Modulation of the immunologic response to acute stress in humans by beta-blockade or benzodiazepines

Autoantibodies against heat shock protein (hsp) 60 have been reported to be detected in sera of non-obese diabetic mice, in an experimental model of IDDM. However, there are only a few studies which have examined IDDM patients for antibodies against mammalian hsp60. We produced murine hsp60 derived from pancreatic beta cells which has high homology to human hsp60 and examined antibodies against the hsp60 in IDDM patients using an enzyme-linked immunosorbent assay. We extended the analysis to patients with other immune-mediated diseases and non-insulin-dependent diabetes mellitus (NIDDM). Positive sera for hsp60 antibody were more frequently detected in 13 out of 84 IDDM (15.5%) and 5 out of 25 rheumatoid arthritis patients (20%), when compared to healthy subjects (1/85; 1.2%, P < 0.001 and P < 0.01, respectively). The levels of hsp60 antibodies of IDDM (0.218 +/- 0.227) and rheumatoid arthritis patients (0.259 +/- 0.191) were significantly higher than those of healthy subjects (0.076 +/- 0.131, P < 0.001, P < 0.01, respectively). Patients with slowly progressive IDDM (n = 26), autoimmune thyroid disease (n = 42), or NIDDM (n = 40) had levels of hsp60 antibodies similar to those in healthy subjects. We found no relationship between the levels of hsp60 antibodies and islet cell antibodies (ICA) or antibodies to glutamic acid decarboxylase (GAD65) in IDDM patients. In conclusion, hsp60 antibodies were detected in Japanese IDDM as well as in rheumatoid arthritis patients. Although the positivity was low, the detection of hsp60 antibodies may be helpful for diagnosis of IDDM especially in GAD65 Ab- or JCA-negative Japanese patients.     

Human B cells secreting immunoglobulin G to glutamic acid decarboxylase-65 from a nondiabetic patient with multiple autoantibodies and Graves' disease: a comparison with those present in type 1 diabetes

Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in a number of autoimmune disorders, such as insulin-dependent (type 1) diabetes mellitus (IDDM), stiff man syndrome, and polyendocrine autoimmune disease. Antibodies to GAD in IDDM patients usually recognize conformation-dependent regions on GAD65 and rarely bind to the second isoform, glutamic acid decarboxylase-67 (GAD67). In contrast, those present in stiff man syndrome and polyendocrine disease commonly target the second isoform (GAD67) and include antibodies that are less dependent on the conformation of the molecule. By immortalizing peripheral blood B cells with Epstein-Barr virus, we have generated three human IgG autoantibodies, termed b35, b78, and b96, to GAD65 from one patient with multiple autoantibodies to endocrine organs and Graves' disease. All three autoantibodies are of the IgG1 isotype, with islet cell activity, and do not react with GAD67. The regions on GAD65 recognized by the three autoantibodies have been investigated by immunoprecipitation with a series of chimeras, by binding to denatured and reduced antigens, and using protein footprinting techniques. Using chimeric GAD proteins, we have shown that b35 targets the IDDM-E1 region of GAD65 (amino acids 240-435) whereas both b78 and b96 target the IDDM-E2 region of GAD65 (amino acids 451-570). Furthermore, examination of binding to recombinant GAD65 and GAD67 by Western blotting revealed some differences in epitope recognition, where only b78 bound denatured and reduced GAD65. However, b35, b78, and b96 autoantibodies had different footprinting patterns after trypsin treatment of immune complexes with GAD65, again indicating different epitope recognition. Our results indicate that antibodies to GAD65 present in nondiabetic patients with multiple autoantibodies to endocrine organs show similarities to those in IDDM (by targeting IDDM-E1 and IDDM-E2 regions of GAD65) as well as subtle differences in epitope recognition (such as binding to denatured and reduced GAD65 and by protein footprinting). Thus, the GAD65 epitopes recognized by autoantibodies in different autoimmune diseases may overlap and be more heterogeneous than previously recognized.     

Radioimmunoassay for glutamic acid decarboxylase (GAD65) autoantibodies as a diagnostic aid for stiff-man syndrome and a correlate of susceptibility to type 1 diabetes mellitus

OBJECTIVE: To establish and validate a double-antibody radioimmunoassay (RIA) for detecting serum auto-antibodies against glutamic acid decarboxylase (GAD65). This enzyme catalyzes synthesis of the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic islet cells. MATERIAL AND METHODS: We compared the frequency of GAD65 and other "thyrogastric" autoantibodies in adult patients with stiff-man (Moersch-Woltman) syndrome, type 1 diabetes, or polyendocrine disorders and in healthy subjects. The frequency of pancreatic islet cell antibody (ICA) detection was also assessed. The GAD65 RIA was validated by testing blinded samples, by confirming the specificity of low-titered positive results by "cold" antigen inhibition, and by comparing the RIA results with results of a kit assay incorporating staphylococcal protein A as immunoprecipitant. Recombinant GAD65 protein labeled with 125I was used as antigen, and a combination of anti-human IgG and IgM was used as immunoprecipitant. Seropositivity was determined for ICA and gastric parietal cell antibodies by indirect immunofluorescence assays and for thyroid peroxidase (microsome) and thyroglobulin antibodies by agglutination assays. RESULTS: We detected GAD65-specific antibodies in all but 1 of 46 local patients with stiff-man syndrome (98%); 16 had evidence of diabetes. Positive values exceeded 20 nmol/L in 96%, and 89% were ICA-positive; 76% had additional thyrogastric antibodies. Of 41 patients with type 1 diabetes (17 local and 24 workshop serum specimens), 33 were GAD65 antibody-positive (80%); 85% of these positive values were 20 nmol/L or lower. Only 18% of sera from patients with type 1 diabetes were ICA-positive, but 59% had other thyrogastric autoantibodies. Of 20 patients with autoimmune endocrinopathies without diabetes or stiff-man syndrome, 35% were GAD65 antibody-positive, 5% were ICA-positive, and 90% were thyrogastric antibody-positive. Of 117 healthy control subjects, 8% were GAD65 antibody-positive, and a third of those had other thyrogastric antibodies (14% overall); none was ICA-positive. CONCLUSION: Seropositivity in the double-antibody RIA for GAD65 autoantibody is a sensitive and specific marker of predisposition to type 1 diabetes and related organ-specific autoimmune disorders. As such, this RIA is complemented by assays for thyroid and gastric parietal cell autoantibodies.     

Identification of mimicry peptides based on sequential motifs of epitopes derived from 65-kDa glutamic acid decarboxylase

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88-99), presented by HLA-DRB1*0101, and GAD65 (248-257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248-257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.     

Autoimmune diabetes induced by the beta-cell toxin STZ. Immunity to the 60-kDa heat shock protein and to insulin

Administered at a suitably low dose, the toxin streptozotocin (STZ) can trigger an autoimmune process leading to destruction of the beta-cells of the pancreatic islets. In this study, we examined specific immunological reactions in mice before and during the development of STZ-induced autoimmune diabetes. We now report that the development of spontaneous autoantibodies to insulin can serve as a marker of susceptibility to a low dose of STZ. Susceptible male mice of the C57BL/KsJ strain manifested such anti-insulin antibodies, and resistant female mice did not. Administration of a low dose of STZ (five daily doses each of 30 mg/kg) induced transient hyperglycemia approximately 20-30 days later, which temporarily remitted but was followed by intractable diabetes approximately 2.5 months later. The diabetogenic process triggered by the low dose of STZ was associated with an increase in the level of anti-insulin antibodies bearing the Dana and Micha (DM) idiotype, later followed by the appearance of anti-idiotypic antibodies that peaked before the onset of diabetes. Antibodies and T-cells reactive to hsp60 (heat shock protein) were triggered by the low-dose STZ administration and persisted throughout the period that preceded clinical diabetes. T-cells reactive to the p277 peptide of hsp60 were also observed. Finally, active immunization to hsp60 caused transient hyperglycemia by itself and also aggravated the hyperglycemia induced by low-dose STZ. Thus, autoantibodies to insulin can indicate susceptibility to a toxic trigger of diabetes, and a low dose of a toxin can activate the insulin and hsp60 autoimmunity that has been detected previously in the spontaneous autoimmune diabetes of NOD strain mice.     

A case of an old woman with Sj?gren's syndrome associated with insulin-dependent diabetes mellitus

Insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-dependent beta cells of the pancreas. Sj?gren's syndrome is also an autoimmune disease characterized by the destruction of the lacrimal and salivary glands that leads to keratoconjunctivitis sicca and xerostomia. But it is a very rare case that a patient with Sj?gren's syndrome developed IDDM. We present a case of a 65-year old woman with Sj?gren's syndrome who developed diabetic ketoacidosis due to IDDM. Recent studies have revealed that there was molecular mimicry between glutamic acid decarboxylase (GAD) and coxsackievirus. Furthermore, some reports have shown that sialadenitis was represented in IDDM model mice. This case shows the possibility that a causal relationship between IDDM and Sj?gren's syndrome may exist.     

Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.     

Analysis of a kanamycin resistance gene (kmr) from Streptomyces kanamyceticus and a mutant with increased aminoglycoside resistance

The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes. The dominant epitope, VIVMLTPLVEDGVKQC (aa 805-820), which elicited the highest T-cell responses in all at-risk relatives, has 56% identity and 100% similarity over 9 amino acids (aa) with a sequence in VP7, a major immunogenic protein of human rotavirus. Both peptides bind to HLA-DR4(*0401) and are deduced to present identical aa to the T-cell receptor. The contiguous sequence of VP7 has 75% identity and 92% similarity over 12 aa with a known T-cell epitope in glutamic acid decarboxylase (GAD), another autoantigen in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity over 8-14 aa to sequences in Dengue, cytomegalovirus, measles, hepatitis C, and canine distemper viruses, and the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 aa to herpes, rhino-, hanta- and flaviviruses. Two others are 80-82% similar over 10-11 aa to sequences in milk, wheat, and bean proteins. Further studies should now be carried out to directly test the hypothesis that T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and (for rotavirus) GAD.     

Cytochromes P450 and uridine triphosphate-glucuronosyltransferases: model autoantigens to study drug-induced, virus-induced, and autoimmune liver disease

Enzymes of phase I (cytochromes P450) and phase II (UDP [uridine diphosphate]-glucuronosyltransferases) of drug metabolism are targets of autoimmunity in the following chronic liver diseases of different etiology: 1)autoimmune hepatitis (AIH); 2) hepatitis associated with the autoimmune polyendocrine syndrome type 1 (APS-1); 3) virus-induced autoimmunity; and 4) drug-induced hepatitis. AIH is diagnosed by the following: the absence of infection with hepatitis viruses; the presence of a threshold of relevant factors, including circulating autoantibodies, hypergammaglobulinemia, female sex (female/male ratio 4:1), human leukocyte antigen (HLA) B8, DR3, or DR4; and benefit from immunosuppression. Patients with autoimmune hepatitis type 2 (AIH-2) are characterized by antibodies directed against liver and kidney microsomes, by an early onset of autoimmune hepatitis, which is a more aggressive course of the disease, and by a higher prevalence of autoimmunity directed against other organs. The major target of autoimmunity in patients with AIH-2 is cytochrome P450 2D6. Epitope mapping experiments revealed four short linear epitopes on cytochrome P450 2D6, recognized by liver/kidney microsomal autoantibodies type 1 (LKM-1) in patients with AIH-2. In addition, about 10% of the patient sera contain autoantibodies that detect a conformational epitope on UDP-glucuronosyltransferases (UGTs) of family 1. Presently, LKM-1 autoantibodies are used as diagnostic markers for AIH-2. It is unclear whether these autoantibodies have a pathogenetic role. Hepatitis is found in some patients with APS-1. Presumably this also is an autoimmune liver disease. APS-1 patients with hepatitis may develop autoantibodies directed against microsomal P450 enzymes of the liver; however, these autoantibodies do not recognize cytochrome P450 2D6, but they do recognize cytochrome P450 1A2. Autoimmunity in patients with APS-1 usually is directed against several organs simultaneously, and several organ specific autoantibodies may exist. Interestingly, APS-1 patients may produce various anti-cytochrome P450 antibodies. In addition to the hepatic anti-cytochrome P450, 1A2 autoantibodies are directed against steroidogenic cytochromes P450, namely P450 c21, P450 scc, and P450 c17. These autoantibodies correlate with adrenal and ovarian failure and often these steroidal cell autoantibodies precede the manifestation of adrenal or ovarian dysfunction. Whether anti-P450 1A2 autoantibodies have a similar predictive value is not yet known. LKM autoantibodies are further found in association with chronic hepatitis C and D. In chronic hepatitis C, the major target of LKM autoantibodies is cytochrome P450 2D6. Predominantly, conformational epitopes are recognized by LKM-1 sera of patients with chronic hepatitis C. In 13% of patients with chronic hepatitis D, LKM-3 autoantibody is detectable. The target proteins are UGTs of family 1 and in a minority of sera UGTs of family 2. The epitopes are conformational. All hepatic diseases discussed earlier have in common that autoimmunity, which is directed against enzymes of drug metabolizing multigene families. Each disease is characterized by a specific pattern of autoantibodies, with apparently little overlap. For example, LKM-1 autoantibodies, which are directed against P450 2D6, seem to overlap between AIH and chronic hepatitis C. However, a close examination of these autoantibodies shows differences between LKM-1 autoantibodies from patients with chronic hepatitis C and with AIH. In AIH, LKM autoantibodies are more homogenous, titers are higher, and major autoepitopes on cytochrome P450 2D6 are small and linear. LKM autoantibodies in viral hepatitis C are more heterogeneous and there are multiple epitopes, many of which are conformational. These differences indicate the different mechanisms that are involved in the generation of autoimmunity. (ABSTRACT TRUNCATED)     

Chronic autoimmunity of type I diabetes

A considerable body of data supports the hypothesis that type I diabetes is a chronic progressive autoimmune disorder. Individuals with very high probability of progressing to diabetes can now be readily identified. Assays for autoantibodies reacting with insulin (IAA), glutamic acid decarboxylase (GAD65AA), and the neuroendocrine tyrosine phosphatase ICA512/IA-2 (ICA512AA) allow for the identification of more than 95% of individuals developing type I diabetes. The expression of a single autoantibody does not indicate high risk for diabetes and in general, prediabetic individuals express a series of biochemically defined autoantibodies. Levels of such autoantibodies are usually stable over years of follow-up. Unusual variants of autoantibody expression (e.g. GAD-ICA with high titers of GAD65 autoantibodies as the sole autoantibody) have low prognostic significance. Given the presence of multiple autoantibodies, low first phase insulin secretion (following intravenous glucose) is the best predictor of time to diabetes onset. Measurement of autoantibodies can now be automated and applied to large populations such that screening and prediction in the general population is now feasible. We favor the hypothesis that insulin may be the primary autoantigen for type I diabetes, and therapies which after the immune response to insulin may lead to safe and effective preventive modalities.     

Combined use of autoantibodies (IA-2 autoantibody, GAD autoantibody, insulin autoantibody, cytoplasmic islet cell antibodies) in type 1 diabetes: Combinatorial Islet Autoantibody Workshop

The aim of this workshop was to assess the ability of individual autoantibody (ab) assays and their use in combination to discriminate between type 1 diabetic and control sera. Coded aliquots of sera were measured in a total of 119 assays by 49 participating laboratories in 17 countries. The sera were from 51 patients with new onset type 1 diabetes and 101 healthy control subjects with no family history of diabetes. In the final analysis, data on diabetic sera were restricted to 43 subjects younger than age 30 years. The laboratories were asked to report results for these sera using their currently available anti-islet autoantibody assays. In addition, they were asked to combine information from their assays to classify sera as having high, moderate, or low probability of originating from a patient with type 1 diabetes. Actual strategies for combining assays were determined by each laboratory. There were no significant differences in sensitivity among 19 radioimmunoassays (RIAs) for IA-2 autoantibodies (cytoplasmic islet cell antibody [ICA] 512) using different constructs that included the intracellular portion of the molecule (mean sensitivity 73%). However, an enzyme-linked immunosorbent assay (ELISA) using the extracellular portion of the IA-2 molecule did not discriminate between diabetic and control sera. Among GAD autoantibody assays that achieved sensitivity >70%, 26 were RIAs and one was an ELISA. When the sera were ranked according to their autoantibody levels, the concordance for insulin autoantibodies (IAAs) in different laboratories was markedly less than for IA-2ab and GADab. Using a combination of autoantibody assays, several laboratories achieved excellent discrimination between diabetic and control sera (sensitivity up to 80% with false-positive rate of 0%). A variety of strategies for combining information from different assays were successful (e.g., those including and excluding ICA), and no one strategy emerged as clearly superior. In conclusion, IA-2/ICA512 autoantibodies are a marker of type 1 diabetes and can be measured consistently by most assays. Several different strategies for combining assays achieved high sensitivity with a low false-positive rate.     

Antilactoferrin antibodies in autoimmune liver diseases

OBJECTIVE: Lactoferrin, an immunoregulatory protein in mucosal secretions, is one of the target antigens to perinuclear antineutrophil cytoplasmic antibodies (P-ANCAs). Circulating lactoferrin is cleared in the liver, but little is known about the implication of lactoferrin in hepatic inflammation. To evaluate the implication of immunological response to lactoferrin, we examined antilactoferrin antibodies in autoimmune liver diseases. METHODS: Fourteen patients with primary biliary cirrhosis (PBC), 14 with autoimmune hepatitis (AIH), five with autoimmune cholangitis (AIC), six with chronic hepatitis C, and five with chronic hepatitis B were studied. We evaluated autoantibodies to lactoferrin in the sera of the patients by the Western Immunoblotting method. RESULTS: Sera of five of the 14 patients (35.7%) with PBC, four of the 14 patients (28.6%) with AIH, and five of the five patients (100%) with AIC contained autoantibodies to human lactoferrin, but none with hepatitis B or C had them. The higher prevalence of serum antibodies to human lactoferrin was shown to be higher in patients with AIC than with hepatitis B (p < 0.01), hepatitis C (p < 0.01), PBC (p < 0.05), and AIH (p < 0.05). CONCLUSION: Lactoferrin located in bile ducts and liver cells is one of the candidates of target antigens in autoimmune liver diseases, especially in AIC.     

Correlation between chlamydial infection and autoimmune response: molecular mimicry between RNA polymerase major sigma subunit from Chlamydia trachomatis and human L7

L7 is one of the ribosomal proteins frequently targeted by autoantibodies in rheumatic autoimmune diseases. A computer search revealed a region within the immunodominant epitope of L7 (peptide II) that is highly homologous to amino acid sequence 264-286 of the RNA polymerase major sigma factor of the eubacterium Chlamydia trachomatis. Anti-L7 autoantibodies affinity purified from the immunodominant epitope were able to recognize this sequence as they reacted with purified recombinant sigma factor. Immunofluorescence labeling experiments on C. trachomatis lysates revealed a punctate staining pattern of numerous spots when incubated with the affinity-purified anti-peptide II autoantibodies. Binding of autoantibodies to peptide II was inhibited by the homologous sigma peptide. This is the first demonstration of epitope mimicry between a human and a chlamydial protein on the level of B cells. Antibody screening revealed a significant correlation between the presence of anti-L7 autoantibodies and C. trachomatis infection in patients with systemic lupus erythematosus and mixed connective tissue disease. Our results suggest that molecular mimicry is involved in the initiation of anti-L7 autoantibody response and may represent a first glance into the immunopathology of Chlamydia with respect to systemic rheumatic diseases.     

Association between HLA and islet cell antibodies in diabetic patients with a mitochondrial DNA mutation at base pair 3243

Islet cell antibodies (ICA), autoantibodies to glutamic acid decarboxylase (GAD) and HLA genotypes were examined in 31 patients with diabetes and a mitochondrial gene mutation located at base pair 3243 (mtDNA 3243 mutation). ICA was detected in 42% (13/31) of these patients compared to 0 of 90 among healthy control subjects. The ICA showed a "non-restricted" pattern of staining in all 13 ICA-positive patients. In a sensitive radioligand assay only 2 of 31 (6%) diabetic patients with the mutation were positive for both GAD65 autoantibodies and ICA, while the remaining 29 patients were GAD65 antibody negative. The ICA-positive patients had an increased frequency of the HLA-DQA1*0301 allele compared to control subjects (p < 0.05). Of the diabetic patients with the mutation 45% (14/31) had progressive clinical course of beta-cell failure. These results indicate that patients with an mtDNA 3243 mutation may develop islet autoimmunity associated with ICA and GAD autoantibodies. We hypothesize that the presence of HLA-DQA1*0301 in individuals with the mtDNA 3243 mutation increases the risk for diabetes and associated autoantibodies against islet cell antigens.     

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.     

Phenytoin penetration into brain after administration of phenytoin or fosphenytoin

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that occurs in association with underlying neoplasms. PNP patients develop characteristic autoantibodies directed against multiple antigens, mostly identified as members of the plakin family of cytoplasmic proteins (desmoplakin I and II, bullous pemphigoid antigen I, envoplakin, and periplakin). HD1/plectin, another member of the plakin family, has not previously been detected in the characteristic PNP antigen complex, which may relate to practical difficulties associated with its large size (molecular weight approximately 500 kDa). In this study, a combination of immunoprecipitation and immunoblot is used to demonstrate that HD1/plectin is also recognized by sera from PNP patients. Thirteen of 16 PNP sera tested were positive for HD1/plectin compared with none of 43 control sera (11 pemphigus vulgaris, 11 pemphigus foliaceus, 11 bullous pemphigoid, and 10 normal individuals). Combined with our recent finding that desmoglein 3 and desmoglein 1 are cell surface target antigens in PNP, this demonstration of plectin/HD1 as another component of the antigen complex in PNP confirms that PNP is an autoimmune disease against desmoglein and plakin family molecules.     

Dependence of apparent diffusion coefficients on axonal spacing, membrane permeability, and diffusion time in spinal cord white matter

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that occurs in association with underlying neoplasms. Patients with PNP develop characteristic IgG autoantibodies directed against multiple antigens, most of which have been identified as cytoplasmic proteins of the plakin family (desmoplakin I, II, BPAG1, envoplakin, and periplakin). This study identified cell surface target antigens of PNP. We focused on desmoglein (Dsg) 3 and Dsg1, the autoantigens of pemphigus vulgaris and pemphigus foliaceus. ELISA using baculovirus-expressed recombinant Dsgs (rDsg3, rDsg1) has revealed that 25 out of 25 PNP sera tested were positive against Dsg3 and 16 of 25 were positive against Dsg1. All of 12 PNP sera tested immunoprecipitated Dsg3. Removal of anti-Dsg3 autoantibodies by immunoadsorption was sufficient to eliminate the ability of PNP sera to induce cutaneous blisters in neonatal mice in vivo. Furthermore, anti-Dsg3-specific antibodies that were affinity purified from PNP sera were proven to be pathogenic and caused blisters in neonatal mice. These findings indicate that Dsg3 and Dsg1 are the cell surface target antigens in PNP and that IgG autoantibodies against Dsg3 in PNP sera play a pathogenic role in inducing loss of cell adhesion of keratinocytes and causing blister formation.     

Autoantibody profiles in canine ANA-positive sera investigated by immunoblot and ELISA

Canine systemic lupus erythematosus (SLE) has a similar disease expression as human SLE, but the serological characterisation of the canine disease is as yet incomplete. In the present study, we examined the specificity of antinuclear antibodies (ANA) in indirect immunofluorescence (IIF) positive canine sera. Sixty-four canine IIF ANA positive sera were characterised using HeLa cell nuclear extract immunoblots and recombinant U1-70K ELISA. We compared these results with a previously shown concordance between indirect immunofluorescence and immunodiffusion in canine SLE serological diagnosis. One canine serum reacting with Sm proteins was observed, and five canine sera presented anti-RNP autoantibodies against the antigens 70K, A, C, and/or B/B'. The autoantigen most frequently recognised was a 43 kDa nuclear protein, previously described as hnRNP G. This prominent canine autoantigen was missing in the commercially available extract designed for immunodiffusion testing of human sera. Other prominent canine autoantigens were found not to be identical with the principal human ones, thus making present human test systems deficient for the use in canine systemic connective disease diagnosis. The development of antigenic extract designed for canine autoimmune autoantigens is necessary in order to make immunodiffusion a useful method in canine diagnosis. The anti-RNP positive canine sera were examined in more detail and we found that the human major antigenic region of the most prominent RNP antigen, the U1-70K protein, also is targeted by canine autoantibodies. Thus, the response against the RNP antigen seems to be conserved between man and dog.     

Bilateral diffuse hypoperfusion of posterior temporoparietals and occipitals in Tc-99m HMPAO brain SPECT in a patient with noncommunicating hydrocephalus

We measured class-specific antibodies to the mycobacterial hsp70 protein in 67 patients with diabetes mellitus (27 type 1 and 40 type 2) with or without vascular complications. Using ELISA, the levels of IgG and IgM antibodies in the sera of diabetic patients did not significantly differ either from those of healthy control subjects or between both types of diabetes, regardless of gender, disease duration, HbA1 level, or type of vascular complication. In patients with type 2 diabetes, the mean serum IgA levels were significantly higher than those in their matched controls [274(71) mg/dl vs 208(88) mg/dl; P < 0.01]. In this group of patients, the IgA antibody titer was significantly correlated to the serum IgA level (r = 0.334; P < 0.01). Serological autoimmunity (IgG or IgM type) to hsp70 protein is common in both the normal and the diabetic population. The increased IgA levels and anti-hsp70 IgA titers in the sera of diabetics suggest a possible role of IgA in the pathogenesis of the vascular complications of diabetes mellitus.     

Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.