Autoimmune polyendocrine syndrome type 1: an Italian survey on 158 patients.

BACKGROUND: Autoimmune Polyglandular Syndrome type 1 (APS-1) is a rare recessive inherited disease, caused by AutoImmune Regulator (AIRE) gene mutations and characterized by three major manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CH) and Addison's disease (AD). METHODS: Autoimmune conditions and associated autoantibodies (Abs) were analyzed in 158 Italian patients (103 females and 55 males; F/M 1.9/1) at the onset and during a follow-up of 23.7 ± 15.1 years. AIRE mutations were determined. RESULTS: The prevalence of APS-1 was 2.6 cases/million (range 0.5-17 in different regions). At the onset 93% of patients presented with one or more components of the classical triad and 7% with other components. At the end of follow-up, 86.1% had CH, 77.2% AD, 74.7% CMC, 49.5% premature menopause, 29.7% autoimmune intestinal dysfunction, 27.8% autoimmune thyroid diseases, 25.9% autoimmune gastritis/pernicious anemia, 25.3% ectodermal dystrophy, 24% alopecia, 21.5% autoimmune hepatitis, 17% vitiligo, 13.3% cholelithiasis, 5.7% connective diseases, 4.4% asplenia, 2.5% celiac disease and 13.9% cancer. Overall, 991 diseases (6.3 diseases/patient) were found. Interferon-ω Abs (IFNωAbs) were positive in 91.1% of patients. Overall mortality was 14.6%. The AIRE mutation R139X was found in 21.3% of tested alleles, R257X in 11.8%, W78R in 11.4%, C322fsX372 in 8.8%, T16M in 6.2%, R203X in 4%, and A21V in 2.9%. Less frequent mutations were present in 12.9%, very rare in 9.6% while no mutations in 11% of the cases. CONCLUSIONS: In Italy, APS-1 is a rare disorder presenting with the three major manifestations and associated with different AIRE gene mutations. IFNωAbs are markers of APS-1 and other organ-specific autoantibodies are markers of clinical, subclinical or potential autoimmune conditions.

Autoimmune polyendocrine syndrome type 1 (APECED) in the Indian population: case report and review of a series of 45 patients.

BACKGROUND: Autoimmune polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) or autoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive genetic disease due to mutations in the AIRE (AutoImmune REgulator) gene. The clinical diagnosis is classically based on the presence of at least two of the three main components: chronic mucocutaneous candidiasis, hypoparathyroidism and primary adrenal insufficiency. Patients often suffer from other endocrine or non-endocrine autoimmune conditions throughout life. APECED etiopathogenesis is mediated by T lymphocytes. Autoantibodies against proteins of the affected organs are found in the serum of APECED patients as well as neutralizing antibodies against cytokines. We report here the clinical and genetic characteristics of 45 Indian APECED patients in comparison to Finnish, Sardinian, Turkish and North/South American cohorts from their published results. We also report a new case of APECED of Indian origin, a 2-year old child suffering from chronic mucocutaneous candidiasis since the age of 8 months, with confirmatory AIRE homozygous mutation c.274C > T (p.R92W). CONCLUSION: With the inherent limitations of a retrospective study, analysis of Indian APECED patients suggested that compared to classic criteria, application of Ferre/Lionakis criteria validated in North/South American patients could help in earlier diagnosis in 3 of 8 (37.5%) patients for whom adequate information for evaluation was available.

Family history and ethnicity influencing clinical presentation of type 1 diabetes in childhood.

BACKGROUND: A different clinical presentation of type 1 diabetes (T1DM) could be supposed in children belonging to different ethnicities, with or without family history of autoimmunity. OBJECTIVE: This study investigates the effect of ethnicity and family history of T1DM on clinical characteristics at presentation in a group of T1DM children. METHODS: One hundred ninety-six T1DM children <18 years, consecutively diagnosed during the years 2011-2014, were studied including 91 % of Caucasians of Italian ancestry and 9 % of non-Caucasian origin. RESULTS: Children with 1st or 2nd degree relatives affected by T1DM were younger at disease onset (p = 0.005) and showed lower HbA1C levels (p = 0.002), and higher IAA levels (p = 0.01). Non-Caucasian children were younger at disease onset (p = 0.029), and showed more severe hyperglycemia (p = 0.008) and ketoacidosis (pH p < 0.001). HbA1C levels were negatively related to positive family history of T1DM (p = 0.01), fasting C-peptide levels (p = 0.003), IAA levels (p = 0.03), and IA-2 levels (p = 0.003). The level of pH was positively influenced by fasting C-peptide (p = 0.004), and negatively impacted by C-reactive protein (p = 0.01) and non-Caucasian ethnicity (p = 0.03). CONCLUSION: The milder metabolic decompensation in children with a positive family history of T1DM is probably explained by the awareness of the families in terms of early symptoms of T1DM, while the younger age at onset and the higher levels of autoantibodies may suggest a stronger genetic susceptibility, associated with a more aggressive autoimmune process. The younger age in non-Caucasian children is probably explained by the higher genetic susceptibility in subjects belonging to ethnic groups with a low T1DM incidence. Social aspects and poor living conditions probably predominate in determining the increased severity of metabolic decompensation at onset in children from non-Caucasian ethnicities.

Role of caspase-8 in thymus function.

The thymus is the primary organ responsible for de novo generation of immunocompetent T cells that have a diverse repertoire of antigen recognition. During the developmental process, 98% of thymocytes die by apoptosis. Thus apoptosis is a dominant process in the thymus and occurs through either death by neglect or negative selection or through induction by stress/aging. Caspase activation is an essential part of the general apoptosis mechanism, and data suggest that caspases may have a role in negative selection; however, it seems more probable that caspase-8 activation is involved in death by neglect, particularly in glucocorticoid-induced thymocyte apoptosis. Caspase-8 is active in double-positive (DP) thymocytes in vivo and can be activated in vitro in DP thymocytes by T-cell receptor (TCR) crosslinking to induce apoptosis. Caspase-8 is a proapoptotic member of the caspase family and is considered an initiator caspase, which is activated upon stimulation of a death receptor (e.g., Fas), recruitment of the adaptor molecule FADD, and recruitment and subsequent processing of procaspase-8. The main role of caspase-8 seems to be pro-apoptotic and, in this review, we will discuss about the involvement of caspase-8 in (1) TCR-triggered thymic apoptosis; (2) death receptor-mediated thymic apoptosis; and (3) glucocorticoid-induced thymic apoptosis. Regarding TCR triggering, caspase-8 is active in medullary, semi-mature heat-stable antigen(hi) (HAS(hi) SP) thymocytes as a consequence of strong TCR stimulation. The death receptors Fas, FADD, and FLIP are involved upstream of caspase-8 activation in apoptosis; whereas, Bid and HDAC7 are involved downstream of caspase-8. Finally, caspase-8 is involved in glucocortocoid-induced thymocyte apoptosis through an activation loop with the protein GILZ. GILZ activates caspase-8, promoting GILZ sumoylation and its protection from proteasomal degradation.

Peptide immunotherapies in Type 1 diabetes: lessons from animal models.

Insulin dependent diabetes mellitus (Type 1 diabetes, T1D) is a chronic autoimmune disorder characterized by the destruction of insulin-producing pancreatic beta cells by proinflammatory autoreactive T cells. In the past, several therapeutic approaches have been exploited by immunologists aiming to regulate the autoimmune response; this can occur by deleting lymphocyte subsets and/or re-establishing immune tolerance via activation of regulatory T cells. The use of broad immunosuppressive drugs was the first approach to be explored. Subsequently, antibody-based immunotherapies failed to discriminate between autoreactive versus non-autoimmune effectors. Antigen-based immunotherapy is a third approach developed to manipulate beta cell autoimmunity. This approach allows the selective targeting of disease-relevant T cells, while leaving the remainder of the immune system intact. Animal models have been successfully employed to prevent or treat T1D by injection of either the self proteins or peptides derived from them. Peptide immunotherapies have been mainly experimented in the NOD mouse spontaneous model of disease. In this review we therefore report the main approaches that rely on the use of peptides obtained from relevant autoantigens such as glutamic acid decarboxylase, isoform 65 (GAD65), insulin, proinsulin and islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP). Protective peptides have proven to be effective in treating or delaying the diabetic process. We also highlight the main difficulties encountered in extrapolating data to guide clinical translational investigations in humans.

Unravelling the role of infectious agents in the pathogenesis of human autoimmunity: the hypothesis of the retroviral involvement revisited.

The incidence of autoimmune disorders is increasing worldwide. Several theories have been proposed to explain how the breakdown in the balance between autoregulatory immune pathways and pathogenic autoreactivity generate autoimmunity. On the basis of a large body of epidemiological, clinical and experimental evidence, it has been suggested that an unfortunate interplay of genetic susceptibility and environmental factors must play an important role in generating the abnormal autoimmune response. Although genetic factors have been well dissected, the environmental agents, that may be causative of disease are still under investigation. Their discovery is obviously relevant because they enable us to device preventive and therapeutic strategies in trying to halt the progress and ultimately treat this category of disorders. Among the environmental factors, infectious agents have been proposed as the best candidate triggers in the autoimmune pathogenesis. The observation that a long preclinical period often precedes the clinical onset of disease, in analogy to the clinical symptoms of AIDS, led to propose exogenous and endogenous retroviruses as suspected culprits for organ and non-organ specific autoimmune disorders. The hypothesis is revisited in this article in the light of our research experience over the past years and of relevant literature emerging in the field.

Identification and characterization of a novel expandable adult stem/progenitor cell population in the human exocrine pancreas.

It is a general opinion that tissue-specific stem cells are present in adult tissues but their specific properties remain elusive. They are rare in tissues and heterogeneous; in addition, their identification and the characterization of their progeny has encountered technical difficulties. In particular, the existence of pancreatic stem cells remains elusive because specific markers for their identification are not available. We established a method for the isolation of a population of stem/progenitor cells from the human exocrine pancreas, and propose it as a model for other human compact organs. We also used markers that identified and finally characterized these cells. Spheroids with self-replicative potential were obtained from all specimens. The isolated population contained a subset of CD34+ CD45- cells and was able to generate, in appropriate conditions, colonies that produce insulin. We obtained evidence that most freshly isolated spheroids, when co-cultured with the c-kit positive neuroblastoma cell line LAN 5, produced a c-kit positive progeny of cells larger in their cytoplasmic content than the original spheroid population, with elongated morphology resembling the neuronal phenotype. We identified a novel predominant functional type of stem/progenitor cell within the human exocrine pancreas, able to generate insulin-producing cells and potentially non-pancreatic cells.

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.

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.

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.

Osteopontin is an autoantigen of the somatostatin cells in human islets: identification by screening random peptide libraries with sera of patients with insulin-dependent diabetes mellitus.

Random peptide libraries (RPLs) screening with IDDM sera has identified 5 disease-specific 'mimotopes' displayed on phage (phagotopes). We characterised one phagotope (CH1p), by raising a rabbit antibody against the peptide insert on phage, which was employed in immunohistochemistry, Western blotting and cDNA libraries screening. The CH1p mimotope was detected in somatostatin cells of human islets and experimentally raised anti-osteopontin antibodies or human sera positive for the phagotope, detected a similar subpopulation of islet cells. The screening of cDNA library identified a clone corresponding to human osteopontin. In summary, RPLs proved to be successful in the identification of a novel islet-related autoantigen (osteopontin), whose significance in disease remains to be established.

The effect of staphylococcal enterotoxin B on thyrocyte HLA molecule expression.

Microbial superantigens have been implicated in the pathogenesis of human autoimmune diseases. In autoimmune glands, thyrocytes inappropriately express HLA-DR molecules and these cells may function as antigen presenting cells (APC) We studied the effect in vitro of staphylococcal enterotoxin B (SEB) on HLA molecule expression on thyrocytes obtained from autoimmune and non-autoimmune glands by immunofluorescence. HLA class I and class II upregulation could be detected by FACS analysis on thyrocytes. Anti-IFN-gamma neutralizing antibodies markedly affect both class I and class II upregulation on thyrocytes. FRTL5 cells were not responsive to SEB. Similarly, a human thyroid cell strain maintained in culture in a conditioned medium was not induced to express HLA products by SEB stimulation. The addition of autologous intrathyroidal lymphocytes caused reestablishment of the SEB effect.

Identification of a novel type 1 diabetes-specific epitope by screening phage libraries with sera from pre-diabetic patients.

We used random peptide libraries displayed on phage to search for ligands to insulin dependent diabetes mellitus-related antibodies and were able to identify several candidate disease-related peptides. One of them, clone 92, showed a significant difference in the frequency of reactivity with the sera of patients and normal controls. Human immunoglobulins immunopurified on phage 92 specifically stained the islets on human pancreatic sections. When injected into rabbits, the selected peptide elicited antibodies that also stained human and rat pancreatic sections, with a pattern similar to that observed with immunoglobulins purified from the sera of patients. No reactivity was observed in other tissues. Our results indicate that the peptide identified in this work mimics a novel, diabetes-related self-antigen.

Selection of phage-displayed peptides mimicking type 1 diabetes-specific epitopes.

Phage display technology represents a powerful tool for the identification of peptides reacting with disease-related antibodies present in human sera. The application of this technology to type 1 diabetes could provide a set of novel reagents for diabetes prediction and could also lead to the identification of novel autoantigens or even of environmental factors possibly causing the disease. In the present study, sera of prediabetic and high risk individuals were used to select candidate peptides from phage-displayed random peptide libraries. Diabetes specific phage clones were then identified from these through screening and counter screening, using sera from diabetic and non-diabetic individuals. The results presented in this paper demonstrate the feasibility of this methodology to identify peptides reacting preferentially with antibodies present in the serum of diabetic patients.

Calcitonin and somatostatin containing C cells in rat and human thyroid. Immunohistochemical study by a double-staining method.

C cells of the thyroid probably exert a paracrine control on follicular cells through secretion of peptides such as calcitonin and somatostatin. The aim of the present study was to investigate the expression of different peptides produced by C cells in rat thyroid and in the different morphological and pathological conditions of the human thyroid. Therefore we employed the immunohistochemical double-staining method using anti-calcitonin and anti-somatostatin antibodies. The results of this study show the presence of C cells containing calcitonin and C cells containing somatostatin exclusively in the rat and the human thyroid. This distinction with prevalence of one peptide on the other is maintained in the different morphological and pathological conditions of the human thyroid.

[Concentration of C cells in the thyroid of rats treated with mercaptoimidazole or levo-thyroxine]. / Concentrazione delle cellule C nella tiroide di ratti trattati con mercaptoimidazolo o con levo-tiroxina.

Concentration of C cells in the thyroid of rats treated with Mercaptoimidazole or Levo-thyroxine. The aim of the present study was to elucidate the possible functional relationship between follicular and parafollicular cells of the thyroid gland; therefore we have investigated the behaviour of calcitonin producing cells and serum calcitonin concentration in rats both in resting and hyperstimulation conditions of follicular cells. Our results showed that with regard to follicular mass the concentration of the C cells was reduced in the two groups of rats treated compared to control rats. C cell concentration decrease was associated with reduced serum calcitonin concentration. In conclusion C cell activity is independent of TSH and thyroid hormones circulating levels.

Distribution of calcitonin- and somatostatin-containing cells in thyroid lymphoma and in Hashimoto's thyroiditis.

Eleven cases of thyroid lymphoma were studied by the immunoperoxidase avidin-biotin technique with calcitonin and somatostatin rabbit antisera. In 6 cases of non-Hodgkin lymphoma, in thyroid tissue residual to the lymphomatous infiltration, the C cell density was markedly increased and clustering was often observed; the C cells often took part in the follicular lining, frequently with polar distribution; these elements displayed a strong positivity for calcitonin, while the number of somatostatin-containing cells was lower and the staining less intense. In the only case of Hodgkin's lymphoma of the thyroid gland the staining was negative; in other 4 cases of non-Hodgkin lymphoma no residual thyroid tissue was found and the staining was also negative. As Hashimoto's thyroiditis is often associated with thyroid lymphoma, 13 cases of Hashimoto's thyroiditis were also studied; no C cells were observed and both stainings were negative. These data show that an increase in the C cell number may be a hallmark of thyroid lymphoma and that hyperplastic C cells show an intensive positivity for calcitonin. On the other hand, C cell hyperplasia is not present in Hashimoto's thyroiditis, in spite of the close association with thyroid lymphoma. Furthermore, we provide evidence that somatostatin-containing cells are present both in normal thyroid glands and in thyroid lymphoma.