Insulin autoimmune syndrome: a rare cause of spontaneous hypoglycaemia in non-diabetic individuals.

Insulin autoimmune syndrome (IAS) is a rare cause of hypoglycaemia, characterised by recurrent hypoglycaemic episodes secondary to insulin autoantibodies in individuals who are not exposed to exogenous insulin. We are reporting a case of IAS in a 64-year-old gentleman, who presented with predominant postprandial hypoglycaemic episodes. On biochemical evaluation, he was found to have hyperinsulinemic hypoglycaemia. Localisation studies with MRI abdomen and endoscopic ultrasound (EUS) were negative for pancreatic tumour. Tests sent for insulin antibody levels were elevated. The patient was treated with frequent meals, acarbose and glucocorticoids. Patient condition improved and did not experience hypoglycaemia on follow-up.

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 µL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1µL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.

Insulin autoimmune syndrome in an occidental woman: a case report and literature review

SUMMARY Insulin autoimmune syndrome (IAS, Hirata's disease) is a rare hypoglycemic disorder characterized by spontaneous hypoglycemia associated with extremely high circulating insulin levels and positive anti-insulin antibody results. Thus far, most cases have been reported in Asian countries, notably Japan, with few cases reported in western countries. As a possible cause, it is associated with the use of drugs containing sulfhydryl radicals, such as captopril. This report refers to a 63-year-old female Brazilian patient with a history of postprandial hypoglycemia. After extensive investigation and exclusion of other causes, her hyperinsulinemic hypoglycemia was considered to have likely been induced by captopril. Most cases of IAS are self-limiting. However, dietary management, corticosteroids, plasmapheresis, and rituximab have already been used to treat patients with IAS. In our case, after discontinuation of captopril, an initial decrease in insulin autoantibody levels was observed followed by improvement in episodes of hypoglycemia. Although it is a rare disease, IAS should be considered in the differential diagnosis of endogenous hyperinsulinemic hypoglycemia. Patients with suspected IAS must be screened for autoimmunity-related drugs for insulin. Initial clinical suspicion of IAS can avoid unnecessary costs associated with imaging examinations and/or invasive surgical procedures.

Anti-Insulin B Cells Are Poised for Antigen Presentation in Type 1 Diabetes.

Early breaches in B cell tolerance are central to type 1 diabetes progression in mouse and man. Conventional BCR transgenic mouse models (VH125.Tg NOD) reveal the power of B cell specificity to drive disease as APCs. However, in conventional fixed IgM models, comprehensive assessment of B cell development is limited. To provide more accurate insight into the developmental and functional fates of anti-insulin B cells, we generated a new NOD model (VH125SDNOD) in which anti-insulin VDJH125 is targeted to the IgH chain locus to generate a small (1-2%) population of class switch-competent insulin-binding B cells. Tracking of this rare population in a polyclonal repertoire reveals that anti-insulin B cells are preferentially skewed into marginal zone and late transitional subsets known to have increased sensitivity to proinflammatory signals. Additionally, IL-10 production, characteristic of regulatory B cell subsets, is increased. In contrast to conventional models, class switch-competent anti-insulin B cells proliferate normally in response to mitogenic stimuli but remain functionally silent for insulin autoantibody production. Diabetes development is accelerated, which demonstrates the power of anti-insulin B cells to exacerbate disease without differentiation into Ab-forming or plasma cells. Autoreactive T cell responses in VH125SDNOD mice are not restricted to insulin autoantigens, as evidenced by increased IFN-γ production to a broad array of diabetes-associated epitopes. Together, these results independently validate the pathogenic role of anti-insulin B cells in type 1 diabetes, underscore their diverse developmental fates, and demonstrate the pathologic potential of coupling a critical ß cell specificity to predominantly proinflammatory Ag-presenting B cell subsets.

Fulminant Type 1 Diabetes Mellitus Accompanied by Positive Conversion of Anti-insulin Antibody after the Administration of Anti-CTLA-4 Antibody Following the Discontinuation of Anti-PD-1 Antibody.

An 80-year-old woman with malignant melanoma received 20 cycles of anti-programmed death 1 (PD-1) antibody (nivolumab) treatment and showed normal glucose tolerance. Three weeks after switching to anti-cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) antibody (ipilimumab), her plasma glucose level was elevated to 639 mg/dL, her HbA1c was 7.7%, and her fastening serum C-peptide immunoreactivity was undetectable. Anti-glutamic acid decarboxylase and insulinoma-associated protein-2 antibodies were negative. She was diagnosed with fulminant type 1 diabetes mellitus (F1DM). Remarkably, her anti-insulin antibody was positively converted, and her Sialylated Carbohydrate Antigen, Krebs von den Lungen-6 levels increased after ipilimumab therapy. She possessed F1DM-susceptible Human Leukocyte Antigen-DR4. A fluorescence activated cell sorting analysis showed an altered T-cell population. This case of F1DM highlights specific mechanisms underlying pancreatic beta cell immunity.

Insulin Autoimmune Syndrome Accompanied by Multiple Myeloma.

In 1981, a 48-year old man was diagnosed with insulin autoimmune syndrome. In 2005, he experienced a substantial increase in his monoclonal insulin antibody levels; in 2006 and 2007, serum monoclonal gammopathy and an 11% marrow plasmacyte ratio were confirmed. In 2012, asymptomatic multiple myeloma was diagnosed based on an increased γ-globulin fraction and serum M-protein (IgG) levels. The insulin antibody binding rate was 75.4% in 2005 and 78.8% in 2012. In 2012, he was hospitalized for ileus and died. Autopsy identified multiple myeloma and no endocrinological tumors in the pancreas.

Reversing Tolerance in Isotype Switch-Competent Anti-Insulin B Lymphocytes.

Autoreactive B lymphocytes that escape central tolerance and mature in the periphery are a liability for developing autoimmunity. IgG insulin autoantibodies that predict type 1 diabetes and complicate insulin therapies indicate that mechanisms for tolerance to insulin are flawed. To examine peripheral tolerance in anti-insulin B cells, we generated C57BL/6 mice that harbor anti-insulin VDJH-125 site directed to the native IgH locus (VH125(SD)). Class switch-competent anti-insulin B cells fail to produce IgG Abs following T cell-dependent immunization of VH125(SD) mice with heterologous insulin, and they exhibit markedly impaired proliferation to anti-CD40 plus insulin in vitro. In contrast, costimulation with LPS plus insulin drives robust anti-insulin B cell proliferation. Furthermore, VH125(SD) mice produce both IgM and IgG2a anti-insulin Abs following immunization with insulin conjugated to type 1 T cell-independent Brucella abortus ring test Ag (BRT). Anti-insulin B cells undergo clonal expansion in vivo and emerge as IgM(+) and IgM(-) GL7(+)Fas(+) germinal center (GC) B cells following immunization with insulin-BRT, but not BRT alone. Analysis of Igκ genes in VH125(SD) mice immunized with insulin-BRT reveals that anti-insulin Vκ from the preimmune repertoire is selected into GCs. These data demonstrate that class switch-competent anti-insulin B cells remain functionally silent in T cell-dependent immune responses, yet these B cells are vulnerable to reversal of anergy following combined BCR/TLR engagement that promotes Ag-specific GC responses and Ab production. Environmental factors that lead to infection and inflammation could play a critical yet underappreciated role in driving loss of tolerance and promoting autoimmune disease.

NFATc2 (NFAT1) assists BCR-mediated anergy in anti-insulin B cells.

NFAT transcription factors play critical roles in both the activation and repression of T and B lymphocyte responses. To understand the role of NFATc2 (NFAT1) in the maintenance of tolerance for anti-insulin B cells, functionally inactive NFATc2 (NFATc2(-/-)) was introduced into C57BL/6 mice that harbor anergic anti-insulin 125Tg B cells. The production and peripheral maturation of anti-insulin B cells into follicular and marginal zone subsets was not altered by the absence of functional NFATc2. Surface B cell receptor expression levels, important for tonic signaling and altered by anergy, were not altered in any spleen B cell subset. The levels of anti-insulin antibodies were not different in 125Tg/B6/NFATc2(-/-) mice and the anti-insulin response remained silenced following T cell dependent immunization. However, studies addressing in vitro proliferation reveal the anergic state of 125Tg B cells is relieved in 125Tg/B6/NFATc2(-/-) B cells in response to BCR stimulation. In contrast, anergy is not released in 125Tg/B6/NFATc2(-/-) B cells following stimulation with anti-CD40. The relief of anergy to BCR stimulation in 125Tg/B6/NFATc2(-/-) B cells is associated with increased transcription of both NFATc1 and NFATc3 while expression of these NFATs does not change in anti-IgM stimulated 125Tg/B6/NFATc2(+/+) B cells. The data suggest that NFATc2 plays a subtle and selective role in maintaining anergy for BCR stimulation by repressing the transcription of other NFAT family members.

Reduced diabetes in btk-deficient nonobese diabetic mice and restoration of diabetes with provision of an anti-insulin IgH chain transgene.

Type 1 diabetes results from T cell-mediated destruction of insulin-producing beta cells. Although elimination of B lymphocytes has proven successful at preventing disease, modulation of B cell function as a means to prevent type 1 diabetes has not been investigated. The development, fate, and function of B lymphocytes depend upon BCR signaling, which is mediated in part by Bruton's tyrosine kinase (BTK). When introduced into NOD mice, btk deficiency only modestly reduces B cell numbers, but dramatically protects against diabetes. In NOD, btk deficiency mirrors changes in B cell subsets seen in other strains, but also improves B cell-related tolerance, as indicated by failure to generate insulin autoantibodies. Introduction of an anti-insulin BCR H chain transgene restores diabetes in btk-deficient NOD mice, indicating that btk-deficient B cells are functionally capable of promoting autoimmune diabetes if they have a critical autoimmune specificity. This suggests that the disease-protective effect of btk deficiency may reflect a lack of autoreactive specificities in the B cell repertoire. Thus, signaling via BTK can be modulated to improve B cell tolerance, and prevent T cell-mediated autoimmune diabetes.

The natural history of type 1A diabetes

We can now predict the development of Type 1A (Immune Mediated) diabetes primarily through the determination of four biochemically characterized islet autoantibodies [insulin, GAD65, IA-2 (ICA512) and (Znt8)]. Prediction is possible because beta-cell destruction is chronically progressive and very slow in most, but not all individuals. We can also prevent type 1A diabetes in animal models and a major goal is the prevention of type 1A diabetes in man with multiple clinical trials underway.

Atualmente o desenvolvimento do diabetes melito tipo 1 A( imune mediado) pode ser predito através da determinação de quatro auto-anticorpos antiilhotas [antiinsulina, anti-GAD65, anti-IA2 (ICA512) e (anti-Znt8)] caracterizados bioquimicamente. A predição dessa doença é possível devido a destruição das células-beta, não em todos os indivíduos mas na sua maioria, ser crônica e lentamente progressiva. Também é possível prevenir o DM1 A em modelos animais e o objetivo maior é a prevenção dessa doença em humanos, para os quais vários protocolos clínicos estão em andamento.

Variation in enterovirus receptor genes.

The increased incidence of a enterovirus infections observed in patients with type 1 diabetes preceding the development of the clinical disease could be partially explained by variation in the genes coding for enterovirus receptors. We carried out sequence analysis of the most common enterovirus receptor molecules in 21 diabetic children and 20 healthy adults. DNA was isolated from the leukocytes, and gene regions known to code for virus-recognizing domains in major enterovirus receptors were amplified and sequenced. Heterozygous single-nucleotide polymorphism (SNP), Ala 67 (GCG) --> Thr (ACG), was detected in the poliovirus receptor gene in four individuals in the diabetes group, but not in the control group. However, serological studies could not confirm that this substitution would convey different susceptibility to poliovirus infection. A heterozygous SNP, Lys 29 (AAG) --> Met (ATG), was found in the intracellular adhesion molecule-1 (ICAM-1) (receptor for rhinoviruses and some coxsackie A viruses) in one individual in both groups. A silent SNP in the alpha2 integrin subunit gene (echovirus 1 receptor) was frequently found in both groups, a silent heterozygotic SNP in coxsackievirus-adenovirus receptor (coxsackie B virus receptor) gene was seen in one individual in the diabetes group, whereas no variation was found in the DAF (echovirus receptor) and beta3 integrin subunit sequences (receptor for coxsackievirus A9) studied. In conclusion, both synonymous and nonsynonymous sequence variability of genes coding for enterovirus and rhinovirus receptors was shown to occur, but no pattern directly specific for type 1 diabetes was found. =

Perfil de auto anticuerpos y lactancia materna en pacientes diabéticos tipo 1 / Auto-antibody levels and history of breast-feeding in type 1 diabetic patients

Background: Islet cell-specific autoantibodies such as islet cell antibody (ICA), antiinsulin (IAA), anti-glutamic acid decarboxylase (GAD) and anti-tyrosine phosphatase (IA2) can be present in patients with type I diabetes. Breast feeding duration and the early exposure to milk substitutes are environmental factors associated to etiology of type 1 diabetes. Aim To study the frequency of the anti-GAD, anti-IA-2 e ICA antibodies in Chilean type 1 diabetic patients and determine the possible modulator effect of the breast feeding. Patients and methods: One hundred thirty four type I diabetic patients, aged one to 15 years old, were studied at the moment of their diagnosis. Patients were classified according to the duration of exclusive breast feeding. IA-2 and GAD were determined by radio immuno assay and ICA by means of indirect immunofluorescence. Results: Subjects with three months or less and those with more than three months of breast feeding were positive for ICA in 78.8 and 90.6 per cent of cases respectively, for GAD in 75 and 54.6 per cent of cases respectively (p=0.024) and for IA-2 in 73 and 43.8 per cent of cases respectively (p=0.001). All three antibodies were positive in 53.9 and 21.8 per cent of children with less or more than three months of breast feeding (p=0.001). Conclusion: Both IA-2 and GAD antibodies are less frequently positive in type 1 diabetic patients who have been breast fed for more than three months. These findings suggest a possible attenuating role of exclusive breast feeding on pancreatic aggression events in patients with type 1 diabetes

Peptide-specific cytotoxicity of T lymphocytes against glutamic acid decarboxylase and insulin in type 1 diabetes mellitus.

Cytotoxic T lymphocytes (CTL) against pancreatic beta-cells probably play a major role in the etiology of type 1 diabetes mellitus (DM). CTLs recognize a complex formed between MHC class I and antigenic peptides fragments derived from intracellular processing of proteins. However, the exogenous peptides, which show strong affinities to MHC class I, can be presented. In this study, we focused on the cytotoxic activity of peripheral lymphocytes in patients with type 1 DM against the peptides of glutamic acid decarboxylase (GAD) and insulin, which can bind MHC class 1 A24. Lymphocytes were isolated from peripheral blood of 12 type 1 DM patients and eight healthy control subjects. The effector cells were cultured with peptides, IL-2 and IL-7, restimulated weekly by autologous antigen presenting cells, which were cultured with IL-4 and GM-CSF. On day 21, CTL activities of cultured effector cells were tested against autologous EB-blast cells as target cells pulsed with the stimulating peptides using 51Cr release assay. The results showed that cytotoxicity against insulin peptide binding to MHC class I A24 was observed in lymphocytes of four out of ten patients with type 1 DM. The mean cytotoxicity was 46.0% of the maximum release. The antibody against HLA-class I inhibited this effect. Cytotoxicity against GAD peptide which bind MHC class I A24 was not observed in seven patients. None of healthy controls showed cytotoxicity against GAD or insulin peptides was observed. This is the first report describing the cytotoxic activity of CD8+ T lymphocytes against insulin in type 1 DM.

Severe insulin-resistant diabetes mellitus associated with hypereosinophilic syndrome.

We describe a 52-year-old male manifesting severe insulin resistance associated with hypereosinophilic syndrome (HES). Diabetes mellitus was initially well-controlled by an oral hypoglycemic agent, and thereafter by human insulin. Due to the progression of hypereosinophilia, hepatosplenomegaly and peripheral lymphoadenopathy, severe insulin resistance associated with diabetic ketoacidosis occurred repeatedly, despite intravenous administration of over 1,000 U per day of human insulin. A high plasma insulin-binding capacity as determined by Scatchard analysis was consistent with insulin antibody-mediated resistance. The diagnosis of HES was made due to the persistent elevation of eosinophil count and associated liver and cardiac damage. Glucocorticoid therapy successfully achieved both reducing clinical symptoms and improving glycemic control.

Associaçäo entre auto-anticorpos anti-pro-insulina e anti-insulina com diabetes mellitus do tipo I / The association between insulin and proinsulin autoantibodies with tipe I Diabetes Mellitus

O presente trabalho compara a incidência de auto-anticorpos anti-insulina (IAA) e anti-pró-insulina (PAA) em diabéticos do tipo I de início recente e em seus parentes de primeiro grau. Foram estudados 33 indivíduos normais (grupo I), 16 diabéticos do tipo I de início recente (grupo II) e 141 parentes em primeiro grau de diabéticos do tipo I (grupo III). Os IAA e PAA foram determinados pelo método de radioensaio, sendo considerados anormais níveis de IAA acima de 0,584 pmol/L e de PAA acima de 0,441 pmol/L. Näo foram observadas diferenças significantes quanto a idade e sexo entre os 3 grupos. Nos indivíduos normais os níveis de PAA foram significantemente menores do que os de IAA. Entre os diabéticos de início recente foi encontrada uma incidência de IAA de 37,5 por cento e de PAA de 25,0 por cento, näo ocorrendo, entretanto, diferenças significantes entre os níveis destes dois anticorpos. Entre os parentes em primeiro grau a incidência de IAA foi de 3,5 por cento e de PAA de 7,8 por cento, näo ocorrendo também diferenças entre os dois testes. Houve uma correlaçäo significante entre os níveis dos IAA e dos PAA no grupo de diabéticos de início recente (r=0,64; p < 0,05). Os IAA e PAA parecem estar dirigidos contra o mesmo determinante antigênico e, portanto, têm o mesmo valor preditivo para o DM tipo I

Insulin autoantibodies and immune response to human insulin therapy in 24 type 1 (insulin-dependent) diabetic children: superiority of radio binding assay over solid phase assay.

To evaluate the immunization pattern against human insulin, 24 newly diagnosed diabetic children (12 females, 12 males; mean age: 7 +/- 4 years) were treated from diagnosis onwards with semisynthetic human insulin (NOVO). Informed consent was obtained from all parents. Blood samples were taken before, 1, 2, 3, 4, 6 and 8 weeks after the start of therapy and, thereafter, at monthly intervals for 2 years. Insulin (auto) antibodies (I(A)A) were measured by radio binding assay (RBA) and by enzyme-linked immunosorbent assay (ELISA). IAA, determined by RBA, were detected in eight children. Using ELISA, IgM IA were not detected after onset of therapy. By contrast, IgG IA were found in 8 children after 2 weeks of treatment and in 12 after 1 month. Using RBA, all children had IA after 2 months of therapy, whereas with ELISA, IA remained undetectable during the study period in 8 out of 24 patients. These results confirm previous observations suggesting that the 2 methods are not interchangeable and yield different estimations of the insulin immune reaction, not only before but also after the start of insulin therapy. In addition, the detection of IA by RBA in all treated patients unambiguously demonstrates that human insulin is immunogenic in man.

Generation of human monoclonal antibodies by transformation of lymphoblastoid B cells with ras oncogene.

Human monoclonal antibodies (hu-mAbs) of predetermined specificity and isotype are potentially important for a variety of applications, including therapy and diagnosis. Their efficient generation, however, is still hampered by technical difficulties. Even the most established approaches to the generation of hu-mAbs, i.e., B cell immortalization by Epstein-Barr virus (EBV) and/or fusion with appropriate myeloma cell lines, are characterized by a relatively low efficiency. It has been shown that expression of activated Ha- or N-ras oncogenes causes the malignant transformation and plasmacytoid differentiation of EBV-immortalized lymphoblastoid cell (LC) lines, suggesting that activated ras oncogenes can convert LC lines into effective hu-mAb producers. We have used retroviral vector-mediated gene transfer to introduce an activated Ha-ras (v-ras) oncogene into four distinct LC lines producing hu-mAbs of different classes (IgM and IgG) and specificities (to human insulin, human thyroglobulin and rabies virus glycoprotein). The cloning efficiency and antibody secretion of these ras-transformed LC (ras-LC) lines were compared with those of the hybrid LC (hyb-LC) lines generated by fusing the same parental LC lines with the Ig non-secretor F3B6 human-mouse hybrid cells. ras-LC lines were comparable to their hybrid counterparts in either parameter tested. This, together with the relatively higher efficiency of the method, suggests that ras transformation may constitute a valid alternative to the currently available technologies for hu-mAbs production from LC lines.

Antigeno HLA, anticuerpos anti-islotes, anti-insulinicos y anti-virales en niños venezolanos con diabetes mellitus tipo I recien diagnosticados / HLA antigen, anti-islets antibodies, anti-insulin and anti-virals in venezuelan children with newly diagnosed diabetes mellitus type I

En Venezuela no han sido reportados previamente los marcadores inmunológicos y genéticos en niños con Diabetes Mellitus tipo I durante el debut de la enfermedad. Nosotros tuvimos la oportunidad de estudiar a 27 niños recién diagnosticados con Diabetes Mellitus tipo I, con una edad promedio de 9.4+/- 3.2 años (Rango 1.3-14.9), 11 hembras y 16 varones. Ochenta y nueve por ciento tenían una historia familiar de Diabetes Mellitus (26 por ciento tipo I) 56.7 por ciento habían presentado una infección respiratoria superior previo al diagnóstico y 12.7 por ciento habían padecido de parotiditis o varicela. El pico de incidencia de la enfermedad fue durante los meses de febrero y octubre. Ochenta y nueve por ciento (24 pacientes) tenían HLD-DR3 y/o DR4 versus 37 por ciento observado en la población venezolana general; 86.1 por ciento (23 pacientes) fueron HLA-DQW2 y/o HLA DQS. Anticuerpos anti-islotes (ICA) fueron positivos en 37 por ciento (10 pacientes) y 4 de los mismos presentaron la prueba de fijación de complemento (CF/ICA) positiva. Tres pacientes presentaron anticuerpos anti-insulinicos. Solo 1 de 9 hermanos HLA idénticos al propósito presentó ICA positivo y CF/ICA positivo, y ésta niña desarrolló diabetes tipo I posteriormente. Se detectaron títulos de anticuerpos anti-virales significativamente positivos para citomegalovirus en 11 pacientes (40.7 por ciento); y títulos positivos para sarampión, parotiditis, herpes y varicela en un menor número de casos. No encontramos serotipos positivos a Coxsackie y Rubéola. Estos resultados confirman que la mayoría de nuestros diebéticos tipo I tiene HLA-DR3 oDR4 y que los fenotipos heterozigotos DR3/DR4 están significativamente aumentados en esta población. También podemos concluir que los haplotipos DR3DQW2 y DR4DQWS8 están asociados con un alto riesgo a desarrollar Diabetes Mellitus tipo I en nuestra población venezolana de origen étnico mixto