Biomarker profiles in serum and saliva of experimental Sj?gren's syndrome: associations with specific autoimmune manifestations

Introduction  

Sj?gren's syndrome (SS) is a systemic autoimmune disease that mainly targets the exocrine glands. The aim of this study was to investigate the involvement of 87 proteins measured in serum and 75 proteins analyzed in saliva in spontaneous experimental SS. In addition, we intended to compute a model of the immunological situation representing the overt disease stage of SS.     

Cytostatic drug testing in human leukemias by means of multiparametric flow cytometry

Human bone marrow cells from 20 patients as well as the permanent human B-cell lines RPMI 1788, Raji, Daudi, T-cell lines Molt, CEM, Jurkat and the promyelocytic line HL 60 were assayed by means of a newly developed in vitro flow cytometric cytostatic drug assay. The cells were exposed to cytosine-arabinoside, L-asparaginase, daunorubicin, prednisone or vincristine. Surviving cells were stained after an incubation period of 2 to 7 days with esterase and pH-indicator dye ADB (1,4-diacetoxy-2,3-dicyanobenzene), dead cells with DNA-dye PI (propidium iodide). Dose-response curves were established using percent surviving cells. It was possible to evaluate bone marrow samples from 16 out of 20 patients. Seven samples were leukemic (acute myeloid leukemia (AML) n = 6, Non-Hodgkin's Lymphoma (NHL) n = 1). Nine samples were from patients either in complete remission or with benign diseases. Daunorubicin and cytosine-arabinoside were cytotoxic in both groups, whereas vincristine was effective mainly in the leukemic group (p less than 0.05). There was significant heterogeneity in the reactivity of AML-marrow cells from different patients to different drugs. The cell lines exhibited different patterns of sensitivity. Vincristine arrested cells in G2/M-phase, cytosine-arabinoside caused an increase of cells in the S-phase.     

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Cleavage of a hydrophilic C-terminal domain increases growth-inhibitory activity of oncostatin M.

Oncostatin M is a polypeptide cytokine, produced by normal and malignant hematopoietic cells, that has several in vitro activities, including the ability to inhibit growth of cultured carcinoma cells. Here we present a structural and functional comparison of two oncostatin M-related proteins (Mr 36,000 and 32,000) secreted by COS cells transfected with oncostatin M cDNA. The smaller of these forms lacked a hydrophilic C-terminal domain comprising predominantly basic amino acids. This domain was also absent from native oncostatin M produced by U937 cells. The 32,000-Mr form of oncostatin M was not produced by cells transfected with plasmids (G195 and G196) in which a potential trypsinlike cleavage site within the hydrophilic C-terminal domain was altered by site-directed mutagenesis. A 32,000-Mr fragment was produced by trypsin treatment of the 36,000-Mr form of oncostatin M. These observations suggest that the 32,000-Mr form of oncostatin M was derived from the 227-amino-acid propeptide by proteolytic cleavage at or near the paired basic residues at positions 195 and 196. Pro-oncostatin M was equally active in radioreceptor assays as the processed form but was 5- to 60-fold less active in growth inhibition assays. Likewise, nonprocessed mutant protein encoded by plasmid G196 was equally active in the radioreceptor assays as the processed form but was five- to ninefold less active in growth inhibition assays. Thus, the highly charged C-terminal domain of pro-oncostatin M is not required for receptor binding or growth-inhibitory activity but may alter the functional properties of the molecule. Propeptide processing of oncostatin M may be important for regulating in vivo activities of this cytokine.