A rabbit polyclonal antibody raised against human being CD95L and a mouse monoclonal antibody against human being CD3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). analyzed. In both instances aberrant overexpression of the soluble CD95 receptor isoform and deviations from normal TCR V-gene utilization normalized in parallel with the medical improvement. Furthermore, soluble CD95 was identified as a survival element for eosinophils rescuing eosinophils from apoptosis in the TIMP1 absence of growth factors Given the part of eosinophils as effector cells in CSS, these findings suggest that soluble CD95 may be mechanistically involved in the disease. Churg-Strauss Syndrome (CSS) was for the first time explained by Lotte Strauss and Jacob Churg in 1951 in associated with peripheral polyneuropathy. Consecutive immunosuppressive treatments led to medical improvement within one month. Patient WI was recently described 16 and is a 48-year-old female having a 22-yr history of asthma. She offered in 1992 for the first time with severe alveolar hemorrhage. During the following two years there were three recurrences of pulmonary hemorrhage due to lung infarctions. In 1995, multiple cutaneous ulcerations appeared together with episodes of severe gastrointestinal bleeding. In 1997, twelve ulcerations of the top gastrointestinal tract and four ulcerations of the colon were found. The ulcerations showed considerable eosinophilic infiltrates and granulomatous lesions. In parallel to the eosinophilic vasculitis and eosinophilic lung infiltrates, the counts of eosinophils in the peripheral blood were significantly improved ( 10%). Because of severe gastric hemorrhage due to multiple ulcerations, gastrectomy and Y-Roux reconstruction were performed. The patient was treated with corticosteroids (prednisone 1 mg/kg/day time) for 14 days, followed by significant medical improvement. After discharge, immunosuppressive treatment with azathioprine (5 mg/kg/day time) was continued for about 6 months. In 1998 the patient relapsed with ulcerations of the skin and an esophageal residue of gastric mucosa. Cells and Blood Samples From patient WI, five independent cells samples were from normal gastric mucosa and from ulcerative gastric mucosa at the time of gastrectomy. After recurrence of the disease 1 year later on, a biopsy from an ulcerative esophageal residue of gastric mucosa was acquired. In peripheral blood from eight CSS individuals and seven healthy volunteers, erythrocytes were selectively lysed using an erythrocyte lysis buffer (Qiagen, Hilden, Germany) and lymphocytes were recovered by centrifugation. From individuals WI and LM blood samples were also available during and after immunosuppressive treatment. From patient PF peripheral blood eosinophils were isolated and purified as explained below. In addition, peripheral blood eosinophils were purified from one 24-year-old male patient (DA) Clemizole with infectious eosinophilia (25% eosinophilic counts) like a control and from three healthy donors. Anti-neutrophil cytoplasmic antibody (ANCA) titers were determined by using Clemizole immunofluorescence test. Purification of Peripheral Blood Eosinophils Eosinophils were purified from peripheral blood from nonallergic healthy donors (= 3), one individual with infectious eosinophilia (individual DA, 25% eosinophilic counts) and one CSS-patient (individual PF, 32% eosinophilic counts) using Percoll (Pharmacia, Uppsala, Sweden) denseness gradient centrifugation and bad selection with an anti-CD16 monoclonal antibody and immunomagnetic beads coated with goat anti-mouse IgG (Miltenyi Biotec, Bergisch Gladbach, Germany) relating to Hansel et al. 18 The purity of eosinophils based on light microscopic examination of the purified cells after staining with Diff-Quick (American Scientific Products, McGraw Park, IL) was 95% and viability was 98% as assessed by trypan blue (Sigma, Deisenhofen, Germany) exclusion. After purification, eosinophils were suspended in RPMI-1640 medium and supplemented with 10% fetal bovine serum (FBS). Reagents and Antibodies RPMI 1640 medium and FBS were purchased from Biochrom (Berlin, Germany), phytohemagglutinin (PHA) from Seromed (Berlin, Germany), and all other chemicals from Sigma (Deisenhofen, Germany). Oligonucleotides were synthesized by MWG-Biotech Clemizole (Ebersberg, Germany). A rabbit polyclonal antibody raised against human CD95L and a mouse monoclonal antibody against human being CD3 were purchased from Santa Cruz.
A rabbit polyclonal antibody raised against human being CD95L and a mouse monoclonal antibody against human being CD3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
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