Children who develop type 1 diabetes early in life show low levels of carnitine and amino acids at birth: does this finding shed light on the etiopathogenesis of the disease?

BACKGROUND: Children and adolescents with overt type 1 diabetes (T1D) have been found to show an altered carnitine profile. This pattern has not previously been analyzed in neonates before onset of the disease. MATERIALS AND METHODS: Fifty children who developed T1D during the first 6 years of life, born and living in the Tuscany and Umbria Regions of Italy, were identified and 200 controls were recruited into the study. All newborns were subjected to extended neonatal screening by mass spectrometry at 48-72 h of life. Four controls for each of the 50 index cases were taken randomly and blinded in the same analytical batch. The panel used for neonatal screening consists of 13 amino acids, free carnitine, 33 acyl-carnitines and 21 ratios. All Guthrie cards are analyzed within 2 days of collection. RESULTS: Total and free carnitine were found to be significantly lower in neonates who later developed T1D compared with controls. Moreover, the concentrations of the acyl-carnitines - acetyl-L-carnitine (C2), proprionylcarnitine (C3), 3-hydroxyisovalerylcarnitine (C5OH), miristoylcarnitine (C4), palmitoylcarnitine (C16) and stearoylcarnitine (C18) - were also significantly low in the cases vs controls. Furthermore, total amino-acid concentrations, expressed as the algebraic sum of all amino acids tested, showed a trend toward lower levels in cases vs controls. CONCLUSIONS: We found that carnitine and amino-acid deficit may be evident before the clinical appearance of T1D, possibly from birth. The evaluation of these metabolites in the neonatal period of children human leukocyte antigen genetically at 'risk' to develop T1D, could represent an additional tool for the prediction of T1D and could also offer the possibility to design new strategies for the primary prevention of the disease from birth.

Serum transforming growth factor ß1 during diabetes development in non-obese diabetic mice and humans.

Recent data show that regulatory cells with transforming growth factor (TGF)-ß1-dependent activity are able to restore self-tolerance in overtly diabetic non-obese diabetic (NOD) mice. Thus, TGF-ß1 seems to have a relevant role in protection from autoimmune diabetes. Our aim was to investigate the possible significance of serum TGF-ß1 measurement in the natural history of diabetes in NOD mice, as well as in children positive for at least one islet-related antibody. Serum TGF-ß1 (both total and active) was measured by enzyme-linked immunosorbent assay at monthly intervals in 26 NOD mice during the spontaneous development of diabetes and, on a yearly basis, in nine siblings of patients with type 1 diabetes (T1D) with a follow-up of 4 years. Diabetes appeared between the 12th week of age and the end of the study period (36 weeks) in 17 mice. TGF-ß1 serum level variations occurred in the prediabetic period in both NOD mice and humans and diabetes diagnosis followed a continuing reduction of active TGF-ß1 (aTGF-ß1) serum levels. In mice, aTGF-ß1 serum levels measured at 4 weeks of age correlated positively with severity of insulitis, and negatively with percentage of insulin-positive cells. Our findings suggest that in NOD mice serum TGF-ß1 levels during the natural history of the diabetes reflect the course of islet inflammation. The measurement of aTGF-ß1 in islet-related antibody-positive subjects may provide insights into the natural history of prediabetic phase of T1D.

From the fetal liver to spleen and gut: the highway to natural antibody.

The film of sIgA lining the intestinal epithelium plays a role in the regulation of the commensal microflora and prevention of pathogen invasion. We show that, in the absence of intentional immunization, all sIgA in the gut is produced by B-1a B cells. We also show that B-1a B cells and sIgA derive from lineage-negative precursors found in the fetal liver and located in the spleen after birth. The splenic precursors do not generate B cells of the adaptive immune system in bone marrow, spleen, and lymph nodes, but efficiently replenish the cells producing the natural antibodies. Therefore, B-1a B cells with their splenic progenitors and their progeny of plasma cells fill the same function of the primordial immune system of lower vertebrates. The natural antibodies in the serum and on the intestinal epithelium may be an evolutionary ancient tool for the immediate protection against commensal and pathogenic bacteria.

Length of gestation and gender are associated with HLA genotypes at risk for Type 1 diabetes (Italian DIABFIN 3).

AIM: The aim of this study, which is part of the ongoing DIABFIN project, was to correlate HLA class II genotypes, classified for their effect on susceptibility to Type 1 diabetes (T1D), with various risk factors during pregnancy and the neonatal period. METHODS: Cord blood was collected from 4349 neonates; 1.0% were at high HLA risk (HR), 9.0% at moderate HLA risk (MR), and 90.0% at low HLA risk (LR) for T1D. Information about the mother's pregnancy, type of delivery, the neonates' clinical features at birth, and family history for autoimmune diseases were collected. RESULTS: Significant correlations were found between the different HLA risk categories and length of gestation, even when adjusted for sex, weight and length at birth of the neonate, birth order and mother's age (adjusted P = 0.007). The male : female ratio tended to increase from the LR to the HR category, from 1.00 and 1.21, respectively, in the LR and MR groups, to 1.62 in the HR group (P = 0.05). CONCLUSIONS: Length of gestation is inversely correlated with HLA risk categories for T1D. The higher the HLA risk for T1D, the shorter the gestational age, especially in male neonates.

IA-2 antibody prevalence and risk assessment of early insulin requirement in subjects presenting with type 2 diabetes (UKPDS 71).

AIMS/HYPOTHESIS: Established autoimmune markers of type 1 diabetes, including islet cell autoantibodies (ICA) and autoantibodies to glutamic acid decarboxylase (GADA) have been used to screen people presenting with type 2 diabetes for latent autoimmune diabetes in adults. We have examined the prevalence of autoantibodies to protein tyrosine phosphatase isoforms IA-2 (IA-2A) and IA-2beta/phogrin (IA-2betaA) in a cohort of adult UKPDS patients thought to have type 2 diabetes, and investigated the possible role of these autoantibodies in predicting requirement for insulin therapy. METHODS: IA-2A and IA-2betaA were measured by a validated radioimmunoassay with human recombinant autoantigens in 4,169 white Caucasian patients aged 25-65 years and newly diagnosed with type 2 diabetes. The clinical requirement for insulin therapy within 6 years was examined in 2,556 patients not randomised to insulin. RESULTS: IA-2A and IA-2betaA were present in 2.2 and 1.4%, respectively, of these patients. IA-2A were more prevalent in younger patients (p for trend <0.00001), more often associated with the HLA-DR4 allele (26.3 vs 8.0%, p<0.0001), and their presence increased the likelihood of insulin therapy requirement within 6 years from diagnosis [relative risk (95%CI) 12.2 (9.8-15.3)]. The presence of IA-2A together with GADA increased the relative risk of requiring insulin therapy from 5.4 (4.1-7.1) for GADA alone to 8.3 (3.7-18.8) and the corresponding positive predictive value from 33 to 50%. CONCLUSIONS/INTERPRETATION: In type 2 diabetes, the presence of IA-2A is infrequent, associated with the HLA-DR4 haplotype, and highly predictive of future need for insulin therapy. The measurement of IA-2betaA does not provide additional information.

Islet autoantibodies in clinically diagnosed type 2 diabetes: prevalence and relationship with metabolic control (UKPDS 70).

AIMS/HYPOTHESIS: We examined the prevalence of islet autoantibodies and their relationship to glycaemic control over 10 years in patients diagnosed clinically with new-onset type 2 diabetes. METHODS: Patient clinical characteristics and autoantibody status were determined at entry to the UK Prospective Diabetes Study (UKPDS) before randomisation to different glucose control policies. Patients were followed for 10 years. RESULTS: Data available on 4,545 of the 5,102 UKPDS patients showed that 11.6% had antibodies to at least one of three antigens: islet cell cytoplasm, glutamic acid decarboxylase and islet autoantibody 2A (IA-2A). Autoantibody-positive patients were younger, more often Caucasian and leaner, with lower beta cell function and higher insulin sensitivity than autoantibody-negative patients. They also had higher HbA1c, and HDL-cholesterol levels, and lower blood pressure, total cholesterol and plasma triglyceride levels. Despite relative hyperglycaemia, autoantibody-positive patients were less likely to have the metabolic syndrome (as defined by the National Cholesterol Education Program Adult Treatment Program III), reflecting a more beneficial overall risk factor profile. Of 3,867 patients with post-dietary run-in fasting plasma glucose (FPG) values between 6.0 and 14.9 mmol/l and no hyperglycaemic symptoms, 9.4% were autoantibody-positive, compared with 25.1% of 678 patients with FPG values of 15.0 mmol/l or higher, or hyperglycaemic symptoms. In both groups, no differences were seen between those with and without autoantibodies in changes to HbA1c over time, but autoantibody-positive patients required insulin treatment earlier, irrespective of the allocated therapy (p<0.0001). CONCLUSIONS/INTERPRETATION: Autoantibody-positive patients can be treated initially with sulphonylurea, but are likely to require insulin earlier than autoantibody-negative patients.

Long term follow up of children with myocarditis treated by immunosuppression and of children with dilated cardiomyopathy.

OBJECTIVE: To describe the treatment and long term outcome after immunosuppressive treatment of children with myocarditis. METHODS AND RESULTS: 114 patients with newly diagnosed dilated cardiomyopathy were divided into three groups, according to the histological pattern: group A, acute myocarditis; group B, borderline myocarditis; and group C, non-inflammatory cardiomyopathy. Groups A and B were treated with cyclosporine and prednisone in addition to conventional treatment. Survivors of the whole cohort were analysed for 13 year transplant-free survival and assessed for left ventricular function. Event-free survival at 13 years was 97 (3)% for group A, 70 (8)% for group B, and 32 (7)% for group C (p < 0.0001). It was 96 (4)% at one year and 83 (5)% at 13 years for the cumulated myocarditis group (A and B). Cardiac function recovered completely in 79% of survivors in group A, 64% in group B, and 36% in group C. The rate of complete recovery in the cumulated group (A and B) was 70%. CONCLUSIONS: The high long term survival rate of this cohort of children with myocarditis is probably due to the effect of short term immunosuppression. This result differs from previously published series of conventionally treated children, whose survival probability at one year was about 60%.

The genetic basis of immune and autoimmune responses.

HLA class I and class II molecules play a major role in the presentation of short, pathogen-derived peptides to T cells, a process that initiates the adaptive cellular and humoral immune responses. However, the factors governing a cell's ability to respond or not to particular peptides are still not completely understood. Taking the example of a viral infection, in tissues infected with a virus, viral particles are taken up by antigen-presenting cells and uncoated. The viral DNA or RNA enters the nucleus, where it replicates. mRNA enters the cytosol and is transcribed into proteins. These proteins are degraded in proteasomes and the resulting peptides (8-10 residues) are loaded onto class I molecules for export to the surface of the cells. In the meantime, the groove of the class II molecules is also preparing to accommodate peptides (12-24 residues) generated by the endocytic protein-processing pathway. The surface of the infected cell then becomes adorned with peptide-loaded human leukocyte antigen (HLA) molecules. CD4+ T helper lymphocytes engage class II molecules and elicit responses from B cells, which will ultimately lead to antibody production, whereas CD8+ T lymphocytes become cytotoxic T cells. As a consequence, the virus is eliminated from the body. However, certain mysteries and challenges remain. How can, as an exception to this rule, an autoimmune response be the escape from the perfect machinery? This review offers some hypotheses on how to see the problem through to its solution.

Distinct antigenic features of linear epitopes at the N-terminus and C-terminus of 65 kDa glutamic acid decarboxylase (GAD65): implications for autoantigen modification during pathogenesis.

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

The Continuous Glucose Monitoring System (CGMS) in type 1 diabetic children is the way to reduce hypoglycemic risk.

BACKGROUND: Diabetic children treated with intensive insulin therapy are showing a dangerous increase in severe hypoglycemic episodes. The Continuous Glucose Monitoring System (CGMS) allows glycemic profiles to be monitored over a 72-h period. The aim of the present study was to evaluate whether this system is sufficiently sensitive to detect asymptomatic hypoglycemia, and to determine if its periodic application would help to minimize the hypoglycemic risk in children with type 1 diabetes mellitus (T1DM). METHODS: Twenty-seven T1DM children (age range 6-13.1 years) were enrolled in the study. The sensor was inserted subcutaneously in each patient and the standard four or five registrations of capillary glycemia per day were performed. Eighteen patients continued in the study and the glucose sensor was again inserted after a 6-week interval. At the beginning and end of the study, fructosamine, glycosylated hemoglobin (HbA(1c)), median glycemia, number and duration of hypoglycemic events and insulin requirement were evaluated. RESULTS: A significantly higher number of asymptomatic hypoglycemic events was revealed by CGMS in comparison with the standard system (3.6 +/- 2.3 vs 0.7 +/- 0.9; p < 0.0001). In patients who continued in the study, insulin therapy adjustments reduced the incidence of hypoglycemic events (2.5 +/- 1.7 vs 3.9 +/- 2.2; p < 0.05). At the 6-week point, the fructosamine level was reduced (330 +/- 30 vs 349 +/- 24 micro mol/l; p < 0.05). CONCLUSIONS: The CGMS is a useful device not only for detecting unrecognized hypoglycemia, but also for modifying insulin therapy in order to reduce hypoglycemic events. The system appears to be useful in avoiding long exposure to hypoglycemia.

The number of markers of pancreatic autoimmunity is proportional to the risk for type 1 diabetes mellitus in Italian and English patients with organ-specific autoimmune diseases.

An 11-year prospective study was carried out in 226 patients with organ-specific autoimmune disease (OSAD) coming from northern Italy and southern England. Patients were investigated for diabetes-related autoantibodies (ICAs, GADAbs, and IA2Abs) in order to evaluate the best immunological combination in predicting type 1 DM. One hundred twenty-eight patients were ICA positive (77 Italian and 51 English), and 98 were ICA negative. ICAs were detected by immunofluorescence technique on human pancreas, whereas GADAbs and IA2Abs were found by immunoprecipitation assay. During follow-up, 33 of 128 (25.8%) ICA(+) (26% of Italian and 25.5% of English) and 2 of 98 (2%) ICA(-) patients developed type 1 DM (17 with acute-onset, and 18 with non-acute-onset disease). Among ICA(+) patients, three subgroups were considered: ICA(+) alone; ICA and GADAb(+); ICA, GADAb, and IA2Ab(+). Patients who were only ICA(+) had a predictive value for type 1 DM of 4.7%, with an annual incidence of 0.7%, and a cumulative risk of 6%. ICA and GADAb(+) patients had a predictive value of 17.5%, with an annual incidence of 2%, and a cumulative risk of 20%. ICA, GADAb, and IA2Ab(+) patients had a predictive value of 72, with an annual incidence of 13%, and a cumulative risk of 87%. Patients having three immunological markers revealed a prevalence increased in HLA-DR3 and/or -DR4, but reduced in HLA-DR2 haplotypes. The risk for type 1 DM increased proportionally with the number of diabetes-related antibodies, which were also related to the presence of genetic markers of disease susceptibility.

The sardinian autoimmunity study. 4. Thyroid and islet cell autoantibodies in sardinian pregnant women at delivery: a cross-sectional study.

A high incidence of autoimmune Type 1 diabetes mellitus (DM) has been clearly established in Sardinia. Although systematic epidemiological studies are still not available, an increased prevalence of thyroid autoantibodies (ATA) has been documented in the Sardinian adult population as compared to other Italian regions, suggesting that thyroid autoimmune disease may also have increased. We carried out a preliminary study with the aim of determining the prevalence of serological markers of thyroid (anti-thyroperoxydase antibodies, TPOAb) and islet cell (ICA) autoimmunity in a large number (no.=2249) of sera obtained from cord-blood of Sardinian pregnant women at delivery. The prevalence of TPOAb was 11.9%, while ICA were detected in 59 cases (2.6%). A higher prevalence of TPOAb (6/17=35.3%) was found in sera with high ICA titers (> or = 20 JDF-U), as compared to sera with low ICA titers (5-19 JDF-U) and to ICA-negative sera (3/42=7,1%; chi2=5.4, p=0.02 and 258/2190=11,8%; chi2=6.8, p=0.009 respectively). Fourteen women (all ICA-negative) were diabetic: 4 had Type 1 and 10 had gestational DM; due to the low number, no correlation could be established between DM type and TPOAb prevalence and/or titer. These preliminary data indicate that ATA are frequently observed in the general population of Sardinian pregnant women at term. As a consequence, even the frequency of postpartum thyroiditis is expected to be high. Although ATA were not increased in women with clinical overt diabetes, a higher prevalence of ATA was found in women with high titers of circulating ICA. Our results also confirm that Sardinia represents, perhaps for its peculiar genetic characteristics, an ideal place to study organ-specific autoimmunity.

The Sardinian Autoimmunity Study: 3. Studies on circulating antithyroid antibodies in Sardinian schoolchildren: relationship to goiter prevalence and thyroid function.

The relationship among iodine intake, goiter prevalence, and thyroid autoimmunity remains controversial. In the present article, we report the prevalence of antithyroid antibodies (ATA) in relation to iodine intake, frequency of goiter, and thyroid function in a large group of Sardinian schoolchildren living in areas with borderline iodine sufficiency, or mild to moderate iodine deficiency. A total of 8,040 schoolchildren (4,194 males, 3,846 females, ages 6-15 years) from 29 communities were examined between 1986-1994. Thyroid size was assessed by palpation, according to the Pan American Health Organization (PAHO) criteria. In all cases antimicrosomal (MAb) or antithyroid peroxidase antibodies (TPOAb) and thyrotropin (TSH) were assayed. Urinary iodine was determined in a subgroup of 820 children. ATA was detected in 235 (2.92%) sera (88 males, 2.12%; 147 females, 3.82%; chi2 = 20.41, p < 0.0001). ATA prevalence ranged between 0.0%-7.3% in the 29 communities without any geographical correlation with goiter prevalence and urinary iodine excretion. However, ATA was more frequently detected in goitrous children, especially in females. The presence of ATA was not age-dependent in males, whereas a significant increase of ATA was observed in females older than 11 years of age. Seventy-seven (0.96%) children showed borderline to slightly increased serum TSH (>5.2-32 mU/L). Increased serum TSH concentration was more frequently found in children with ATA, especially at higher titers. In summary, our study in Sardinian schoolchildren indicates: (1) ATA display geographical heterogeneity, which seems to be unrelated to goiter prevalence and/or to iodine supply; (2) ATA are more frequently detected in females older than 11 years of age, suggesting that puberty has a role in determining the predominance in females of thyroid autoimmunity; (3) although most goitrous children are ATA-negative, the prevalence of ATA is increased in children with enlarged glands; (4) ATA is associated with an increased prevalence of subclinical hypothyroidism.

A genomewide scan for loci predisposing to type 2 diabetes in a U.K. population (the Diabetes UK Warren 2 Repository): analysis of 573 pedigrees provides independent replication of a susceptibility locus on chromosome 1q.

Improved molecular understanding of the pathogenesis of type 2 diabetes is essential if current therapeutic and preventative options are to be extended. To identify diabetes-susceptibility genes, we have completed a primary (418-marker, 9-cM) autosomal-genome scan of 743 sib pairs (573 pedigrees) with type 2 diabetes who are from the Diabetes UK Warren 2 repository. Nonparametric linkage analysis of the entire data set identified seven regions showing evidence for linkage, with allele-sharing LOD scores > or =1.18 (P< or =.01). The strongest evidence was seen on chromosomes 8p21-22 (near D8S258 [LOD score 2.55]) and 10q23.3 (near D10S1765 [LOD score 1.99]), both coinciding with regions identified in previous scans in European subjects. This was also true of two lesser regions identified, on chromosomes 5q13 (D5S647 [LOD score 1.22] and 5q32 (D5S436 [LOD score 1.22]). Loci on 7p15.3 (LOD score 1.31) and 8q24.2 (LOD score 1.41) are novel. The final region showing evidence for linkage, on chromosome 1q24-25 (near D1S218 [LOD score 1.50]), colocalizes with evidence for linkage to diabetes found in Utah, French, and Pima families and in the GK rat. After dense-map genotyping (mean marker spacing 4.4 cM), evidence for linkage to this region increased to a LOD score of 1.98. Conditional analyses revealed nominally significant interactions between this locus and the regions on chromosomes 10q23.3 (P=.01) and 5q32 (P=.02). These data, derived from one of the largest genome scans undertaken in this condition, confirm that individual susceptibility-gene effects for type 2 diabetes are likely to be modest in size. Taken with genome scans in other populations, they provide both replication of previous evidence indicating the presence of a diabetes-susceptibility locus on chromosome 1q24-25 and support for the existence of additional loci on chromosomes 5, 8, and 10. These data should accelerate positional cloning efforts in these regions of interest.

Lack of detection of retroviral particles (HIAP-1) in the H9 T cell line co-cultured with thyrocytes of Graves' disease.

Evidence for a possible aetiopathogenetic role of endogenous and/or exogenous retroviruses (RVs) in organ- and non-organ-specific autoimmune diseases is circumstantial in both humans and animal models. Intracisternal A type particles, antigenically related to HIV, have been reported in H9 cells co-cultured with homogenates of salivary glands obtained from patients with Sjögren syndrome and with synovial fluid of patients with rheumatoid arthritis. In order to identify a possible transfer of a putative 'infective RV agent' involved in the pathogenesis of human thyroid autoimmune disease, the H9 T cell line was co-cultured not only with thyroid homogenates, but also with viable thyrocytes, both prepared from glands of patients with Graves' disease. At the end of a prolonged co-culture period (24 weeks), no RV particles could be detected by electron microscopy in the H9 cells co-cultured with both thyroid preparations. These data seem to exclude the involvement of HIAP-1 in the aetiopathogenesis of human autoimmune thyroid disease.

Genetic and immunological characteristics of Type I diabetes mellitus in an Indo-Aryan population.

AIMS/HYPOTHESIS: Our aim was to characterise the genetic and immunological features associated with Type I (insulin-dependent) diabetes mellitus in a cohort of Indo-Aryan children resident in the United Kingdom. METHODS: Children with Type I diabetes (n = 53), unaffected first-degree relatives (n = 146) and unrelated healthy control children (n = 54) were typed for alleles of the HLA-DRB1, HLA-DQA1 and HLA-DQB1 genes. Islet cell antibodies and antibodies to glutamic acid decarboxylase, protein tyrosine phosphatase-2 (IA-2ic) and insulin were measured in the diabetic and control children. RESULTS: The DRB1*03.DQA1*05.DQB1*02 haplotype was positively associated with the disease, occurring in 78% of diabetic children compared with 22.6 % of healthy children (p(c) < 2.4 x 10(-5)). In simplex families, this haplotype was transmitted more frequently to the diabetic children than to their unaffected siblings (p < 1 x 10(-4)). The DRB1*04.DQA1* 03.DQB1*0302 haplotype was also transmitted preferentially to the diabetic probands (p < 0.025) but was not associated with disease in the case control study. Islet-related autoantibodies were detected in 89.6 % of diabetic patients compared with 11.8 % of control children (p < 1 x 10(-6)). Although protein tyrosine phosphatase-2 autoantibodies were detected more frequently among DRB1*04-positive diabetic patients compared with patients lacking this allele, the overall frequency of these autoantibodies was lower than observed in Europid diabetic subjects. This could reflect the absence of a disease association with DRB1*04 in the Indo-Aryan cohort. CONCLUSION/INTERPRETATION: Type I diabetes in our Indo-Aryan cohort is similar to the disease observed in Anglo-Europeans but has important immunogenetic differences. The low frequency of protein tyrosine phosphatase-2 autoantibodies among the Indo-Aryan diabetic children could have important implications for the design of future strategies for disease prediction in this population.