Persistent expression of a soluble form of Fas/APO1 in continuously activated T cells from a patient with SLE.

OBJECTIVE: To report a patient with SLE whose T cells expressed disproportionally increased amounts of an alternatively spliced form of Fas/APO1 transcript and secreted a soluble form of Fas. METHODS: We established continuously activated, short-term T cell lines from 16 patients with SLE and from 6 normal controls. The structure, expression and function of Fas was examined using RT-PCR and sequencing, flow cytometry (surface expression of Fas), ELISA (measurement of soluble Fas) and a PI-based cytotoxicity assay (functional analysis). RESULTS: A soluble form of Fas which originates from an alternatively spliced transcript and lacks the transmembrane domain of the original molecule was the dominant product of the Fas-gene in one line (S18B) derived from a patient with very active SLE. Compared to a control line, the S18B cells displayed decreased surface Fas expression but increased accumulation of Fas inside the cell. The amount of soluble Fas in the culture supernatant of S18B was found to be 1.8 times higher than that of a control line. Culture supernatants from S18B cells inhibited anti-Fas mAb-medicated T cell death. CONCLUSION: Continuously activated T cells from one patient with SLE displayed increased amounts of soluble Fas that inhibits anti-Fas mediated cell death. Although the frequency of this abnormality among patients with SLE and other diseases is unknown, increased production of soluble Fas may have contributed to the pathogenesis of SLE in the patient presented here.

Evidence for a factor in normal human serum that induces human neutrophilic granulocyte end-stage maturation in vitro.

The end-stage maturation of neutrophilic granulocyte precursor cells isolated from normal human bone marrow by Ficoll density centrifugation was studied in a liquid culture assay system used previously to study the maturation of guinea pig granulocyte precursors. Dialyzed normal human serum induced end-stage morphological maturation of human granulocyte precursors and this induction was proportional to a serum level of up to 5.0% in the assay medium. At serum concentrations greater than 5.0% a pronounced inhibition of maturation was observed. Passage of serum through a DEAE-Fractogel 650S column equilibrated with 0.01 M phosphate buffer (pH 7.0) resulted in the binding of the end-stage granulocyte maturation factor to the column. The activity eluted from the column in a fraction containing 17% of the starting serum protein that was inhibitor-free and was also capable of inducing the appearance of granulocyte alkaline phosphatase, a specific biochemical marker for granulocyte end-stage maturation. GMF is most likely a protein since it was destroyed by protease digestion. The data also indicate that neither purified human transferrin nor human recombinant granulocyte colony-stimulating factor can substitute for human serum GMF as a granulocyte end-stage maturation factor in this assay system. It was observed, however, that purified human transferrin greatly potentiated the effect of GMF suggesting that transferrin plays a supporting role in the end-stage maturation of human granulocytes in vitro. To our knowledge the evidence presented here indicates for the first time the existence of a neutrophilic granulocyte end-stage maturation factor in normal human serum.

Progressive loss of phenotypic proteins in mature granulocytes before the onset of blast crisis in human chronic myelogenous leukemia.

A combined high-performance liquid chromatography-sodium dodecyl sulfate-gel electrophoresis method for the study of the phenotypic protein patterns of mature blood granulocytes was previously described. With the use of this method in the present study, the progression of human chronic myelogenous leukemia (CML) from the stable to the blast crisis stage was shown to be accompanied by a progressive decrease in the amounts of cell membrane and granule phenotypic proteins in mature granulocytes. Survival time from the initial diagnosis was significantly shorter for CML patients whose levels of granulocyte phenotypic proteins were below the normal range compared with survival time for those patients whose levels were normal or higher than normal. The data suggest that these changes in mature granulocytes serve as useful diagnostic indicators of an impending blast crisis in CML patients.

Direct analysis of differentiation proteins in normal and leukemic human granulocytes by high-performance liquid chromatography.

The feasibility of the use of reverse-phase high-performance liquid chromatography (RP-HPLC) for detecting differences in the protein phenotypes of highly purified fractions of normal and chronic myelogenous leukemic (CML) mature granulocytes isolated from peripheral blood was determined. At least 21 protein peaks were consistently and reproducibly detected in the RP-HPLC profiles of acetonitrile-trifluoroacetic acid extracts of normal granulocytes. This assay takes only 60 minutes to perform and can be done on 4 X 10(6) granulocytes (approximately the number of granulocytes in 1 ml normal blood). Furthermore, sodium dodecyl sulfate-gel electrophoresis of the RP-HPLC fractions provides a second dimension to the analysis of the polypeptide pattern of these cell lysates. Analyses of subcellular fractions by these methods revealed that most of the major peaks in the RP-HPLC profiles of intact granulocytes originate mainly from the granule and membrane fractions. Although the protein phenotypes of mature granulocytes were remarkably uniform among normal individuals, those of mature granulocytes obtained from the blood of CML patients were consistently abnormal and varied considerably among individual patients. The results indicate that the approach used here could have useful application in the study of abnormal granulocyte differentiation in leukemia.

Comparison of folylpolyglutamate hydrolases of mouse liver, kidney, muscle and brain.

The folylpolyglutamate hydrolase activities of mouse liver, kidney, muscle and brain were examined by incorporation of methylenetetrahydrofolate polyglutamate reaction products into a stable ternary complex with tritiated fluorodeoxyuridylate and L. casei thymidylate synthetase. Complexes were separated electrophoretically on the basis of charge associated with the polyglutamyl moieties to determine distribution of chain lengths throughout the time course of the reaction. Tissue folylpolyglutamate hydrolase activities were allowed to utilize endogenous folylpolyglutamate as substrates by incubating crude tissue extracts at pH 7.4 ang pH 4.5. Kidney and muscle contained relatively reactive hydrolases which were capable of generating intermediates of essentially all chain lengths from folylpentaglutamate, the predominant endogenous species. The relatively low activity in brain also gave rise to all possible intermediates. Liver contained a high concentration of methylenetetrahydrofolate but little hydrolase activity. The activity present in liver gave rise to essentially no intermediates but yielded only the monoglutamate form of the cofactor. When purified lysosomal preparations from liver and kidney were allowed to react with synthetic folylpolyglutamates, the same specificity with regard to reaction products was observed as with endogenous substrates.

Splenectomy in chronic myelogenous leukemia.

There were no deaths or major complications in three chronic myelogenous leukemia patients in group 1 who were selected under well controlled conditions and had the spleen removed. Intensive chemotherapy in the treatment of chronic myelogenous leukemia patients who undergo splenectomy remains to be resolved. Well controlled prospective clinical trials are now being conducted to evaluate the value of splenectomy in chronic myelogenous leukemia.

Direct counting and sizing of mitochondria in solution.

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 micro aperture is 0.48 micro diameter, or 0.056 micro(3) particle volume The mean volume of rat liver mitochondria was 0.42 micro(3) or 0.93 micro in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10(3), and per gram of rat liver was about 11 x 10(10). These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 micro(3) and from rat kidney cortex, 0.23 micro(3). These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a / a(3)), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement