Characterization of zinc transporter 8 (ZnT8) antibodies in autoimmune diabetic patients from Argentinian population using monomeric, homodimeric, and heterodimeric ZnT8 antigen variants.

OBJECTIVE: In order to gain further knowledge of the structure of zinc transporter 8 (ZnT8) epitopes, we studied the role of the amino acid at position 325 in the antigen and its dimeric conformation for autoantibodies to ZnT8 (ZnT8A) recognition. METHODS: For this purpose, several ZnT8 C-terminal domain variants were designed: monomer carrying Arg325 or Trp325, homo-dimers ZnT8-Arg-Arg325 and ZnT8-Trp-Trp325, and hetero-dimer ZnT8-Arg-Trp325. Two groups of Argentinian diabetic patients were subjected to analysis using [(35)S]-ZnT8 variants by radioligand binding assay (RBA): i) 100 new-onset, insulin-dependent, type 1 diabetic patients and ii) 282 slowly progressing to insulin requirement, non-obese adult-onset diabetic patients. In addition, 50 type 1 diabetic patients and 100 normal control sera provided by the American Diabetes Association (ADA) were evaluated in order to calculate the sensitivity and specificity of ZnT8A assays for each antigenic variant. Other routine ß-cell autoantibodies were also tested by RBA. RESULTS: Of the 100 Argentinian type 1 diabetic patients, 65 were ZnT8A+. Out of them, 8 patients recognized all recombinant forms of ZnT8 and most patients (56) reacted against the heterodimer. Additionally, out of 282 non-obese adult-onset diabetic patients 46 were ZnT8A+, whereas 29 patients recognized only dimers. Besides, exclusive reactivity against ZnT8A was found in 9.0% for type 1 diabetes mellitus and 10.3% for non-obese adult-onset diabetic patients. CONCLUSIONS: Significantly higher signal values in RBA were obtained with the heterodimeric variant. An increased detection of humoral autoimmunity was found in both groups when ZnT8A was employed in combination with the other ß-cell autoantibodies. The inclusion of homodimeric immunoreactive peptides revealed the existence of quaternary structure-defined epitopes probably resembling the actual state of the autoantigen in vivo. Finally, the differential profiles of ZnT8A exhibited by type 1 and non-obese adult-onset diabetic patients suggest the different nature of autoimmune processes underlying both pathologies.

CD226 rs763361 is associated with the susceptibility to type 1 diabetes and greater frequency of GAD65 autoantibody in a Brazilian cohort.

CD226 rs763361 variant increases susceptibility to type 1 diabetes (T1D) in Caucasians. There is no data about CD226 variants in the very heterogeneous Brazilian population bearing a wide degree of admixture. We investigated its association with T1D susceptibility, clinical phenotypes, and autoimmune manifestations (islet and extrapancreatic autoantibodies). Casuistry. 532 T1D patients and 594 controls in a case-control study. Initially, CD226 coding regions and boundaries were sequenced in a subset of 106 T1D patients and 102 controls. In a second step, two CD226 variants, rs763361 (exon 7) and rs727088 (3' UTR region), involved with CD226 regulation, were genotyped in the entire cohort. C-peptide and autoantibody levels were determined. No new polymorphic variant was found. The variants rs763361 and rs727088 were in strong linkage disequilibrium. The TT genotype of rs763361 was associated with TID risk (OR = 1.503; 95% CI = 1.135-1.991; P = 0.0044), mainly in females (P = 0.0012), greater frequency of anti-GAD autoantibody (31.9% × 24.5%; OR = 1.57; CI = 1.136-2.194; P = 0.0081), and lower C-peptide levels when compared to those with TC + CC genotypes (0.41 ± 0.30 ng/dL versus 0.70 ± 0.53 ng/dL P = 0.0218). Conclusions. The rs763361 variant of CD226 gene (TT genotype) was associated with susceptibility to T1D and with the degree of aggressiveness of the disease in T1D patients from Brazil. Ancestry had no effect.

Autoantibodies for gastrointestinal organ-specific autoimmune diseases in rheumatoid arthritis patients and their relatives.

BACKGROUND: Clustering of autoimmune diseases is common and may be due to genetic background and exposition to environmental triggers. OBJECTIVE: The aim is to carry out a laboratory and clinical study of the prevalence of gastrointestinal organ-specific autoantibodies in rheumatoid arthritis (RA) patients and their relatives. METHODS: Serum samples of 156 RA patients, 200 relatives, and 100 healthy controls were studied for anti-smooth muscle antibody (ASMA), anti-mitochondrial (AMA), anti-parietal cell (APCA), anti-liver-kidney microsome (LKM), and anti-endomysium antibodies (IgA-EmA) by indirect immunofluorescence. RESULTS: A total of eight out of the 156 (5.1%) RA patients were positive for the autoantibodies (ASMA = 1; AMA = 2, APCA = 5). In the relative group, 12/200 (6%) had at least one positive autoantibody (ASMA = 1; AMA = 2, APCA = 7, IgA-EmA = 2). In the control group, two out of the 100 (2%) healthy controls were positive (ASMA = 1, APCA = 1). No statistical difference was found between RA patients, their relatives, and controls in relation to the frequency of autoantibodies evaluated. CONCLUSION: Although RA patients and their relatives have positivity of AMA, ASMA, and APCA without statistical difference in relation to healthy individuals, the findings may be of value for adequate clinical approach of these subjects.

Liver autoantibodies in patients with scleroderma.

Association between autoimmune liver diseases and scleroderma has been described. The purpose of this study was to study the prevalence of antimithocondrial antibody (AMA), antismooth muscle antibodies (SMA), and liver-kidney-microsomal (LKM-1) autoantibody in a cohort of 63 scleroderma patients and 100 healthy controls. The autoantibodies AMA, SMA, and LKM were determined by indirect immunofluorescence. Patients' charts were reviewed for demographic data, scleroderma form, and clinical and anti-nuclear antibody profile, aiming a comparison between patients with and without liver autoantibodies. Nine patients (14.3%) were positive for at least one of the liver autoantibodies; only one patient had both AMA and SMA positive. Antibody SMA was positive in 6.4% (4/63) patients; AMA was present in 9.52% (6/63) of them; none were positive to LKM-1. In the control group just one patient (1%) was SMA positive; the other autoantibodies were negative. There is an increased prevalence of liver autoantibodies in patients with scleroderma than in control population. These patients should be carefully followed for liver dysfunction.

Anticuerpos antitiroperoxidasa y antitransglutaminasa en familiares de primer grado de personas con diabetes tipo 1 y su relación con algunas características clínicas, bioquímicas e inmunológicas / Antithyroperoxidase and antitransglutaminase antibodies in first degree relatives of type 1 diabetes persons and its relation to some clinical, biochemical and immunological features

Los anticuerpos antitiroperoxidasa (AcTPO) y antitransglutaminasa (ATGt) son útiles marcadores de enfermedad tiroidea autoinmune y enfermedad celíaca, respectivamente. Su presencia en familiares de primer grado de personas con diabetes tipo 1 no se ha descrito en Cuba. Objetivo: determinar las frecuencias de los AcTPO y ATGt en familiares de primer grado de personas con diabetes tipo 1 y su relación con algunas características clínicas, bioquímicas e inmunológicas. En un grupo de 285 sujetos se realizó la medición del AcTPO y en 262 individuos la de ATGt. Se incluyeron casos entre los 2 y 65 años de edad. Se registraron datos sobre edad, sexo, color de la piel, antecedentes personales, historia familiar de obesidad, diabetes tipo 2, enfermedad tiroidea y enfermedad celíaca. Se interrogaron síntomas y exploraron signos clínicos de enfermedad celíaca y enfermedad tiroidea autoinmune. Se determinó glucemia, insulinemia, AcTPO, ATGt y autoanticuerpos asociados a diabetes tipo 1 (AGAD y AIA-2), así como la resistencia a la insulina mediante el índice HOMA-IR. RESULTADOS: las frecuencias de AcTPO y ATGt positivos fueron 5,3 y 1,9 por ciento, respectivamente. La historia familiar de enfermedad tiroidea, el temblor muscular fino y el exoftalmos se relacionaron con la presencia de AcTPO. Malabsorción intestinal, diarrea persistente, dolor abdominal recurrente y antecedente personal de hepatopatía se asociaron con la presencia de ATGt. Se encontró asociación entre los ATGt y el AIA-2. La resistencia a la insulina no se asoció con la presencia de AcTPO ni de ATGt. En los familiares de primer grado de personas con diabetes tipo 1 las frecuencias de AcTPO y ATGt son bajas. Algunos antecedentes, síntomas y signos vinculados con enfermedad celíaca y enfermedad tiroidea autoinmune pueden ser indicadores prácticos previos a la indicación de estos autoanticuerpos(AU)

The antithyroperoxidase (TPOAb) and antitransglutaminase (tTGAb) antibodies are useful markers of autoimmune thyroid disease and celiac disease, respectively. Its presence in first-degree relatives of type 1 diabetes patients has not been described in Cuba. Objetive: to determine the TPOAb and tTGAb frequencies in first-degree relatives of type 1 diabetes patients and its relation to some clinical, biochemical and immunological features. In a group of 285 subjects we measured TPOAb and in 262 subjects we measured tTGAb. The cases included aged between 2 and 65. Data were registered on age, sex, skin color, personal backgrounds, and a family history of obesity, type 2 diabetes, thyroid disease and celiac disease. Symptoms were look for and clinical signs of celiac disease and autoimmune thyroid disease were explored. Fasting glucose, fasting insulin, TPOAb, tTGAb and type 1 diabetes associated autoantibodies (AGAD and AIA-2) were determined as well as the insulin resistance according the HOMA-IR index. RESULTS: the frequencies of positive TPOAb and tTGAb were of 5,3 and 1,9 percent, respectively. The family history of thyroid disease, slight muscular tremor and exophthalmos are related to presence of TPOAb. Intestinal malabsorption, persistent diarrhea, recurrent abdominal pain and personal background of liver disease were associated with presence of tTGAb. There was an association between tTGAb and AIA-2. Insulin resistance was not associated with the presence of both antibodies. In first-degree relatives of type 1 diabetes patients, frequencies of TPOAb and tTGAb are low. Some backgrounds, symptoms and signs linked to celiac disease and autoimmune thyroid disease may be practical indicators previous to perform these autoantibodies(AU)

Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus.

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)). In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65. Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients. It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, beta-cell antigens.

Antiribosomal-P autoantibodies from psychiatric lupus target a novel neuronal surface protein causing calcium influx and apoptosis.

The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P-mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.

Regulation of m1 muscarinic receptors and nNOS mRNA levels by autoantibodies from schizophrenic patients.

In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), can act as an inducer of m1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M1 synthetic peptide (with identical aminoacid sequence to human M1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M1 mAChR. The corresponding affinity-purified anti M1 peptide IgG (anti M1 peptide IgG) from schizophrenic patients by stimulation of M1 mAChR exerted an increase in m1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (alpha = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.

Phase I/II clinical trial of the humanized anti-EGF-r monoclonal antibody h-R3 labelled with 99mTc in patients with tumour of epithelial origin.

AIM: To evaluate the biodistribution, internal radiation dosimetry and toxicity of the humanized MAb h-R3 labelled with Tc in humans. METHODS: Twenty-five patients with suspected epithelial-derived tumours were included in this study and divided into two groups: group I consisted of 10 patients who received 3 mg/1110 MBq (3 mg/30 mCi); and group II consisted of 15 patients who received 6 mg/2220 MBq (6 mg/60 mCi). Single photon emission computed tomography (SPECT) and planar images, and multiple blood and urine samples were collected up to 24 h after injection. Haematological parameters and adverse effects were classified according to the WHO criteria. Biodistribution, human anti-mouse antibody (HAMA) response and absorbed doses were estimated and reported. RESULTS: Liver, spleen, kidneys and heart were identified as source organs. Their higher uptakes were 53.3+/-6.4%ID, 2.0+/-1.4%ID, 9.8+/-4.3%ID and 2.8+/-0.9%ID, respectively. The urinary bladder and large intestine also had a significant uptake. The mean urinary excretion was around 22%ID. The liver received the highest absorbed doses followed by the kidneys and the urinary bladder wall. There were no haematological or biochemical abnormalities with clinical significance related to the product. No patient developed HAMA response. Preliminary analysis of clinical results showed a sensitivity of 76.5% and a specificity of 100%. CONCLUSIONS: The results of this study suggest that Tc-h-R3 could be used in patients in a safe and effective way, for the diagnosis of epithelial-derived tumours at the two evaluated dose levels.

Anti-beta 2-glycoprotein-I antibodies in scFv format.

Phage display was introduced almost 20 years ago. It has been used to produce large amounts of diverse proteins, to analyze protein-ligand interactions, to improve the affinity of proteins for their binding receptors, and to characterize antibody binding sites. The recombinant version of the antibody Fv is termed single-chain variable fragment (scFv). Many large phage libraries have been developed that have yielded antibodies to several hundred antigens, but only 5 human anti-beta2-glycoprotein-I and three anti-prothrombin antibodies in scFV have been so far characterized. Antibodies to beta2GP-I thus generated show 92-94% homology with their nearest germ line genes. Their mutations frequently appear to be independent of antigen. Two anti-prothrombin antibodies show strong crossreactivity with beta2GP-I. Four mouse anti-beta2GP-I scFV show less binding properties than their original counterparts, but had the same capacity of inducing experimental antiphospholipid syndrome. This pathogenicity appears to reside in the V(H)DJ(H)C(H) region of the scFv since the V(H)DJ(H)C(H) regions of pathogenic scFV combined with irrelevant V(L) J(L)C(L) regions retained their pathogenicity while the opposite failed to do so.

Autoantibodies against Cajal bodies in systemic lupus erythematosus.

BACKGROUND: Cajal bodies (CB) are distinct sub-nuclear domains rich in small nuclear ribonucleoprotein particles (snRNPs); they are involved in pre-mRNA processing. Lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production against different nuclear molecules, including those involved in pre-mRNA processing. The aim of the present investigation is to assess the presence of anti-CB autoantibodies in a cohort of SLE sera. MATERIAL/METHODS: Antinuclear antibodies (ANA) were screened by indirect immunofluorescence in a batch of 190 sera from patients who met the ACR criteria for SLE classification; fine specificity was determined by Western blot using HEp-2 cells or rat hepatocyte extracts purified by ion exchange chromatography. RESULTS: Four sera had anti-Cajal body (CB) autoantibodies. Interestingly, all of these patients had intermittent extensive oral and esophageal ulceration. The autoantibodies to CB were of the IgG class, and by Western blot these sera had reactivity against an 80 kDa protein (coilin) associated with Sm proteins. CONCLUSIONS: Anti-CB autoantibodies constitute an uncommon specificity of SLE; therefore it seems that anti-CB antibody specificity is associated with extensive mucous ulceration.

Occurrence, properties, and function of asymmetric IgG molecules isolated from non-immune sera.

We have previously demonstrated that 10-20% of the IgG isolated from non-immune sera is asymmetrically glycosylated, in such a way that it fails to trigger immune effector mechanisms. As a result, a major portion of the non-immune asymmetric IgG molecules of the host could be self-specific, acting as auto-protective antibodies. In order to test this hypothesis, we investigated whether asymmetric IgG molecules are capable of recognizing self-antigens. About 40% of F(ab')2 fragment from normal rat IgG was able to react specifically with autologous rat cells. Moreover, upon being purified from normal rat sera, 78% of the asymmetric IgG sub-population showed self-reactivity. We demonstrated that about 14% of rat asymmetric IgG-F(ab')2 fragments was able to react with bacteria isolated from the intestine of uninfected rats. Lastly, in order to test whether there is a correlation between the decline of immune responses during ageing and asymmetric antibody production, we assayed IgG isolated from sera of young and old rats. There was an increase in the asymmetric:symmetric IgG ratio with ageing. We therefore suggest that asymmetric antibodies may exert a beneficial action by protecting self-antigens as well as normal intestinal flora from a deleterious immune response.

Three-dimensional structure of the Fab from a human IgM cold agglutinin.

Cold agglutinins (CAs) are IgM autoantibodies characterized by their ability to agglutinate in vitro RBC at low temperatures. These autoantibodies cause hemolytic anemia in patients with CA disease. Many diverse Ags are recognized by CAs, most frequently those belonging to the I/i system. These are oligosaccharides composed of repeated units of N:-acetyllactosamine, expressed on RBC. The three-dimensional structure of the Fab of KAU, a human monoclonal IgM CA with anti-I activity, was determined. The KAU combining site shows an extended cavity and a neighboring pocket. Residues from the hypervariable loops V(H)CDR3, V(L)CDR1, and V(L)CDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hypervariable loops V(H)CDR1 and V(H)CDR2. This fact could explain the V(H)4-34 germline gene restriction among CA. The KAU combining site topography is consistent with one that binds a polysaccharide. The combining site overall dimensions are 15 A wide and 24 A long. Conservation of key binding site residues among anti-I/i CAs indicates that this is a common feature of this family of autoantibodies. We also describe the first high resolution structure of the human IgM C(H)1:C(L) domain. The structural analysis shows that the C(H)1-C(L) interface is mainly conserved during the isotype switch process from IgM to IgG1.

Cholesterol sulphate-reactive autoantibodies are specifically increased in chronic chagasic human patients.

An antibody reactive with cholesterol sulphate (CS) was characterized in human sera by ELISA, erythrocyte and liposome absorption. This antibody was found evenly distributed between the IgA and IgM classes, and whilst this was present at low titres in the serum of 16% of healthy individuals studied, it was significantly elevated in 78% of Trypanosoma cruzi-infected subjects. No association was found between antibody levels and the degree of myocardial damage. No significant difference in immunoreactivity was found between healthy and chagasic subjects using dehydro-epiandrosterone sulphate and pregnenolone sulphate and cholesterol, ergosterol, lanosterol, stigmastanol, beta-stigmasterol, pregnenolone, prednisolone and dehydroepiandrosterone as antigens, suggesting that in chagasic sera the whole sterol molecule is important for optimal antibody binding. CS-reactive antibodies were easily purified by absorption either with CS-bearing liposomes or with dextran sulphate gel and further elution with 1.5 M NaCl. The optimal pH of CS-antibody interaction was 4.0 with 85% binding at pH 7.0. Polylysine strongly decreased the binding of these antibodies to the corresponding antigen. Furthermore, these antibodies were strongly absorbed by rabbit and guinea pig erythrocyte but not by rat or human erythrocyte. In contrast with anti-sulphatide antibodies, no significant increase in CS-reactive antibodies was found in dilated cardiomyopathies. Whilst CS itself was not detected in T. cruzi lipid extracts, there is an unidentified sulphated sterol, which migrates close to standard CS and which strongly binds chagasic but not control sera. This latter sterol might be acting in chagasic patients as a powerful antigen, triggering specific autoantibody production.