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HLA class I and class II molecules play a major role in the presentation of short, pathogen-derived peptides to T cells, a process that initiates the adaptive cellular and humoral immune responses. However, the factors governing a cell's ability to respond or not to particular peptides are still not completely understood. Taking the example of a viral infection, in tissues infected with a virus, viral particles are taken up by antigen-presenting cells and uncoated. The viral DNA or RNA enters the nucleus, where it replicates. mRNA enters the cytosol and is transcribed into proteins. These proteins are degraded in proteasomes and the resulting peptides (8-10 residues) are loaded onto class I molecules for export to the surface of the cells. In the meantime, the groove of the class II molecules is also preparing to accommodate peptides (12-24 residues) generated by the endocytic protein-processing pathway. The surface of the infected cell then becomes adorned with peptide-loaded human leukocyte antigen (HLA) molecules. CD4+ T helper lymphocytes engage class II molecules and elicit responses from B cells, which will ultimately lead to antibody production, whereas CD8+ T lymphocytes become cytotoxic T cells. As a consequence, the virus is eliminated from the body. However, certain mysteries and challenges remain. How can, as an exception to this rule, an autoimmune response be the escape from the perfect machinery? This review offers some hypotheses on how to see the problem through to its solution.

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

Aims/hypothesis: Juvenile-onset, insulin-dependent diabetes is associated with islet cell antibodies and with specific "high-risk" HLA-DRB1 and HLA-DQB1 genotypes. Patients with Type II (non-insulin-dependent) diabetes mellitus can have islet-related antibodies, but the genotypic associations at different ages of onset have not been evaluated. Our aim was to determine (i) the prevalence of DRB1 and DQB1 genotypes in patients at diagnosis of Type II diabetes at different ages from 25 to 65 years compared with the general population, and (ii) whether the presence of islet cell antibodies (ICA) or glutamic acid decarboxylase antibodies (GADA) or both by age is associated with different DRB1 and DQB1 genotypes., Methods: The antibodies to islet cells and those to glutamic acid decarboxylase were measured in 1712 white Caucasian diabetic subjects at diagnosis of diabetes and they were genotyped for HLA DRB1*03 and DRB1*04 and the high-risk DRB1*04-DQB1* 0302 haplotype. To assess over-representation of high-risk alleles for Type I (insulin-dependent) diabetes mellitus, the prevalence of high-risk alleles in diabetic patients was expressed relative to the prevalence of low-risk alleles, non-DR3/non-DR4, that provided a reference denominator in both the diabetic patients and in 200 non-diabetic control subjects. The prevalence of ICA or GADA or both in patients with different HLA genotypes was assessed in those diagnosed in four age groups, 25-34 years, 35-44 years, 45-54 years and 55-65 years., Results: In Type II diabetic patients presenting at ages 25-34, 35-44 and 45-54 years, there was an increased prevalence of DR3/DR4 compared with the general population with approximately 6.5-fold, 2.9-fold, 2.1-fold over-representation, respectively (p < 0.0001, < 0.01, < 0.05) but this was not found in those aged 55-65 years old. In the group aged 25-34 years, 32 % of patients with ICA or GADA or both had DRB1*03/DRB1*04-DQB1*0302 compared with 10% in those aged 55-65 years and expected 3% prevalence. Conversely, only 14% of those aged 25-34 years with antibodies had non-DR3/non-DR4, compared with 35 % in those aged 55-65 years. There was thus pronounced age heterogeneity in DRB1 and DQB1 predisposition to Type II diabetes. The antibodies displaced DRB1 or DQB1 genotypes in the multivariate model for requiring insulin therapy by 6 years of follow-up., Conclusion/hypothesis: The age of presentation of Type I diabetes in adulthood was in part dependent on the DRB1/DQB1 genotype. Islet cell antibodies and glutamic acid decarboxylase antibodies were strongly associated with DRB1*03/DRB1*04-DQB1*0302 in early adulthood but showed little relation with specific HLA genotypes after the age of 55 years.

The "IDDM-Sardinia project" started in the beginning '90s and this main objective was, and still is, to clarify the epidemiological aspects of insulin-dependent diabetes mellitus in Sardinia, an island with a high incidence of the disease. Initially, the project included three main aims: 1) to continue monitoring the incidence of the disease and to design maps of geographical distribution of it in the island; 2) to study the pre-diabetes period, by assaying islet-related autoantibodies (ICA, GADA and IA-2icA), and to design models of prediction in a general population from a large cohort of 10,000 schoolchildren, and 3) to investigate the natural history of the disease by monitoring the appearance of islet-related autoantibodies in a cohort of 19,000 newborn. Most recently, new research lines branched from these main topics, and now the project is also investigating other autoimmune diseases, in particular coeliac disease and autoimmune thyroiditis. In this paper we still summarise and discuss the state-of-the-art of the whole project.

Purpose: We investigated the immunological features and possible autoimmune basis of Peyronie's disease., Materials and Methods: The sera of 100 patients with Peyronie's disease were tested for circulating autoantibodies, including anti-penis antibodies, by indirect immunofluorescence. Antibody deposition and the immunological activity in Peyronie's plaque tissue from patients with early and long-standing disease were also assessed., Results: Circulating anti-penis antibodies were not found in any patient although antinuclear antibodies were present in 24%. Patients with early Peyronie's disease had IgM antibody deposition, marked T lymphocytic and macrocytic infiltration in the sub-tunical space, increased expression of adhesion molecules by endothelial cells and an increased human lymphocyte antigen class 2 expression by the cellular infiltrate, indicating cellular immune activation., Conclusions: These results show that some of the features of autoimmunity, in particular the cell mediated response, are present in Peyronie's disease.

Organ-specific antibodies are found in patients with autoimmune disease and their symptom-free relatives many years before clinical onset. Organ-specific cardiac antibodies can be found in patients with dilated cardiomyopathy (DCM) and their relatives, which supports the idea that DCM is an autoimmune disease. We did non-invasive cardiological assessment and antibody screening in 342 symptom-free relatives (170 male, 172 female, mean [SD] age 31 [16] years). 177 relatives were from 33 families with more than 1 affected individual (familial DCM) and 165 relatives from 31 families with only 1 affected member (non-familial DCM). The frequency of cardiac antibodies was higher among relatives of DCM patients than in controls (20% vs 3.5%, p = 0.0001). In 37 (58%) of the families studied, cardiac antibodies were found in the proband and/or in at least 1 family member and were more common in familial than in non-familial DCM (24% vs 15%, p = 0.036). Antibody-positive relatives were younger (26 [15] vs 33 [17] years, p = 0.01) and had a larger mean echocardiographic left ventricular end-systolic dimension (35 [6] vs 32 [6], p = 0.01 mm) and reduced percentage fractional shortening compared with antibody-negative relatives (31 [6] vs 34 [6], p = 0.008). Presence of cardiac-specific autoantibodies in symptom-free DCM relatives provides evidence of autoimmunity in a subset of our patients (58%), including familial and non-familial forms of DCM. These antibodies are associated with mild left ventricular systolic dysfunction on echocardiography and may be early markers for relatives at risk of DCM.

The contribution of glutamate decarboxylase (Mr 65000) antibodies to the reactivity of islet cell cytoplasmic antibodies with the 'whole' islet staining pattern from patients with newly diagnosed Type I diabetes was investigated. Diluted sera (n = 10) were preincubated with increasing concentrations of purified recombinant human islet glutamate decarboxylase (Mr 65000) and the change in islet cell cytoplasmic antibody binding was evaluated by quantitative immunocytochemistry. Binding to islet cells was partially blocked by glutamate decarboxylase in 9/10 diluted sera; the maximum blocking obtained at high concentrations of glutamate decarboxylase (5 micrograms/ml) was 36% (median, range 24-61%). In contrast, binding to islet cells in three diluted sera (two polyendocrine patients without Type I diabetes and one patient with newly diagnosed Type I diabetes) with the 'selective' islet staining pattern was totally blocked by glutamate decarboxylase. The concentration of glutamate decarboxylase required to achieve maximum blocking was less for the 'whole' islet (0.4-8.0 micrograms/microliters undiluted serum) compared to the 'selective' islet (20-645 micrograms/microliters undiluted serum) positive sera. All sera were positive for glutamate decarboxylase antibodies in an immunoprecipitation assay using 35S-methionine labelled extract of baby hamster kidney cells transfected with glutamate decarboxylase. However, the binding activity of these antibodies was less in the sera positive for the 'whole' islet compared to the 'selective' islet staining pattern. In conclusion, glutamate decarboxylase antibodies contribute partially to the reactivity of islet cell cytoplasmic antibodies of the 'whole' islet staining pattern in the sera of newly diagnosed patients with Type I diabetes, and totally to reactivity of the 'selective' islet staining pattern. The antigens recognized by the other antibodies contributing to the 'whole' islet reactivity remain to be defined.

Organ-specific cardiac antibodies are serological markers of autoimmunity in dilated cardiomyopathy (DCM). HLA-DR4 and possibly DR5 are immunogenetic markers of susceptibility in DCM, but it is not known whether they are associated with autoantibody production. We studied the frequency of HLA-DR antigens and the presence of organ-specific cardiac antibodies in 80 DCM Caucasian patients from Northern Italy. HLA-DR typing was performed by serology; 289 healthy blood donors from the same region were tested as controls. HLA-DR frequencies in DCM were also compared with VIII International Workshop control data for Italy. Cardiac antibodies were detected by indirect immunofluorescence on human heart. Skeletal muscle was used to identify cross-reacting antibodies. The prevalence of cardiac antibodies in DCM was: organ-specific 34% and skeletal muscle cross-reactive 30%. The previously reported positive association between DCM and HLA-DR4 was confirmed using either the controls from the same region (21.25% vs 10.73% p = 0.02, relative risk = 2.30) or from all of Italy (21.25% vs 12.3%, p = 0.03). HLA-DR5 frequency was slightly but not significantly higher in DCM than in controls from the same region (46.25% vs 31.49% p = 0.02, relative risk of 1.87, p corrected = NS) or from all of Italy (46.25% vs 35.8% p = NS). HLA-DR3 frequency was lower in DCM than in controls from the same region (12.50% vs 29.41% p = 0.003, relative risk of 0.36, p corrected = 0.03). This negative association was not confirmed using the control data from the whole of Italy (12.50% vs 16.5% p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

The cytoplasmic islet cell antibody patterns of sera from islet cell antibody positive non-diabetic and diabetic endocrine autoimmune patients, and newly-diagnosed Type 1 (insulin-dependent) diabetic patients were characterised using four layer immunofluorescence with monoclonal anti-proinsulin or anti-glucagon antibodies. Two distinct islet cell antibody types were identified. One gave a diffuse cytoplasmic staining in both Beta and Alpha cells ('whole' islet pattern), and was not affected by pre-incubation with rat brain homogenate. The other had a granular appearance with staining restricted predominantly to Beta cells ('selective' islet pattern) and was completely inhibited by pre-incubation with rat brain homogenate. Some sera appeared to have a 'mixed' islet pattern, in which glucagon-positive cells gave a weaker cytoplasmic staining than proinsulin-positive cells. The granular 'selective' pattern was found in sera from 19 (79%) of 24 non-diabetic endocrine autoimmune patients, in two (22%) endocrine autoimmune patients who developed Type 1 diabetes (p less than 0.0001 vs non-diabetic endocrine autoimmune patients), and in none of 19 newly-diagnosed diabetic patients. The 'whole' islet pattern was found only in sera from patients who had, or who subsequently progressed to, Type 1 diabetes. This study has identified a novel islet cell antibody specificity and demonstrates that in islet cell antibody positive endocrine autoimmune patients, only islet cell antibodies which stain both Beta and Alpha cells are associated with progression to Type 1 diabetes.

The expression of adhesion molecules by cells of the small intestinal mucosa was compared in gut biopsies from children with autoimmune small intestinal enteropathy and normal controls and related to HLA-DR expression by the same tissue. Jejunal biopsies were stained by IFL with monoclonal antibodies to LFA-1 (TS1/22 and CD11a/25.3.1) and ICAM-1 (RR1/1 and 84H10) molecules. LFA-1 and ICAM-1 positive cells were observed in the lamina propria in all cases and the counts were increased in autoimmune enteropathy compared with controls. In addition, in 4 of 7 cases of autoimmune enteropathy crypt enterocytes were positives for ICAM-1 when stained with RR1/1 and 3 of the 4 were also positive for LFA-1 when stained with both LFA-1 reagents. We speculate on the role of adhesion molecule expression in autoimmune enteropathy.

Aetiology and pathogenesis of idiopathic dilated cardiomyopathy (DCM) are uncertain. The two major pathogenetic hypotheses are: 1) autoimmunity; 2) persistent viral infection. Indirect evidence for virus association comes from the finding of raised titres of antibody to coxsackievirus in DCM, but infectious virus has never been isolated in myocardium from DCM patients. Bowles et al. using the slot-blotting technique reported that enteroviral RNA was commonly detectable in the myocardium of patients with myocarditis (53%) and with DCM (52%). Other groups using this as well as more refined hybridization techniques have failed to confirm such a high prevalence. Detection of enteroviral genomic RNA in cardiac tissue does not, however, imply active infection or pathogenicity. Thus the mechanisms of chronic myocardial damage in the absence of whole competent infectious virus remain uncertain. The other major pathogenetic hypothesis in DCM involves autoimmune mediated damage to myocytes. Circulating organ specific autoantibodies have been reported in a quarter of a group of patients with idiopathic DCM. This suggests that there may be autoimmune mechanisms operating at least in this subset of patients, but the exact relation of these antibodies to the pathogenesis and prognosis needs to be defined. The abnormal expression of major histocompatibility complex class II antigens on cardiac microvascular endothelium in endomyocardial biopsy tissue from DCM patients, and the reported association with HLA-DR4 phenotype lend further support to the autoimmune hypothesis. The viral and the autoimmune hypothesis in chronic myocarditis and in DCM are not mutually exclusive. In experimentally murine virus-induced myocarditis infectious virus can no longer be recovered from the myocardium after two weeks, although nucleic acid sequences of the viral genome are still detectable. The development of chronic inflammation takes place only in mice with a predisposing genetic background. Chronic myocyte damage is associated with the production of circulating heart-specific autoantibodies and autoreactive lymphocytes. In this animal model chronic myocarditis appears to be a virus-triggered or precipitated autoimmune disease, rather than a persistent viral infection with tissue damage due to active virus synthesis and replication. A similar transition from acute myocarditis into DCM may occur in man.

Insulin dependent diabetes (IDDM) has an autoimmune pathogenesis. Included is the presence of antibodies to pancreatic islet cells. The first identified were islet cell antibodies (ICA), detected by indirect immunofluorescence, and which react with all cells within islets. Importantly, the autoantibodies are found several years prior to disease and although a pathogenic role for the autoantibodies is unclear, they have become useful markers of prediabetes. A number of studies of twins discordant for IDDM and of first degree relatives of IDDM patients have established that there is an increased risk for disease in individuals who have ICA, especially when ICA levels are high. This high predictive value of ICA decreases in the general population where the incidence of IDDM is lower than in first degree relatives, and both ICA and the disease risk associated with ICA, appear to be influenced by a genetic susceptibility. This has been sustained in a study of patients with endocrine autoimmunity and ICA (Polyendocrine Study) where the predictive value of very high levels of ICA is less than 50% in patients without a first degree relative with IDDM. Hence, there remain a substantial number of patients with ICA who do not develop disease. From these patients, it was demonstrated that ICA include at least two distinct specificities, one of which is beta cell specific and is not associated with a high risk for IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

In an 11-yr screening program carried out on serum samples sent to an autoimmune serology laboratory, 158 patients with clinical or subclinical autoimmune endocrine manifestations and islet cell antibodies (ICAs) in the absence of overt diabetes were identified and followed for the development of insulin-dependent (type I) diabetes. Twenty-two (13.9%) developed type I diabetes in a follow-up of up to 12 yr (mean +/- SE 4.8 +/- 3.2 yr). The probability of being free of type I diabetes was 69.8% at 10 yr after the first detection of ICAs. Progression to disease was influenced by 1) the amount of ICAs represented by high titers (63% of those with ICAs greater than or equal to 20 Juvenile Diabetes Foundation units being free of type I diabetes at 10 yr), ICA persistency (59% being free of type I diabetes; P less than 0.02 vs. nonpersistent ICA), and complement-fixing (CF)-ICAs (63% being free of type I diabetes; P less than 0.05 vs. non-CF-ICA); 2) the coexistence of insulin autoantibodies (IAAs) (25% being free of type I diabetes; P less than 0.005 vs. IAA-); and 3) a positive family history (1st-degree relative) for type I diabetes (32% being free of type I diabetes; P less than 0.005 vs. no family history). There was a trend for diabetes to develop earlier in males of a younger age. No relationships were found with the number, type, or clinical expression of the associated autoimmunities or with a family history of such disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating organ-specific autoantibodies are serological markers of destruction or impairment of the relevant endocrine tissue cells and may be associated with abnormal hormone levels with or without clinical evidence of overt disease. We sought organ-specific cardiac antibodies in patients with autoimmune polyendocrinopathy because of increasing evidence that the heart has endocrine characteristics (secretion of atrial natriuretic peptide [ANP] and other peptide hormones). Serum samples from 166 patients with polyendocrinopathy, 80 with autoimmunity confined to one gland, and 200 healthy blood donors were tested for these antibodies by means of immunofluorescence on human heart. Skeletal muscle was used to identify cross-reacting antibodies. Organ-specific cardiac antibodies were detected in significantly more of the patients with autoimmune polyendocrinopathy (28 [17%]) than of those with autoimmunity confined to one gland (1 [1%]) or of normal subjects (7 [3.5%]; p = 0.0001). Among the patients with autoimmune polyendocrinopathy, the prevalence of systemic hypertension was higher in those with cardiac autoantibodies than in those without (5/28 [18%] vs 2/80 [3%]; p = 0.01); the same was true for a family history of hypertension (11 [42%] vs 5 [7%]; p = 0.0001). There were no significant differences in mean basal or stimulated ANP concentrations between patients with or without antibodies or between patients and controls. 5 of the 22 antibody-positive patients had ANP concentrations outside the normal range, but these disturbances were not associated with systemic hypertension or a family history of the disorder. Patients with autoimmune polyendocrinopathy can have organ-specific cardiac antibodies, which may represent novel serological markers for an autoimmune form of systemic hypertension in the absence of overt cardiac disease.

Children with protracted diarrhoea, circulating enterocyte autoantibodies, and an enteropathy showing features of inappropriate HLA molecule expression on the jejunal crypt epithelium, often present with persistent blood and mucus in their stools. Eight children with autoimmune enteropathy were investigated for the presence of associated colonic disease. Six children with protracted diarrhoea, no circulating autoantibodies, and an enteropathy (in five of them) undergoing colonoscopy were used as control subjects. In all eight patients, but not in the control subjects, there was macroscopic and microscopic evidence of an accompanying colitis of variable severity, thus indicating that a more generalised intestinal disorder was present, which might affect the whole intestine. Aberrant expression of DR molecules on the colonic surface and crypt epithelium was also detected. Autoimmunity may play a role in the colitis.

The observations emerged from the pancreatic transplant experiments in identical twins indicate that Type I diabetic patients maintain memory cytotoxic T cells for several years and these can be "re-awakened" when Class I identical beta cells are re-introduced into the diabetic melieu. In addition, these data have shown that these lymphocytes can (in a matter of a few weeks) cause rapid irreversible decompensation of beta cell function. From this, an important lesson is learnt: cytotoxic T cells, when generated in sufficient number against beta cells do not leave much "breathing space" for these cells. This has important implications for explaining the long latency period preceding the acute onset of Type I diabetes. ICA, when produced, seem unable to cause gross damage to beta cells. They persist for several years in the blood, but beta cell function remains apparently unaltered. It is only when cytotoxic T cells, with fine specificity for beta cells are generated that the 'killing cycle' is completed. Whether these cells are present all the time, and kept under tight control by active suppressor mechanisms or whether they appear only after an environmental trigger (e.g., retroviruses) is unknown. If the former is the case, this would give strong support to the suggested important role of suppressor T cells in the pathogenetic circuit. Some evidence for this has been produced (rev. in [116]) but, obviously, it requires confirmation (see debate which followed [116]). If, on the other hand, the latter is experimentally confirmed, one can return to the theory that cytotoxic T cells acquire the characteristics of autoreactivity by expressing receptors on their surface in a configuration which enables combination with self-autoantigens (rev. in [45]). In summary, the study of the etiopathogenesis of Type I diabetes and human autoimmunity in general has attracted a great deal of interest among immunologists. It is only by further dissecting the various limbs of the undesirable immune response against beta cells and, by trying to formulate novel hypotheses, sometimes against accepted dogmas [32], that the complete picture will be finally disclosed. At this stage it will be possible to design effective therapy trials, so that Type I diabetes and other related autoimmune disorders ultimately may be prevented.

The cell surface expression of the thyroid microsomal antigen is confined to the apical microvilli of the polarized cells in intact or semi-intact thyroid acini. This makes it possible to envisage a pathogenetic role for the commonest antibodies found in thyroid autoimmunity by mechanisms involving complement- and antibody-dependent cytotoxicity. TSH rereceptor antibodies can now be differentiated into those that stimulate hormone synthesis and release (TSI), and those which mimic TSH in its growth-promoting function (TGI). TGI were demonstrated in goitrous Graves' disease and in some patients with euthyroid non-toxic goitres, diffuse or nodular, defining a new variety of thyroid autoimmunity. In patients with non-toxic goitre there is hardly any lymphoid thyroiditis and a low incidence of thyroglobulin or microsomal antibodies, the main expression of autoimmunity being the impetus to thyroid hyperplasia and hypertrophy. TSH receptor antibodies of both types, TSI and TGI, can either be stimulating or have blocking effects (TSI block and TGI block). Atrophy of the thyroid gland in myxoedema is due to blocking of the normal pituitary-controlled repair mechanism. In goitrous thyroiditis cell re-growth as a result of increased TSH can occur, as there are no blocking antibodies. In some patients with 'simple' euthyroid goitres, TSI blockers prevent the onset of hyperthyroidism while growth-promoting antibodies give rise to the goitre.

A method is described for culturing intact human thyroid follicles, based on the study of 40 thyroidectomy specimens from normal (n = 18) and diseased glands (n = 22). Reversal of the normal polarity of thyrocytes, whereby the microvilli move from the colloid edge to the vascular pole of the cells, occurs gradually when the amount of fetal calf serum (FCS) is changed from 0.5% to 10%. The translocation of thyroid 'microvillar' antigens, (surface expression of 'microsomal' and a separate surface antigen) from the follicular to the vascular pole of thyrocytes was assessed by indirect immunofluorescence with human sera containing microsomal antibodies, as well as by electron microscopy. In normal and diseased thyroid glands up to 80% of follicles became reversed after 5-10 days in high FCS and the microsomal/microvillar antigen persisted for about twice as long as in monolayer cultures. Spontaneous reversal of polarity was observed in six of eight glands from patients with Graves' thyrotoxicosis or toxic nodular goitre in freshly dispersed tissues or after 2 days in 0.5% FCS, unlike normal tissues where only a trace of reversal appeared after 7 days of culture under these conditions. It is postulated that polarity reversal may play a role in human thyroid autoimmunity as the normally secluded 'microvillar' antigens becomes transposed to the vascular pole of thyroid follicles where they are in direct contact with cytotoxic antibodies or sensitized immunocytes. This could initiate lesions in intact follicles. Inappropriate HLA-DR expression on thyrocytes, either stimulated by phytohaemagglutinin (PHA) or appearing spontaneously as an early marker of thyroiditis, did not correlate with reversal of polarity.

Autoantibodies to vasopressin-secreting cells of human hypothalamus were detected by means of indirect immunofluorescence (IFL) in 13 patients with diabetes insipidus (DI). 11 of 30 patients (36 . 7%) with "idiopathic" and 2 of 32 (6 . 3%) with symptomatic DI were positive, and 139 control patients were negative. The specificity of the reaction vasopressin cells was demonstrated with a 4-layer double-fluorochrome IFL test in which the second sandwich consisted of rabbit antivasopressin or anti-oxytocin counterstained with rhodaminated anti-rabbit immunoglobulin. 5 patients had also antibodies to oxytocin-producing cells. The antibodies reacted with cytoplasmic components distinct from the hormone; they were of IgG, IgA, or IgM class or a combination of these classes, and half of them fixed complement. Maximum titres were 1:32, and the antibodies could not be absorbed out by incubation with vasopressin, oxytocin, neurophysin I, or neurophysin II. Some sera stained as yet unidentified small cells in the hypothalamus. This report suggests that autoimmunity extends to the hypothalamus. Vasopressin-cell antibodies may prove to be useful markers for the diagnosis of an autoimmune variant of diabetes insipidus.

Thirty-nine patients with idiopathic cranial diabetes insipidus (DI) and 81 secondary to hypothalamic lesions were investigated for the presence both of associated autoimmune diseases and autoantibodies. Eleven (28%) of the idiopathic but none of the secondary DI cases had an overt autoimmune disease. A further two patients with idiopathic DI had associated organ-specific autoantibodies. Autoantibodies to vasopressin (AVP)-secreting hypothalamic cells were detected in 12 patients with idiopathic DI (31%). Seven out of 13 cases of DI secondary to histiocytosis X (HX) were also positive (54%), whereas only two (3%) of the other 68 sera from patients with secondary DI reacted with AVP cels. Of the 13 patients with DI associated with frank organ-specific autoimmune diseases or autoantibodies alone, eight (62%) were positive for AVP-cell antibodies. The finding of associated autoimmune diseases in a patient with idiopathic DI is therefore suggestive of an autoimmune origin of DI, and this can be supported by the detection in the serum of AVP cell antibodies. In cases of HX, the new finding of the presence of AVP-cell antibodies reflects hypothalamic infiltration by HX cells, and suggests that DR + 'Langerhans-like' cells play more than a passive role in the hypothalamic lesion.

This study was prospectively carried out in order to clarify if an aberrant expression of HLA-DR molecules could take part in the pathogenesis of chronic pancreatitis. Pancreatic specimens from 12 chronic pancreatitis patients and nine controls were examined. Strong HLA-DR expression was observed in 6/12 chronic pancreatitis patients and in 1/9 controls. Moreover, lymphocytic foci with large numbers of activated cells were found only in chronic pancreatitis. The four HLA-DR - patients had a marked increase of fibrous tissue with small portions of acinar tissue, whereas the six patients with strong positivity had the greatest dilatation and hyperplasia of the ducts. These findings are similar to those observed in immune diseases, such as thyroiditis and primary biliary cirrhosis (PBC), and suggest that autoimmune phenomena are involved in chronic pancreatitis.

After the death of a 12-year old girl with newly discovered insulin-dependent diabetes mellitus, we used monoclonal antibodies in an effort to identify the cells invading the pancreas. The majority of infiltrating lymphocytes were of the T cytotoxic/suppressor phenotype, but other T-cell subpopulations were present. Some of the T cells were "activated" (positive for HLA-DR antigen, and the interleukin-2 receptor). Immunocytes bearing IgG were scattered in the gland, and complement-fixing IgG antibodies were deposited in some islets. Increased expression of Class I (HLA-A, B, and C) molecules was observed in the affected islet cells, and in damaged islets showing scant lymphocytic infiltration, some beta cells (still producing insulin), but not glucagon or somatostatin cells, were HLA-DR positive. The capillary endothelium was markedly dilated and strongly HLA-DR positive. These findings may contribute to an understanding of the sequence of events leading to the destruction of beta cells in classic Type I diabetes mellitus.

Autoantibodies that react exclusively with the gastrin-secreting cell of human antrum have been detected by immunofluorescence in eight of 106 patients with histologic evidence of chronic atrophic gastritis, Type B, involving mainly the antrum. These antibodies were of the IgG class and of low titer. However, follow-up studies one to two years later showed persistently positive reactions, despite symptomatic treatment. These data support the concept of an autoimmune variant of chronic "antral" gastritis, Type B.

Sixty-eight patients with longstanding diabetes and persistent islet-cell antibody and 35 with coexistent diabetes and Graves's disease or primary myxoedema were studied with particular reference to the HLA system and autoantibody patterns. A higher incidence of HLA-B8 than normal was observed in the two groups. An additive relative risk exists when type I diabetes and autoimmune thyroid disease coexist, indicating that different HLA-linked genes may confer susceptibility to the pancreatic and thyroid disorders. Other characteristics, including female predominance, a later onset of diabetes, and a strong family history of autoimmune endocrinopathy, provide further evidence that this form of diabetes is aetiologically distinct from that generally seen in children. These results support the hypothesis of a primary autoimmune type of diabetes mellitus.

Anti-perinuclear factors (APF), IgM-, IgG- and IgA-rheumatoid factors have been detected in six, five, one and three out of 20 patients with primary myxoedema, respectively. We were unable to find APF in 22 patients with Hashimoto's thyroiditis nor in 20 patients with Graves' thyrotoxicosis. APF appears to be a marker for non-organ specific autoimmune diseases.

Sixty-two consecutive patients with sporadic euthyroid goiter (57 women and 5 men) from noniodine-deficient areas, including 15 patients with diffuse goiter, 39 patients with multinodular goiter, and 8 patients with a single nodule, were studied for the presence of serum thyroid growth-stimulating immunoglobulins (TGI) by the ultrasensitive cytochemical bioassay based on DNA cytophotometry (Feulgen-cytochemical bioassay). Using strictly specified conditions, 43 patients (67%) were positive. Values tended to be high in diffuse goiter, nodular goiters reccuring after partial thyroidectomy, and those with recent growth. Thirty-seven individual immunoglobulin (Ig)-rich fractions obtained by ammonium-sulfate precipitation from 20 normal subjects, 13 atrophic thyroiditis patients, and 4 dyshormonogenetic goiter patients were tested similarly, and only 3 gave positive growth assays. These results lend further support to our concept that a majority of patients with sporadic nontoxic simple goiters have a variant of thyroid autoimmune diseases separate from lymphocytic thyroiditis. With regard to assays thought to reflect activities of TSH receptor antibodies, none of 20 tested Ig preparations stimulated thyroid cAMP production. The TSH binding inhibition assay gave weak positive activity, but failed to correlate with either TGI or TSH unresponsiveness to TRH. These findings suggest that sporadic goiter TGI is not directed to the TSH-binding site. Dose-response studies performed with Igs of patients with nontoxic goiters and with human TSH standard and goitrous hyperthyroid Graves Igs as controls all revealed bell-shaped responses. Similar maximal values were reached regardless of the growth stimulus applied. However, approximately 10 times more Ig was needed to reach maximal responsiveness in sporadic goiter than in goitrous Graves' disease (i.e. 125-500 micrograms vs. 15-125 micrograms Ig/ml culture fluid). The optimal dose of human TSH ranged from 0.01-1.0 microU/ml. The assays in the present series of the 62 consecutive patients with nontoxic diffuse or nodular goiter were all carried out with a fixed amount of 125 micrograms Ig/ml and considering a value above 5% of cells in the S-phase as a positive assay. Some Ig preparations negative for TGI at this concentration may contain TGI when tested using other doses, and these are a prerequisite to assess the potency of growth antibodies in individual patients.

Immune responses are initiated by HLA-DR+ cells, which present antigen to T cells. Observations that HLA-DR may be experimentally induced on thyroid epithelium and that HLA-DR occurs on thyrocytes in autoimmune thyroid diseases suggest a mechanism of autoimmunity with special relevance to organ-specific diseases. This involves the local aberrant expression of HLA-DR antigens by epithelial cells and their subsequent capacity to present autoantigens occurring on their surfaces to T lymphocytes. For autoantigens which T cells recognise infrequently because of their restricted tissue location and low concentration in the circulation, T-cell tolerance is unlikely, and so induction of autoreactive T cells would occur. Because interferon is the best known inducer of DR antigen expression and viral infections may predate endocrine autoimmunity, the following sequence seems likely: local viral infection which causes interferon production, or other local environmental factors which would induce DR expression, presentation of autoantigens, and subsequent autoimmune T-cell induction. These T cells would activate effector B and T cells. Whether the initial induction of autoimmune T cells leads to autoimmune disease would depend on factors such as abnormalities of the suppressor T-cell pathway, reported to coexist with autoimmunity and necessary to induce autoimmune disease in mice. This mechanism of autoimmune disease induction explains vague associations with viral infections and long latency periods before disease becomes manifest and gives a simple explanation for the well-documented association between HLA-DR and autoimmune diseases in man.

The presence of organ-specific cell surface-reactive antibodies in sera of patients with autoimmune thyroid diseases (ATD) has been detected by indirect immunofluorescence on viable blood group O normal thyroid cultures. The cell surface determinants involved in this reaction have been identified as the thyroid 'microsomal' antigen which is thus also represented on the outer surface of the plasma membrane. The reactivity persists when F(ab')2 fragments from positive sera are used. Non-thyroid cells present in the monolayers, as well as human adrenal or fibroblast cultures, do not show positive staining. Human thyroid cells progressively lose both cell surface and intracytoplasmic antigenic components during the first week of culture. The exposure and reactivity of this antigen on the cell surface do not depend upon unmasking effects of proteolytic enzymes used for dispersal of the cells. The 'microsomal' antigen on the cell surface is also involved in the complement-mediated cytotoxic effect of sera from ATD patients on freshly dispersed thyroid cells. Thyroglobulin antibodies failed to stain viable thyroid monolayers, even when these were previously incubated with purified human thyroglobulin, indicating that this protein or possible receptors for it cannot be detected on the cell surface under these conditions. Similarly, some thyrotoxicosis sera containing thyroid-stimulating but not microsomal antibodies, gave negative reactions on viable cultures.

Alpha-interferon has been administered as an adjuvant treatment for women with loco-regional relapse of breast cancer. During the course of treatment 5/10 (50%) of women receiving interferon developed de novo thyroid autoantibodies. Three patients became clinically myxoedematous, with biochemical evidence of hypothyroidism which responded to thyroxine replacement therapy. The leucocyte alpha-interferon preparations used in the trial enhanced Class I but not Class II MHC antigens on thyrocytes in vitro. These data strongly suggest that patients receiving alpha-interferon therapy should be closely monitored for the possible development of thyroid dysfunction and that thyroid antibody determination can greatly help to predict overt thyroid clinical abnormalities.

In addition to thyroglobulin autoantibodies, some Obese strain (OS) chickens gave organ-specific reactions with the cytoplasm of thyroid acinar cells when the sera were tested by immunofluorescence. The staining pattern was similar to that seen with human antibodies to thyroid microsomes. A proportion stained the proventricular glands of the chicken stomach in a manner indistinguishable from that of pernicious anaemia sera containing parietal cell antibodies. Isolated examples of organ-specific reactions with adrenal and exocrine pancreas were also recorded. These findings strengthen the notion that the OS chicken represents an authentic model for human organ-specific autoimmune disease. The high incidence of non-organ specific reactions complicates but does not necessarily invalidate this view since normal chickens show a propensity to develop such antibodies. However the OS chicken appears to differ from the human in being hyper-responsive to antigens in general.

81 patients with spontaneously acquired myasthenia gravis (MG) were investigated for the presence of autoimmune (AI) diseases and their sera were tested for a range of organ-specific autoantibodies. 77 of the patients were HLA-phenotyped. Antibody titres to acetylcholine receptors (AChR) were higher in non-thymomatous patients who possessed HLA-B8 (p less than 0.05) and/or -DR3 (p less than 0.05) as compared to patients lacking these HLA antigens. 3 out of 20 (15%) patients with ocular MG, 7/23 (30%) with generalized MG of early onset, 11/23 (48%) generalized MG of late onset and 5/14 (35%) patients with thymoma had either overt AI diseases or significant titres of organ-specific autoantibodies suggesting subclinical AI disease. In ocular MG, low titres and an infrequent finding of antibodies to AChR (32%) as well as the low prevalence of associated autoantibodies and AI diseases indicate that this subgroup of MG consists of patients with restricted AI reactivity. HLA-B8 and -DR3 were present in all the patients with associated AI disorders in the young onset group but in none of the patients with old age of onset. In the young group, 6 out of 7 patients with associated AI conditions were women whereas the sex ratio was about equal in the older cases in both, patients with and without associated AI diseases or autoantibodies. We conclude from these observations that ageing provides conditions that allow the breakdown of self tolerance. The simultaneous presence of HLA B8, DR3 and female sex provide important additional factors for early expression of MG.

Complement-fixing islet-cell antibodies (CF-ICA) were found in 20 out of 685 unaffected first-degree relatives of children with type 1 diabetes. During a 5-year follow-up, 7 of the 20 became diabetic, 1 continued to show the antibodies without any abnormality of glucose tolerance, and 12 subjects lost them without the disease developing. Although CF-ICA are useful as a marker of active insulitis they should not at present be used to define subjects who might benefit from preventive immunosuppression.

The thyroid glands of patients with autoimmune diseases such as Graves' disease and certain forms of goiter contain infiltrating activated T lymphocytes and, unlike cells of normal glands, the epithelial follicular cells strongly express histocompatibility antigens of the HLA-DR type. In a study of such autoimmune disorders, the infiltrating T cells from the thyroid glands of two patients with Graves' disease were cloned in mitogen-free interleukin-2 (T-cell growth factor). The clones were expanded and their specificity was tested. Three types of clones were found. One group, of T4 phenotype, specifically recognized autologous thyroid cells. Another, also of T4 phenotype, recognized autologous thyroid or blood cells and thus responded positively in the autologous mixed lymphocyte reaction. Other clones derived from cells that were activated in vivo were of no known specificity. These clones provide a model of a human autoimmune disease and their analysis should clarify mechanisms of pathogenesis and provide clues to abrogating these undesirable immune responses.

Circulating autoantibodies to enterocytes were detected by indirect immunofluorescence in 14 out of 25 patients with idiopathic protracted diarrhoea of infancy. Similar specificities were not found in 50 control children with nongastroenterological disorders. The immunofluorescence pattern was more accentuated on the apical border of mature enterocytes. Enterocyte autoantibodies were mainly of IgG class (13/14), but 11 sera were positive for IgM and IgA classes, and five out of 14 positive sera also had the ability to fix complement. Absorption of sera positive for autoantibodies with an IgA coupled immunoabsorbent did not modify the intensity of the staining, indicating that these antibodies were not directed against secretory IgA. High titres and the complement fixing ability of enterocyte autoantibodies indicated a poorer prognosis despite the use of immunosuppressive drugs. Organ specific and non-organ specific autoimmune diseases or corresponding autoantibodies or both were often found in children with enterocyte autoantibodies and their family. These data show the existence of an autoimmune variant of protracted diarrhoea of infancy, despite the rare occurrence of autoimmune diseases in childhood.

The inappropriate expression of HLA Class II molecules by the target cells of endocrine autoimmune diseases is a recent observation that has been intensively studied in thyroid autoimmunity and type I diabetes mellitus. In vitro studies have shown that interferon-gamma can induce Class II expression, either alone, as in thyrocytes, or in combination with other mediators like tumour necrosis factor or lymphotoxin, as in islet cells, pointing to possible mechanisms operating in vivo. Endocrine cells expressing Class II molecules are able to present their autoantigens to helper T cells, thus possibly inducing the autoimmune process. However, until now it is still unclear if the expression of Class II molecules by the target cells is the primary immune phenomenon, which might possibly be triggered by a latent viral infection of the endocrine cell. Alternatively, it might be a secondary response in an ongoing autoimmune process. Particularly data obtained in the diabetic pancreas favour the first possibility, but only progress in our understanding of the role of HLA antigens in immunoregulation will make it possible to interpret the phenomenon properly.

Adult primary myxoedema is usually due to an autoimmune thryoiditis characterized by progressive shrinking of the thyroid gland, loss of epithelium, dense infiltration by sensitized lymphocytes and plasma cells with final replacement of the gland by a fibrous scar. Antibodies directed against thyroglobulin (TgHA) and microsomal antibodies (McHA), detectable years before the onset of hormonal failure, and cell-mediated immune mechanisms contribute to the pathogenesis. When 90% of the gland is destroyed, the secretion of thyroid hormones (T3/T4) falls below normal needs and pituitary thyrotrophs react by producing over 100 times the normal amount of thyroid-stimulating hormone (TSH) which should normally lead to regrowth of the gland as happens in goitrous Hashimoto thyroiditis. The inability of the thyroid in primary myxoedema to respond to the trophic action of TSH suggested that blocking antibodies exist in this disease which compete with TSH for its receptors. The trophic effect of TSH can be assayed in vitro by measuring DNA-synthesis by Feulgen cytophotometry; elevated pentose-shunt oxidative activity correlates with DNA synthesis. Here we report that immunoglobulins from patients with adult, primary myxoedema block the trophic effect of TSH, as assessed by this in vitro system. This is in marked contrast to the growth stimulation induced by immunoglobulins from patients with thyrotoxic Graves' disease with prominent goitres.

In most of the cases, once a given endocrine gland is involved, the corresponding specific autoantibody may be detected; for example, anti-islet cell antibodies are produced within the first few years, after onset of symptoms in insulin-dependent diabetes (DID). Accompanying autoantibodies are quite frequently found in the patient himself. In Schmidt's syndrome (thyroid and adrenal glands are involved and associated to IDD in 30% of the cases) thyroid microsomal antibodies are found in 38% of the cases, thyroglobulin antibodies in 11% of the cases, islet-cell antibodies in 7% of the cases and steroid cell antibodies in 17% of the cases. Associations are also possible in patient family members. Aberrant expression of HLA-DR molecules at the membrane of follicular thyroid cells (as of any other endocrine gland), following a viral aggression, could well account for the endocrinopathy combinations, but alternative mechanisms should be discussed.