Activation of the immune system and coronary artery disease: the role of anti-endothelial cell antibodies.

On the basis of the role of immuno-mediated inflammation in atherosclerosis we investigated, (1) the prevalence of anti-endothelial cell antibodies (AECA) in ischaemic heart disease (IHD); (2) if beta2-glycoprotein I (beta2-GPI) was the target antigen of AECA; (3) the relationship between AECA, tissue factor (TF) and tissue factor pathway inhibitor (TFPI). In 93 consecutive IHD patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and 105 controls AECA were detected by ELISA on human umbilical vein endothelial cells (HUVEC). AECA positive sera were evaluated for anti-beta2-GPI antibodies by ELISA. TF and TFPI plasma levels were assessed by ELISA. Twelve of 93 (12.9%) IHD patients and only one of 105 controls (0.95%) were AECA positive. The prevalence of AECA was higher in unstable angina (UA) than in effort angina (EA) (P=0.01). Three of 12 AECA positive sera resulted positive for anti-beta2-GPI and showed a marked decrease in EC-binding when tested on HUVEC cultured in serum-free medium. The binding was restored by the addition of beta2-GPI. TF and TFPI levels were similar in AECA positive and AECA negative patients. The rate of angiographically documented clinical recurrences was 66.7% in the AECA positive and 14.8% in the AECA negative group (P=0.0004) with a significant relationship between restenosis and AECA (P<0.0001), unchanged by the inclusion of cardiovascular risk factors in the regression model. Our results suggest a 'role' for AECA in the immune-mediated inflammation in UA beta2-GPI is not the only AECA target antigen. AECA are not responsible for high TF and TFPI levels. The high rate of clinical recurrences after PTCA, confirmed by angiography, in AECA positive patients is in line with such a role and suggests further large-scale 'ad hoc' studies.

Clinical study on Alternaria spores sensitization.

BACKGROUND: sensitization to fungi spores is often associated with sensitization to other allergens and so it is difficult to estabilish the exact cause of illness. OBJECTIVE: the aim of the study was to evaluate a group of patients monosensitized to Alternaria and to establish the prevalence, periodicity and clinical profile of this kind of sensitization. METHODS: 37 subjects who were monosensitized to Alternaria (prick-test positive), according to a previous epidemiological study were evaluated. Clinical symptoms and the period of their occurrence were taken into consideration as well as immunological parameters (RAST). RESULTS: 20 patients (over 50%) included in the study proved to be affected by asthma associated with other allergic symptoms and 22 patients (60%) presented perennial symptoms. The RAST carried out on 34 monosensitized subjects proved positive in 11 and negative in 23. CONCLUSIONS: Alternaria sensitization is characterized by a perennial periodicity with severe respiratory symptoms (asthma) which occur primarily in children. Prick test is preferable to and more reliable than RAST as a diagnostic test.

Clinical study on Alternaria spores sensitization

Background: sensitization to fungi spores is often associated with sensitization to other allergens and so it is difficult to estabilish the exact cause of illness. Objective: the aim of the study was to evaluate a group of patients monosensitized to Alternaria and to establish the prevalence, periodicity and clinical profile of this kind of sensitization. Methods: 37 subjects who were monosensitized to Alternaria (prick-test positive), according to a previous epidemiological study were evaluated. Clinical symptoms and the period of their occurrence were taken into consideration as well as immunological parameters (RAST). Results: 20 patients (over 50%) included in the study proved to be affected by asthma associated with other allergic symptoms and 22 patients (60%) presented perennial symptoms. The RAST carried out on 34 monosensitized subjects proved positive in 11 and negative in 23. Conclusions: Alternaria sensitization is characterized by a perennial periodicity with severe respiratory symptoms (asthma) which occur primarily in children. Prick test is preferable to and more reliable than RAST as a diagnostic test (AU)

Introducción: la sensibilización a esporas de hongos se asocia frecuentemente con la sensibilización a otros alergenos, con lo que es difícil establecer la causa exacta de la enfermedad. Objetivo: la finalidad del estudio fue evaluar a grupos de pacientes monosensibilizados a Alternaria y establecer la prevalencia, periodicidad y perfil clínico de esta clase de sensibilización. Métodos: se estudiaron 37 pacientes monosensibilizados a Alternaria (prick test positivo) seleccionados en un estudio epidemiológico previo. También se consideraron los síntomas clínicos y su frecuencia, así como algunos parámetros inmunológicos (RAST). Resultado: 20 pacientes (por encima del 50 por ciento) padecían asma asociada con otros síntomas alérgicos y 22 pacientes (60 por ciento) presentaban síntomas perennes. El RAST llevado a cabo en 34 pacientes monosensibilizados, fue positivo en 11 y negativo en 23. Conclusiones: la sensibilización a Alternaria se caracterizó por ser perenne, cursar con síntomas respiratorios graves (asma) y tener lugar principalmente en niños. Es preferible y más fiable como test diagnóstico el test cutáneo que el RAST. (AU)

Five-year follow-up of 165 Italian patients with undifferentiated connective tissue diseases.

OBJECTIVE: To study those conditions with a proven or hypothesised immunologic pathogenesis and denominated under a working definition of undifferentiated connective tissue diseases (UCTD). METHODS: A multicentre prospective study was organised involving 10 tertiary referral centers of internal medicine in Italy, with the aim of describing the natural history of UCTD and the prevalence of its different clinical and immunological manifestations. RESULTS: After a five-year follow-up period, data on 165 patients were available for analysis. UCTDs occur mainly in females in their fourth decade of life. Articular and mucocutaneous features and Raynaud's phenomenon represent the most common findings. Nevertheless, we also detected a relatively high incidence of permanent major organ damage. Regarding the immunologic parameters, we documented some conflicting results in the correlation between serologic abnormalities and clinical features. In 10 patients UCTD evolved to a major disease, generally systemic lupus erythematosus or Sjögren's syndrome. CONCLUSION: A low rate of evolution to a defined autoimmune disease, the limited use of steroid or immunosuppressive therapy, and a favourable course in the majority of cases are the main characteristics of patients with UCTDs.

High prevalence of anti-beta2 glycoprotein I antibodies in patients with ischemic heart disease.

Ischemic cardiac manifestations have been reported in a various percentage of patients with anti-phospholipid antibodies. As concerns the relationship between anti-beta2 glycoprotein I antibodies (anti-beta2-GPI) and ischemic heart disease (IHD), it was investigated in only one coronary primary prevention study. We investigated the prevalence of anti-beta2-GPI in a well characterized group of patients with different clinical manifestation of IHD. Sera from 37 patients (mean age 62.7 +/- 9.9) with IHD (20 with unstable angina-UA and 17 with effort angina-EA) and from 40 healthy subjects, matched for age and sex, were tested for the presence of IgG and IgM anti-beta2-GPI using an ELISA technique. Eleven/37 patients (29.7%) resulted positive for anti-beta2-GPI. A positivity for IgG anti-beta2-GPI was found in 10 patients, 1 patient was positive for IgM and 1 for both isotypes. The prevalence of anti-beta2-GPI in the control group resulted significantly lower (2.5%; p < 0.005) than in patients with IHD. Positivity for anti-beta2-GPI was found in 9/20 (45%) patients with UA and only in 2/17 patients (11.8%) with EA (p = 0.0365). IgG anti-beta2-GPI levels (median 7.7U/ml, range 2.6-24.1) were significantly higher in patients with UA compared to patients with EA (median 4.6 U/ml, range 2.3-11.5; p = 0.02) and controls (median 3.15 U/ml, range 2.3-9.0; p < 0.0001); also IgM levels resulted higher in patients with unstable angina. A positivity for anti-beta2-GPI was observed in 4/13 patients (30.8%) with a previous myocardial infarction (MI) and in 7/24 (29.2%) patients without a previous MI. Our findings suggest that anti-beta2-GPI could represent an expression of the T-cell activation detectable in patients with unstable angina. The lack of a significant difference in the prevalence of these antibodies in patients with or without a previous MI suggests that anti-beta2-GPI are not induced by tissue necrosis.

Central nervous system involvement in systemic lupus erythematosus patients without overt neuropsychiatric manifestations.

OBJECTIVE: To verify whether features of CNS involvement can be detected in SLE patients without overt neuropsychiatric manifestations. METHODS: 114 SLE patients who had never received a diagnosis of neuropsychiatric lupus (never-NPSLE) were studied and compared to 65 SLE patients with known neuropsychiatric involvement (NPSLE). The study relied on evaluation of neurocognitive functions by means of a battery of neuropsychological tests, on psychiatric and neuropsychological assessments and on neuroimaging studies (computed tomography, magnetic resonance, single photon emission computed tomography (SPECT)). RESULTS: Clinical features, including disease duration/activity and pharmacological therapy, of never-NPSLE and NPSLE patients were similar. Short-term and long-term memory, visuo-spatial and verbal information processing were similarly compromised in never-NPSLE and in NPSLE patients; only attention was significantly more compromised in NPSLE patients. Psychiatric morbidity was higher than expected in never-NPSLE patients, although less than in the control neuropsychiatric group. Ischemic lesions, multiple small high intensity lesions and cortical atrophy, detected by CT and MR scans, as well as abnormal SPECT were also frequently detected in never-NPSLE patients. Interestingly, left parietal and occipital area hypoperfusion by SPECT was significantly more frequent in the patients with impaired visuo-spatial intelligence and short-term memory. CONCLUSIONS: Most abnormalities detected by available diagnostic tools and characteristics of neuropsychiatric SLE are also present in non-symptomatic patients. They may derive from an unexpected widespread involvement of the CNS and are not per se sufficient, in the absence of clinical manifestations, for a diagnosis of neuropsychiatric SLE.

Determination of antibodies to extractable nuclear antigens by commercial kits: a multicenter study.

Several enzyme immunoassays for serum antibodies to extractable nuclear antigen have recently become available. The aim of this study was to evaluate the results obtained with: (1) the same kit under different conditions; (2) different enzyme immunoassays; (3) Western blot and enzyme immunoassays. Twenty-five sera from patients with autoimmune disorders were tested in five different laboratories by one Western blot and four enzyme immunoassay commercial kits. The different methods produced comparable qualitative results. However, semiquantitative evaluation, based on a cut-off value (index), yielded different results due both to laboratory conditions and to the kits employed. Standardization of commercial products and methods should be improved so that the results of different laboratories can be compared and large-scale and follow-up studies conducted. Western blot analysis could also be useful to analyze complex reactivities, although greater experience is necessary to interpret these results correctly.

Prevalence of sensitization to Alternaria in allergic patients in Italy.

BACKGROUND: The actual prevalence of sensitization to Alternaria is not known, partly due to the unreliability of diagnostic extracts. OBJECTIVE: To assess skin positivity to extracts of Alternaria in a wide population of Italian patients suffering from respiratory symptoms using a biologically standardized extract. METHODS: A total of 2942 patients were skin prick tested with Alternaria, and a panel of common inhalant allergens. Blood samples for specific IgE quantitation were taken both from patients positive and from patients negative (control group) to Alternaria extract. RESULTS: Three hundred six patients (10.4%, ranging from 1.8% in Turin to 29.3% in Cagliari) were positive to Alternaria; 37 were sensitized to only this mold, while the remaining 269 were sensitized to at least one other allergen. Of the Alternaria-positive patients, 79.7% suffered from rhinitis and 53.3% from asthma, either alone or associated with other symptoms. CONCLUSIONS: We suggest that, at least in Italy and in countries with similar climatic and environmental situations, standardized Alternaria extract should be included in the panel commonly used in investigating the allergen responsible in patients suffering from respiratory allergy.

Some anti-thyroperoxidase antibodies positive sera give a pANCA pattern on ethanol-fixed human neutrophils: cross-reactivity or false positives?

Fifty-six sera from patients with autoimmune thyroiditis and 33 sera from patients with MPO-ANCA were examined in order to ascertain whether a cross reactivity between MPO-ANCA and anti-thyroperoxidase (aTPO) was present. Sera from 20 healthy donors aTPO and aMPO negative were used as control. About 95% of heat inactivated sera from patients with autoimmune thyroiditis and from controls gave positive results (atypical pANCA pattern) on ethanol-fixed neutrophils. The prevalence of positive results was significantly lower when unheated aTPO positive sera were used (17.8%). On the other hand, only 9% of sera with MPO-ANCA were positive on cryostatic sections of human thyroid. Indirect immunofluorescence tests (IF) on human neutrophils with MPO defect were negative with sera from patients with MPO-ANCA, but uninactivated sera with aTPO and positive for pANCA on normal neutrophils showed a very high prevalence of positive results (90%). According to our data only few sera positive for aTPO recognize "normal" MPO, but the majority of sera from patients with autoimmune thyroiditis and positive for pANCA on normal neutrophils recognize also an "abnormal" MPO. On the other hand MPO-ANCA usually recognize epitopes presently only on the normal enzyme, a small proportion of these autoantibodies can react with TPO. Heat inactivated sera give false positive results for pANCA on ethanol fixed human neutrophils.

Antiphospholipid antibodies (aPL) increase the potential monocyte procoagulant activity in patients with systemic lupus erythematosus.

Lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are frequently detected in sera from patients affected by systemic lupus erythematosus (SLE). However, the role of antiphospholipid antibodies (aPL) in thrombus formation has not been defined as yet. Twenty-two patients affected by SLE, all fulfilling the 1982 ARA revised criteria, and twenty healthy subjects were investigated for the presence of LA, aCL and other aPLs. Monocyte procoagulant activity-PCA (Tissue Factor production) was evaluated by one stage plasma recalcification time. In all patients the plasma levels of F1 + 2 and of plasminogen activator inhibitor (PAI) were also determined. Monocyte PCA was significantly higher in SLE patients with LA and/or aCL in comparison to SLE patients without LA and/or aCL (p < 0.01) and to controls (p < 0.05). However, no connection was observed between PCA expression by mononuclear cells and LA or aCL levels. No differences in F1 + 2 and PAI plasma levels were found between SLE patients with or without aPL and controls. In our SLE patients LA and/or aCL positivity appears strictly related to an increased monocyte activation that could play an important role in the occurrence of thrombotic events.

MRI and SPECT investigations of the CNS in SLE patients.

Twenty-two SLE patients were examined with Magnetic Resonance Imaging (MRI) and Single Photon Emission Computed Tomography (SPECT). Multifocal areas of cerebral blood flow (CBF) defects were found in 81.8% of the patients. These alterations were present in patients with severe neurological disorders, in those with neuropsychiatric symptoms only, and also in asymptomatic cases. Anticardiolipin antibodies (aCL) were detected in all patients, but no correlation was found between serum aCL positivity and neurological involvement. The high incidence of cerebral blood flow disorders found in asymptomatic patients, and their poor correlation with the MRI alterations suggest a primary defect of CBF in these patients.

Prevention of miscarriage in antiphospholipid syndrome.

Recurrent adverse pregnancy outcome may be the final result of different causes, including autoimmune diseases, as the Antiphospholipid Syndrome. Antiphospholipid antibodies (lupus anticoagulant and/or anticardiolipin antibodies) were found in 16% of 197 patients with prior unexplained recurrent miscarriages. During our study 22 out of 32 antiphospholipid antibodies positive women became pregnant again. To prevent abortion relapses, 16 of them were treated with acetylsalicylic acid (50 mg x 2/day) and/or fluocortolone (20 mg/day for 5 days/week). Such therapy started as soon as pregnancy was diagnosed in 14 patients. Two patients began the therapeutic regimen during the third month of gestation. Six patients, who didn't accept this therapeutic approach, represent our control group. All the 14 early treated patients ended pregnancy with success. The 2 women that began the therapy later presented abortion relapses. Among the 6 not treated patients, 5 presented spontaneous abortion and only one gave birth to a baby. No side effect was observed neither in treated mothers nor in their babies. In conclusion, even if further studies would be necessary to standardise a therapeutic protocol, our results encourage the clinical care of patients with antiphospholipid antibodies and adverse pregnancy outcomes.

Immunological abnormalities in a group of patients with limited cutaneous systemic sclerosis and prominent vascular disease.

Antinuclear antibodies, circulating immune complexes, rheumatoid factors and anticardiolipin antibodies were detected in the sera of 17 patients affected by the limited cutaneous subset of systemic sclerosis and marked clinical evidence of ischaemic cutaneous lesions (fingertip ulcerations). This study was designed to evaluate the possible role of anticardiolipin (aCL) antibodies and other immunological disorders in the endothelial damage characteristic of the disease. ACL antibodies were found in 41% of the patients. With the exception of a significant connection with positive rheumatoid factor tests (RIA), no notable associations between anticardiolipin antibodies and antinuclear antibodies, circulating immune complexes (CIC), and other serological abnormalities were found. ACL antibodies did not significantly correlate with the presence of vascular lesions in our patients. However, a role of these antibodies in endothelial damage cannot be excluded, possibly in association with other serum factors such as immune complexes and antinuclear antibodies. A positive connection between the incidence of CIC and the severity of lung perfusion impairment was observed, and the previously reported relationship between anticentromere antibodies and calcinosis was indirectly confirmed.

Leukocytes, eosinophils and complement function during hemodialysis with polysulphone and polymethylmethacrylate membranes: comparison with cuprophan and polyacrylonitrile.

The biocompatibility of the two new dialysis membranes, polysulphone (PS) and polymethylmethacrylate (PMMA), was evaluated versus cuprophan (CUP) and polyacrylonitrile (PAN) by studying the in vivo effects of the four different membranes on leukocyte counts, eosinophil levels and complement function both in the presence and absence of dialysis fluid. Complement function was also examined in vitro by studying the generation of chemotactic factors, whole complement activity and C3d serum conversion. Passive absorption of complement fractions by membranes has completed in vitro studies. PS, PMMA and PAN showed a higher biocompatibility than CUP, even if slight differences can be observed: PS showed a PAN-like biocompatibility pattern with a relatively high absorption of complement factors by the membrane and without complement activation. On the other hand, PMMA showed a CUP-like pattern and caused complement activation, even though to a lower intensity than CUP. PMMA biocompatibility appears to stand in-between CUP and the other two synthetic membranes PS and PAN. Our results confirm the important role played by membrane-induced complement activation on hemodialysis leukopenia. Dialysis fluid does not have a significant influence on membrane biocompatibility, but represents the major factor in determining intradialytic eosinopenia. Eosinophils seem to represent a more important marker of dialysis than of membrane biocompatibility.

Autoantibodies to human nuclear antigen(s)-HNA-in connective tissue diseases and other disorders.

Autoantibodies reacting with nuclear antigen(s) on human cells (HNA) with weak or without reactivity on nuclei of other species have been found by the indirect immunofluorescence technique used in routine tests for the diagnosis of autoimmune diseases. Precipitin lines were obtained by counterimmunoelectrophoresis (CIE) only when human lymphocyte extracts were used and not with rabbit thymus acetone powder. By comparison with reference sera, the autoantibodies directed to HNA were found to be different from SSA/Ro antibodies and did not give the fluorescence pattern of anti nuclear mitotic apparatus (NuMA) antibodies on HEp-2 cells. The prevalence of sera with anti-HNA antibodies not associated with other antinuclear antibodies (ANA) is low (about 0.7% of ANA found in routine assay). In association with ANA of other specificities, the prevalence of anti-HNA antibodies, demonstrated after absorption of sera with rat liver acetone powder, was higher (about 1% of ANA positive sera). By treatment with physicochemical agents and enzymes, the HNA was found to be a DNA (glyco)-protein complex extractable with saline solution, resistant to 56 degrees C for 6 h and stable at pH values ranging from 3 to 10. Anti-HNA antibodies were found in patients with mild connective tissue diseases, but also in idiopathic interstitial pneumonia and in chronic hepatitis.

Plasma exchange in the treatment of acute systemic lupus erythematosus without circulating immune complexes.

One patient affected by systemic lupus erythematosus with recurrent phlebothrombosis and peripheral neuropathy is described. False positive VDRL test, anticardiolipin antibodies, and high titres of antinuclear antibodies were present. Circulating immune complexes were not found. The effect of plasma exchange on clinical symptoms and on serological abnormalities was rapid and striking.

A solid phase immunoradiometric assay for gastric parietal cell antibodies.

We recently described a solid phase immunoradiometric assay (IRMA) for anti-thyroglobulin and anti-thyroid microsomal antibodies. In the present study a similar IRMA for gastric parietal cell antibodies (PCA) has been developed. Samples to be tested are incubated within wells of polyvinyl microtitre plates coated with solubilized gastric microsomal antigen. After removal of unbound material, PCA is detected by adding purified 125I-anti-human IgG antibody. A good correlation was found with the results of PCA assays obtained by indirect immunofluorescence. In contrast, negative PCA by IRMA were consistently obtained in sera containing high titres of several other organ specific and non-organ specific autoantibodies. PCA determinations by IRMA were than carried out in a series of normal controls and patients with autoimmune or non-autoimmune thyroid disorders. Positive results were obtained in three of 70 (4.3%) apparently normal subjects, in 16 of 87 (18.4%) patients with Hashimoto's thyroiditis, in 10 of 48 (20.8%) with idiopathic myxoedema, in 25 of 95 (25.6%) with Graves' disease and in five of 64 (7.8%) with other non-autoimmune thyroid disorders. Preliminary results showed that quantitative measurements of PCA by IRMA could be performed using a serum containing high levels of PCA as standard reference. In conclusion, PCA may be easily and specifically detected using the same IRMA procedure previously developed for anti-thyroid antibodies. We therefore suggest that the present IRMA may be proposed as a general technique for the detection of different organ specific autoantibodies.