Distinct phenotypes in mixed connective tissue disease: subgroups and survival.

The aim of the present study was to assess the autoantibody profile, dominant clinical symptoms and cluster characteristics of different mixed connective tissue disease (MCTD phenotypes. Two-hundred-and-one patients with MCTD were followed-up longitudinally. Five clinical parameters, Raynaud's phenomenon, pulmonary artery hypertension (PAH), myositis, interstitial lung disease (ILD), erosive arthritis and five auto-antibodies besides anti-U1RNP, antiendothelial cell antibodies (AECA), anti-CCP, anti-cardiolipin (anti-CL), anti-SSA/SSB and IgM rheumatoid factor (RF) were selected for cluster analysis. The mean age of patients was 52.9 ± 12.4 years and the mean follow-up of the disease was 12.5 ± 7.2 years. Patients were classified into three cluster groups. Cluster 1 with 77 patients, cluster 2 with 79 patients and cluster 3 with 45 patients. In cluster 1 the prevalence of PAH (55.8%; p < 0.001), Raynaud's phenomenon (92.2%; p < 0.001) and livedo reticularis (24.6%, p < 0.001) was significantly greater than in cluster 2 and 3. In cluster 2, the incidence of ILD (98.7%; p < 0.001), myositis (77.2%; p < 0.001), and esophageal dysmotility (89.8%; p < 0.001) was significantly greater than that in cluster 1 and 3. In cluster 3, anti-CCP antibodies were present in 31 of 45 patients (68.8%) with erosions. Anti-CCP antibodies were present in 37 of 42 patients (88.0%) with erosions. PAH, angina, venous thrombosis was observed in cluster 1 and pulmonary fibrosis in cluster 2, musculosceletal damage, gastrointestinal symptoms and osteoporotic fractures were most frequent in cluster 3. Cumulative survival assessment indicated cluster 1 patients having the worst prognosis. Cluster analysis is valuable to differentiate among various subsets of MCTD and useful prognostic factor regarding the disease course.

Assessment of IgG antibodies to oxidized LDL in patients with acute coronary syndrome.

OBJECTIVES: Circulating IgG antibodies to oxidized low-density lipoprotein (anti-oxLDL) have been implicated in the development of atherosclerotic plaques. In this study, we investigated the prognostic value of IgG anti-oxLDL antibodies in patients with acute coronary syndrome (ACS). METHODS: In total 54 patients with ACS and 41 matched healthy controls were involved in this prospective study. Serum IgG anti-oxLDL levels were assessed by ELISA. RESULTS: Higher IgG anti-oxLDL levels were found in patients with ACS versus controls (22.8 ± 23.3 vs. 7.5 ± 5.27 EU/ml, p < 0.0001). IgG anti-oxLDL concentrations were significantly higher in ACS patients with unstable clinical complications (circulatory insufficiency, malignant arrhythmias, recurring ischaemic pain, positive stress-test, need for urgent coronary intervention or sudden cardiac death) versus those without such complications (30.0 vs. 11.7 EU/ml, p < 0.001). Twelve patients (22%) were taking statins. Patients on statins had a significant reduction in clinical complications (33%) versus patients not receiving statin therapy (61%). IgG anti-oxLDL levels were also different in these two groups (11.4 vs. 25.8 EU/ml, respectively; p = 0.03). Serum IgG anti-oxLDL levels correlated with the subsequent development of unstable coronary events. Levels of anti-oxLDL significantly decreased in response to statin therapy, independently of its lipid-lowering effect. CONCLUSIONS: Anti-oxLDL antibodies are involved in ACS. The association of anti-oxLDL with unstable clinical complications may indicate the role of this antibody in plaque destabilization.

Characteristics of interstitial lung disease in SS-A positive/Jo-1 positive inflammatory myopathy patients.

The strongest predictive factor for the development of interstitial lung disease (ILD) in myositis (IIM) patients is the presence of different antisynthetase antibodies. The aim of this study was to compare the clinical characteristics, radiological findings and therapeutic response between the anti-SS-A positive and negative antisynthetase syndrome (ASS) patients. A prospective study of 315 IIM patients was conducted including 27 anti-Jo-1 positive ASS patients. Mean disease duration was 46.6 (range 4-198) months. All patients fulfilled the classification criteria for IIM. All patients underwent chest radiography, pulmonary function tests and HRCT at he time of diagnosis and 6 months after the immunosuppressive therapy. Routine laboratory tests, RF, ANA, anti-ENA, anti-SS-A, anti-histidyl-transfer RNA antibody (Jo-1) measurements were performed in all patients. ILD was found to be present in 70.4% of ASS patients. The anti-SS-A negative ASS group had a more frequent association with alveolitis and responded well to immunosuppressive therapy (p < 0.05). HRCT scan showed more fibrosis in the SS-A positive group. 15.8% of patients died due to pulmonary or cardiac complications. In conclusion, coexistence of anti-SS-A and anti-Jo-1 antibody may be a good predictor for a more coarse and severe ILD in IIM patients who require a more aggressive approach in therapy.

Diagnostic significance of HLA-DQ typing in patients with previous coeliac disease diagnosis based on histology alone.

BACKGROUND: Coeliac disease is strongly associated with human leukocyte antigen (HLA)-DQ2 or DQ8 genotypes. The diagnosis is based on demonstrating crypt-hyperplastic villous atrophy, endomysial or transglutaminase antibodies and correlation of disease activity with gluten intake. AIM: To evaluate the clinical utility of HLA-DQ typing, when coeliac disease diagnosis had previously been established solely by histology. METHODS: HLA-DQ alleles, endomysial and transglutaminase antibodies were investigated and histology slides reviewed in 70 patients diagnosed 2-25 years earlier by small-intestinal biopsy but without measuring endomysial or transglutaminase antibodies. Patients without DQ2 or DQ8 or without unequivocal villous atrophy were followed-up on free diet by using serology and biopsies. RESULTS: All 40 endomysial/transglutaminase antibodies positive patients carried DQ2 or DQ8, and 39 of them had severe villous atrophy. Only 56% of patients without endomysial or transglutaminase antibodies positivity had DQ2 or DQ8 (P < 0.001). Seropositivity and relapse developed in 4 of 11 DQ2 positive but in none of 15 DQ2 and DQ8 negative patients on long-term gluten exposure. CONCLUSIONS: Coeliac disease diagnosis based solely on histology is not always reliable. HLA-DQ typing is important in identifying DQ2 and DQ8 negative subjects who need revision of their diagnosis, but it does not have additive diagnostic value if endomysial positivity is already known.

CR1 density polymorphism and expression on erythrocytes of patients with systemic lupus erythematosus.

The present study investigated the expressed number of CR1 on erythrocytes (E) in relationship of the CR1 density genotype from 46 patients with systemic lupus erythematosus (SLE) and 47 healthy volunteers. The CR1 genotype was determined by a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of 1.8 kb separated by HindIII endonuclease digestion and agarose gel electrophoresis. Our data supported the earlier results that the number of binding sites/E for monoclonal anti-CR1 decreased among SLE patients compared with normal individuals having the same alleles for the CR1/E density. At the same time the novelty of our recent results was that the decreased expression of CR1 on E correlated significantly with kidney involvement in patients homozygous for the CR1/E high density allele (HH). These data suggest that the deficiency of the detectable number of CR1 on erythrocytes is acquired in this SLE population.

Serum complement activation of SLE patients during plasmapheresis.

The erythrocyte complement receptor 1 (ECR1)-immune complex binding assay is a sensitive method for the determination of complement fragments which can be activated by bovine serum albumin (BSA)-anti-BSA in vitro. When the C3b/C4b containing bovine serum albumin (BSA)-anti-BSA was formed in the presence of the serum of patients with systemic lupus erythematosus (SLE) its binding to ECR1 was found to be lower than that formed in sera of normal volunteers. The plasmapheresis of SLE patients homozygous for the CR1/E high density allele displays a beneficial effect on the formation of C3b/C4b containing BSA-anti-BSA and its binding to ECR1. There was no significant correlation between the serum C3/C4 level and the percentage of C3b/C4b containing BSA-anti-BSA binding to the ECR1 of SLE patients during plasmapheresis. At the same time, there was an inverse correlation between the serum immune complex level and the ECR1 binding, which was significant in 3 of 5 cases. These data suggest that, besides the determination of different components of complement activation, the functional assay of complement activation might be useful in monitoring the effect of plasmapheresis in SLE.

Anti-platelet antibodies against gpIIb/IIIa in systemic sclerosis.

An enzyme-linked immunosorbent assay and western immunoblotting analysis were used to detect anti-platelet antibodies in the sera of 50 patients with systemic sclerosis. Eleven (22%) positive sera were found by ELISA. Serial investigations showed that the presence of these antibodies was often transient. Immunoblotting analysis demonstrated that the antibodies were directed against a 114 kDa antigen. Using monoclonal antibody, anti-platelet antibodies directed against gpIIIa were found in 4 cases. Similar to immune thrombocytopenia and lupus erythematosus, a significant portion of the anti-platelet antibodies were also directed against the glycoprotein complex IIb/IIIa in systemic sclerosis.

Anti-endothelial cell antibodies in mixed connective tissue disease: frequency and association with clinical symptoms.

OBJECTIVE: Anti-endothelial cell antibodies (AECA) have been described in a number of systemic autoimmune-inflammatory diseases. However, little is known about the relationship of AECA with mixed connective tissue disease (MCTD). METHODS: Using an ELISA, the presence of AECA was evaluated in the sera of 33 patients with MCTD and of 30 healthy subjects as controls. Serum levels of AECA were correlated with clinical activity, as well as the existence of various organ manifestations. RESULTS: Significantly increased AECA production was observed in MCTD patients (OD = 0.337+/-0.193) compared to controls (OD = 0.136+/-0.065). In addition, patients with active MCTD exerted significantly elevated serum AECA levels (OD = 0.487+/-0.090) than did patients with inactive MCTD (OD = 0.135+/-0.040) or controls. MCTD patients with pulmonary hypertension had a tendency of increased serum AECA levels (OD = 0.452+/-0.080) compared to patients without this manifestation (OD = 0.307+/-0.039). Sera of MCTD patients with AECA concentrations higher or lower than the mean serum AECA level in controls+2SD (OD = 0.266) were considered as AECAhigh (n = 19/33) and AECAlow (n = 14/33), respectively. Interestingly, all patients with active disease had AECAhigh, while all inactive MCTD patients had AECAlow sera. IgG purified from ten MCTD sera (OD = 0.415+/-0.290) showed a tendency to up-regulate E-selectin expression on cultured human umbilical vein endothelial cells (HUVEC) compared to IgG from control sera. In addition, AECAhigh MCTD sera exerted significantly increased stimulatory effect on endothelial E-selectin expression (OD = 0.651+/-0.190) compared to AECAlow (OD = 0.178+/-0.110) or control sera (OD = 0. 131+/-0.080). CONCLUSION: AECA may activate endothelial cells by the up-regulation of E-selectin expression and thus may be implicated in the pathogenesis of MCTD. Furthermore, serum AECA may be a useful marker of endothelial activation and clinical activity in this disease.

Recombinant human erythropoietin modulates erythrocyte complement receptor 1 functional activity in patients with lupus nephritis.

Deposition of immune complexes (IC) is an important step in the pathogenesis of lupus nephritis. Impairment of IC-clearance contributes to the accumulation of IC. It may be partly attributed to decreased complement containing immune complex (ICC) binding by erythrocytic complement receptor 1 (ECR1). Stimulating erythropoiesis with recombinant human erythropoietin (rHuEPO) may enhance the IC-clearance as increasing ECR1 expression and/or functional activity. Ten anemic patients with lupus nephritis were treated with 50 IU rHuEPO (Eprex) per kg body weight three times a week during a five week period. ICC-binding capacity of ECR1 was determined with 125I-labelled, C3ib containing BSA-anti-BSA complexes. In addition to effective correction of anemia, indicated by increased red blood cell count (RBC), hemoglobin concentration and reticulocyte ratio, rHuEPO significantly improved decreased ECR1 functional (ICC-binding) activity in patients with lupus nephritis. This improvement correlated with the increase in reticulocyte ratio. Although patients were kept on their previous therapy during Eprex administration, their clinical condition also improved. That was shown by a decrease in Westergreen ratio, serum creatinine concentration and anti-dsDNA level and also by an increase in creatinine clearance. Results suggest a beneficial immune modulatory effect of rHuEPO in lupus nephritis.

[Anti-Saccharomyces cerevisiae antibodies in patients with Crohn's disease]. / Saccharomyces cerevisiae elleni antitest elófordulása Crohn-betegségben.

Inflammatory Bowel Diseases are a group of diseases with chronic inflammation of the gastrointestinal tract, but without proven etiology. Immunologic, environmental, infective and genetic factors equally can play role in their development. Antibodies to an oligomannose epitope of the Saccharomyces cerevisiae demonstrated in 60-70% of the patients with Crohn's disease. The origin and the clinicopathological role are not clarified. It is important that there are no surveys with patients suffering in gluten sensitive enteropathy in the literature. As there are no ASCA survey in Hungary, the aim of this study was to determine the prevalence of the ASCA. The authors examined at their patients the ASCA's occurrence and compared with the clinical picture of the Crohn's disease. The results supported the theory that ASCA positivity correlates with small intestines' Crohn's disease and in these cases both the IgG and IgA type antibodies proved. The antibodies in the sera at the analyzed ASCA positive cases prove a systemic immune response against Saccharomyces cerevisiae and the authors suggest the end of the oral tolerance against the yeast's antigens. The diet restriction (elemental diet, total parenteral nutrition, and fecal diversion) may ameliorate the status of the patients with Crohn's disease. It is speculated that the yeast-free diet as a part of the therapy for the ASCA positive patients can be reasonable: moreover the permanent "forbidding" of the yeast can be an acceptable alternative in case of getting well.

Investigation of monoclonal gammopathy of undetermined significance: a single-centre study.

Summary The main characteristic of monoclonal gammopathies (MG) is the presence of an increased amount of both electrophoretically and immunologically homogeneous immunoglobulins (M component). According to the WHO classification, monoclonal gammopathies are classified among the non-Hodgkin's lymphomas as 'plasma cell dyscrasias'. The unknown behaviour state, so-called MGUS (monoclonal gammopathy of undetermined significance), is distinguished from the malignant diseases. We investigated the frequency and features of MG and MGUS by reviewing the serum immunochemistry protein analyses between 1998 and 2004. Among 18,642 analyses, MG was found in 1,983 cases (10.39%) derived from one or more samples of 416 patients. Case histories of 340 patients were analysed. A malignant lymphoproliferative disease was proved in 171 cases, while in 169 cases the behaviour of the gammopathy was unknown. In 65 cases, the disease was possibly not related to MG. Mean follow-up time of the 65 patients with MGUS was 42 (9-81) months. During the follow-up period seven patients progressed into a malignant lymphoproliferative disorder-- mean probability of the malignant transformation was 3.07%/year and it occurred more frequently in the presence of immunoglobulin A isotype. There was no correlation between the progression of the disease and other laboratory findings. Besides the analyses of MG-related diseases, we focus on the malignant transformation of MGUS and on the importance of regular clinical and laboratory control examinations.

Clinical and immunoserological characteristics of mixed connective tissue disease associated with pulmonary arterial hypertension.

We investigated the clinical characteristics and immunoserological alterations in patients with mixed connective tissue disease (MCTD) associated with pulmonary arterial hypertension (PAH). Anti-U1RNP autoantibodies, anti-endothelial cell antibodies (AECA) and serum thrombomodulin (TM) as well as von Willebrand factor antigen (vWFAg) concentrations were measured in 25 patients with MCTD associated with PAH and in 154 MCTD patients without PAH. The results showed that the probability of survival was lower in MCTD patients with PAH than in the 154 MCTD-non-PAH patients (5-year survival rate in MCTD with PAH: 73%, versus 96% in MCTD-non-PAH; P < 0.01). AECA were more frequently present in the sera of MCTD patients with PAH than in MCTD-non-PAH (P < 0.001). Serum TM and vWFAg levels were higher in MCTD-PAH patients than in MCTD-non-PAH patients (TM: P < 0.001; vWFAg: P < 0.001). Significant correlation was noticed between the quantity of AECA and TM level (r = 0.466) as well as the quantity of AECA and vWFAg level (r = 0.550). In conclusion, our results suggest that in MCTD the presence of AECA and endothelial cell activation may play a role in the development of PAH and in the maintenance of obliterative vascular processes.

Antibodies against extractable nuclear antigen in non-Hodgkin lymphoma patients.

Autoantibodies are found at higher frequency in malignant lymphoproliferative diseases and also the association of these diseases with autoimmunity is documented. However precise mechanisms are not yet understood beyond these findings. We measured anti-extractable nuclear antigen (ENA) antibodies in non-Hodgkin's lymphoma patients before, during and after chemotherapy and compared these values to healthy controls. Sixty six lymphoma patients' data were compared with 30 healthy patients' data. ENA levels were significantly elevated in untreated lymphoma patients compared with healthy controls (1.85 U/l versus 0.68 U/l, P < 0.05). This increase could be observed during and after treatment as well. Those patients who responded well to initial chemotherapy were demonstrated with gradually increasing ENA antibody titers compared with the rest of patients, where a gradual decrease in titer was found. These findings are not yet statistically significant, but may help us further understand immunological reactions beyond the treatment of malignant lymphomas.

Evaluation of interstitial lung disease in mixed connective tissue disease (MCTD).

OBJECTIVE: Interstitial lung disease (ILD) may be a characteristic, often serious, manifestation of mixed connective tissue disease (MCTD). In this retrospective study, the frequency and clinical picture of ILD were determined in patients with MCTD using two diagnostic tests: high-resolution computed tomography (HRCT) and inhaled aerosol clearance times of (99m)Tc-labelled diethylene-triamine pentaacetate ((99m)Tc-DTPA). In addition, pulmonary function, effects of therapy and a variety of immunoserological markers were also assessed. METHODS: One hundred and forty-four consecutive patients with MCTD were selected from the clinic, irrespective of the presence or absence of ILD. All patients underwent a detailed clinical assessment, chest HRCT scanning, chest radiography, inhaled aerosol of (99m)Tc-DTPA clearance times, and all pulmonary function tests. Patients who had active ILD received corticosteroid (CS) or CS in combination with cyclophosphamide (CPH). All investigations were repeated after 6 months of immunosuppressive therapy. RESULTS: Ninety-six out of 144 MCTD patients (66.6%) had active ILD, 75 of this group (78.1%) showed ground glass opacity, 21 patients (21.8%) ground glass opacity with mild fibrosis with HRCT. Forty-five patients with active ILD received 2 mg/kg/day CS for 6-8 weeks alone and 51 patients CS in combination with CPH (2 mg/kg/day). Six months later, after therapy, 67 out of 96 MCTD patients with ILD (69.8%) showed a negative HRCT pattern, ground glass opacity with mild fibrosis developed in 15 patients (15.6%), and fibrosis was detected in 13 patients (13.5%). Only one patient showed subpleural honeycombing. (99m)Tc-DTPA was rapid in all 96 MCTD patients with active ILD (28.7 +/- 8.2 min, normal value >40 min). After therapy the (99m)Tc-DTPA was normalized, 79 out of 96 patients (82.3%). Carbon monoxide diffusion capacity (DLCO) was reduced in 33 out of 96 MCTD patients with active ILD (34.3%), while there were no significant differences in the pulmonary function tests between the active versus inactive stage of ILD or versus patients without ILD. The sera of 96 MCTD patients with active ILD contained a high level of immune complexes (ICs), and the total haemolytic complement levels (CH50/ml U) decreased. After 6 months of therapy, the IC levels decreased and CH50/ml levels normalized (MCTD patients before and after active ILD: IC optical density = 355 +/- 227 vs 206 +/- 92, P<0.001; CH50/ml, 38.0 +/- 12.6 U vs 64.3 +/- 13.0 U, P<0.001). CONCLUSIONS: HRCT is the gold standard for diagnosis of ILD. However, we used another method, (99m)Tc-DTPA, in order to compare this technique with HRCT. This latter technique has not been studied previously in MCTD. The elevated levels of IC and increased complement consumption indicated that IC-mediated alveolocapillary membrane damage and tissue injury might play a role in the pathogenesis of ILD in MCTD.

A sensitive simple ELISA for quantitation of sensitizing IgG from dissolved erythrocytes.

A simple enzyme-linked immunosorbent assay has been developed for quantitating the rabbit IgG present on the surface of sensitized red blood cells. A suitable cell lysis was carried out by an alkaline buffer, which dissolved the erythrocytes without forming any precipitate and without disruption of IgG, and facilitated the dissociation of the immune complexes, i.e. the erythrocyte-anti-erythrocyte rabbit IgG. In this alkaline buffer of pH 11.4 the IgG adsorbed directly into wells of microtitration plates unprecoated with anti-rabbit IgG. The detection was performed with alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate as substrate. Constructing a calibration curve from rabbit IgG made possible the calculation of molecules of IgG bound per red cell. The method was sensitive for the detection of fewer than 500 bound IgG molecules per erythrocyte.

Triggering of respiratory burst by phagocytosis in monocytes of patients with systemic lupus erythematosus.

The triggering of the respiratory burst by phagocytosis via different receptors in monocytes of patients with systemic lupus erythematosus (SLE) was investigated. The superoxide anion synthesis was assayed by reduction of ferricytochrome C that was inhibited by superoxide dismutase. The mononuclear cell suspensions were triggered by IgG-coated latex, C3 complement fragment-coated and uncoated yeast (Saccharomyces cerevisiae). Superoxide generation induced by phagocytosis via Fc gamma R was decreased in monocytes of patients with SLE. On the other hand, MoAbs against Fc gamma RI, Fc gamma RII and especially CR3 could also induce superoxide anion synthesis. At the same time, superoxide generation induced by anti-CR3 could be inhibited with C3-coated yeast.

Measurement of complement components and alpha 1-antitrypsin during plasma exchange.

Therapeutical plasma exchange can be carried out by using blood cell separators based upon centrifugation or in equipments with membranes. We investigated whether activation of the complement system took place in polyvinylchloride tubes used in the blood cell separator. We also examined the changes in the classical and alternative complement pathways by an analysis of functional haemolytic titers, as well as the levels of C4, C3D, C4, albumin and alpha 1-antitrypsin. No measurable activation of the complement system was found. The decreased levels of the C3, C3d, C4, albumin and alpha 1-antitrypsin in the sera after PE could be a consequence of the haemodilution.

Flow cytometric assay of phagocytic activity of human neutrophils and monocytes in whole blood by neutral red uptake.

In a new, simple, and fast flow-cytometric method for the simultaneous measurement of phagocytic activity of human neutrophils and monocytes in whole blood, the fluorescence capability of the well-known vital stain, neutral red was used. The incubation of 0.5 ml heparinized human blood with the phagocytic stimulus of zymosan dose- and time-dependently increased the percentage and the red fluorescence intensity of both neutrophils (4.3 +/- 1.2 times) and monocytes (2.7 +/- 0.7 times) measured cytofluorimetrically. Decreased uptake of neutral red was observed in a patient with phagocytic disorder, based upon impaired engulfment of particles and production of reactive oxygen species. In a patient with chronic granulomatosis, however, no decrease of neutral red uptake was measured. Platelet activating factor and phorbol myristate acetate were also able to increase the uptake of neutral red by both monocytes and neutrophils, but to a lesser extent than zymosan. The advantage of this method is the possibility for the simultaneous measurement of phagocytic activities of monocytes and neutrophils stimulated by either particles or soluble activators. This method is suitable for the selective measurement of activation processes not related to the production of free radicals in the phagocytes.