T-cell depletion of bone marrow does not inhibit in vitro hematopoiesis

One approach to overcome the problem of histoincompatibility in bone marrow transplantation is to use T cell depleted marrow from a haploidentical donor in an attempt to ameliorate graft-versus-host disease. Since the T cell requirements for normal hematopoiesis are uncertain, experiments were performed to study the effects of E rosette-T cell depletion on in vitro growth of hematopoietic progenitor cells. Marrow mononuclear cells were cultured in a modified CFU-GEMM assay before and after T cell depletion. The number of 7 day granulocytic and erythrocytic colonies, and 14 day granulocytic, erythrocytic and mixed colonies were enumerated and expressed in terms of colonies per 10(5) non T cells plated. T cell depletion did not result in decreased proliferation of any of these progenitors save possibly for 14 day granulocytic colonies in one of four experiments. In two cases, T cell depletion resulted in increased growth of progenitor cells. Three of four patients transplanted with T cell depleted haploidentical marrow cells engrafted. It is concluded that E rosette depletion of T cells from marrow does not decrease the potential of these cells to establish hematopoiesis in vitro or in vivo.     

Purine pyrophosphorylase as a selective genetic marker in a mouse lymphoma, P388, in cell culture

It is possible to use the purine pyrophosphorylase in mammalian cell culture systems as a genetic marker in selecting small numbers of enzyme positive cells from large populations of pyrophosphorylase negative cells of the mouse lymphoma line P388 in medium containing amethopterin, hypoxanthine, glycine and thymidine. Conversely, it is readily possible to obtain pyrophosphorylase-deficient cells by treatment with 8-Azaguanine. We were unsuccessful in demonstrating DNA-mediated transformation using DNA from enzyme positive cells incubated with cells which were enzyme negative.     

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.     

Immunochemical Characterization of Plaque Mutants of Simian Virus 40

Analysis of large and small plaque mutants of simian virus 40 using antisera prepared against each has revealed quantitative and possibly qualitative antigenic differences for each plaque type. A sensitive micro radioisotope precipitation test permitted evaluation of immunochemical similarities and differences of capsid antigens by inhibition of precipitation.     

A new enzyme-coupled spectrophotometric method for the determination of arginase activity

A spectrophotometric method for the determination of arginase (EC 3.5.3.1) is presented. Arginase is coupled to urease and glutamate dehydrogenase and the decrease in absorbance at 340 nm due to the oxidation of NADPH is followed. The method is rapid, is sensitive, is economical and permits continuous monitoring. The initial velocities were directly proportional to the enzyme concentrations between 0.06 and 0.30 units per 0.5 ml. The Lineweaver-Burk plot yielded positive allosteric behavior for the tetrameric enzyme. The K' and the Hill coefficient, n, calculated from Hill plot were found to be 4.7 mM and 1.26 (r = 1.00), respectively. These values are in good agreement with the literature.