig 5-6-8 BSA (0.8)/BI (0.2)
125
5.5.2 Heat processing of native and modified bovine serum albumin with bovine
insulin
Compared to the unmodified and water bath heat processed BSA, changes in the
absorbance maximum of mBI-BSA in reference to the not heat processed mBI-BSA were
substantial. This was temperature and exposure time dependent (Figure 5-5 and 5-6).
While BSA has exhibited obvious spectral changes at 50ºC/5min, and 60ºC/5min, the
highest was at 70 ºC/5min (36% at �275nm, and 33% at �280nm).
Modified BI-BSA has also shown substantial changes in the absorbance maximum at
40ºC /10min, and 60ºC /15min (97.7%, 48.4% at �275nm, and 100.3%, 49.3% at �280nm
respectively); the highest change was at 70ºC /10min (119.5% at �275nm, and 122.3% at
�280nm). Both BSA and mBI-BSA have shown a steady increase in their absorbance
spectrum as subjected to higher temperatures (80, 90, and 100ºC /5min). This was
substantially higher in mBI-BSA absorbance spectrum activity with a slight milky
discolouration.
126
Figure 5-5 Percent change in the effect of water bath heat processing at various
temperatures and exposure time on the absorbance maximum of bovine serum albumin
(BSA) and BSA modified with bovine insulin (mBI-BSA) under physiological conditions (37
ºC/24h/25 rpm). The percent change in the absorbance maximum of heat processed BSA
and mBI-BSA calculated in reference to that of untreated BSA and untreated mBI-BSA
respectively. Absorbance determined at �280nm.
-5%
15%
35%
55%
75%
95%
115%
mBI-BSA BSA
Modification and water bath heat processing
Change in
absorbance
spectrum
100ºC / 5min
80ºC / 5min
70ºC / 5min
60ºC / 15min
60ºC / 5min
40ºC / 10min
37ºC /24h /25rpm
17ºC/24h /25rpm
127
Figure 5-6 Effect of water bath heat processing at various temperatures and exposure time
on the chromatographic profiles of bovine serum albumin (BSA), and BSA modified with
bovine insulin (mBI-BSA) under physiological conditions (37 ºC/24h/25 rpm). Column C18,
isocratic mobile phase solvents (100% acetonitrile and 0.1% trifluoroacetic acid in double-
deionised water). Flow rate 1ml/min. Detection at 220nm.
Fig. 5-8-1 BSA 60ºC/15min
Fig.5-8-2 BI-BSA 60ºC/15min
Fig. 5-8-3 BSA 70ºC/10min
Fig. 5-8-4 mBI-BSA 70ºC/10min
Fig. 5-8-5 BSA 80ºC/5min
Fig. 5-8-6 mBI-BSA 80ºC/5min
128
5.5.3 Water bath versus hot plate heat treatment
To replicate home and industrial heat processes, milk proteins were heat processed by
both water bath and hot plate heat processing. Figure 5-7 shows that all examined BMPs
have shown considerable changes in their absorbance maximum as subjected to water
bath heat processing with considerable variation among them. Infant starter formulae
(ISFK and ISFS) have yielded the highest absorbance maximum, whereas enzymatically
digested ISFN has shown a negative change in its absorbance.
While the highest positive percent change in the absorbance maximum of water bath heat
processed BMPs was observed in ISFK, ISFN has exhibited the lowest percent change in
its absorbance maximum with a negative trend throughout all temperatures applied (40-
100ºC/15min). Figure 5-9 also exemplifies that while only RWPBM has yielded a lower
absorbance maximum as the water bath heat processing was replaced with hot plate heat
processing procedure, ISFK and ISFS have yielded the highest change in their
absorbance maximum as the processing method changed, whereas enzymatically digested
(ISFN) formula has shown relatively lower change than that of ISFK and ISFS.
129
Figure 5-7 Effect of water bath heat processing (WB) at (40-100 ºC for 15 min) and hot
plate heat processing (HP) at (63 ºC for 15 and 30 min) procedures on the percent change in
the absorbance maximum of bovine serum albumin (BSA), raw whole pooled bovine milk
(RWPBM), canned whey dominant bovine-based starter infant formulae (ISFS), and
canned enzymatically hydrolysed whey protein infant formula (ISFN). The percent change
in absorbance calculated in reference to that of not heat processed milk protein.
Absorbance determined at �280nm.
-0.25
-0.05
0.15
0.35
0.55
0.75
0.95
1.15
BSA RWPBM ISFS ISFN
Milk proteins
% C
han
ge a
bso
rba
nce a
t �280n
m
WB 100 ºC/min
WB 70 ºC/min
HP 63 ºC/15min
HP 63 ºC/5min
WB 60 ºC/15min
WB 40ºC/min
130
5.6 Discussion
Bovine milk is a very complex system with a minimum of 100,000 different molecular
varieties. Many factors are capable of affecting the constituents of milk, for instance
breed, cow to cow and breed to breed variations that encompass feeds, seasonal and
geographic variations (Goff and Hill 1993). The appearance of both milk and milk
products is influenced by their spectral characteristics that provides the basis for many
rapid indirect methods of analysis, such as proximate analysis (Goff and Hill 1993; Goff
2004).
Infants experience rapid growth during their first year of life. Human breast milk is the
ideal initial food for them (Wold and Adlerberth 2000; Oddy 2002). Mature and early
human milk is heterogeneous (Viverge et al. 1990; Coppa et al. 1993; Newburg 2000)
and its composition differs considerably from that of bovine milk and bovine milk-based
infant formulae, which contain protein at a very low concentration as well as a high
concentration of lactose (Goff and Hill 1993; National Health and Medical Research
Council 2003).
Modern infant formulae require both large scale and home food processing that can bring
about extreme alteration in the chemical composition of food products (Rechcigl 1982).
Requirements to assimilate human milk high lactose content have made infants formula
one of the most difficult-to-dry foods. Bovine milk is a complex polydispersion system
and individual components of milk have various impact on the physical properties of
milk (Pisecky 1997). The physical properties, viscosity, density and heat stability of milk
are the most important properties to produce canned milk formulae (Pisecky 1997).
131
In spite of all efforts, it is not possible to duplicate the quality of human milk as well as
the concentration of both macro and micronutrients content (Mayor 2004). Bovine milk
contains more than 25 separate proteins, 80% of these proteins are heterogeneous casein
occurring as a micellar complex. Whey proteins form the remaining 20% of the milk
protein component (for example -lactoglobulin (BLG), �-lactalbumin (ALA) and BSA)
(Pisecky 1997; National Health and Medical Research Council 2003). Both adult and
infant formulae require various processing procedures to meet ingredient and nutritional
requirements.
Heat processing is one of the most frequently applied methods in milk-based foods
processing technology. Thermal processing attributes to some undesirable possibly severe
quality defects to bovine milk, a heat sensitive liquid, including the denaturation of the
proteins (Pisecky 1997). Protein functional properties are completely dependent on its
three dimensional structure (Kimball 2004). Heat treatment (Pisecky 1997), the presence
or absence of co-denaturation associate, industrial thermal pre-treatment (Bertrand-Harb
et al. 2002), and pH (Law and Leaver 2000) are all factors capable of disrupting protein
structure.
132
Bovine milk proteins are widely implicated as an allergenic factors in infants (Goldman
et al. 1963; Kletter et al. 1971b; May et al. 1982; Bahna and Gandhi 1983) as well as a
potential triggering factors of the pancreatic autoimmune process (Karjalainen et al.
1992b; Saukkonen et al. 1994; Levy-Marchal et al. 1995a). It contains a minimum of 25
proteins capable of inducing immune responses (Bahna and Gandhi 1983). The
antigenicity of certain milk proteins altered because of heat processing, BSA for example
loses most of its antigenicity at 70 ºC (Hanson and Mansson 1961; Strand 1994); it
renders protection to casein (Pisecky 1997), a heat resistant milk protein (Hanson and
Mansson 1961). Heat processed albumin go through reversible and irreversible stages of
structural changes (Kuznetsov et al. 1975; Lin and Koenig 1976; Oakes 1976). Heat
processing above 65ºC is regarded as an irreversible stage but does not necessarily result
in a complete destruction of the ordered structure (Wetzel et al. 1980).
The pH of milk fluctuates within (6.5 to 6.7, 25 °C) with casein and phosphate acting as
the main buffers (Goff and Hill 1993). Changes in the rate of heat denaturation with
whey proteins at certain pH are complex and dependent on the rates of unfolding and
aggregation phases (Law and Leaver 2000). Given that various heating processing
methods affect the physical nature of milk proteins (Saperstein and Anderson 1962) as
well as causing protein antigens to aggregate, (Hanson and Mansson 1961; Harlow and
Lane 1988; Strand 1994; Alting et al. 1997), small changes in the structure of an antigen
can influence intensely the vigour of the immune response (Harlow and Lane 1999).
Unlike pasteurised whey protein or skimmed milk, antibodies produced against extremely
heat processed whey protein were very low or undetected. If detected antibodies were
residual casein specific that indicates an extensive denaturation of these proteins
(Kilshaw et al. 1982; Heppell et al. 1984).
133
The modification and thermal treatment of various proteins can lead to the formation of
new epitopes. This can alter the immune response to recognise the newly formed epitopes
of the modified molecule rather than the native epitopes of the molecule. Bovine serum
albumin is a thermo-labile milk protein and loses most of its antigenicity beyond critical
temperatures 70 to 80ºC (Hanson and Mansson 1961; Karjalainen et al. 1992b; Savilahti
et al. 1993; Boza et al. 1994; Alting et al. 1997). Modification of both BSA (Teale and
Benjamin 1976) and bovine insulin (Speth and Lee 1984) with various binding molecule
as well as heat processing have a substantial effect on their immunogenic response
(Hanson and Mansson 1961; Karjalainen et al. 1992a; Alting et al. 1997). While the
receptor activity of BSA is enhanced significantly if the modification was first of the
amine terminal of arginine (Leibowitz and Soffer 1971), the modification of BSA with
methoxypolyethylene has masked the antigenic sites of the modified BSA and was not
able to induce any immunogenic response (Teale and Benjamin 1976). Modified bovine
and porcine insulin have shown a high intramolecular mobility (Bak et al. 1967).
The findings of the present study are consistent with previous studies and provide further
evidence that thermal processing of BMP can cause substantial changes in their spectral
characteristics. Changes in the spectral profiles of milk proteins may be indicative of
changes in the denaturation behaviour of milk proteins and indicative of the initiation of
the aggregation process of heat processed milk proteins (Dalgleish 1990; de Frutos et al.
1992; Ferreira et al. 2001). Reports are helpful in representing the relative behaviour of
different proteins; nonetheless data do not necessarily foretell the functionality of such
proteins in real food systems which reflect the fact that in foods, substantial interactions
with other ingredients may take place, resulting in modified behaviour of the proteins
(Kinsella and Whitehead 1989).
134
5.7 Conclusion
The identification of bovine milk protein solutions by their characteristic spectral profile
has provided further evidence on their heterogeneity. Both modification and thermal
processing of bovine milk proteins have caused substantial changes in their spectral
profiles that were concentration, temperature, and exposure times dependent. Whether
these changes are indicative of changes in the immunogenicity of the modified and heat
processed milk proteins require further investigation. It does seem encouraging to suggest
that humanisation of the spectral profile of infant formulae may open a window to
resolving controversy encircling the diabetogenicity of bovine milk proteins.
135
6 A newly developed saliva-based ELISA to determine
immune response to milk proteins in young diabetic
Jordanians: Lack of association for infantile feeding
practices
6.1 Abstract
Objective: There is growing worldwide interest in the identification of potential
environmental factors that may trigger the pancreatic autoimmune diabetes in genetically
susceptible individuals. The possible immunogenicity of bovine serum albumin (BSA)
and modified and heat processed BSA with bovine insulin (mBI-BSA) was examined.
Design and methods: Fifty diagnosed diabetic children under the age of 14 years and
their unrelated age and gender matched control subjects were identified. Serum and saliva
samples were collected and IgG and secretory IgA antibodies to BSA as well as to native
and heat processed mBI-BSA were determined. Data on participants’ breastfeeding and
early infant feeding practices were collected using constructed three-part questionnaire
developed previously.
136
Results: Heated mBI-BSA (70ºC/5min) has shown the highest relative change in the both
IgG and sIgA titre levels, whereas mBI-BSA has also shown a substantial relative change
in antibody titre levels compared to that of untreated BSA. Mean log effect of mBI-BSA
on the titre levels of both IgG and sIgA antibodies in both DC and NDC has increased by
approximately 1.3 fold of that of unheated or modified BSA (P = 0.000, and 0.114
respectively). The mean log effect of heat processed mBI-BSA at (70ºC/5min) on IgG
and sIgA titre levels in both groups has also increased by nearly 1.5 of that of native BSA
(P = 0.000), and 1.1 fold of that of native mBI-BSA (P = 0.000). There were no immune
responses to mBI-BSA heated at 70 ºC/10min and 80ºC/5min) and 60ºC/5min). There
were significant correlations between DC (60%) and NDC (25%) for the positivity of
CRP (P = 0.05).
Conclusion: The saliva-based ELISA system may be a useful proxy of immune
responses and may constitute new grounds for the ongoing dietary intervention trials. If
bovine milk proteins are true diabetogens, the modification and thermal processing
procedure of BSA is a merely sophisticated system in eliciting immune responses and is
neither limited to diabetic patients nor associated with breastfeeding or early infant
feeding practices, and may reflect unspecific defect of the immune system.
137
6.2 Introduction
None of the potential primary determinants, either genetic or environmental factors, of
type 1 autoimmune diabetes has been established unequivocally (Kimpimaki et al. 2001;
Akerblom et al. 2005; Kent et al. 2005; Nakayama et al. 2005). Nonetheless, a number of
different complex mechanisms of pancreatic beta cell destruction are operative in type 1
autoimmune diabetes (Kawasaki et al. 2004).
Although bovine insulin is implicated as a putative primary autoantigen in animals
(Atkinson et al. 1986; Srikanta et al. 1986; Komulainen et al. 1999; Kimpimaki et al.
2002; Nakayama et al. 2005), it is a small protein (hapten) (Harfenist and Craig 1952;
Sanger 1958) and is unable to elicit immune response by itself. Rather it requires a
linkage to a large immunogenic carrier, such as bovine serum albumin (BSA) (Carter and
Ho 1994; Harlow and Lane 1999; Roitt et al. 2001).
The modification of either BSA (Teale and Benjamin 1976) or bovine insulin (Speth and
Lee 1984) with different binding molecules as well as exposure to heat processing
(Hanson and Mansson 1961; Karjalainen et al. 1992a; Strand 1994; Alting et al. 1997)
can cause a substantial effect on their immunogenic response. Substantial interactions
with other ingredients in food systems may result in the modified behaviour of the
proteins (Kinsella and Whitehead 1989). Changes in the mucosal architecture as well as
concomitant interactions between different dietary (Teale and Benjamin 1976; Speth and
Lee 1984) and non-dietary constituents (Clarke 1975; Sharma et al. 1995b) coexisting in
the gut environment can either boost or suppress the autoimmune process (Scott et al.
1996b).
138
The present study focuses on determining immune responses to modified and heat
processed milk proteins particularly BSA and bovine insulin by using a newly developed
saliva-based enzyme linked immunosorbent system (ELISA) in young Jordanian diabetic
children. This will be with regard to their breastfeeding and early feeding practices of
participants. In addition, this study may verify whether changes in the spectral profiles of
BMP are indicative of alteration in their immunogenicity (Al-Domi et al. 2004).
6.3 Research design and methodology
6.3.1 The population sample
The cohort of this retrospective case-control study consisted of 50 (36 females and 14
males) Jordanian diabetic children (DC) aged 14 years or less. Participants were
identified by The National Centre for Diabetes, Endocrinology and Genetics, Jordan, and
50 (28 females and 22 males) unrelated, age and sex matched non-diabetic children
(NDC) were identified by Princess Rahmah Children’s Hospital, and Al-Zahrawi Medical
Laboratory, Jordan. Diagnosis of diabetes was confirmed by the medical history of the
diabetic children, including the presence of clinical symptoms and the need for insulin
therapy right after diagnosis. Participants were unrelated to those with diabetes or to each
other. Control subjects were age and gender matched. Data on breastfeeding and early
infant feeding practices were collected using a constructed questionnaire. Ethical
approval was granted by the Human Research Ethics Committee of the University of
Western Sydney as well as by the authorities of all Jordanian participating institutions.
All subjects, or their parents when appropriate, gave either written informed consent or a
witnessed verbal consent when appropriate.
139
6.3.2 Breastfeeding and early infant feeding practices
Data on breastfeeding and early infant feeding practices were modified from a previous
study using a constructed questionnaire (Chapter 3, appendix 10.1 and appendix 10.2).
6.3.3 Blood samples collection
A licensed phlebotomist drew 10ml venous blood samples during routine consultation
visits to the Hospital Outpatients Clinics by venipuncture into a clot (red top) test tube.
Whole blood samples were stored overnight at 4ºC and then centrifuged at 4ºC/1000
rpm/15 min. Serum aliquots were stored frozen at -20ºC (Jonstone and Thrope 1996).
Two hemolyzed blood serum samples were discarded.
6.3.4 Saliva samples collection
Twenty saliva samples were collected from age-matched DC and their age and sex
matched controls. Participants with periodontal diseases or who had undertaken major
dental work and/or those prone to respiratory infections were excluded. To minimize the
potential of saliva contamination particularly from dairy products, whole unstimulated
samples were collected at least 30 minutes after the last meal or drink. Participants were
asked to rinse their mouth thoroughly with water 10 minutes prior to sample collection.
140
Participants were encouraged to relax and visualize their favourite foods. Participants
were seated comfortably (Miletic et al. 1996) and saliva samples (5-10 ml) were drawn
by either passive slavering into a sterile swap tube or by a sterile plastic transfer suction
pipette. Saliva samples were clarified by centrifugation at 4ºC/1000 rpm/15min (Castro et
al. 2004). Aliquots were stored frozen at -20ºC. To break down mucopolysaccharides
saliva samples were exposed to a single freeze-thaw cycle (Worthman et al. 1990;
Shirtcliff et al. 2000).
6.3.5 C - reactive protein (CRP)
Blood serum of participants who had donated saliva samples were tested for total serum
CRP by slide agglutination (CRP-Latex test) obtained from (Plasmatec Laboratory
Products Ltd, UK). Specimens were tested for CRP positivity and negativity. The
presence of agglutination indicates a level of CRP in the sample ( 6 mg/l).
Manufacturer’s specifications were maintained. Positive and negative controls were
tested in parallel with each batch of tests.
6.3.6 Preparation of the testing antigens
Bovine serum albumin (BSA) and bovine insulin (BI) (Sigma-Aldrich MO, USA) were
reconstituted in double deionised ultrafiltered water (0.18µm) and were filtered through
0.22µm filter (Millipore, UK). Bovine serum albumin (1mg/ml) was modified with an
equal volume of BI (1mg/ml) under strictly aseptic physiological conditions
(37ºC/24h/25rpm, pH 7.4).
141
Modified BSA with BI (m-BI-BSA) was subjected to water bath heat processing under
the same conditions at 40ºC/10 min, 50ºC/5 min, 60ºC/5min, 60ºC/15min, 70ºC/5 min,
70ºC/10min and 80ºC/5min as described in chapter five (Al-Domi et al. 2004). Since this
study was carried out in Jordan and to verify our developed spectrophotometric assay, the
modification and heat processing of mBI-BSA were conducted strictly under the same
condition (for more details see chapter 5) (Al-Domi et al. 2004).
6.3.7 Enzyme linked immunosorbent assay (ELISA)
A newly developed ELISA was used to determine serum IgG and sIgA tire levels. In
brief, 96-well, high-binding flat-bottom microtitre plates (Greiner-Bio-One GmbH,
Germany) were coated with 50 l of (1 g/ml) native BSA, native mBI-BSA, and mBI-
BSA heat processed at different temperatures and various exposure times (40ºC/10 min,
50ºC/5 min, 60ºC/5min, 60ºC/15min, 70ºC/5 min, 70ºC/10min and 80ºC/5min) in (0.1M
phosphate-buffered saline (PBS), pH 7.4, buffer 1) (Sigma-Aldrich, St Louis, USA).
Plates were incubated for 12 hours at 4 ºC.
The coated plates were washed with 0.05% Tween 20 in buffer 1 (buffer 2) five times.
The plates were blocked with 100l (1%) normal sheep serum (DakoCytomation,
Denmark) thermally treated at 50ºC/5min in buffer 2 and were incubated for one hour at
37 ºC) then were washed with buffer 2 three times. Serum and saliva specimens were
thawed and plates were coated with 50l of both serum and saliva specimens. Two-fold
dilution in buffer 1 was carried out for each of the specimens under the same conditions
on the same plate this to avoid interassay variability. Coated plates were sealed and
incubated for one hour at 37ºC followed by washing with buffer 2 three times.
142
Plates were coated with 50l polyclonal alkaline-phosphatase-conjugated affinity purified
rabbit anti-human IgG specific for Gamma-chains and alkaline-phosphatase-conjugated
affinity purified rabbit anti-human IgA specific for Alpha-chains antisera
(DakoCytomation, Denmark) diluted 1/3000 in buffer 2, incubated for one hour at 37ºC
and washed with buffer 2 four times. Liquid p-nitrophenylphosphate substrate (50l)
(Sigma-Aldrich, St Lois, MO) was added and the plates were incubated for six hours at
37ºC). Reaction was stopped by adding 1 M NaOH and the end-point was measured at
405nm (ELX 800, Bio-Tek Instruments Inc.; Kcjunior software). Duplicates varying by
more than 5% error were retested. All plates were sealed prior to incubation, and
incubation was undertaken in a humidified incubator with a preset temperature.
Optical densities were subjected to point-to-point analysis. The cut-off point was
determined as blank optical density [OD] *2. Serum and saliva titres were expressed as
the 50% of the reciprocal dilution factor of OD just above the cut-off point. Titres less
than or equal to the cut-off point were assumed zero.
6.3.8 Statistical analysis
All data were coded, computer entered and analysed using the Statistical Package for
Social Sciences (SPSS). Assuming normally distributed errors, the data fit a linear
regression model and the bivariate correlations procedure was used to compute Pearson’s
correlation coefficient and Paired-Samples T-test, independent-Samples T-test and
multivariate analysis of variance (ANOVA) parametric statistics were computed for
serum and saliva logarithmic (log) titre levels.
143
Based on the values of a set of predictor variables, the logistic regression model was used
to estimate odds ratios (OR) with 95% confidence interval (CI) for each of the
independent variables in the model. Data were considered statistically significant at
P<0.05.
6.4 Results
Figure 6-1 delineates relative change in the serum IgG and secretory IgA antibody titre
levels produced to native bovine serum albumin (BSA), and native and heat processed
modified BSA with bovine insulin (mBI-BSA) in both DC and NDC, with reference to
that of BSA. While mBI-BSA has shown a substantial relative change in antibody titre
levels, different heat treatment temperatures and exposure times have exhibited a wide
range of changes in IgG and sIgA immune responses. Heat processed mBI-BSA
(70ºC/5min) has shown the highest relative change in both IgG and sIgA antibody titre
levels. Heat processing of mBI-BSA at 40ºC/10min, 60ºC/15min, 70ºC/10min and
80ºC/5min has shown a negative relative change in the IgG and sIgA antibody titre
levels, whereas heat processing of mBI-BSA at 50ºC,60 and70ºC for five minutes has
shown a positive trend.
144
Figure 6-1 Percent change (*100) in serum IgG and secretory IgA (sIgA) titre levels of
diabetic children produced to modified and heat processed bovine serum albumin (BSA)
with bovine insulin (mBI-BSA) at different temperatures and exposure durations
(70C/5min, 60ºC/15min, 60ºC/5min, 50ºC/5min, 40ºC/10min and untreated mBSA-BI).
Relative change calculated in reference to that produced against untreated BSA. Percent
change calculated in reference to IgG and sIgA antibody titre levels produced against
untreated BSA.
-20%
0%
20%
40%
60%
80%
100%
Change in
serum IgG and
secretory IgA
titre levels*
100
Patients
IgG
(n=48)
Patients
sIgA
(n=20)
Water bath heat
treatmnet (ºC/min)
mBSA-BI (70/5)
mBSA-BI (60/15)
mBSA-BI (60/5)
mBSA-BI (50/5)
mBSA-BI (40/10)
mBSA-BI
145
Figure 6-2 compares logarithmic (log) mean effect that native bovine serum albumin
(BSA) and native and heat processed mBI-BSA have on both serum IgG and secretory
IgA antibody titre levels in diabetic and non-diabetic Jordanian children aged 14 years or
less. The mean log effect of mBI-BSA on the antibody titre levels of both IgG and sIgA
antibodies in DC and NDC has increased by approximately 1.3-fold of that of unheated
or modified BSA (P = 0.000, and 0.114 respectively). The mean log effect heat processed
mBI-BSA at 70ºC/5min has on IgG and sIgA antibody titre levels in both DC and NDC
has also increased by approximately 1.5-fold of that of native BSA (P = 0.000) as well as
1.1-fold of that of native mBI-BSA (P = 0.000).
In addition, there was a significant difference for the increase in mean log serum IgG
antibody titre levels produced to mBI-BSA heat processed at 50 and 60ºC/5min (P =
0.000) as well as to sIgA antibody titre levels produced against mBSA-BI heat processed
at 50ºC/5min (P = 0.001). This was not significant to mBI-BSAS heat processed at
60ºC/5min (P = 0.155). Interestingly there were neither humoral nor secretory immune
responses to mBI-BSA heated at 70 ºC/10min and 80ºC/5min. Furthermore, there were
significant correlations between DC (60%) and NDC (25%) for the positivity of CRP (P
= 0.05, Phi = 0.21), given the normal levels of 6 mg/l (Hayashi et al. 1970; Roitt et al.
2001).
146
Figure 6-2 Comparison between the sample paired test mean effect that native bovine
serum albumin (BSA) and modified and heat processed BSA with bovine insulin (mBI-BSA)
have on both logarithmic (log) serum IgG and secretory IgA (sIgA) antibody titre levels in
both diabetic and non-diabetic Jordanian children aged 14 years or less. Mean effect was
statistically significant at P<0.05 for all heat and modified BSA and BI.
0
0.5
1
1.5
2
2.5
3
BSA mBI-
BSA*
mBI-
BSA
(40/10)
mBI-
BSA
(60/15)*
mBI-
BSA
(70/5)
Modification and heat processing
(ºC/min)
Mean
effect on
DC and
NDC
IgG (n = 48)
sIgA (n = 20)
* The effect of mBI-BSA (60ºC/15 was statistically insignificant on sIgA antibody titre
levels at P<0.05
147
Titre levels of IgG antibodies produced to native and heated mBI-BSA at 70ºC/5min
were strongly associated with the diabetic and control group (r = -0.78 and -0.65, P =
0.000, respectively); IgG antibody titre levels to heat processed mBI-BSA at (50 and
60ºC/5min were also positively associated (r = -0.45, -0.43 and -0.32, P = 0.00,
respectively). Similarly, sIgA antibody titre levels produced to heated mBI-BSA at
70ºC/5min were strongly associated with the diabetic and control group (r = -0.77, P =
0.000) that was not significant for native mBI-BSA.
Table 6-1 shows that there were statistically significant differences for tests of between-
subjects effect in DC and NDC for interactions between IgG and sIgA antibody titre
levels produced to unheated and not modified BSA, and native and heat processed mBI-
BSA at different temperatures and exposure times. Modified BI-BSA has a strong
negative association with log IgG antibody titre levels (r = -0.65, P = 0.000) that was (-
0.25, P = 0114) for sIgA in both the diabetic and control group, whereas IgG and sIgA
log antibody titre levels has the strongest association with mBI-BSA heat processed at
70ºC/5 min in both groups (r = -0.78 and -0.77, P = 0.000 respectively). The logistic
regression analysis yielded that IgG and sIgA log antibody titre levels produced to
untreated BSA, mBI-BSA and heat processed mBI-BSA in both DC and NDC was
associated with a relatively similar risk of developing diabetes.
148
Table 6-1 Multivariate analysis of tests between-subjects; effects for native bovine serum
albumin (BSA) and modified and heat processed BSA with bovine insulin (mBI-BSA) on
serum IgG and secretory IgA (sIgA) titre levels in both diabetic and non-diabetic Jordanian
children aged 14 years or less. Data were considered statistically significant at P<0.05.
Between-antigens effects F P-Value
Anti-BSA sIgA and
anti mBI-BSA IgG
5.211 0.029
Anti-mBI-BSA sIgA and
anti-mBI-BSA IgG (60 ºC / 5 min)
4.836 0.035
Anti-mBI-BSA (50 ºC / 5 min) sIgA and
anti-mBI-BSA IgG (50 ºC / 5 min)
7.028 0.013
Anti-mBI-BSA (60 ºC / 5 min) sIgA and
anti-mBI-BSA IgG (60 ºC / 15 min)
4.511 0.042
6.5 Discussion
To the best of the researcher’s knowledge, this study constitutes the first study on the
immune response in young Jordanian DC aged 14 years or less with regard to their
breastfeeding and early feeding practices. Worldwide, this study is also the first to
develop a new saliva-based ELISA to determine the salivary IgA antibodies to dietary
proteins particularly bovine milk proteins in diabetic children. This assay is non-invasive,
rapid, accurate, valid and highly reproducible if proper standards and conditions are
controlled to avoid unsystematic errors.
149
This includes number of factors. 1), age-matching, given that the levels of sIgA that
follow the development and maturation of the salivary glands have a parabolic rapport
with age (Smith et al. 1987; Ben-Aryeh et al. 1990; Kugler et al. 1992; Miletic et al.
1996; Percival et al. 1997); 2), saliva sample collection by means other than cotton
(Dabbs 1991; Shirtcliff et al. 2001); 3), exclusion of patients with either periodontal
diseases (Alaluusua 1983) or respiratory infections (Lehtonen et al. 1987; Atis et al.
2001); and 4), avoidance of contamination with dairy products (Magnano et al. 1989).
Saliva collection is a non-invasive, stress-free, simple method, and can be easily collected
and stored (James-Ellison et al. 1997). Therefore, sIgA has been widely used as a
biomarker in a number of fields. 1), biobehavioural studies (Ben-Aryeh et al. 1984;
Worthman et al. 1990; Hertsgaard et al. 1992; Shirtcliff et al. 2000; Shirtcliff et al. 2001);
2), toxins and pollutants (Gilfrich et al. 1981; Bauer et al. 1983); 3), as diagnostic tool
(Behets et al. 1991; Ciclitira and Ellis 1991; Kozlowski and Jackson 1992); and 4), to
determine the levels of antibodies produced to gliadin in patients with celiac autoimmune
disease (Ciclitira and Ellis 1991; Fasano et al. 2003).
Salivary IgA antibodies are one dominant characteristic humoral factor of the local
immune system in the body secretions; therefore, it can act as first-defence line against
local infections and as well as preventing the access of foreign antigenic factors to the
immune system (Roitt et al. 2001). The high synthesis rate of the T cells dependent sIgA
antibodies and the direct association with foreign factors as well as half-life and direct
association with the activation of both T and B cells (Smith et al. 1987; Paul 1993) have
made sIgA a better immune marker over measuring B and T cell in blood serum (Miletic
et al. 1996).
150
Antibody immune responses at various mucosal effector sites are antigen type and dosage
dependent. The whole mucosal immune system distant from the sensitisation site can be
engaged by the sensitisation of a specific site of the system that may present a thorough
view of the performance of the entire mucosal immune system (Jertborn et al. 1986;
Externest et al. 2000). Changes in dietary ingredients, microbial flora (Sharma et al.
1995b; Sharma et al. 1995a), bacterial metabolites (Fontaine et al. 1996) and enteric viral
infections (Hiltunen et al. 1997; Honeyman et al. 2000; Salminen et al. 2004) as well as
interaction between these factors coexisting in the gut system may significantly change
the mucosal architecture, the mucosal adaptation (Sharma and Schumacher 1995), the
epithelial cell production (Clark et al. 1981), and the regional number of the
enteroendocrine cells (Sharma and Schumacher 1996).
Therefore, stabilisation of the mucosal barrier may become an important factor important
in gut health maintenance (Kleessen et al. 2003) that may affect gut permeability to
possible foreign, dietary and non-dietary antigens and that impair secretory immunity.
Substantial interactions with other ingredients in food systems may result in modifying
the behaviour of proteins (Kinsella and Whitehead 1989).
151
The immunogenicity of antigens can often be profoundly altered by slight changes in the
physical and chemical nature of the antigen; many molecules can be made more
immunogenic by denaturation. Given that aggregate-free antigens may inhibit the
immune responsiveness (Kletter et al. 1971b; Harlow and Lane 1988; Saukkonen et al.
1994), protein antigens aggregate caused by heat processing are usually more
immunogenic. Heat treatment changes the structure of many compounds particularly
proteins and expose new epitopes. This modification can either alter the regions of the
immunogen to present better sites for T-cell binding or expose new epitopes for B-cell
binding. While antibodies can differentiate between conformations of protein antigens,
small structural changes of the epitope can prevent the recognition of the antigen.
Antibodies can differentiate between conformations of protein antigens (Harlow and
Lane 1988).
Bovine serum albumin particularly ABBOS peptide (Karjalainen et al. 1992a; Saukkonen
et al. 1994), and insulin and proinsulin (B: 9-23 peptide) (Wegmann et al. 1994; Abiru et
al. 2000; Alleva et al. 2000; Wong et al. 2002; Kent et al. 2005; Nakayama et al. 2005)
are among the most putative environmental dietary proteins implicated in setting of the
beta cell destruction. Insulin is one of the most strongly implicated as a potential primary
autoantigen capable of initiating the destruction of insulin secreting beta cells sanctioning
other pancreatic proteins to become the primary target leading to the disease. It has also
been anticipated that insulin becomes a target ensuing the initiation of the autoimmune
process by another autoantigen (Alleva et al. 2000; Juvenile Diabetes Research
Foundation International 2005b; Nakayama et al. 2005).
152
Bovine insulin, differs from human insulin only at three amino acid residues (Yip et al.
1998), is a small hapten (5,808 Daltons) (Harfenist and Craig 1952; Sanger 1958) and
cannot elicit an immune response by itself. For insulin to initiate an immune response, it
requires coupling to a carrier protein such as BSA (66430.3 Daltons) (Harlow and Lane
1988; Carter and Ho 1994). The immunogenicity of antigens can often be profoundly
altered by slight changes in the physical nature of the antigen; many molecules can be
made more immunogenic by denaturation.
Given that aggregate-free antigens may inhibit the immune responsiveness (Kletter et al.
1971b), protein antigens aggregate caused by heat processing are usually more
immunogenic. Heat treatment changes the structure of many compounds particularly
proteins and expose new epitopes. This modification can either alter the regions of the
immunogen to present better sites for T-cell binding or expose new epitopes for B-cell
binding. While antibodies can differentiate between conformations of protein antigens,
small structural changes of the epitope can prevent the recognition of the antigen.
Antibodies can differentiate between conformations of protein antigens (Harlow and
Lane 1988).
153
The modification and thermal processing of proteins can lead to the formation of new
epitopes, which can alter the immune response to recognise the newly formed epitopes of
the modified molecule rather than the native epitopes of the molecule. Modification of
both BSA (Teale and Benjamin 1976) and bovine insulin (Speth and Lee 1984) with
various binding molecule as well as heat processing have a substantial effect on their
immunogenic response (Hanson and Mansson 1961; Karjalainen et al. 1992a; Alting et
al. 1997). While the receptor activity of BSA is enhanced significantly if the modification
was first of the amine terminal of arginine (Leibowitz and Soffer 1971), the modification
of BSA with methoxypolyethylene has masked the antigenic sites of the modified BSA
and was not able to induce any immunogenic response (Teale and Benjamin 1976).
Modified bovine and porcine insulin have shown a high intramolecular mobility (Bak et
al. 1967). Porcine insulin was capable of activating and binding to phosphatase to form a
complex, whereas insulin chains as well as BSA were not capable of either the activation
process or the binding process (Speth and Lee 1984).
Modern infant formulae require both large scale and home food processing, which can
bring about extreme alteration in the chemical and physical nature of food products
(Rechcigl 1982). Although the detection of antibodies associated with diabetes is
technically demanding, antibodies to BSA are common in the general population (Levy-
Marchal et al. 1995a). Bovine serum albumin has many epitopes capable of inducing a
broad range of high and low affinity general antibodies population which can recognise
both conformational epitopes as well as changes due to the denaturation of BSA
(Karjalainen et al. 1992b; Savilahti et al. 1993; Saukkonen et al. 1994).
154
Bovine serum albumin is a thermo-labile milk protein and loses most of its antigenicity
beyond critical temperatures 70 to 80ºC (Hanson and Mansson 1961; Karjalainen et al.
1992b; Savilahti et al. 1993; Boza et al. 1994; Alting et al. 1997). Thermal processing of
canned infant formulae as well as liquid formulae at different temperatures and various
exposure times has rendered milk free of BSA (Hanson and Mansson 1961; Monte et al.
1994; Strand 1994; Alting et al. 1997). Patients sensitive to BSA might tolerate BMP
subjected to heat treatment, and to some extent, they are less tolerated by those who are
responsive to other protein fractions (Crawford 1960). Animal studies (Kilshaw et al.
1982; Heppell et al. 1984) have shown that unlike pasteurised whey proteins or skimmed
milk, antibodies produced to extremely heat treated whey proteins (100 or 115ºC for 30
minutes) were very low or undetected; if detected antibodies were residual casein specific
that indicates an extensive denaturation of these proteins.
Studies on the thermo lability of insulin and its role in the diabetogenicity of insulin
remain scarce. Nonetheless, its widely accepted that for insulin to maintain its viability,
insulin-dependent diabetic patients are encouraged not to stored insulin vials under
extreme temperatures (<2 or >30ºC) (American Diabetes Association 2002).
Furthermore, insulin suspensions exposed to temperatures more than 25ºC for extend
periods may become difficult to homogenise, whereas espousing insulin to 50ºC or more
leads to the coagulation of the insulin suspensions (Brange 1987) studies on the thermo
lability of insulin and its role in altering the immunogenicity of the insulin molecule
remain scarce. It is, therefore, pivotal to examine the effect of both modification and
thermal processing on the diabetogenicity of insulin, given that canned infant milk
formulae undergo a wide range of both large and home scale heat processing (Pisecky
1997).
155
Home scale preparation methods of infants liquid bottle milk feeds vary between the
application of severe heat processing temperature and extended exposure times (boiling
for an extended exposure times) to a less severe heat processing temperature and shorter
exposure times, for example the application of microwave fields (Singh et al. 1998).
Recent indications on the possibility to manipulate the initiation of pancreatic
autoimmune process by dietary intervention in infancy implies that different modification
as well as processing procedures of bovine milk proteins (conventional bovine-based
infant formula versus casein hydrolysate-based infant formula) may affect its
immunogenicity (Akerblom et al. 2005). Therefore, controlling the variability in protein
composition and protein modification as well as the degree of protein denaturation
requires further processing procedures appropriation and investigation.
Numerous physiological consequences are caused by adverse reactions to food that can
be either immunologic ally or non-immunologically mediated reactions resulting in a
wide range of signs and symptoms (Miller 1998). The majority of people with high
circulating levels of antibodies produced against a number of common microorganisms
often with highly immunogenic antigens (Miller 1998) such as viruses which among a
wide range of microorganisms can be also found in foodstuffs including milk products
(Vela 1997; Miller 1998). While circulating antibodies to food proteins are also common
in the general population (Kletter et al. 1971a), antibodies to bovine milk proteins are
weaker and less common in adults than in children (Kletter et al. 1971a). The
development of a state of unresponsiveness to dietary proteins such as bovine serum
albumin in adults can be a result of prolonged minimal antigenic stimulation (Korenblat
et al. 1968).
156
Antibodies to bovine milk protein in neonates are often very low or absent, if present it
may originate from the maternal circulation (Kletter et al. 1971a). While children born to
diabetic mothers have maternally acquired antibodies that can continue to at least nine
months after birth, increased islet autoantibody levels in infants is most likely a sign of de
novo production of autoantibodies associated with T1ADM (Ziegler et al. 1999). The
production of the antibodies in neonates can be initiated within eight days of exposure
reaching the highest levels at three to fifteen months of age where the production of
antibodies start to decrease with age (Kletter et al. 1971a).
On the surface it would appear to be a logical association with the risk or protection from
diabetes, nevertheless evidence on the use of breastfeeding or early infant feeding
restrictions to prevent or minimise the disease occurrence is of limited strength and
should not be misinterpreted by parents and the public (Atkinson and Gale 2003). Human
retrospective studies remain discrepant and controversial, and are featured by a number
of factors. First, differences between low and high risk populations examined (Esfarjani
et al. 2001). Second , the actual exposure to certain dietary and non-dietary diabetogenic
factors (Norris et al. 2003). Third, the timing of exposure to triggering factors
(Kimpimaki et al. 2001). Fourth, ethical implications particularly in preventive trials
(Rosenbloom et al. 2000). Finally, the design and analysis methods used in studies
(Karjalainen et al. 1992b).
157
The determination of the circulating antibodies to bovine milk proteins by ELISA
remains a convenient and rapid method. Nevertheless, given that the findings of the
studies on the diabetogenicity of dietary proteins remain discrepant and that the valence
of certain proteins, for instance BSA in solutions is 5-fold higher than that on solid phase
immunoassays (Harlow and Lane 1988; Pesce and Michael 1992), a number of problems
can be identified with regard to the validity and reliability of the method (Miller 1998).
These include; 1), variation between assays methods (Rowe et al. 1972; Matthews et al.
1980; Karjalainen et al. 1992b); 2), limitations of steric and diffusion activity (Pesce and
Michael 1992); 3), the lack of an accepted golden standard to measure against; 4),
binding to solid phase may denature the native epitopes that may cause unintended
formation of different type of binding characteristics; 5), antigen absorption and
desorption from the surface is a time and surface-dependent, which affects the quantity
and stability of a bound protein; and 6) the absence of a reliable and valid method to
determine the number of non-specific antigen-antibody interactions (Miller 1998).
Therefore, findings on the immune responses to non-genetic determinants of type 1
autoimmune diabetes require cautious interpretation.
The findings of the present study indicates that immune responses were neither limited to
diabetic patients nor associated with the breastfeeding or early infant feeding practices. A
higher proportion of diabetic children received no bottle-feeding in their early infancy
reflecting an unspecific defect of the immune system in young Jordanian children.
Variation between low and high-risk populations requires further investigation.
Immune responses were neither limited to diabetic patients nor associated with the
breastfeeding or early infant feeding practices. A higher proportion of diabetic children
received no bottle-feeding in their early infancy reflecting an unspecific defect of the
immune system in young Jordanian children. Variation between low and high-risk
populations requires further investigation.
158
6.6 Conclusion
The newly developed saliva-based ELISA is non-invasive, rapid, and convenient method
and constitutes a useful proxy of immune response studies in young diabetic children.
Heat processing of proteins at various temperatures and exposure times cause substantial
change in the secretory and humoral immune responses with significant variations. This
verifies that changes in the spectral profiles of proteins are useful indicators for altered
immunogenicity of proteins caused by physical and chemical treatment (Al-Domi et al.
2004).
159
7 Future prospects
Type 1 autoimmune diabetes is a lifelong degenerative disease. Even with good health
care systems, the risk of developing long-term retinopathy, neuropathy, and nephropathy
complications is increased. Given the knowledge that the prevention of the disease is not
yet possible, minimising or delaying the destruction of the beta cell remains of great
benefits particularly to individuals at high risk. Achieving ample insights into the
immunopathogenesis of the disease, and the identification of individuals for prevention
trials may be directed to a more efficient early diagnosis prior to the manifestation of the
disease. Success in the ongoing research on preventing the disease in animals and the
exciting findings about its causes are imperative for diagnosis, treatment, and minimising
or even preventing the onset of the disease in humans.
The prevention of any disease requires identification of individuals at risk, identification
of the causative factors and understanding of the mechanisms of the aetiopathogenesis of
the disease (Becker et al. 2000). The aetiopathogenesis of type 1 autoimmune diabetes
remains unknown. Nonetheless, a complex interplay between a number of mechanisms of
beta cell destruction are operative in the disease (Kawasaki et al. 2004). This requires
both genetic and non-genetic determinates for potentially harmful events to direct the
autoimmune process (Ellis and Atkinson 1996; Akerblom et al. 1997; Visvanathan and
Zabriskie 2000; Rich and Concannon 2002). Hitherto, none of these determinants that
may either boost or weaken the immune response has been identified unequivocally
(Kimpimaki et al. 2001; Akerblom et al. 2005; Kent et al. 2005; Nakayama et al. 2005).
160
The findings of the present study have demonstrated that the modification of dietary
proteins particularly bovine milk proteins has caused substantial variations in the immune
responses of both diabetic and non-diabetic children. Similarly, heat processing of the
modified proteins at different temperature and exposure times has significantly altered
the immune responses with wide variations. These findings are consistent with the
knowledge indicating that substantial interactions with other ingredients in the food
systems can result in modifying the behaviour of the proteins (Kinsella and Whitehead
1989). Even slight changes in antigen structure can profoundly affect the immunogenic
interactions (Harlow and Lane 1999).
The present study was also able to develop two simple tools that can constitute new
grounds for future studies on the modified immunogenic behaviour of dietary
diabetogens in human trials. The first tool was used to identify changes in spectral
profiles of modified and heat processed milk proteins, and the second tool was a new,
non-invasive, rapid and convenient saliva-based enzyme linked immunosorbent assay to
determine the secretory immune response to these proteins retrospectively. It is pivotal to
examine the effect of both large scale and home heat processing procedures as well as the
modification of various milk proteins on the diabetogenicity of milk proteins in suitable
animals models and consequently in longitudinal human studies. This can play a crucial
role in our understanding of the disease aetiopathogenesis.
161
If dietary proteins particularly milk proteins are true diabetogens (Harrison and
Honeyman 1999; Wegmann and Eisenbarth 2000; Nakayama et al. 2005), bovine insulin
and bovine serum albumin remain among the most putative non-genetic dietary
diabetogenic factors implicated in the initiation of beta cell destruction. Nonetheless,
given the evidence on the possibility of manipulating pancreatic beta cells autoimmunity
by dietary interventions in infancy by using infant formulae subjected to complex heat
and enzymatic processing procedures (Akerblom et al. 2005), investigation of the nature
and effect of receptor behaviour of the coexisting milk proteins in their natural milk
system, the nature and effect of both large and home scale heat processing procedure on
the diabetogenicity of dietary proteins should provide novel insights into the detrimental
role these factors may have in the aetiopathogenesis of the disease in humans.
A number of factors can influence the development of type 1 autoimmune diabetes.
These factors include: (1) variations in amount (Westrom et al. 1985; Scott 1990; Dahl-
Jorgensen et al. 1991; Gerstein 1994) and type of milk proteins consumed (Thorsdottir et
al. 2000), (2) cleanliness of the environment (Harrison and Honeyman 1999), (3) mucosal
mechanisms and exposure to various dietary constituents, (4) substantial interactions with
other ingredients in food systems which can result in a modified behaviour of proteins
(Kinsella and Whitehead 1989), (5) changes in the mucosal architecture, and (6)
interaction between different dietary (Teale and Benjamin 1976; Speth and Lee 1984) and
non-dietary constituents (Clarke 1975; Sharma et al. 1995b) coexisting in the gut
environment. All can influence both the immunogenicity of these factors and the
maturation of the gut mucosa and therefore the macromolecular uptake of possible
foreign diabetogens (Sharma et al. 1995b; Sharma et al. 1995a; Fontaine et al. 1996;
Hiltunen et al. 1997; Honeyman et al. 2000; Westerholm-Ormio et al. 2003; Salminen et
al. 2004). This process can either boost or suppress the autoimmune process (Scott et al.
1996b) and can play, among other factors and mechanisms, a critical role in the
destruction of pancreatic beta cell (Harrison and Honeyman 1999; Kawasaki et al. 2004).
162
Although on the surface it would appear to be a logical association with the risk or
protection from diabetes, evidence on the use of breastfeeding or early infant feeding
restrictions to prevent or minimise the disease occurrence is of limited strength and
should not be misinterpreted by parents and the public (Atkinson and Gale 2003).
Emerging findings on gene therapy (Zalzman et al. 2003; Sapir et al. 2005) and cellular
immunotherapy (Ogasawara et al. 2004; Tang et al. 2004; Tarbell et al. 2004) as well as
studies on the regeneration of insulin-producing pancreatic beta cells may be harnessed
as novel therapeutic targets for type 1 autoimmune diabetes (Juvenile Diabetes Research
Foundation International 2004). Eventually, if the genetic and non-genetic determinants
of the disease are established unequivocally; the benefits to individuals and the world
society will be enormous. Therefore, studies embarking on identifying the nature and
timing of the primary environmental factors that may predispose to the initiation of the
disease should continue (Kin et al. 2005; Mirenda et al. 2005). Mind map III exemplifies
the aspects of future studies.
163
7.1 Mind Map III: Aspects of future studies
164
8 Conclusion
The present study has addressed the heterogeneity of milk proteins and the effect
modification and heat processing have on altering the immunogenicity of dietary proteins
particularly bovine serum albumin and bovine insulin in Jordanian diabetic children aged
14 years or less with regard to their breastfeeding and early infant feeding practices and
has drawn the following conclusions:
• Global geographic variation in the disease occurrence among children aged 14
years or less is significantly associated with worldwide variations in both mean
and relative change in both total milk imports excluding butter and the amount of
milk proteins consumption and is influence by the socioeconomic status of
populations. Whether these variations are indicative of variation in the
diabetogenicity of bovine milk proteins requires further longitudinal studies.
• Breastfeeding in Jordan is common and is usually extended for a minimum of one
year for the majority of children. No significant associations were established for
neither breastfeeding nor early infant feeding practices between both DC and
NDC Jordanian children aged 14 years or less. A higher proportion of diabetic
children received no bottle-feeding in their infancy. The majority of children were
diagnosed with diabetes in winter at approximately six years of age and were
female predominant.
165
• Dietary compliance in diabetic children was poor with signs of undernutrition in
both groups as well as signs of obesity among NDC. For better nutrition
compliance, there does seem a need for better dietary monitoring and professional
dietary counselling programmes for both diabetic and non-diabetic children as
well as establishing a national diabetes registry and developing national dietary
guidelines.
• Blood group O+ is significantly predominant in young Jordanian children with
diabetes and is associated with a greater risk of developing diabetes. A higher
proportion of NDC has Rh (D) negative blood groups with gender variation.
Whether certain blood groups confer a protective effect from diabetes requires
further investigations on the secretory status of humans with type 1 autoimmune
diabetes.
• The identification of bovine milk protein solutions by their characteristic spectral
profile has ascertained their heterogeneity. Both modification and thermal
processing of bovine milk proteins have caused substantial changes in their
spectral profiles. These changes were concentration, temperature, and exposure
times dependent. Changes in the spectral profile of proteins are indicative of
changes in the immunogenicity of processed proteins. Humanisation of the
spectral profile of infant formulae may open a window to resolving controversy
encircling the diabetogenicity of bovine milk proteins.
• Newly developed saliva-based ELISA is non-invasive, rapid, and convenient
method and constitutes a useful proxy of immune response studies in young
diabetic children.
166
• The modification as well as heat processing of bovine serum albumin with bovine
insulin (mBSA-BI) at various critical heating temperatures and exposure times
has triggered different secretory and humoral immune responses with substantial
variation. Various temperatures and exposure times caused different immune
responses. While secretory and humoral immune responses to mBSA-BI heat
processed at 70C/5 min has enhanced substantially, heat processing at
70ºC/10min and 80ºC/5min has masked the immune responses. These findings
verify that changes in the spectral profiles of proteins are useful indicators for
altered immunogenicity of proteins caused by physical and chemical treatment.
• The immune response in young Jordanian children is neither limited to diabetic
patients nor associated with the breastfeeding or early infant feeding practices. A
higher proportion of diabetic children received no bottle-feeding in their early
infancy reflecting an unspecific defect of the immune system. Variations between
low and high-risk populations require further investigation.
The concomitant interactions of the coexisting dietary and non-dietary factors,
interpopulation, and individual variations seem to be a more complex process than
previously anticipated. Changes in the mucosal architecture as well as interactions
between different dietary (native, modified, and thermally processed), and non-dietary
(viral, normal flora) factors coexisting in the gut environment may either boost or
suppress the pancreatic autoimmune process. Therefore, the examination of these factors,
which may intervene concomitantly in a given individual(s) or in a population(s) as
independent factors per se, may constitute a major source of error in the understanding
and interpretation of the aetiopathogenesis of the pancreatic autoimmune process.
Changes in these factors would need a longer period of time to change and would occur
concomitantly in different individuals as well as in different populations, therefore
extended longitudinal studies are necessary�
167
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10 Appendices
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10.1 Appendix A: Information sheet, consent, and survey (English)
10.1.1 Participant information sheet (English)
Dietary proteins & the development childhood type 1 (insulin-dependent) diabetes mellitus among newly diagnosed Jordanian children
Principal researchers: Dr. Mark R. JONES and Dr. Jim BERGAN
Investigator: Hayder AL-DOMI
Investigator’s contact details: School of Science, Food & Horticulture, University of Western Sydney, Australia, Richmond Campus, K-12, Locked Bag 1797 Penrith DC NSW 1797, Australia. Telephone: 0796498736 (Mobile / Jordan). E-mail: [email protected]
Note: This research has been approved by the University of Western Sydney Human Ethics Committee (HEC 02/162). If you have any complaints or reservations about the ethical conduct of this research, you may contact the Human Research Ethics Committee through the Research Ethics officers (telephone: 0061-2 4570 1136). Any issues you raise will be treated in confidence and investigated fully, and you will be informed of the outcome.
Questions and inquiries: Please if you have any questions or inquiries do not hesitate to contact the researcher at any time convenient for you.
THANK YOU
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Introduction
Infancy growth
In any culture, for each infant, child, or adolescent, food nurtures both the physical and the personal “grow up” process. Food, feeding, and eating during these highly significant years of childhood do not and cannot exist apart from the broader overall process of growth and development. The whole process produces the whole person. Infancy growth is rapid during the first year of life. Most infants double their weight by the time they are 6 months of age and triple it by the age of 1 year. The ideal first food for the infant is human milk. It provides essential nutrients in quantities uniquely suited for optimal infant growth and development. Positive growth and development of healthy children depend on optimal nutritional support. From birth, as children grow older, their nutritional needs change with each unique growth period. Infants experience rapid growth. Human milk is preferred as their first food, with solid foods delayed until about 6 months of age.
Breastfeeding
Breastfeeding can be successfully started and maintained by most mothers who try, have support, and have an increased diet for lactation. Breasts are prepared for lactation during pregnancy. As the infant grows, the breast milk develops and adapts in composition to match the needs of the developing child.
Advantages of breastfeeding
There are many physiological and practical advantages to breastfeeding, including: (1) fewer infections, (2) fewer allergies, (3) ease of digestion and (4) convenience and economy. If the mother does not choose breastfeeding, or some condition in either mother or baby prevents it, bottle-feeding of an appropriate formula, usually cow’s milk-based formula is an accepted alternative.
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Diabetes mellitus
Diabetes is a disease characterized by high blood glucose levels, resulting from either insufficient or no insulin release by the pancreas. Type 1 (insulin-dependent) diabetes is a form of diabetes in which the person with the disease requires insulin therapy. Type 1 diabetes often begins in late childhood, but can occur at any age. It runs in certain families, indicating a genetic link. Most cases of type 1 diabetes begin with an immunological disorder. Our own immune system fails to distinguish between its own body and foreign agents leading to variety of autoimmune disease, for example rheumatoid arthritis, rheumatoid fever, multiple sclerosis and type 1 diabetes. In type 1 diabetes, the autoimmune disorder causes destruction of insulin producing cell in the person’s pancreas. This disorder is, most likely triggered by a foreign virus or protein foreign to our bodies setting off the destruction. In response to pancreatic cell destruction, eventually the pancreas loses its ability to synthesize insulin and the clinical stage of the disease begins. Cow’s milk-based infant formula is the first foreign proteins introduced into our children’s diet. Thus, it has undergone extensive and intensive investigations. Nonetheless, no causal links between introduction of foreign proteins and the development of the disease have yet been established unequivocally, there is no scientific evidence to support this hypothesis.
Magnitude of Diabetes among Jordanian children
Despite the great advances in control and treatment, diabetes mellitus remains a major cause of morbidity and mortality throughout the world. Recent estimates suggest that more than 100,000 inhabitants in the Middle East suffer from type 1 insulin-dependent diabetes and that about 6000 subjects in the region develop the disease each year, and of these some 3000 are children below the age of 15 years. In 1997, there were around 5200 Jordanians experiencing the disease. Of them, some 700 are children below 15 years old. The occurrence of type 1 insulin-dependent diabetes mellitus in Jordanian children aged 0-14 years is among the lowest in the region, but is increasing. Data indicate that between 1992 and 1996 the occurrence of type 1 insulin-dependent diabetes has increased from 2.8 per 100,000 children in 1992 to 3.6 per 100,000 children in 1996.
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Aim and benefits of the study
The increasing occurrence of the disease among Jordanian children has encouraged us to study whether the introduction of certain dietary proteins at a particular age has any effect on the initiation of insulin producing pancreatic cells or no. Thus, the main aim of this study was to identify the changes that persevere up to the initiation of insulin-producing pancreatic cells autoimmunity process, and to study the possible role cows’ milk proteins may have in provoking the pancreatic autoimmunity process in Jordanian young children. That is in order not only to minimizing or stopping progression of the ongoing process, but also to prevent the initiation of beta-cell destruction.
Objectives of the study
The main objectives of the study are:
• to establish a general dietary profile for young diabetic Jordanian children compared with non-diabetic children, and to study possible changes in feeding practices if the child develops the disease; and
• to study the possible role cows’ milk proteins may have in the development of the disease by determination of saliva and/or blood serum antibody levels may produced against certain cows’ milk proteins.
Importance of your participation
The rational of the study hinged upon improving our understanding of the disease. You are participating in this research to help scientists understand the reasons behind the increasing level of the diabetes in children throughout the world and in Jordan as well. Our understanding will help in setting new preventive measures to control or minimise the occurrence of the disease.
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Your part in the study
• Your permission is required for your child to participate in this study. You will be asked to sign a consent form.
• Filling in a questionnaire that will help us to establish a dietary profile for your child, and to study your feeding practices if the child develops the disease.
• Donating a saliva sample. That will enable us to determine antibodies, which might be produced against certain cows’ milk proteins. Please consider not
feeding the child at least 30 minutes before giving the saliva sample, and
rinsing the child’s mouth with water just before the collection.
• To compare the levels of antibodies that may be present in the child’s saliva with that in the child’s blood; upon your agreement, a licensed blood drawer will collect a blood sample. Primarily only one saliva and/or blood sample will be collected. The total process will take approximately 20 minutes.
Harms and risks in participation
There will be little risk or harm in participating in this study. A licensed blood drawer will obtain the blood sample; nonetheless, you may notice a slight redness at the site of the needle prick. The procedure will be carried in a clean room, and only one-use needles will be used. The site of needle prick will be cleaned properly before and after the needle insertion. A false needle prick is not expected, but if it does happen, a repeat blood drawing will take place ONLY with your approval. Psychologically, the infant may encounter uneasiness and fear; therefore, parents will be asked to and involved in relaxing, reassuring and calming the infant. The blood drawer will take every professional effort to draw the sample swiftly. The room will be as comfortable as possible. It is unforeseen that collecting saliva samples will result in any stress/distress. Nonetheless, the participant may feel discomfort. Thus, we will make sure that the participant is relaxed before swabbing the sample with his/her parent(s) assistance.
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Privacy & confidentiality
We will maintain the highest degree of confidentiality, personal, and information privacy. No individuals will be identified at any stage during the research or any future publication of the information. All documents related to the informed consent will be stored in a safe place separately from the data, which will be kept securely in a safe place for the mandatory years then destroyed. You are completely free to stop participating in this study at any time or to withdraw prematurely. In addition, there are no disadvantages/penalties or adverse consequences for not participating or for stopping participation in this research.
New findings
If new and conclusive information becomes known during the research, you will be informed if you have requested such information. The results of the research will be published in relevant scientific journals. Nonetheless we are pleased upon you request to supply you with a results summary report. This research summary is your copy and you may keep along with a copy of the
Consent Form
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10.1.2 Consent to participate in research (English)
Title of Research Project: The Relationship Between Dietary Proteins and the Development of Type 1 (Insulin-Dependent) Diabetes Mellitus in Young Jordanian Children by Examining Susceptible Blood Groups and the Levels of IgA and IgG Name of Researcher: Hayder AL-DOMI
• I understand that Mr. Al-Domi is a PhD student studying the effect dietary proteins might have on the development of insulin-dependent diabetes mellitus among Jordanian children. The researcher will conduct this study in a manner conforming with ethical and scientific principles set out by the University of Western Sydney-Human Research Ethics Committee (UWSHREC), Australia.
• I acknowledge that I have read, or have read to me the Participant Information Sheet relating this study. I acknowledge that I understand the Participant Information Sheet. I acknowledge that the general purpose, methods, demands and possible risks and inconveniences that may occur to me during the study have been explained to me by __________________________ (“the researcher”).
• I understand that since my child is under the age of 14 years, I have to sign on behalf of my child.
• I acknowledge that I have been given time to consider the information and to seek other advice.
• I realize that the study will take approximately 20 minutes of my time, and will involve filling in a questionnaire.
• I understand the general purposes, methods, demands and possible risks and inconveniences that may occur during the study.
• I understand that participation in the study might cause some physical and/or psychological stress to my child.
• I acknowledge that my participation in this study is strictly voluntary, and I may withdraw at any time.
214
• I know that I have the right to withdraw at any time and that the care of my family member and my relationship with the health care team will not be affected.
• I acknowledge that this research has been approved by the UWSHREC.
• I acknowledge that I have received a copy of this form and the Participant Information Sheet.
• If I have any question about the study or about being a subject, I know I can call Mr. Al-Domi. I may reach him at (++961-2 -45703216 [Work], ++9611-2-968 75816 [home] and ++961-405543209 [mobile], Australia, and at (02-7046118 [home] and 0796498736 [mobile] Jordan.
• I agree to participate in this study, and I have been assured that the highest degree of confidentiality, personal and information privacy will be observed. My identity and my child’s identity will not be revealed while the study is being conducted or when the study is published.
• I am informed clearly that all documents related to the survey and the signed informed consent will be stored in a safe and confidential place separately from the data for the university’s five mandatory years; then documents will be destroyed according to the university’s policy.
Date:
__________________________________________________________________
Name of Participant: _________________________ Date of Birth:
_______________
Address of Participant:
___________________________________________________
Signature of Participant (if applicable): ___________ Date:
_____________________
Name of Participant’s Parent:
_____________________________________________
Address of Participant’s Parent (if different)
_________________________________
Relationship to participant:
_______________________________________________
215
Signature of Participant’s Parent: _______________ Date:
_____________________
Researcher’s Signature:
__________________________________________________
216
10.1.3 Survey form (English)
Title of Research Project: The Relationship between Dietary Proteins and the Development of Type 1 (Insulin-Dependent) Diabetes Mellitus in Young Jordanian Children by Examining Susceptible Blood Groups and the Levels of IgA and IgG. Dear Sir /Madam, I am Hayder Al-Domi, a PhD student at University of Western Sydney studying the possible effect dietary proteins have on the development of type 1 (insulin-dependent) diabetes mellitus among Jordanian children. This study has been approved by the University of Western Sydney Human Research Ethics Committee (HEC 02/162). If you have any complaints or reservations about the ethical conduct of this research, you may contact the ethics Committee through the Research Ethics Officers (phone: 0061-2-4570 1136). Any issues you raise will be treated in confidence and investigated fully, and you will be informed of the outcome. Please find attached a research summary (participant Information Sheet). It is your copy and you may keep it along with a copy of the Consent Form. The attached survey form consists of six parts, please tick, or fill in as appropriate. If you have any questions, or you want to know more about the study and its outcomes, please do not hesitate to contact the researcher using the following contact details.
Researcher’s Contact details:
Telephones: 0796498736 (Mobile/Jordan), 0061-2-96875816 (Home/Sydney), and
0061-405543209 (Mobile/Sydney) E-mail: [email protected] Mailing address: Richmond Campus, K-12, Locked Bag 1797, Penrith DC NSW 1797
Thank you for participation
217
Part 1: General
Child’s name (optional): -------------------------------------------------------------------
Mother’s name (optional): ----------------------------------------------------------------
Address (optional): ---------------------------------------------------------------------------
Telephone (optional): -----------------------------------------------------------------------
-
Please tick or fill in as appropriate
1. Who is completing this survey? The mother of the participant. The father of the participant.
2. Do you have a child with diabetes? Yes No, please go to question 4.
3. If YES to question (2), do you agree to: Filling in a questionnaire. Yes No Allow swapping a saliva sample. Yes No And/or drawing a blood sample. Yes No
4. If NO to question (2), do you like to include your child as a control? Yes No
If YES to question (4), do you agree to: Filling in a questionnaire. Yes No Allow swapping a saliva sample. Yes No
And/or drawing a blood sample. Yes No
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Part 2: Participant Demographic Data
Age
(Months)
Sex Weight
(kg)
Height
(cm)
Birth weight
(kg)
Birth order
Male Female
Participant Blood group
I do not know
A+
A�
B+
B�
AB+
AB�
O+
O�
Part 3: Parents Demographic Data
Age (years) Weight (Kg) Height (cm) Blood group
Mother
Father
Education
Under 4 years
4-10 Years
High school Undergraduate Postgraduate
Father
Mother
Occupation
Father
Mother
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Part 4: Family Medical History
4-1 Did any of the participant’s family members have diabetes mellitus? Yes. No.
If yes, please specify how many they are?
1 person 2 persons 3 persons More than 3 persons What is the relationship between them and the participant?
Brother. Sister. Father. Mother. Parental grandfather. Maternal grandfather. Parental grandmother. Maternal grandmother.
What type of diabetes did they have?
Type 1 insulin-dependent diabetes mellitus. Type 2 non-insulin-dependent diabetes mellitus. Gestational diabetes. Other, please specify if known------------------------------------------------------
4-2 Did the child’s mother have diabetes during pregnancy? Yes. No.
Part 5: Participant Medical History
5-1 At what age was the child diagnosed with diabetes? At birth. More than 1 week to 1 month. More than 1 month to 2 months. More than 2 month to 3 months. More than 3 months to 6 months.
More than 6 months, please specify years, and months.
5-2 In which month was the child diagnosed?
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
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5-3 Did the child show any signs or symptoms of diabetes, such as frequent urination, increased thirst, before being diagnosed with diabetes?
No, he/she was diagnosed on birth. No, he/she was diagnosed on a regular hospital visit. No, somebody else brought it to my attention. Yes, I recognised some of the signs and symptoms.
5-4 Does the child have any other chronic (lifelong) disease(s)? No. Yes, please tick one of the following. Rheumatic fever. Rheumatoid arthritis. Multiple sclerosis. Systemic lupus erythematosus. Other, pleas specify if known----------------------------------------------
5-5 Does the child have any food allergy(s)? No. Yes, please specify ------------------------------------------------------------------
Does the child have any gastrointestinal disease(s)?
No. Yes, please specify ------------------------------------------------------------------
Part 6: Participant Dietary History
6-1 Was the child breastfed? No. Yes.
If yes, for how long was the child breastfed?
Less than 1 week. More than 1 to 2 weeks. More than 2 weeks to 1 month. More than 2 weeks to 3 months.
More than 3 months, please specify years, and months.
221
6-2 How old was the child when it first received bottle-feeding? Less than 1 week. More than 1 to 2 weeks. More than 2 weeks to 1 month. More than 2 weeks to 3 months.
More than 3 months, please specify years, and months.
6-3 What type of milk formula did you first introduced to your child? Cows’ milk-based formula. Soybeans-based formula. Fresh cows’ milk. Nutramigen®. Others, please specify-----------------------------------------------------------
6-4 After the child was diagnosed with diabetes; did you change the type of bottle-feeding formula?
No. Yes.
If so, what is the type of the new bottle formula that you have introduced after the diagnosis?
Cows’ milk-based formula. Soybeans-based formula. Fresh cows’ milk. Nutramigen®. Others, please specify-----------------------------------------------------------
6-5 How old was the child when it first received an extra food supplement? 3 months 3-6 months 6-9 months
More than 9 months, please specify years, and months.
6-6 What is the type of the extra solid food supplement you first introduced to your child?
Wheat Rice Soybeans Others, please specify ---------------------------------------------------------------
222
6-7 What was the procedure you used to prepare your child bottle milk formula? Boil water then allow cooling and then adding the milk powder Adding milk powder to cold water and then boil them together Adding milk powder to cold water and then heating in microwave Others, please specify ---------------------------------------------------------------
Do you have any extra information you want to add? -------------------------------------------------------------------------------------------------------- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Thank you for your time
223
10.2 Appendix B: Information sheet, consent, and survey (Arabic)
10.2.1 Participant information sheet (Arabic)
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