Hans-Michael Dosch

Dietary Intervention in Infancy and Later Signs of Beta-Cell Autoimmunity

BACKGROUND Early exposure to complex dietary proteins may increase the risk of beta-cell autoimmunity and type 1 diabetes in children with genetic susceptibility. We tested the hypothesis that supplementing breast milk with highly hydrolyzed milk formula would decrease the cumulative incidence of diabetes-associated autoantibodies in such children. METHODS In this double-blind, randomized trial, we assigned 230 infants with HLA-conferred susceptibility to type 1 diabetes and at least one family member with type 1 diabetes to receive either a casein hydrolysate formula or a conventional, cows-milk-based formula (control) whenever breast milk was not available during the first 6 to 8 months of life. Autoantibodies to insulin, glutamic acid decarboxylase (GAD), the insulinoma-associated 2 molecule (IA-2), and zinc transporter 8 were analyzed with the use of radiobinding assays, and islet-cell antibodies were analyzed with the use of immunofluorescence, during a median observation period of 10 years (mean, 7.5). The children were monitored for incident type 1 diabetes until they were 10 years of age. RESULTS The unadjusted hazard ratio for positivity for one or more autoantibodies in the casein hydrolysate group, as compared with the control group, was 0.54 (95% confidence interval [CI], 0.29 to 0.95), and the hazard ratio adjusted for an observed difference in the duration of exposure to the study formula was 0.51 (95% CI, 0.28 to 0.91). The unadjusted hazard ratio for positivity for two or more autoantibodies was 0.52 (95% CI, 0.21 to 1.17), and the adjusted hazard ratio was 0.47 (95% CI, 0.19 to 1.07). The rate of reported adverse events was similar in the two groups. CONCLUSIONS Dietary intervention during infancy appears to have a long-lasting effect on markers of beta-cell autoimmunity--markers that may reflect an autoimmune process leading to type 1 diabetes. (ClinicalTrials.gov number, NCT00570102.).

Dietary manipulation of beta cell autoimmunity in infants at increased risk of type 1 diabetes: a pilot study

Aims/hypothesisWe aimed to assess the feasibility of a dietary intervention trial with weaning to hydrolysed formula in infants at increased risk of type 1 diabetes and to study the effect of the intervention on the emergence of diabetes-associated autoantibodies in early childhood.MethodsWe studied 242 newborn infants who had a first-degree relative with type 1 diabetes and carried risk-associated HLA-DQB1 alleles. After exclusive breastfeeding, the infants underwent a double-blind, randomised pilot trial of either casein hydrolysate (Nutramigen; Mead Johnson) or conventional cow’s milk-based formula until the age of 6–8 months. During a mean observation period of 4.7 years, autoantibodies to insulin, anti-glutamic acid decarboxylase and insulinoma-associated antigen-2 were measured by radiobinding assays, and islet cell antibodies (ICA) by immunofluorescence.ResultsThe feasibility of screening and identifying a cohort of first-degree relatives with HLA-conferred disease susceptibility, enrolling them in a dietary intervention trial and following them for seroconversion to autoantibody positivity is established. The cumulative incidence of autoantibodies was somewhat smaller in the casein hydrolysate vs control formula group, suggesting the need for a larger well-powered study. After adjustment for duration of study formula feeding, life-table analysis showed a significant protection by the intervention from positivity for ICA (p=0.02) and at least one autoantibody (p=0.03).Conclusions/interpretationThe present study provides the first evidence ever in man, despite its limited power, that it may be possible to manipulate spontaneous beta cell autoimmunity by dietary intervention in infancy.

Diagnosis of nasopharyngeal carcinoma by DNA amplification of tissue obtained by fine-needle aspiration.

BACKGROUND In nasopharyngeal carcinoma the primary lesion is often difficult to find. Metastatic lesions occur frequently but are difficult to distinguish from other head and neck tumors. The viral genome of the Epstein-Barr virus (EBV) can be identified in the cells of this carcinoma. METHODS We used the polymerase chain reaction (PCR) to test for the presence of EBV genomes in 15 samples of metastatic squamous-cell carcinoma of the neck obtained by fine-needle aspiration and in 26 samples obtained by biopsy of lymph nodes. For controls we used disease-free lymph nodes from 10 patients with various head and neck tumors, tonsillar tissue from 46 subjects, blood from 59 EBV-seropositive blood donors, and mononuclear cells from 8 patients with fatal lymphoproliferative lesions. RESULTS Of the 41 malignant lesions examined, only the nine nasopharyngeal carcinomas (one primary lesion and eight metastases) contained EBV genomes. None of the 20 nodes with other types of cancer, the 10 disease-free nodes, or any of the 105 normal control samples contained detectable EBV. In two patients with suspected metastases from occult primary tumors, the presence of EBV was predictive of nasopharyngeal carcinoma; in both cases overt nasopharyngeal carcinoma developed within one year. CONCLUSIONS In patients with suspected nasopharyngeal carcinoma, fine-needle aspiration can provide tissue for diagnosis by DNA amplification of EBV genomes. The presence of EBV in metastases from an occult primary tumor is predictive of the development of overt nasopharyngeal carcinoma.

Milk Proteins in the Etiology of Insulin-Dependent Diabetes Mellitus (IDDM)

The etiology of insulin-dependent diabetes mellitus (IDDM) is multifactorial. The final cause of the disease, the specific destruction of the islet beta-cells, is the result of a cellular/humoral autoimmune process that operates in individuals with a particular genetic background in response to an external triggering factor(s). The most likely environmental triggers are virus infections and dietary factors. Among the latter group dietary proteins, mainly cow milk proteins, have been found to be important. Elimination of intact cow milk proteins from the diet significantly reduced the incidence of IDDM in the spontaneously diabetic BB rat, the elimination being most effective when it occurs during the pre-weaning period. Conversely, in newly discovered diabetics (both rats and children) increased levels of antibodies to cow milk proteins as compared with non-diabetic controls were found. These higher titres of antibodies were against beta-lactoglobulin and anti-bovine serum albumin. In further studies we found that antibodies to bovine serum albumin cross-react with a beta-cell membrane protein of Mr 69,000 and that this protein is likely induced by interferon. At the molecular level, a region of the bovine serum albumin has distinct homology to the beta-subunits of the MHC class II proteins Ia, DQ and DR, and antibodies raised against this bovine serum albumin region identified the same 69K beta cell membrane protein, in the same manner as antibodies to the third hypervariable region of DR-beta did. Our hypothesis is that bovine milk proteins (mainly bovine serum albumin) might be an important environmental factor providing specific peptides that share antigenic epitopes with host cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of an Intrathymic Defect in a Case of Severe Combined Immunodeficiency Disease

An in vitro system has been developed to elucidate the nature of the cellular defect in primary immunodeficiency diseases. Incubation, on human thymic epithelial monolayer cultures, of peripheral blood lymphocytes and bone-marrow cells from a child with documented severe combined immunodeficiency disease resulted in the appearance of a population of cells that formed rosettes with sheep erythrocytes. The same cell preparation permitted the synthesis of antigen-specific, complement-dependent antibodies after in vitro education, as demonstrated in a plaque assay system. In addition, thymic tissue from the same child gave morphologic and functional evidence of maturation when cultured in vitro. The experimental results suggest that in this case, lymphoid precursor cells were present in the bone marrow but failed to differentiate to functional maturity due to a defect in maturation of thymic tissue.

Type I Diabetes and Multiple Sclerosis Patients Target Islet Plus Central Nervous System Autoantigens; Nonimmunized Nonobese Diabetic Mice Can Develop Autoimmune Encephalitis

Type I diabetes and multiple sclerosis (MS) are distinct autoimmune diseases where T cells target either islet or CNS self-proteins. Unexpectedly, we found that autoreactive T cells in diabetic patients, relatives with high diabetes risk, nonobese diabetic (NOD) mice, and MS patients routinely target classical islet as well as CNS autoantigens. The pathogenic potential of CNS autoreactivity was testable in NOD mice. Pertussis holotoxin, without additional Ags or adjuvants, allowed development of an NOD mouse-specific, autoimmune encephalitis with variable primary-progressive, monophasic, and relapsing-remitting courses. T cells from diabetic donors transferred CNS disease to pertussis toxin-pretreated NOD.scid mice, with accumulation of CD3/IFN-γ transcripts in the brain. Diabetes and MS appear more closely related than previously perceived. NOD mouse-specific, autoimmune encephalitis provides a new MS model to identify factors that determine alternative disease outcomes in hosts with similar autoreactive T cell repertoires.

Immunological Aspects of Nutritional Diabetes Prevention in NOD Mice: A Pilot Study for the Cow's Milk–Based IDDM Prevention Trial

Human epidemiological studies delineated early exposure to intact dietary protein (e.g., most infant formulas) as an environmental risk factor for the development of IDDM. The Trial to Reduce IDDM in the Genetically at Risk (TRIGR), an international IDDM prevention trial, has been designed to determine if avoidance of intact dairy protein in high-risk infants ≤6 months of age can reduce the subsequent diabetes incidence. We here studied the casein hydrolysate-based trial diet (Nutramigen) in NOD mice. When given either continuously or for 10 weeks after weaning, the test diet was highly effective in preventing autoimmune diabetes (32-week incidence: 4.6 vs. 58.8%) and in preserving pancreatic insulin levels, with little effect on islet inflammation. Spleen cells from protected NOD mice failed to adoptively transfer diabetes into irradiated syngeneic recipients. When co-transferred with splenocytes from diabetic donors, cells from diet-protected mice inhibited adoptive diabetes transfer (incidence 50 vs. 94%, P < 0.001). T-cell reactivity to the islet cell autoantigens ICA69 (islet cell antigen 69) and GAD65 developed only in diabetic re recipients of spleen cell grafts, indicating that diabetes protection extends to more than one autoantigen. In protected mice, ICA69 T-cell reactivity was not detectable spontaneously nor after priming with this autoantigen; however, priming with the cross-reactive non-self-antigen bovine serum albumin recruited T-cells responsive to ICA69. Thus, diabetes prevention with the clinical trial diet is effective in NOD mice, where it affects some T-cell repertoires and allows development of regulatory cells that interfere with destructive autoimmunity.

Cholecalciferol Plus Calcium Suppresses Abnormal PBMC Reactivity in Patients with Multiple Sclerosis

CONTEXT The active metabolite of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)(2)D], is a potent modulator of immune cells in vitro. OBJECTIVE Our objective was to determine whether the sun-dependent nutrient, cholecalciferol, can alter disease-associated cellular immune abnormalities in patients with multiple sclerosis (MS). DESIGN This was an open-label, 12-month, randomized controlled trial. SETTING Patients with MS were recruited from the MS Clinic at St. Michaels Hospital, Toronto. PATIENTS Forty-nine patients were matched (for age, sex, disease duration, disease-modifying drug, and disability) and enrolled (treated n = 25; control n = 24). Four patients were lost to follow-up (n = 2 from each group). INTERVENTION Treated patients received increasing doses of cholecalciferol (4,000-40,000 IU/d) plus calcium (1200 mg/d), followed by equilibration to a moderate, physiological intake (10,000 IU/d). Control patients did not receive supplements. MAIN OUTCOME MEASURES At enrollment and at 12 months, peripheral blood mononuclear cell (PBMC) proliferative responses to disease-associated, MS-relevant, and control antigens were measured, along with selected serum biochemical markers. RESULTS At 12 months, mean serum 25-hydroxyvitamin D [25(OH)D] concentrations were 83 ± 35 nmol/liter and 179 ± 76 nmol/liter in control and treated participants, respectively (paired t, P < 0.001). Serum 1,25(OH)(2)D did not differ between baseline and 1 yr. In treated patients, 12-month PBMC proliferative responses to neuron antigens myelin basic protein and exon-2 were suppressed (P = 0.002). In controls, there were no significant changes in disease-associated PBMC responsiveness. There were no significant differences between groups in levels of selected biomarkers. INTERPRETATION MS-associated, abnormal T cell reactivities were suppressed in vivo by cholecalciferol at serum 25(OH)D concentrations higher than 100 nmol/liter.

Abnormal T‐cell reactivities in childhood inflammatory demyelinating disease and type 1 diabetes

Pediatric‐onset multiple sclerosis offers a unique window into early targets and mechanisms of immune dysregulation. It is unknown whether heightened T‐cell reactivities documented in adult patients, to both target‐organ and environmental antigens, emerge in parallel or develop as early or late events. Our objectives were to determine the presence, pattern, and specificity of abnormal T‐cell reactivities to such antigens in the earliest stages of the multiple sclerosis process.

In vitro induction and measurement of hemolytic plaque forming cells in man.

Following co-cultivation with sheep red cells or ovalbumin, Hypaque-Ficoll-separated human tonsillar lymphocytes were demonstrated to generate specific hemolytic PFC with maximum numbers at day 5-7. PFC were enumerated on poly-L-lysine coupled red cell monolayers in Microtest-II-plates. Plaque formation appeared to be puromycin-sensitive, complement-dependent and showed clear specificity for the antigen present during the inductive culture. Treatment of PFC with mu-chain specific antisera and complement resulted in complete inactivation of PFC; gamma-chain antisera had no effect. The development of such a simple and sensitive assay system permits the analysis of cellular interactions required for the induction of PFC responses in man.