Anti-carbonic Anhydrase II Antibodies in Systemic Sclerosis: Association with Lung Involvement

Carbonic anhydrase II (CAII) is expressed on alveolar epithelium and participates to CO 2 elimination, fluid secretion and post-capillary pH regulation. CAII is overexpressed in animal models of lung fibrosis in sites of epithelial injury. Autoantibodies directed against CAII (anti-CAII) have been described in sera from patients affected by systemic sclerosis (SSc), but no study focused on their clinical associations in this disease. The aim of this study was to assess the presence of anti-CAII in sera of SSc patients and to investigate their association with lung involvement. We performed ELISA to detect anti-CAII in 34 SSc patients who underwent pulmonary function tests (PFT) and Doppler echocardiography. We found increased prevalence and significantly elevated serum levels of anti-CAII in SSc patients affected by restrictive lung disease (RLD) compared to SSc patients without lung involvement and healthy controls. These findings suggest both a possible pathogenic role of anti-CAII in the development of lung damage and a potential clinical utility as serological marker of pulmonary involvement in SSc patients.     

The association of systemic sclerosis with malignant neoplasms]

The rate of concomitance of systemic sclerosis and malignant neoplasm is not still ascertained. Therefore we reviewed 8,327 patients who were diagnosed with systemic sclerosis and received public financial aid from the Ministry of Health, Labour and Welfare of Japan in 1999. The concomitance rate was 3.1% of all patients, however, it was more frequent among men than among women (5.4% for men and 2.8% for women, respectively, P<0.01), and the mean age of patients with complicated malignant neoplasms was significantly higher than that of patients without malignancy (P<0.01). For comparison with the Japanese general population, O/E ratios (ratio of observed-to-expected malignant neoplasms) were calculated, and it was found that both men and women had significantly higher O/E ratios (O/E ratio 2.31, 95% confidence interval [CI] 1.68-2.94, P<0.001 for men and O/E ratio 1.64, 95% CI 1.41-1.86, P<0.001 for women). In addition we also assessed laboratory findings including autoantibodies and respiratory function tests for patients with malignant neoplasms by logistic regression analysis adjusted for sex and age. Decreased DLco (pulmonary CO diffusing capacity) appeared to be a risk factor for the concomitant malignant neoplasms among patients with systemic sclerosis (odds ratio 2.00 for DLco < or = 70%, CI 1.06-3.74, P=0.032). These results may help to elucidate the etiology of systemic sclerosis.     

Anticardiolipin antibodies in systemic sclerosis: immunological and clinical associations

Anticardiolipin antibodies of IgG/IgM class were detected in seven of 28 patients with systemic sclerosis including five of 16 patients severely affected by extensive visceral disease. This severely affected sub-group also showed significant elevations of plasma levels of von Willebrand factor antigen in 10 cases and serum C1q binding activity in seven cases respectively. This triple association raises the possibility that multiple immunological mechanisms are involved in the pathogenesis of systemic sclerosis and its vascular lesions.     

Thrombocytopenia in systemic lupus erythematosus: association with antiplatelet and anticardiolipin antibodies

Serum platelet bindable immunoglobulin G (SPbIgG) and anticardiolipin antibodies (ACA) have been assayed in SLE patients with (group A, n = 8) and without thrombocytopenia (group B, n = 8). Moreover, we studied the binding of serum IgG on platelet proteins using a Western blotting procedure. Increased levels of SPbIgG were found in all thrombocytopenic patients and in five cases of group B. A binding of serum IgG on platelet proteins (80 and 53 kDa) was seen in seven of eight thrombocytopenic patients and in only one case of group B. ACA were present in the serum of four patients in each group and a significant inhibition of ACA IgG by entire washed platelets was achieved in only two severe thrombocytopenic cases. These data suggest that participation of ACA to the platelet destruction can be evoked for a few SLE patients, whereas recognition of platelet protein determinants by lupus autoantibodies is necessary to the thrombocytopenia.     

Elevated serum anti-carbonic anhydrase II antibodies in patients with ulcerative colitis

An autoimmune mechanism has been postulated for the pathogenesis of ulcerative colitis (UC). The aim of this study was to evaluate the presence of anti-carbonic anhydrase (CA) I and anti-CA II antibodies in a series of inflammatory bowel disease (IBD) patients. We studied 58 IBD patients [36 UC patients and 21 patients with Crohn's disease (CD)]. As a control, 24 healthy individuals and 12 patients with non-IBD diarrheal diseases were tested. Serum anti-CA I and anti-CA II antibodies were quantified by enzyme-linked immunosorbent assay. Anti-CA II antibody was detected in 27.8% of UC patients, whereas anti-CA I antibody was detected in only 5.6% of UC patients. Positive rate of anti-CA II antibody was significantly higher in UC patients as compared to the control. In CD patients and non-IBD diarrheal patients, there were no significant increase in positive rate of either anti-CA I or II antibody. These results suggest that autoimmune responses against CA II may be involved in the pathogenesis of UC, and similar mechanism may participate in the development of pancreatic lesions in UC patients.     

Cytokine and chemokine levels in systemic sclerosis: relationship with cutaneous and internal organ involvement

Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive collagen deposition in the skin and internal organs. Several cytokines and chemokines have been implicated in the induction of fibrosis, but a definitive relationship between specific cytokines and organ involvement has not been established yet. Serum samples, PBMC and T cell lines (TCL) obtained from 54 patients affected by SSc and 20 healthy donors (HD) were examined by ELISA for Interferon-gamma (IFN-gamma ), interleukin (IL)-4, IL-6, IL-10, IL-18, Transforming growth factor (TGF)-beta1, Tumour necrosis factor (TNF)-alpha, sCD30, Macrophage derived chemokine (MDC), Monocyte chemoattractant protein (MCP)-1, Macrophage inflammatory protein (MIP)-1alpha and Regulated on activation normal T-cell expressed and secreted (RANTES). In all the SSc serum samples, we found significantly increased levels of IL6, TNFalpha and MCP-1 but reduced amounts of gamma-IFN and MDC. IL6, IL10, IL18, MIP-1alpha and TNFalpha measured in supernatants from PHA-stimulated PBMC and IL6, MCP-1 and RANTES in supernatants from stimulated TCL were also increased in patients. MDC was decreased in all the biological SSc sources studied. TGF-beta1, IL10, and sCD30 were produced at a significantly lower level by SSc TCL. Serum IL6 and sCD30 levels were significantly increased in dc-SSc patients compared to lc-SSc as were levels of MCP-1 produced by PBMC and IL10 from TCL. We observed a strict relationship between pulmonary fibrosis and IL10, MCP-1 (both from TCL) and serum IL6. Kidney involvement was related to serum MCP-1 levels and IL18 production from PBMC. Oesophageal involvement correlated with MDC production from PBMC and IL10 synthesis by TCL. We showed that IL-6, IL-10, MDC and MCP-1 are variably associated with internal organ involvement and allow the discrimination between limited and diffuse forms of the disease.     

Autoimmunity and vascular involvement in systemic sclerosis (SSc)

Endothelial injury, obliterative microvascular lesions, and increased vascular wall thickness are present in all involved organs in scleroderma. The vascular pathology is associated with altered vascular function with increased vasospasm, reduced vasodilatory capacity and increased adhesiveness of the blood vessels to platelets and lymphocytes. The extent of injury and dysfunction is reflected by changes in the circulating levels of vascular markers. The initial triggers for the vascular pathology are not known. Possible viral triggers are visited here, including cytomegalovirus in view of increased levels of anti-CMV antibodies in scleroderma, and the remarkable similarities between CMV vasculopathies and scleroderma vascular disease. Endothelial apoptosis in scleroderma may be related to viral infection, immune reactions to viral or environmental factors, reperfusion injury or to anti-endothelial antibodies. The impact of the vascular pathology on the evolution of tissue fibrosis is not known; still, cytokines (TGFbeta, IL4), vascular factors (endothelin), and growth factors (PDGF) are possibly crucial signals that link the vascular disease to tissue fibrosis. Knowledge of the regulation of these and other factors will provide the opportunity to develop more rational therapeutic approaches to the disease.     

The association of HLA-B8 with visceral disease in systemic sclerosis.

Seventy-one patients with systemic sclerosis (SS) were typed for twenty-seven HLA alleles of the A and B loci, and the findings were related to both the extent of visceral disease and tests of cellular immune competence in a subgroup of fifty-two of these patients. Nineteen pa;ients with widespread visceral involvement and more rapidly progressive disease had an increased frequency of HLA-B8 (relative risk = 4.14; P less than 0.05) when compared to thirty-three less severely affected patients and 3000 controls. Patients with severe and progressive disease also had defective cell-mediated immunity with reductions in both the numbers of circulating thymus-dependent (T) lymphocytes and in the lymphocyte transformation response to phytohaemagglutinin. These findings suggest that a genetic factor, such as an abnormal immune response gene, may be involved in the progression of the disease.     

Clinical and serological associations with anti-RNA polymerase antibodies in systemic sclerosis.

There are three classes of RNA polymerase enzyme (RNAPs I, II and III). In systemic sclerosis (SSc), three main groups of anti-RNAP sera have been characterized by radioimmunoprecipitation techniques: anti-RNAP I/III sera, anti-RNAP I/II/III sera, and a group precipitating both RNAP II and topoisomerase I (topo I). Some sera in this third group precipitate the phosphorylated (IIO) form of RNAP II in the absence of the unphosphorylated (IIA) form. Certain other antinuclear antibodies (ANA) have also been detected in anti-RNAP IIO/IIA/topo I and anti-RNAP IIO/topo I sera. In the present study of 155 SSc patients, clinical features of individuals from each of these antibody groups were assessed and compared with those of patients from other autoantibody-defined groups. The anti-RNAP I/II/III antibody specificity was closely associated with the presence of diffuse cutaneous SSc (dc-SSc) (77.8%; cf. remaining group, 12.4%; P < 0.001; relative risk (RR) 6.3). Patients with anti-RNAP I/III antibodies also had an increased incidence of dc-SSc, but this was not significant (42.9%; cf. remainder, 15.7%). Anti-RNAP+ patients had a significantly increased incidence of renal involvement (29.0%, cf. remainder, 11.3%; P < 0.05; RR 2.6), with 40% of anti-RNAP I/II/III patients having renal disease. Meanwhile, the presence of anti-centromere antibodies (ACA) was associated with limited cutaneous SSc (lc-SSc) (100.0%; cf. remainder, 75.3%; P < 0. 005), together with reduced incidences of both renal disease (2.4%, cf. remainder, 22.1%: P < 0.01) and pulmonary fibrosis (21.4%, cf. remainder, 52.3%; P < 0.005; RR 1.9). Anti-topo I antibodies were associated with the presence of pulmonary fibrosis (69.7%; cf. remainder, 32.6%; P < 0.001; RR 2.1). A majority of anti-topo I sera were from lc-SSc patients, regardless of whether anti-topo I antibodies occurred alone (75.0%) or together with anti-RNAP IIO + IIA antibodies (75.0%), and this was similar to the remainder (86. 5%; NS). However, when anti-topo I+ patients were compared with the ACA group, and then with all anti-RNAP I+ patients (37.5% lc-SSc), significant differences were found in the occurrence of dc- versus lc-SSc (P < 0.005 and P < 0.05, respectively). In conclusion, these results confirm that there are three main groups of SSc sera, each characterized by the presence of a mutually exclusive SSc-specific autoantibody (ACA, anti-topo I or anti-RNAP I), and distinguished by patterns of cutaneous involvement and specific clinical features. It appears that, in each of the three groups of SSc patients, distinct pathological processes are occurring, which are responsible for the characteristic symptoms, for the modification of particular autoantigens and, consequently, for the production of particular autoantibodies. Based on these data, together with our previous results, it is further hypothesized that anti-RNAP II antibodies may be produced in the context of two different immune response pathways.     

Serum anti-carbonic anhydrase II antibodies and oxidant-antioxidant balance in pre-eclampsia

PROBLEM The aim of this study was to investigate the presence of anti-carbonic anhydrase II antibodies (anti-CA II) antibodies in pre-eclampsia and the relationships between the autoantibodies, total antioxidant capacity (TAC) and total oxidant capacity (TOC), malondialdehyde (MDA) and oxidative stres index (OSI) parameters. METHOD OF STUDY We studied 40 early and late onset pre-eclamptic patients and 40 healthy pregnant control and 39 healthy non-pregnant control subjects. Serum CA II antibodies, TAC and TOC, and MDA parameters were studied by ELISA. RESULTS The mean values for TAC, TOC, OSI, MDA, and anti-CA II were significantly increased in patients with pre-eclampsia compared to the other groups. The anti-CA II antibody levels for the pregnant control subjects were 0.129 ± 0.04 and that for the pre-eclamptic patients were 0.282 ± 0.18. In this study, any absorbance value higher than 0.136, the mean absorbance + 2 S.D. of pregnant control subjects, was defined as positive. Positive results were obtained in 29 of 40 pre-eclamptic patients (72.5%). There were significant positive correlations between serum anti-CA II antibodies and TOC, MDA levels, and OSI levels. CONCLUSION The results suggest that anti-CA II antibodies and impairment in oxidant-antioxidant balance may be involved in multifactorial etiology of pre-eclampsia.     

Human leukocyte antigen class II associations with systemic sclerosis in South Africans

Human leukocyte antigen class II typing, using polymerase chain reaction-sequence-specific primers, was performed in 52 Black South Africans with systemic sclerosis (SSc) and 112 controls. Increased frequencies of DR2 in the overall SSc group (OR = 2.4), DRB1*0301 in the limited cutaneous SSc (lcSSc) subset (OR = 9.0), and DQB1*0301/4 in the diffuse cutaneous SSc (dcSSc) subset (OR = 9.0) were observed. Pulmonary fibrosis was associated with DRB1*11 and anti-topoisomerase I antibodies were associated with DPB1*1301 and DRB1*15. Patients with anti-fibrillarin antibodies (AFAs) had increased the frequencies of DRB1*1101 allele group (OR = 16) and DQB1*0603/14 (OR = 13.6). These findings provide new serological and immunogenetic data on a previously unreported population. The association of AFAs with class II alleles merits further investigation.     

Anti-collagen antibodies in systemic sclerosis and in primary Raynaud's phenomenon.

The frequency and specificity of antibodies to native and denatured collagens were evaluated in systemic sclerosis (SSc) and in primary Raynaud's phenomenon (PRP) by direct and competitive ELISA. Antibodies reactive with denatured collagen type I (CI) were found in 43% of the SSc sera, and anti-CIV and anti-CV in 31%. In PRP, anti-CI, anti-CIV and anti-CV antibodies were detected in 8% of patient sera. Anti-CI, anti-CIV and anti-CV antibodies reacted with determinants expressed on the native as well as on the denatured molecule. Anti-CI and anti-CIV were cross-reactive; a reactivity with CII and a lower one with CV were detected. Anti-CV antibodies also reacted with CI and CII and, in a smaller proportion of cases, with CIV. Anti-collagen antibodies, affinity-purified from blotted collagen IV and V and cyanogen bromide (CBr)-digested CI, displayed the cross-reactivities shown by inhibition studies on sera. Moreover, antibodies eluted from a CBr fragment of CI reacted with the other CBr fragments as well. These data show that one-third of SSc sera contain antibodies that react with epitopes expressed on native as well as on heat-denatured CI, CII, CIV and CV, and therefore have the potential to bind collagens in vivo.     

IgG antibodies to human cytomegalovirus late protein UL94 in patients with systemic sclerosis

Human cytomegalovirus (HCMV) has been proposed as an amplifying agent for at least some of the spectrum of systemic sclerosis (SSc; scleroderma). In support of this hypothesis, antibodies to the HCMV late protein UL94 have been detected in the majority of SSc patients in a study involving Caucasian subjects from Italy. The aim of this investigation was to determine whether elevated levels of anti-UL94 antibodies are present in African American and Caucasian SSc patients from the U.S. We further wished to determine whether there was a significant difference in the levels of anti-UL94 antibodies between the diffuse and the limited forms of the disease. IgG antibodies to a UL94 peptide were measured in 254 Caucasian and 90 African American subjects by an enzyme-linked immunosorbent assay (ELISA). In both Caucasian and African American subjects, the mean antibody level in the diffuse form of SSc was significantly higher than that in the respective control subjects (714 vs. 466 ng/ml, p = 0.005; 1226 vs. 512 ng/ml, p < 0.0001). Also, among Caucasian SSc patients, the mean antibody level in the diffuse form of SSc was significantly higher than that in the limited form of the disease (714 vs. 465 ng/ml, p = 0.02). These results show that increased levels of antibodies to the HCMV late protein UL94 are associated with SSc and they may be a marker for the severity of the disease.     

Association of antinuclear and antinucleolar antibodies in progressive systemic sclerosis.

Antinuclear and/or antinucleolar antibodies were demonstrated in the sera of 74 of 76 patients (97%) with progressive systemic sclerosis, using tissue culture cells (HEp-2) as substrate in the indirect immunofluorescent method. Six patterns of nuclear staining and three nucleolar patterns were recognized. The nuclear patterns were centromere, fine speckles, coarse speckles, diffusely grainy, homogeneous and nuclear dots. The nucleolar patterns were speckled, homogeneous and clumpy. The results of digestion studies with ribonuclease, deoxyribonuclease and trypsin suggested that the nuclear antigens are proteins, some of which may be associated with chromatin. The nucleolar antigens appeared to be nucleic acid in nature. Certain characteristic serologic and clinical features associated with staining patterns were observed. The diffusely grainy pattern was seen only in sera containing antibody to Scl-70 antigen. Centromere staining was confirmed to be highly selective for the CREST (Calcinosis, Raynaud's phenomenon, esophageal involvement, sclerodactyly and telangiectasis) variant of progressive systemic sclerosis with rheumatoid factor titres higher in these patients with anti-centromere antibodies.     

Lung involvement in systemic sclerosis sine scleroderma treated by plasma exchange.

Systemic sclerosis sine scleroderma can present in some patients as pulmonary interstitial fibrosis. Until now ten cases with this particular clinical variant, all men, have been reported in the literature. The knowledge of systemic sclerosis sine scleroderma presenting as lung interstitial involvement is important in clinical practice for an early diagnosis and correct therapeutic strategy. This work reports the clinico-serological features of two further cases, one a woman, of systemic sclerosis sine scleroderma with prevalent lung involvement, and describes the effects of therapeutic plasma exchange.