Cochlear, auditory brainstem responses in Type 1 diabetes: relationship with metabolic variables and diabetic complications.

AIMS: Few studies have analysed the presence of hearing abnormalities in diabetes. We assessed the presence of subclinical auditory alterations and their possible association with early vascular and neurological dysfunction in young adults with Type 1 diabetes of long duration. METHODS: Thirty-one patients with Type 1 diabetes (mean age 33 ± 2.3 years, disease duration 25.7 ± 4.2 years) and 10 healthy controls underwent pure tone audiometry (PTA), distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) analyses. Associations with metabolic variables and chronic complications were explored. RESULTS: Compared with healthy controls, patients with diabetes had significantly higher mean hearing thresholds, although still within the normoacusic range. DPOAE intensities at medium frequencies (2.8-4 kHz) were significantly lower in patients with diabetes. In ABR, in addition to waves I, III and V, we observed the appearance of a visible wave IV in patients with diabetes compared with controls (prevalence 61% vs. 10%, P < 0.05), and its appearance was related to a prolonged I-V interval (4.40 ± 0.62 ms vs. 4.19 ± 0.58 ms, P < 0.05). Diastolic blood pressure was higher in people with abnormal DPOAE (P < 0.05), whereas systolic blood pressure correlated with wave V and interpeak I-V interval latencies. A trend towards an association between evidence of wave IV and the presence of somatic neuropathy or abnormal cardiovascular autonomic tests was observed. CONCLUSIONS: Young adults with long-term Type 1 diabetes have subclinical abnormalities in qualitative auditory perception, despite normal hearing thresholds, which might reflect neuropathic and/or vascular alterations.

Diagnosis of type 1 diabetes within the first five years of life influences quality of life and risk of severe hypoglycemia in adulthood.

Progressive adaptation to disease is paramount to improve quality of life (QoL) and other psychological dimensions in type 1 diabetes (T1DM). This study aimed at identifying possible correlations between QoL, locus of control (LoC) and clinical variables in patients with T1DM followed up for 16 years. Fifty-nine patients (27 women) with T1DM, part of a cohort of 112 followed since 1996, accepted to participate. Patients were divided into those in whom onset of T1DM had been during the first 5 years of life (n = 16) or later. They were also stratified into worsened, stable and improved, based on whether their HbA1c had increased/decreased by 1 percentage point between baseline and last follow-up visit. QoL was measured by the Diabetes Quality of Life questionnaire (DQOL), translated into Italian and re-validated. The LoC was measured by the Peyrot- and Rubin-specific questionnaire. Patients who developed T1DM before age 5 had a better total DQOL score than those who developed it later in life, mainly due to the satisfaction dimension and a tendency to decreased fatalism in adult age. All subjects whose HbA1c had worsened from baseline had had their diagnosis after age 5 and reported more frequent episodes of hypoglycemia. Onset of diabetes after age 5 and more frequent hypoglycemia was more likely in subjects with worsened HbA1c (ORs 7.6, p < 0.10 and 20.3, p < 0.01, respectively, from a multivariate logistic model with HbA1c, dichotomized in 'worsened' vs all others, as dependent variable). Onset of T1DM during the first 5 years of life may result in better QoL and less fatalism in the long term. Presumably, these patients have no memory of disease onset, which may reduce trauma and facilitate adaptation to managing life with diabetes.

The ghrelin gene products and exendin-4 promote survival of human pancreatic islet endothelial cells in hyperglycaemic conditions, through phosphoinositide 3-kinase/Akt, extracellular signal-related kinase (ERK)1/2 and cAMP/protein kinase A (PKA) signalling pathways.

AIMS/HYPOTHESIS: Pancreatic islet microendothelium exhibits unique features in interdependent relationship with beta cells. Gastrointestinal products of the ghrelin gene, acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (Ob), and the incretin, glucagon-like peptide-1 (GLP-1), prevent apoptosis of pancreatic beta cells. We investigated whether the ghrelin gene products and the GLP-1 receptor agonist exendin-4 (Ex-4) display survival effects in human pancreatic islet microendothelial cells (MECs) exposed to chronic hyperglycaemia. METHODS: Islet MECs were cultured in high glucose concentration and treated with AG, UAG, Ob or Ex-4. Apoptosis was assessed by DNA fragmentation, Hoechst staining of the nuclei and caspase-3 activity. Western blot analyses and pharmacological inhibition of protein kinase B (Akt) and extracellular signal-related kinase (ERK)1/2 pathways, detection of intracellular cAMP levels and blockade of adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) signalling were performed. Levels of NO, IL-1ß and vascular endothelial growth factor (VEGF)-A in cell culture supernatant fractions were measured. RESULTS: Islet MECs express the ghrelin receptor GHS-R1A as well as GLP-1R. Treatment with AG, UAG, Ob and Ex-4 promoted cell survival and significantly inhibited glucose-induced apoptosis, through activation of PI3K/Akt, ERK1/2 phosphorylation and intracellular cAMP increase. Moreover, peptides upregulated B cell lymphoma 2 (BCL-2) and downregulated BCL-2-associated X protein (BAX) and CD40 ligand (CD40L) production, and significantly reduced the secretion of NO, IL-1ß and VEGF-A. CONCLUSIONS/INTERPRETATION: The ghrelin gene-derived peptides and Ex-4 exert cytoprotective effects in islet MECs. The anti-apoptotic effects involve phosphoinositide 3-kinase (PI3K)/Akt, ERK1/2 and cAMP/PKA pathways. These peptides could therefore represent a potential tool to improve islet vascularisation and, indirectly, islet cell function.

Primary and immortalised human pancreatic islet endothelial cells: phenotypic and immunological characterisation.

AIMS/HYPOTHESIS: Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures. MATERIALS AND METHODS: Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus. RESULTS: Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-alpha) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-gamma) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration. CONCLUSIONS/INTERPRETATION: The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.

Expression of nephrin by human pancreatic islet endothelial cells.

AIMS/HYPOTHESIS: The islet microcirculation has morphological characteristics resembling those of renal glomeruli. Transcription of the nephrin gene, a highly specific barrier protein of the slit diaphragm of podocyte foot processes, has been reported in the pancreas, although its cellular localisation and function remain to be defined. In this study, we purified and characterised microvascular endothelial cells (MECs) isolated from human islets and investigated the expression and distribution of nephrin on these cells. METHODS: Human islet MECs were extracted and purified using anti-CD105-coated immunomagnetic beads and their endothelial characteristics were confirmed by expression of classical endothelial markers and basal high-level expression of intercellular adhesion molecule-1 and TNF-alpha-inducible vascular cell adhesion molecule-1. Nephrin expression was assessed by immunofluorescence, flow cytometric analysis and western blotting on cell lysates, as well as by RT-PCR. RESULTS: Immunofluorescence studies detected nephrin in a fine, punctate, diffuse pattern on cultured islet MECs, and also in human pancreatic islet sections. In both cases nephrin colocalised with endothelial markers. TNF-alpha treatment induced a marked reduction and redistribution of the protein in one or multiple aggregates. Nephrin expression was confirmed by flow cytometry, western blotting and RT-PCR studies. In contrast, nephrin could not be detected at the protein or mRNA level in human macro- and microvascular cells from other sites. CONCLUSIONS/INTERPRETATION: Nephrin is expressed at protein and mRNA levels in islet microendothelium, supporting the hypothesis that islet MECs exhibit distinctive morphological characteristics that indicate functional specialisation of potential pathophysiological importance.

Glutamic acid decarboxylase and ICA512/IA-2 autoantibodies as disease markers and relationship to residual beta-cell function and glycemic control in young type 1 diabetic patients.

Circulating autoantibodies (Ab) to islet autoantigens, glutamic acid decarboxylase (GAD(65)), and tyrosine phosphatase ICA512/IA-2 have been proposed as predictive markers of type 1 diabetes mellitus. To ascertain residual beta-cell function and the clinical relevance for monitoring autoimmunity after clinical manifestation of disease, we studied 63 children at diagnosis of type 1 diabetes (mean SD age 7.5 +/- 4 years) and 91 adolescent patients with type 1 diabetes (age 14.7 +/- 1.6 years) with a mean duration of disease of 7 +/- 3.5) years. Forty-two normal adolescent subjects (age 14.6 +/- 1.8 years) without a family history of diabetes were the control group. Anti-GAD(65) and ICA512/IA-2 Ab were assessed by a quantitative radioimmunoprecipitation assay. The relationship between humoral autoimmunity and clinical parameters was explored. GAD(65) and ICA512/IA-2 Ab were detected in 56% and 63% of newly diagnosed children and the prevalence was not different in relationship to clinical characteristics. Levels of GAD(65) Ab positively correlated with diagnosis age (P <.05). Both Ab were associated with islet cell antibodies (ICA) (P <.05), but one fifth of patients had at least 1 of the 2 Ab and absent ICA. At onset, only age showed a significant relationship to residual C-peptide secretion. Among the cohort of patients with diabetes of short-mid duration, GAD(65) and ICA512/IA-2 Ab were present in 44% and 45% of cases (P >.05 and P <.05 v newly diagnosed children, respectively) and more patients were identified by these Ab (68%) than by ICA alone (34%) (P <.05). In this cohort, levels of ICA512/IA-2 Ab negatively correlated with levels of glycosylated hemoglobin (HbA(1c)) (P <.005) and with daily insulin requirement (P <.05). Moreover, the presence of some residual C-peptide secretion was significantly associated with the presence of ICA512/IA-2 Ab (P <.05). Our findings confirm that positivity for either GAD(65) or ICA512/IA-2 Ab is a highly sensitive marker of type 1 diabetes in the pediatric age group, identifying a group of patients with absent ICA immunofluorescence. The persistence of Ab to islet tyrosine phosphatase possibly represents a marker of better glycemic control and less insulin requirement, indicating residual beta-cell function, thus conferring clinical and prognostic relevance to these Ab, as well as potential usefulness in intervention strategies.

Anti-CD38 autoantibodies: characterisation in new-onset type I diabetes and latent autoimmune diabetes of the adult (LADA) and comparison with other islet autoantibodies.

AIMS/HYPOTHESIS: Serum anti-CD38 autoantibodies (aAbs) have been reported in 17 to 19% of patients with long-standing Type I (insulin-dependent) diabetes mellitus and Type II (non-insulin-dependent) diabetes mellitus. Whether these aAbs are also found in new-onset Type I diabetes and in Latent Autoimmune Diabetes in Adults (LADA) is not known, as is their relationship with conventional islet aAbs. METHODS: These issues were addressed by studying new-onset Type I and LADA diabetic cohorts with a recently developed anti-CD38 enzymatic immuno-assay. RESULTS: Anti-CD38 aAb prevalence among new-onset Type I patients (3.8%) was lower than previously found in long-standing Type I diabetes (11.7%, as defined with the 97.5 percentile cutoff; p=0.01), suggesting a late appearance of these aAbs. Among LADA patients, 14.9% were anti-CD38(+). Anti-CD38 were only associated with anti-GAD aAbs in new-onset Type I diabetes. Although the CD38 target molecule was expressed in human pancreatic islets, anti-CD38 aAbs did not contribute to the islet cell antibody (ICA) immunofluorescence reactivity. All the positive sera analysed for Ca(2+) release were found to mobilise it. In agreement with these agonistic features, anti-CD38(+) new-onset Type I patients showed higher fasting C-peptide values as compared to negative counterparts; the association was stronger when the analysis was limited to the agonistic anti-CD38(+) sera. A similar trend was found among LADA patients. CONCLUSION/INTERPRETATION: Anti-CD38 aAbs are distinct markers of islet autoimmunity which are more prevalent in long-standing disease, as opposed to the other known islet aAbs. Their in vitro agonistic properties could be operating in vivo as well, as they identify sub-groups of patients with higher residual beta-cell function.

Autonomic nervous function in de novo parkinsonian patients in basal condition and after acute levodopa administration.

The aims of this study were to assess autonomic nervous function in subjects with recently diagnosed Parkinson's disease (de novo patients) and to evaluate its changes following acute levodopa administration. In 13 patients (8 males, 5 females) and 13 age-matched control subjects, three cardiovascular autonomic function tests (Deep Breathing, Valsalva, Lying to Standing) were performed, the QT interval was calculated on a 12-lead electrocardiogram, and the response of plasma norepinephrine to standing was assessed in basal conditions. The cardiovascular tests and the measurement of the QT interval were repeated in de novo Parkinsonian patients 90 minutes after the administration of levodopa 200 mg per os. The results of the Deep Breathing and Valsalva tests were worse and the QT interval longer in patients than in control subjects (although the differences were not statistically significant). The heart rate increase at 30 seconds after standing up was significantly higher in Parkinsonian patients than in the control group. The response of plasma norepinephrine to standing was similar in both groups. Levodopa administration produced a slight improvement in the Deep Breathing test, a shortening of the QT interval and increased tachycardia on standing. Our data suggest that a mild subclinical impairment of parasympathetic function can be a feature of de novo Parkinsonian patients and that levodopa therapy could have a beneficial effect on this autonomic dysfunction.

Restricted TCR V beta gene expression and enterovirus infection in type I diabetes: a pilot study.

AIMS/HYPOTHESIS: High frequencies of T-cell receptor (TCR) V beta 7+ T cells were detected among the lymphocytes isolated from pancreatic islets of children at the onset of Type I (insulin-dependent) diabetes mellitus. We assessed whether a preferential expression of certain TCR V beta gene families could also be detected among the peripheral blood mononuclear cells from diabetic patients. METHODS: T-cell receptor repertoires were evaluated by using a semi-quantitative RT-PCR-based technique and confirmed by FACS analysis in peripheral blood mononuclear cells from diabetic patients before, at and after onset of the disease. These patients were also tested for exposure to enteroviruses by RT-PCR and by measuring titres of enterovirus-specific antibodies of the IgA, IgG, and IgM classes. RESULTS: T-cell receptor V beta 7 gene family values were higher in recently-diagnosed diabetic patients (10.5% +/- 3.7) than in age-matched non-diabetic control subjects (5.1% +/- 1.6) (p < 0.001). In a time-course analysis of people who developed diabetes during clinical monitoring (i.e., converters), T-cell receptor V beta 7 gene expression showed values consistently above 10% (p < 0.0005). Long-standing diabetic patients showed lower percentage of V beta 7 expression compared to values measured at disease onset. In the longitudinal study of the converters, multiple acute enterovirus infections were also detected. These infections appeared to be temporally related to increased percentage of V beta 7 gene transcripts. CONCLUSION/INTERPRETATION: The deviation in the T-cell receptor V beta repertoire among circulating T cells from diabetic patients seems to re-emphasize the importance of enterovirus infections in accelerating the progression of Type I diabetes.

Prevalence of autoantibodies to autonomic nervous tissue structures in Type 1 diabetes mellitus.

AIMS: The pathogenesis of diabetic autonomic neuropathy is multifactorial, but recent studies have suggested a link between the presence of autoantibodies to nervous tissue structures and severe, symptomatic autonomic neuropathy. The present study was designed to examine the true prevalence of these autoantibodies in a large clinic-based population of Type 1 diabetic patients compared to nondiabetic controls. METHODS: The presence of complement fixing autoantibodies to vagus nerve (CF-VN), sympathetic ganglion (CF-SG) and adrenal medulla (CF-ADM) was assessed by immunofluorescence in a large cohort of patients (n = 394) of varying duration of Type 1 DM (median 28 years, range 6 months to 73 years) and 160 age and sex-matched nondiabetic control subjects. RESULTS: All three autoantibodies were frequently detected in Type 1 DM (CF-VN, 22.1%; CF-SG, 30.7%; CF-ADM, 13.2%) but only rarely in healthy control subjects (4.4%, 4.4% and 3.1%, respectively; P < 0.0005 for all). There was no association between any of the autoantibodies and retinopathy (fundoscopy), peripheral somatic neuropathy (biothesiometry) or nephropathy (urinary albumin-creatinine ratio). CONCLUSIONS: Our results on this large cohort establish the extensive presence of autonomic nervous tissue autoantibodies in Type 1 DM. Their role in reflecting, causing or predicting autonomic neuropathy remains to be determined.

Idiopathic dilated cardiomyopathy: a superantigen-driven autoimmune disease.

BACKGROUND: Many cases of idiopathic dilated cardiomyopathy (IDC) result from an inflammatory myocarditis. The specific immunological mechanisms are not yet defined. Various autoimmune diseases are associated with superantigen-triggered immune responses, resulting in massive T-cell activation and tissue damage. We studied 3 cases in a search for evidence that such a phenomenon is also implicated in IDC. METHODS AND RESULTS: Myocardial, lymph node, and thymic tissue samples were obtained from IDC patients who were undergoing heart transplantation. Infiltrating immune-cell phenotypes and gene expression of T-cell receptor (TCR) alpha- and beta-chain variable (Valpha and Vbeta) regions were analyzed by immunostaining and polymerase chain reaction. Similar technical approaches were used to assay the tissues for the presence of coxsackievirus B (CVB). In all the specimens analyzed, an overexpression of the TCR Vbeta3, Vbeta7, and Vbeta13.1 gene families was detected among the infiltrating T cells. These tissues were also found to be CVB3-positive. In vitro exposure of peripheral blood mononuclear cells to lysates of cells infected with CVB3 was capable of stimulating expansion of the same TCR Vbeta families. The TCR Valpha repertoire was never found to be skewed. CONCLUSIONS: A superantigen-mediated immune response is involved in human heart disease. CVB3 may directly or indirectly trigger this response, suggesting a possible mechanistic link between CVB infection and myocarditis development progressing to IDC.

Autoimmunity in diabetic autonomic neuropathy: does the immune system get on your nerves?

Symptomatic autonomic neuropathy is a devastating occasional complication of diabetes mellitus, especially Type 1. Although the full-blown clinical syndrome is not common, dysfunction of the autonomic nerves is detectable in up to 40% of Type 1 diabetic patients but its aetiopathogenesis is poorly understood. There is evidence to suggest that the damage to the autonomic nerves may be immune-mediated. This evidence is reviewed in the following article.

Autonomic function and autoantibodies to autonomic nervous structures, glutamic acid decarboxylase and islet tyrosine phosphatase in adolescent patients with IDDM.

Recent studies have linked autoimmunity to nervous tissue structures and diabetic autonomic neuropathy, but data on the early stage of IDDM and on the natural history of this association are not available. For this reason, we investigated autonomic nervous function, and the presence of autoantibodies to sympathetic and parasympathetic nervous structures, to glutamic acid decarboxylase (GAD) and tyrosine phosphatase (IA-2/ICA512) in 85 adolescents with insulin-dependent diabetes mellitus (IDDM) (mean age 14.7+/-1.6 yr, mean duration of diabetes 6.8+/-3.5 yr), and 45 age and sex-matched healthy subjects. Nervous tissues autoantibodies were detected using an indirect immunofluorescent complement-fixation technique, with monkey adrenal gland, rabbit cervical ganglia and vagus nerve as substrates. GAD and IA-2/ICA512 autoantibodies were detected by radioimmunoprecipitation assay. Seven patients (8%) had anti-vagus nerve autoantibodies, 7 other patients (8%) had anti-cervical ganglia autoantibodies, while all controls were negative (P < 0.05). Anti-adrenal medulla antibodies were detected in 16 patients (19%) and in 2 control subjects (P<0.02). None of the patients had autonomic symptoms. When patients were divided according to the presence or absence of autoantibodies, values of the cardiovascular tests (deep breathing, 30:15 ratio, Valsalva ratio) were similar in the two groups and similar to those in healthy subjects. However, when considered together, patients positive for one or more autoantibody showed a trend for lower values of deep breathing test and 30:15 ratio test, compared with healthy control subjects, which failed to reach conventional significance values (P=0.17 and P=0.07, respectively). No correlation was found between cardiovascular parameters and metabolic control or diabetes duration. There was no association between autoimmunity to nervous tissue structures and presence of GAD and IA-2/ICA512 Ab, and no correlation between these two autoantibodies and values of cardiovascular tests. Our data indicate that autonomic dysfunction is not a characteristic of young diabetic patients, but that autoantibodies against autonomic nervous structures are present during the first 1 to 15 yr of diabetes. GAD and tyrosine phosphatase appear to be excluded as target autoantigens within autonomic structures. Follow-up studies are required to evaluate future autonomic dysfunction and symptoms in these patients, and to establish whether the subtle autonomic dysfunction detected and/or the nervous tissue autoantibodies, are predictive of the development of this complication.

Combined analysis of GAD65 and ICA512(IA-2) autoantibodies in organ and non-organ-specific autoimmune diseases confers high specificity for insulin-dependent diabetes mellitus.

There is evidence that insulin-dependent diabetes mellitus (IDDM) may develop in association with other non-beta-cell-specific autoimmune diseases. We aimed to assess whether autoantibodies to the islet cell antigens glutamic acid decarboxylase (Mr 65,000 isoform) (GAD65) and ICA512(IA-2), present alone or in combination, are limited to IDDM or also occur in other organ- or non-organ-specific autoimmune disorders. We determined the frequency of these autoantibodies by radioimmunoassay in 199 sera from patients with autoimmune thyroid diseases (AITD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary biliary cirrhosis (PBC), and compared the results with those from 507 newly diagnosed patients with IDDM and 280 healthy controls. ICA512(IA-2) autoantibodies were detected exclusively in AITD with concurrent IDDM, but not in other autoimmune diseases without IDDM, whereas GAD65 autoantibodies exceeded the limit of normal in 67.7% (21 of 31) of patients with AITD who also had IDDM and in 5.5% (three of 55) of patients with PBC. The frequency of either GAD65 and/or ICA512(IA-2) autoantibodies was significantly higher in patients with AITD who also had IDDM (27 of 31, 87.1%) than in those with AITD alone (one of 53, 1.9%; P<10(-6)), but was not significantly different from those patients with newly diagnosed IDDM (418 of 507, 82.4%). Neither patients with organ- or non-organ-specific autoimmune diseases without IDDM nor healthy controls had autoantibodies against both GAD65 and ICA512(IA-2). Despite the fact that one of the two autoantibodies was occasionally detected in patients with non-beta-cell-specific autoimmune diseases without IDDM, combined determination of GAD65 and ICA512(IA-2) autoantibodies specifically identified IDDM in the majority of patients with AITD. In conclusion, because of the strong association of IDDM with AITD, testing for multiple islet autoanti-bodies could be useful as a predictive marker for risk of progression to IDDM onset amongst patients with autoimmune thyroid disorders.

Autonomic nervous dysfunction in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA): possible pathogenic role of autoantibodies to autonomic nervous structures.

Autonomic nervous dysfunction has been previously reported in SLE, RA and systemic sclerosis, but the pathogenesis of such a complication is poorly understood. In the present study, four standard cardiovascular autonomic function tests were performed in 34 female patients with connective tissue diseases and in 25 healthy control subjects, and results expressed as cardiovascular (CV) test scores. Moreover, in each subject the presence of circulating complement-fixing autoantibodies directed against sympathetic and parasympathetic nervous structures, represented by superior cervical ganglia and vagus nerve, respectively, was simultaneously assessed by an indirect immunofluorescent complement-fixation technique, using rabbit tissue as substrate. None of the patients reported autonomic symptoms. However, an abnormal CV test score (> or = 5) was detected in 15% of the patients and in none of the healthy control subjects, approaching statistical significance (P = 0.07). No correlation was found between CV test results and disease duration, type of therapy or presence of conventional autoantibodies. One or two autoantibodies to autonomic nervous structures were detected in six patients (18%) and not in the control subjects (P < 0.05). Values of deep breathing test were significantly lower in autoantibody-positive patients compared with those amongst the control subjects (P < 0.05), and an abnormal CV test score was significantly associated with the presence of autoantibodies to autonomic nervous structures (P < 0.05). In conclusion, we confirm that autonomic nervous function can be impaired in patients with connective tissue diseases, and suggest that autoantibodies directed against autonomic nervous system structures may play a role in the pathogenesis of the autonomic dysfunction.

Expression of glutamic acid decarboxylase in nervous tissue structures targeted by autoantibodies in patients with diabetic autonomic neuropathy.

We have previously identified an association between symptomatic diabetic autonomic neuropathy (DAN) and autoantibodies to sympathetic and parasympathetic nervous structures. The antigens identified by these autoantibodies are not known, but glutamic acid decarboxylase (GAD) has been suggested as a candidate target, since anti-GAD autoantibodies are present in patients with long-term diabetes and GAD is expressed in a variety of cell types and structures in the nervous system. The aim of this study was to examine GAD expression in sympathetic ganglia and vagus nerve and to compare the distribution of GAD within these tissues with that of anti-sympathetic ganglia and anti-vagus nerve autoantibodies from patients with DAN, using single and double indirect immunofluorescence on tissue sections. The monoclonal antibody GAD-6, specific for GAD65, gave a granular, peripheral, cytoplasmic staining pattern in sympathetic ganglion cells. Dual immunofluorescence demonstrated that serum from a patient with anti-sympathetic ganglion autoantibodies stained the same cells, but homogeneously throughout the cytoplasm. In the vagus nerve, patient's serum stained the fibres only; GAD-6 stained the cytoplasm of parasympathetic ganglion cells but only occasional fibres. In addition, GAD enzymatic activity was detectable in both sympathetic ganglia and vagus nerve. Incubation of sera or GAD-6 overnight with a crude homogenate of human brain as an antigen source abolished staining of the nervous tissues by GAD-6, but not by patients' sera. The different localisation of GAD and the autoantigens targeted by patients' sera indicates that GAD is not the target of the autoantibodies characteristic of DAN. Moreover, absorption studies using human brain homogenate suggest that the targets of anti-sympathetic ganglion and anti-vagus nerve autoantibodies are absent or represented only at low levels in the central nervous system and may be confined to the periphery.

Reproducibility and persistence of neural and adrenal autoantibodies in diabetic autonomic neuropathy.

The presence of autoantibodies to autonomic nervous tissue structures is a feature of patients with symptomatic diabetic autonomic neuropathy. It has not been established whether these autoantibodies cause, contribute to or simply reflect nervous tissue damage. Serum samples were tested for the presence of complement-fixing autoantibodies to adrenal medulla, vagus nerve, and sympathetic ganglion cells, to demonstrate: (a) reproducibility of the technique, (b) persistence of the antibodies, and (c) whether or not they occur in patients with non-insulin-dependent (Type 2) diabetes (NIDDM) with neuropathy. Examination of 37 samples, by different observers 2 years apart, revealed a high degree of concordance of both positive and negative results, demonstrating the method of testing to be highly reproducible. Re-testing of 37 patients (by analysing a second blood sample) between 0.5 and 2.7 years (mean 1.7 years) after their first test also demonstrated that antibodies, once present, normally persist; and that most patients initially negative remained so. Of 17 neuropathic NIDDM patients, 16 were negative for all three antibodies, indicating their rarity in this group of patients.

Identification of 3 beta-hydroxysteroid dehydrogenase as a novel target of steroid cell autoantibodies: association of autoantibodies with endocrine autoimmune disease.

Autoantibodies directed against steroid hormone-producing cells (SCA) detectable by immunofluorescence are typically found in a small proportion of patients with premature ovarian failure (POF) as well as in other endocrine autoimmune diseases. The SCA pattern stains cells in the outer zones of the adrenal cortex, ovary, and testis. To identify the molecular target of SCA, an adrenal complementary DNA expression library was screened using SCA-positive serum, and the steroid enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was identified. Only 1 of 48 (2%) patients with idiopathic POF, not pre-selected for the presence of other autoimmune diseases, had SCA by immunofluorescence, whereas 10 of 48 (21%) had anti-3 beta HSD autoantibodies detectable by immunoblot using recombinant human enzyme compared with 6 of 115 (5%) control subjects (P = 0.002). Absorption of SCA-positive serum with recombinant human 3 beta HSD abolished the immunofluorescence pattern. We also examined the prevalence of anti-3 beta HSD autoantibodies in other endocrine autoimmune diseases. Two of 112 (2%) diabetic patients, but none of the thyroid or Addisonian patients, had SCA by immunofluorescence. Twenty-six (23%) diabetic subjects (P < 0.001 vs. controls), 3 of 18 thyroid patients (P > 0.05 vs. controls), and none of 4 Addisonian patients had anti-3 beta HSD autoantibodies. 3 beta HSD is the first steroid cell autoantigen defined at the molecular level to be associated with idiopathic POF occurring in the absence of other polyglandular diseases. Autoantibodies to 3 beta HSD in patients with other organ-specific autoimmune diseases indicate that the enzyme behaves as a typical target of polyendocrine autoimmunity. Anti-3 beta HSD autoantibodies in patients with POF may provide a marker of those subjects whose ovarian failure is autoimmune in origin and, as recent studies suggest, may be salvageable with glucocorticoid treatment.

Severe sensory-autonomic neuropathy and endocrinopathy in insulin-dependent diabetes.

A patient developed insulin-dependent diabetes mellitus at the age of 9 years, complicated by a sensory/autonomic polyneuropathy which presented with a respiratory arrest at the age of 41 years. The neuropathy increased in severity over the subsequent two decades. At the age of 52 years she had hypopituitarism, hypothyroidism and low normal adrenal function. Autopsy at the age of 59 years revealed loss of pituitary tissue with evidence of hypophysitis, a lymphocytic thyroiditis and severe adrenal atrophy with lymphocytic infiltration of the medulla. The pancreas showed reduced numbers and size of the islets of Langerhans with total loss of immunoreactivity for insulin but intact glucagon-producing cells. These features are consistent with a type 2 autoimmune polyendocrine syndrome, in which lymphocytic hypophysitis has not previously been recorded. There was severe loss of myelinated nerve fibres in the sural nerve and rostrally accentuated fibre degeneration in the gracile fasciculi, but only mild cell loss in the dorsal root ganglia. This combination suggests the presence of a central-peripheral distal axonopathy. The cervical sympathetic ganglia were severely atrophic. Minor inflammatory infiltration was observed in the dorsal root and sympathetic ganglia. Significant vascular abnormalities were not present in the peripheral nerves. This, and the pattern of nerve fibre degeneration, suggest that in this case the neuropathy was likely to have been related to metabolic rather than vascular causes. The inflammatory infiltrates in sensory and sympathetic ganglia raise the possibility of an autoimmune inflammatory contribution to the neuropathy.