Immunoadsorption for collagen and rheumatic diseases.

The field of therapeutics has seen remarkable progress in the recent years, which has made mainstream drug treatment possible for collagen and rheumatic diseases. However, treatment of intractable cases where drug effectiveness is poor is a challenge. Furthermore, organ damage, concurrent illnesses or allergic reactions make adequate drug therapy impossible. For such cases, therapeutic apheresis is very significant, and it is important how this should be valued related to drug therapies. Therapeutic apheresis for collagen and rheumatic diseases involves the removal of factors that cause and exacerbate the disease; the aim of immunoadsorption, in particular, is to improve the clinical condition of patients with autoimmune disease by selectively removing pathogenic immune complexes and autoantibodies from their plasma. Immunoadsorption, in particular, unlike plasma exchange and DFPP, utilizes a high-affinity column that selectively removes autoantibodies and immune complexes, leaving other plasma components intact. There is no need to replenish fresh frozen plasma or blood products such as albumin and gamma globulin preparations. Immunoadsorption is thus superior in terms of safety, as the risk of infection or allergic reaction relating to these preparations can be avoided. We anticipate future investigations of application of synchronized therapy using drugs and therapeutic apheresis, most notably immunoadsorption, in combination to treat intractable clinical conditions such as collagen and rheumatic diseases. In this paper, our discussion includes the indications for immunoadsorption such as collagen and rheumatic diseases, the relevant conditions and types, as well as the latest understanding related to methods and clinical efficacy.

[Molecular diagnosis of collagen vascular diseases and vasculitides]. / Molekulare Diagnostik der Kollagenosen und Vaskulitiden.

Collagen vascular diseases and vasculitides comprise various diseases, which may affect virtually every organ system. Therefore, their diagnosis and management is often an interdisciplinary challenge. Because of the heterogeneous symptoms, these diseases have significant overlap, which interferes with the clinical diagnosis and may require additional investigation. Therefore, a rational and comprehensive diagnostic work-up should be performed at the initial presentation before initiation of therapy. The detection of antinuclear (ANA) or anticell antibodies by indirect immunofluorescence microscopy on Hep2 cells is used to screen for autoantibodies in collagen vascular diseases. The molecular specificity of autoantibodies should be further characterized using immunoassays with recombinant or purified protein. When systemic autoimmune disease is suspected, the function of the frequently affected organs should be evaluated. The immunopathological findings should always be interpreted in the context of clinical, histological, and imaging data. The detection of autoantibodies is helpful for the initial diagnosis, provides prognostic information, may indicate involvement of organs or systems and some parameters may also be used for disease monitoring. The clinical significance of autoantibodies is emphasized by the fact that their detection constitutes diagnostic criteria for most collagen vascular diseases and several vasculitides. The screening for ANCA may be performed using immunoassays with recombinant myeloperoxidase and proteinase 3 or by indirect immunofluorescence microscopy on granulocytes. In this article, the current diagnostic tools and their relevance for the diagnosis and monitoring of systemic autoimmune diseases with primary skin involvement are reviewed.

Risk of cytomegalovirus reactivation in patients with immune-mediated inflammatory diseases undergoing biologic treatment: a real matter?

The use of biological agents has grown exponentially in immune-mediated inflammatory diseases (IMID), often achieving a good control of disease progression and improving patients' quality of life. However, their use resulted in an increased risk of adverse events, including reactivation of chronic/latent infectious diseases. As for the risk of Cytomegalovirus (CMV) reactivation, very few data are available. We reviewed the literature reporting cases of CMV infection in IMID patients during biological therapy. Although the risk of CMV reactivation cannot be excluded, we concluded that there is no evidence to warrant CMV screening before starting a biological agent.

NK cell populations in collagen vascular disease.

OBJECTIVES: Pulmonary involvement of varying etiology is common in collagen vascular diseases (CVDs). Bronchoalveolar lavage fluid (BALF) cell differentials reveal information on the immune mechanisms involved in the CVDs. The aim of the present study was to evaluate BALF cell populations in CVD-associated ILD and to investigate possible correlation with pulmonary function. METHODS: Fifty-seven patients (26 male and 31 female, mean age ± SD: 54.68±12.18 years) with CVD-associated interstitial lung disease were studied. Patients were divided into 6 groups based on underlying CVD. The study population also included a group of 10 healthy controls. BALF was examined in all individuals. Cell density, total cell number and differential cell count were recorded. BALF lymphocyte subsets were analysed by dual flow cytometry. Pulmonary function was assessed in all patients. RESULTS: BALF differential cell count did not differ significantly among the different groups. Scleroderma patients showed the highest percentage of CD19 cells (p<0.001). The NK and NKT cell percentages were significantly higher in systemic lupus erythematosus and in Sjögren, respectively, compared to other CVDs and controls (p=0.001 and p<0.001). Also BALF neutrophil percentage correlated negatively with FVC (r=-0.356, p=0.011) and FEV1 (r=-0.336, p=0.017) and BALF NKT cell percentage correlated negatively with pO2 (r=-0.415, p=0.003). CONCLUSIONS: Important variations observed in BALF cell populations suggest the implication of NK and NKT cells in the pathogenesis of lung involvement in CVDs.

Allogeneic disparities in immunoglobulin-like transcript 5 induce potent antibody responses in hematopoietic stem cell transplant recipients.

In hematopoietic stem cell transplant (HSCT) recipients, the recognition of polymorphic antigens by the donor-derived immune system is an important mechanism underlying both graft-versus-host disease and graft-versus-leukemia (GVL) effect. Here we show that a subset of HSCT recipients (13.9%, n = 108) have antibodies directed to surface molecules of dendritic cells. We have used one such serum in conjunction with retroviral expression cloning to identify the highly polymorphic surface molecule immunoglobulin-like transcript 5 (ILT5) as one of the targets of dendritic cell-reactive antibodies. ILT5 reactive antibodies were found in 5.4% of HSCT patients but not in solid organ transplantation recipients, patients with collagen diseases, multiparous women, or polytransfused or healthy persons. We show that ILT5-specific antibodies can mediate killing of ILT5-bearing cells and furthermore demonstrate ILT5 expression in some leukemic cells, indicating that it might be a target for GVL effects. Thus, our results represent the first description of potent allogeneic antibody responses to a non-major histocompatibility complex cell surface molecule in hematopoietic stem cell transplanted patients and warrant further studies to elucidate the role of antibodies to polymorphic cell surface molecules in GVL and graft-versus-host responses.

Identification of α-tubulin as an autoantigen recognized by sera from patients with neuropsychiatric systemic lupus erythematosus.

In a previous study we found in 50% of patients with neuropsychiatric manifestations of systemic lupus erythematosus (NP-SLE) organ specific antibodies to 45-56 kD proteins in a 100,000 g supernatant (SN) from bovine brain mitochondria. Aim of the present study was to identify the corresponding target antigen. A 100,000 g SN from bovine brain mitochondria was applied to SDS-gel electrophoresis. A 50 kD band recognized by sera from patients with NP-SLE in the Western blot (WB) was excised from the gels and applied to mass spectrometry. The identified protein was expressed in Escherichia coli and retested against sera from eleven patients with NP-SLE (severe symptoms n=6, mild symptoms n=5), 26 SLE-patients without NP manifestations and 53 controls (patients with multiple sclerosis, epilepsy, healthy blood donors). Mass spectrometry of the 50 kD band revealed the presence of α-tubulin. Applying the recombinant α-tubulin in the WB, four of the eleven NP-SLE patients (36%), one of the 26 patients with SLE without NP manifestations (4%) and none of the 53 controls reacted with α-tubulin. The antibodies were more frequently found in patients with severe (50%) than with mild NP-SLE (20%). α-tubulin may be a novel marker autoantigen for a neuropsychiatric manifestation at least in a subgroup of patients with SLE. Whether anti-α-tubulin antibodies are of pathogenetic relevance has still to be clarified.

[Non-specific interstitial pneumonia (NSIP)]. / Nicht spezifische interstitielle Pneumonie.

Non-specific interstitial pneumonia (NSIP) belongs to the group of idiopathic interstitial pneumonias (IIP). However, NSIP can also be found in several other diseases. For example, the NSIP pattern is most commonly found in interstitial lung disease due to connective tissue disease. In this review, the definition and classification, aetiology, pathogenesis and histology, clinical symptoms, serological markers, lung function parameters, radiographic signs, treatment, and prognosis of NSIP are presented. Idiopathic NSIP as a distinct form of NSIP will be discussed separately.

Prognostic indicators related to death in patients with Pneumocystis pneumonia associated with collagen vascular diseases.

The objective of this study is to investigate the clinical markers of life-threatening Pneumocystis pneumonia (PCP) in patients with collagen vascular diseases (CVD). The patients who contracted Pneumocystis jeroveccii were retrospectively selected from our medical charts and conditions related to the patients' death were reviewed. The findings indicated that lower levels of serum albumin and cholinesterase, increased alveolar-arterial oxygen gradient, intratracheal intubation, and necessity to treat in the intensive care unit were significantly related to deaths associated with PCP in CVD. A special attention should be paid to decreased serum albumin and cholinesterase as ominous predictors in PCP occurred in patients with CVD.

[Anti-B-cell strategies in vasculitides and collagenoses]. / Anti-B-Zell-Strategien bei Vaskulitiden und Kollagenosen.

B-cells play a central role in the pathogenesis of autoimmune diseases. As already discussed in other articles, besides the production of potentially pathogenic autoantibodies, B-cells may function as antigen-presenting cells, may induce T-cell activation and produce various cytokines. The feasibility of targeting B-cells in patients with severe and refractory autoimmune disorders, especially in patients with vasculitis or connective tissue diseases, has met growing interest among rheumatologists in recent years. The use of rituximab as a monoclonal antibody directed against CD20 positive B-cells has been reported in case reports and small patient series; however, these are hard to compare as different diseases are described and different doses and schedules of rituximab were used. It has to be considered that positive reports are more likely to be reported than patients who do not improve or experience side effects. Data on only a few indications from randomized, double-blind studies are available; however, even these results should be evaluated critically.

An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus erythematosus.

Double diffusion in agarose was employed for the characterization of a soluble nucleoprotein antigen that gave precipitin reactions with sera from patients with systemic lupus erythematosus. The soluble antigen that was isolated from calf thymus nuclei was demonstrated by enzyme degradation and ultracentrifugation studies, and by immunologic analysis, to be a complex of deoxyribonucleic acid and a moiety susceptible to proteolytic digestion. Antibody to soluble nucleoprotein did not react with the DNA portion of the complex released after proteolytic digestion or with purified calf thymus DNA. Immunologic reaction of identity was obtained between the soluble nucleoprotein antigen and synthesized DNA-histone complex, suggesting that the protein moiety of soluble nucleoprotein might in part be composed of histone. Antibody to soluble nucleoprotein was present in 51% of systemic lupus erythematosus sera examined and was more common than antibody to deoxyribonucleic acid.

Antibodies to cellular antigens in Sjögren's syndrome.

An extract of human lymphocytes from continous cell culture was used as the antigen source to detect antibodies in sera of patients with Sjögren's syndrome (SS). Using double diffusion in agarose, 85 per cent of a selected group of patients had precepating antibodies. Three precipitating antigen-antibody systems were detected and were shown to be different from those described previously in othe systemic rheumatic diseases. The SS precipitating antibodies were temporarily classified as precipitins, A, B, and C. SS patinets with sicca syndrome but without clinical rheumatoid arthritis had precipitin systems A and/or B, and SS patients with associated rheumatoid arthritis had precipitin system C. Serum reactants were demonstrated by immuno-electrophoresis to migrate in the gamma globulin region. The precipitating activity of the serum factors was not destroyed by treatment with 2-mercaptoethanol and was not removed by absorption of rheumatoid factor from the sera. The reactivity of the lympuocyte antigens was destroyed by treatment with trypsin but not by deoxyribonuclease or ribonuclease.

The cytotoxicity of leukocytes and lymphocytes from patients with rheumatoid arthritis for synovial cells.

Unseparated peripheral blood leukocytes obtained from patients with rheumatoid arthritis (RA) were cytotoxic for synovial cells. The cytotoxic reactions produced by RA leukocytes were more frequent and of greater magnitude than cytotoxicity induced by leukocytes from normal persons and patients with other diseases, primarily connective tissue diseases. Furthermore, the cytotoxic activity of RA leukocytes was greater for RA synovial cells than for nonrheumatoid synovial cells, in contrast to the cytotoxicity of other leukocytes, which did not discriminate between synovial cells according to their origin. Tests with purified lymphocytes showed that the cytotoxicity of unseparated leukocytes directed against RA synovial cells was due to lymphocyte cytotoxicity. These data are consistent with the possibility that sensitized lymphocytes from patients with RA recognize a distinctive antigen present on rheumatoid synovial cells.

Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy.

Using a hemagglutination test which can detect antibodies to (a) native and denatured deoxyribonucleic acid (DNA) and (b) an extractable nuclear antigen (ENA), a comparative study of patterns of autoantibody formation has been done in systemic lupus erythematosus (SLE) and related rheumatic diseases. Antibody to native DNA was present in the serum in 96% of patients with active SLE and disappeared during remissions. Antibody to ENA was found in 86% of those patients with SLE nephritis who responded to treatment but in only 8% of those who did not. The highest titers of antibody to ENA were found in patients having a mixed connective tissue disease syndrome with features of SLE, scleroderma, and myositis. The latter syndrome was notable for the absence of renal disease and for a striking responsiveness to corticosteroid therapy. Hemagglutination testing of 277 sera from normal persons and patients with a wide variety of acute diseases other than SLE revealed the presence of antibody to native DNA in only 1.4% and antibody to ENA in only 0.4%. These results yield significant correlations among the pattern of autoimmune reactivity, the clinical form of the rheumatic disease, and responsiveness to treatment. They implicate the qualitative nature of the patient's immune response as a conditioning factor in the type of disease. Together with other correlations they may allow classification of rheumatic diseases into more biologically meaningful groups and lead to more selective methods of therapy.

[Cardiovascular disease in early collagen diseases]. / Alterazioni cardiovascolari nelle fasi precoci delle malattie del connettivo.

AIM: The purpose of this paper was to verify whether there is any sign of involvement of the cardiovascular system in the early stages of collagen diseases. METHODS: Seventeen patients (10 female and 7 male, average age 41.35 +/- 9.85 years) (group A) recruited at the Ambulatory of Internal Medicine for suspected collagen diseases with period of onset of the symptomatology less than 6 months, were analyzed. Ten patients were excluded from the study: 8 had been suffering from systemic lupus erythematosus (SLE) for a number of years, 2 were older than 80 and were suffering from concomitant pathologies (diabetes mellitus and hypertension) which would have invalidated the evaluation of valvular changes like thickening. The patients were followed up for 2 years. Clinical diagnosis was made in many cases many months after the observation using the criteria of the American Rheumatic Association (ARA). All patients were subjected to titration of the following autoantibodies by means of the immuno-fluorimetry method: ANA, anti-ENA (SSA, SSB, SM, SM-RNP, SCL-70, Jo-1), anti-nDNA, anti-histones. The cardiological evaluation was carried out by echography (Cardioline 12 leads) and echocardiographic examination (Aloka 2000 and HP sonos 5500 with 2.5 and 3.5 MHz probe) looking for thickening of both valvular flaps (> 3 mm for the mitral and > 2 mm for the aorta), myocardial involvement by studying global and regional kinesis of the left ventricle; pericardial involvement. The control group consisted of 17 healthy subjects with the same sex and age distribution (10 male, 7 female, average age 40.35 +/- 9.80 years) (group B). RESULTS: Eleven patients (64%) proved to be suffering from SLE, 3 (17%) from mixed collagen diseases (MC), 3 (17%) from systemic sclerosis (SS). Cardiac anomalies were observed in 12 patients: in 3 (17%) mitral valve thickening was observed (2 with SLE, 1 with SS), in 2 (11%) thickening associated with mitral valve insufficiency (with MC), in 1 (5%) isolated mitral valve insufficiency (with SLE), in 1 (5%) thickening and slight aortic insufficiency (with SLE), in 1 (5%) mitral valve vegetations (with SLE), in 2 (11%) pericardial effusion (with SLE), in 2 (11%) diastolic changes (with SS). The parameters relative to wall thickness between the 2 groups showed statistically significant differences (mitral 3.1 +/- 0.7 vs 2.3 +/- 0.4 P = 0.0005; aorta 1.7 +/- 0.2 vs 1.5 +/- 0.3 P = 0.03). CONCLUSIONS: In patients observed in the early stages of collagen diseases, cardiac involvement was observed in 70% of cases, but the data require confirmation in a larger sample. The authors, however, believe that the early identification of such involvement is useful from both the diagnostic point of view and from the point of view of patient treatment.