Cartilage-hair hypoplasia with normal height in childhood-4 patients with a unique genotype.

The manifestations of cartilage-hair hypoplasia (CHH), a metaphyseal chondrodysplasia caused by RMRP mutations, include short stature, hypoplastic hair, immunodeficiency and increased risk of malignancies. Clinical features show significant variability. We report a patient with normal height until age 12.5 years (-1.6 SDS at 11 years) who was diagnosed with CHH at 14 years. RMRP sequencing revealed compound heterozygosity for g.70A>G mutation and a 10-nucleotide duplication at position -13 (TACTCTGTGA). Through the Finnish Skeletal Dysplasia Register, we identified 3 additional patients with identical genotype. Two of them also showed unusually mild growth failure (height SDS -1.6 at 14 years and -3.0 at 12 years, respectively). Three of the 4 patients suffered from recurrent infections; 1 developed progressive bronchiectasis and another died from aggressive lymphoma. Our findings expand the phenotypic variability in CHH to include normal childhood height. The milder growth retardation related to this particular genotype was not associated with less severe extra-skeletal manifestations, emphasizing the need for careful follow-up also in CHH patients with mild-skeletal manifestations.

Effect of coincident enterovirus infection and cows' milk exposure on immunisation to insulin in early infancy.

AIMS/HYPOTHESIS: Insulin autoantibodies appear often as the first autoantibody in children who develop islet-cell autoimmunity. Our recent studies indicate that primary immunisation to insulin is induced in early infancy by exposure to dietary bovine insulin present in cows' milk formulas. As gut-associated lymphoid tissue is also the primary replication site of enteroviruses, we tested whether enterovirus infections could modify the development of immune response to dietary insulin in early infancy. METHODS: We studied the development of IgG-antibodies to dietary bovine insulin by enzyme immunoassay in relation to enteroviral infections determined by T-cell proliferation response to the Coxsackie B4 virus and by serological tests for enterovirus antigens in 57 infants who carried the HLA DQB1(*)02/0302 diabetes risk genotype and participated in a Finnish population-based birth-cohort study (Diabetes Prediction and Prevention Project, DIPP, study). RESULTS: In the infants exposed to cows' milk formulas before the age of 3 months, those who had a T-cell proliferation response to enterovirus antigen at 3 months of age ( n = 12) had higher concentrations of IgG-antibodies to bovine insulin at the age of 6 and 9 months than those who did not have T-cell proliferation response to enterovirus antigen ( n = 25) (median OD were 0.742 vs 0.427, p = 0.04, and 0.477 vs 0.293, p = 0.02, respectively). CONCLUSION/INTERPRETATION: Our results suggest that two epidemiological risk factors of Type I (insulin-dependent) diabetes mellitus, enterovirus infections and exposure to cows' milk formulas, could modify the immunisation to insulin in early infancy.

Breast-feeding and the development of cows' milk protein allergy.

Early feeding with cows' milk (CM) may cause cows' milk allergy (CMA). Breast milk contains many immune factors which compensate for the undeveloped defence mechanisms of the gut of the newborn infant. We studied the effect of supplementary CM feeding at the maternity hospital on the subsequent incidence of CMA, the effects of formula and breast feeding on the subsequent immunologic types of CMA, and the importance of immune factors present in colostrum in the immune responses of infants with CMA. In a cohort of 6209 infants, 824 were exclusively breast-fed and 87% required supplementary milk while in the maternity hospital: 1789 received CM formula, 1859 pasteurized human milk, and 1737 whey hydrolysate formula. The cumulative incidence of CMA, verified by a CM elimination-challenge test, was 2.4% in the CM, 1.7% in the pasteurized human milk and 1.5% in the whey hydrolysate group. Among these infants, exposure to CM at hospital and a positive atopic heredity increased the risk of CMA. Of the exclusively breast-fed infants, 2.1% had CMA. Risk factors for the development of IgE-mediated CMA were: exposure to CM at hospital, breast-feeding during the first 8 weeks at home either exclusively or combined with infrequent exposure to small amounts of CM and long breast-feeding. The content of transforming growth factor-beta1 (TGF-beta1) in colostrum from mothers of infants with IgE-mediated CMA was lower than from mothers of infants with non-IgE-mediated CMA. In infants with CMA, TGF-beta1 in colostrum negatively correlated with the result of skin prick test and the stimulation of peripheral blood mononuclear cells to CM, but positively with infants' IgA and IgG antibodies to CM proteins. Feeding of CM formula at maternity hospital increases the risk of CMA, but exclusive breast-feeding does not eliminate the risk. Prolonged breast-feeding exclusively or combined with infrequent exposure to small amounts of CM during the first 8 weeks induces the development of IgE-mediated CMA. Colostral TGF-beta1 may inhibit IgE- and cell mediated reactions and promote IgG-IgA antibody production to CM in infants prone to developing CMA.

Autoimmunity to glutamic acid decarboxylase in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

Antibodies to glutamic acid decarboxylase (GAD) occur frequently in patients with APECED, although clinical insulin-dependent diabetes mellitus (IDDM) is seen only in a subgroup of the patients. We studied the cellular immunity to GAD, antibodies to GAD and their association with the HLA DQB1 risk alleles for IDDM in patients with APECED. Proliferation responses to GAD were enhanced in the patients with APECED when compared with the control subjects (P = 0.004), but autoimmunity to GAD was not associated with IDDM in APECED. The levels of interferon-gamma (IFN-gamma) secreted by GAD-stimulated T cells were higher in the patients than in control subjects (P = 0. 001). A negative correlation (r = - 0.436, P = 0.03) existed between the antibody levels and the stimulation indices (SIs) to GAD. In 14 non-diabetic patients no difference in insulin secretion was observed in intravenous glucose tolerance test (IVGTT) between the patients with and without T cell reactivity to GAD. We conclude that cellular immunity to GAD detected as T cell proliferation response to GAD or IFN-gamma secretion by GAD-stimulated T cells was frequent in patients with APECED (69%) and was not restricted to the patients with clinically detectable beta-cell damage.

Relation between T-cell responses to glutamate decarboxylase and coxsackievirus B4 in patients with insulin-dependent diabetes mellitus.

BACKGROUND: the role of enteroviruses has been implicated in the etiology of insulin-dependent diabetes mellitus (IDDM). A possible connection between glutamate decarboxylase (GAD) autoimmunity and enterovirus infections in IDDM has been suggested to be based on a homology region between GAD and the non-structural protein 2C of coxsackievirus B4 (CVB4). OBJECTIVES: the aims of the study were to measure the occurrence of cellular immunity to GAD and CVB4 in Finnish patients with newly diagnosed IDDM, and to study the relation between these two responses. T-cell responses to GAD and CVB4 were analyzed in relation to HLA DQB1 risk alleles for IDDM and antibodies to GAD and CVB4. STUDY DESIGN: T-cell and antibody responses to GAD65 and purified CVB4 were measured in patients with newly diagnosed IDDM and in healthy children. The purified CVB4 did not contain the non-structural protein 2C thus lacking the reported homology region with GAD. RESULTS: high proliferative responses of PBMC to both GAD and CVB4 were more frequent in IDDM patients than in the control children (40 vs. 16%, 27 vs. 10%; P = 0.03 and 0.04, respectively; Fisher's exact test), when the cut-off for positivity was three multiples of the median SI in the healthy children. Median SI to GAD was higher in the patients with IDDM than in the control subjects (3.10 vs. 1.55; P = 0.03, Mann-Whitney U-test). T-cell responses to GAD and CVB4 showed a positive correlation in the patients (r = 0.62, P = 0.001), but not in the control children (r = 0.23; P = 0.38). CONCLUSIONS: enhanced T-cell responsiveness to CVB4 in patients with newly diagnosed IDDM support the involvement of enteroviral infections in the development of IDDM. The observed correlation between T-cell reactivity to GAD and CVB4, lacking the crossreactive protein 2C, in patients with IDDM suggests that CBV4 reactivity is associated with GAD autoimmunity in IDDM but does not reflect immunization to GAD.

Transforming growth factor-beta1 in mothers' colostrum and immune responses to cows' milk proteins in infants with cows' milk allergy.

BACKGROUND: Breast milk contains immune factors that compensate for the underdeveloped defenses of the gut of the newborn infant. OBJECTIVE: We sought to study the importance of these factors in the immune responses of infants with cows' milk allergy (CMA) to the proteins in cows' milk (CM). METHODS: We prospectively followed the development of CMA in 6209 healthy infants and collected samples of colostrum from mothers. Samples from mothers of infants with CMA and from control subjects were analyzed for immunoglobulins, CM-specific antibodies, and cytokines. In infants with CMA, correlations between the concentration of transforming growth factor (TGF)-beta1 in colostrum and the extent of the immune response to CM proteins were studied. RESULTS: The concentration of TGF-beta1 in colostrum samples from mothers of infants with IgE-mediated CMA (n = 65) was lower (mean, 589 pg/mL; 95% confidence interval [CI], 413-840) than from mothers of infants with non-IgE-mediated CMA (n = 37; mean, 1162 pg/mL; 95% CI, 881-1531; t = 2.57, P =.012). In 126 control subjects the mean concentration was 807 pg/mL (95% CI, 677-963). In the infants with CMA (n = 96-100), the concentration of TGF-beta1 in colostrum was positively correlated with IgA antibodies to beta-lactoglobulin and IgG antibodies to alpha-casein and whole formula and negatively with the diameter of a skin prick test response to CM and lymphocyte stimulation indices to alpha-casein and beta-lactoglobulin. CONCLUSIONS: In an infant prone to having CMA, the TGF-beta1 content of mother's colostrum may promote IgG-IgA antibody production and inhibit IgE- and cell-mediated reactions to CM.

T-cell reactivity to wheat gluten in patients with insulin-dependent diabetes mellitus.

Dietary wheat gluten has been associated with the risk of diabetes in animal models of human insulin-dependent diabetes mellitus (IDDM). To evaluate the role of wheat gluten as a T cell antigen in human IDDM, we studied the cell-mediated immune response to wheat gluten in patients with IDDM and in control subjects. The cellular response to gluten was measured by the peripheral blood mononuclear cell (PBMC) proliferation test, and the results were expressed as a stimulation index (SI). We observed an enhanced cellular immune response to gluten (SI > or = 3) in seven of 29 patients with newly diagnosed IDDM (24.1%), in six of 39 patients with a longer duration of IDDM (15.4%), and in two of 37 non-diabetic controls (5.4%). Reactivity of T cells to gluten was associated with IDDM at diagnosis (P = 0.03), whereas patients with longer duration of IDDM did not differ from controls (P = 0.16). Responses of T cells to gluten were low in general: the median SI (range) was 2.0 (1-8.6) in patients with newly diagnosed IDDM and 1.5 (1-5.8) in control subjects (P = 0.03). Cellular responsiveness to gluten was not associated with HLA-DQB1 risk alleles for IDDM in patients. Although T cell responses to gluten were slightly increased in newly diagnosed patients the responsiveness was rare, and thus our results do not support a major role of gluten in the pathogenesis of human IDDM.

Interferon-gamma production in antigen specific T cell response: quantitation of specific mRNA and secreted protein.

Interferon gamma (IFN-gamma) production as a measure of cellular sensitization was studied by detection of the cytokine in culture supernatant by enzyme immunoassay (EIA) and by measuring cellular mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR) method. These assays were compared to the standard lymphocyte proliferation assay as a marker of T cell responsiveness to foreign antigens. When blood donors seropositive for herpes simplex virus (HSV) were compared to seronegative donors, all measurements of cellular sensitization separated the groups without overlap. There were significant correlations between the IFN-gamma mRNA titre and the secreted IFN-gamma (r = 0.57, P = 0.03), and the proliferative response and the secreted IFN-gamma (r = 0.78, P = 0.001), as well as between the IFN-gamma mRNA titre and the proliferative response (r = 0.78, P < 0.001). When tetanus toxoid (TT) responses were studied in immunized subjects, a wide range of responsiveness could be seen and correlation between various measurements was poor. However, constant individual levels of the cytokine production were demonstrated. Six people who had received their last TT booster vaccination more than 5 years ago were revaccinated and repeatedly studied. An increase in the levels of produced IFN-gamma could be seen in all subjects and two who lacked a lymphocyte proliferation response developed it after revaccination.

Glutamate decarboxylase-reactive peripheral blood lymphocytes from patients with IDDM express gut-specific homing receptor alpha4beta7-integrin.

Migration of lymphocytes to the pancreas is a prerequisite for insulitis in IDDM. Mucosal vascular addressin (MAdCAM-1), involved in the recirculation of lymphocytes to the gut, has been found in the inflamed islets in NOD mice. In humans, triggers of the gut immune system (e.g., early exposure to cow's milk proteins in infancy, exposure to enteroviral infections) have been associated with IDDM. To study the possible link between the gut immune system and IDDM, we tested the expression of the alpha4beta7-integrin, a homing receptor for MAdCAM-1, on GAD65-reactive lymphocytes. Using immunomagnetic cell sorting, we depleted the lymphocytes with high expression of alpha4beta7-integrin in the peripheral blood mononuclear cell population from IDDM patients and patients with autoimmune polyendocrine disease type 1 (APD-I). The depletion led to a marked decrease (mean 70%) in the cellular response against GAD65 in three of six IDDM patients and in one subject at high risk for IDDM. A decrease of 37% in the GAD response was observed after depletion in the case of one APD-I patient who also had IDDM. Cellular response to tetanus toxoid increased in the majority of patients as well as in three control subjects studied. We demonstrated that a remarkable population of islet cell antigen-reactive lymphocytes express the gut-specific homing receptor, which emphasizes the role of gut immunity in IDDM. The manipulation of the gut immune system is therefore proposed as a tool for modulation of the autoimmunity against pancreatic beta-cells in IDDM.

Cellular immune response to cow's milk beta-lactoglobulin in patients with newly diagnosed IDDM.

Elevated levels of antibodies to cow's milk proteins, i.e., beta-lactoglobulin (BLG) and bovine serum albumin (BSA), have been associated with IDDM. We observed enhanced cellular immune response by a proliferation test of peripheral blood mononuclear cells to BLG in 22 of 40 (55%) patients with newly diagnosed IDDM compared with 7 of 32 healthy children (22%) (P = 0.004, chi 2 test). The median stimulation index to BLG was 3.3 in patients and 1.5 in healthy children (P = 0.003, Mann-Whitney U test). No difference was found in cellular reactivity to other cow's milk proteins, such as BSA or alpha-casein, or to a dietary immunogenic protein, ovalbumin. Cellular responsiveness to BLG was not associated with HLA-DQB1* risk alleles of IDDM, which suggests that immune response to the protein does not only reflect the accumulation of these HLA alleles in the patients with IDDM. We suggest that enhanced cellular immune response to dietary BLG may reflect a disturbance in the regulation of immune response to oral antigens in IDDM. This kind of defect may play a fundamental role in the development of beta-cell autoimmunity in IDDM.