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Biomedical subjects

R Clift

Publications and source records attributed to R Clift.

At least 73 records · Page 4Linked to original sources
PubMed1985

Effects of in vitro depletion of T cells in HLA-identical allogeneic marrow grafts.

We report results of a pilot study designed to evaluate the effects of in vitro depletion of T lymphocytes from donor marrow in patients receiving HLA-identical marrow grafts for treatment of hematologic malignancies. Twenty patients aged 31 to 50 years were prepared for transplantation with cyclophosphamide (120 mg/kg) and fractionated total body irradiation (12.0 or 15.75 Gy). All received cyclosporine after grafting. The donor marrows were treated with a mixture of eight murine monoclonal antibodies and rabbit serum complement in a manner that achieved a 2- to 3-log depletion of T cells in most patients. Initial engraftment occurred promptly in 19 of the patients, and only three had clinically significant acute graft-versus-host disease. Depletion of donor T cells, however, was associated with an increased incidence of graft failure, which occurred as late as 244 days after transplantation. Graft failure was transient in one patient but apparently was irreversible in seven others. Three of the seven patients had cytogenetic but not morphological evidence of leukemic relapse at the time of graft failure. All seven patients with irreversible graft failure have died, six after receiving second bone marrow transplants. Seven of the eight cases of graft failure occurred among the 11 patients prepared for transplantation with 12.0 Gy of total-body irradiation, and only one occurred among the nine patients with advanced malignancies who received 15.75 Gy of total-body irradiation. This association with irradiation dose suggests that host factors were partly responsible for the graft failures. Because graft failure seldom occurs in irradiated recipients of unmodified HLA-identical allogeneic marrow transplants, it appears that T cells in the donor marrow may serve a beneficial function in helping to maintain sustained engraftment possibly by eliminating host cells that can cause graft failure. Optimal application of in vitro manipulation of donor marrow as a method for preventing graft-versus-host disease will require more effective immunosuppression of the recipient in order to assure sustained engraftment and function of donor stem cells.

Adult↗
PubMed1985

Studies on the purification of thrombopoietin from kidney cell culture medium.

A thrombocytopoiesis-stimulating factor (TSF) has been purified from human embryonic kidney (HEK) cell culture medium. In the initial purification step, crude HEK cell culture medium was fractionated with saturated ammonium sulfate (step I). The proteins precipitated by 40% to 60% and 60% to 80% ammonium sulfate saturation increased the percent of sulfur 35 incorporation into platelets of assay mice (P less than 0.01). The ammonium sulfate-precipitated proteins that contained significant TSF activity were further refined on Sephadex G-75 columns (step II). The fraction containing the highest specific activity (greatest 35S incorporation into platelets of assay mice per milligram of protein) was further purified by diethylaminoethyl (DEAE)-cellulose column chromatography (step III). TSF activity was eluted from the columns between 0.3 and 1.0 mol/L NaCl. Additional Sephadex chromatography of post-DEAE-chromatographic preparations further increased the purity of the TSF (step IV). TSF from this four-step procedure was further processed on a DEAE-high-performance liquid chromatography (HPLC) column (step Va) or size exclusion (SE)-HPLC columns (step Vb). After HPLC, the activity was localized in a region corresponding to a retention time of 6 to 8 minutes for the DEAE-HPLC, but longer times were found after SE-HPLC. TSF was further purified by additional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and SE-HPLC (step VI). The final product had significant TSF activity and represented a purification of approximately 500,000-fold. It was also shown that the isoelectric pH of partially purified TSF was 4.7 and the molecular weight of the more highly purified preparation was approximately 32,000. After extraction by a combination of chromatographic procedures, a single homogeneous product was obtained.

Ammonium Sulfate↗
PubMed1983

Transfusion requirements after HLA-identical marrow transplantation in 82 patients with aplastic anemia.

Red blood cell (RBC) and platelet (PLT) transfusion requirements during the first 13 weeks after HLA-identical marrow transplantation were studied in 82 patients with aplastic anemia. On the average, patients were given 9 units of RBCs (1-82) and 44 units of PLTS (6-468). The greatest need for support was during the first 4 weeks postgrafting. A multivariate statistical analysis of 22 variables showed that RBC and PLT requirements increased with age. In addition, RBC requirements increased if the patient had isohemagglutinins against the marrow donor's RBC, i.e., if the marrow transplant was across a major ABO barrier.

ABO Blood-Group System↗
PubMed1982

The effect of granulocyte transfusions on the incidence of cytomegalovirus infection after allogeneic marrow transplantation.

The association of granulocyte transfusions with cytomegalovirus infection and interstitial pneumonitis in allogeneic marrow transplant recipients was reviewed. Data from 387 patients with aplastic anemia or acute leukemia receiving transplants from human leukocyte antigen-matched siblings were analyzed. Acquisition of cytomegalovirus infection was higher in recipients of prophylactic and therapeutic granulocytes than in control subjects. The incidence was significantly different, however, only in cytomegalovirus seronegative patients who received granulocytes from seropositive granulocyte donors. There was no significant increase in the total incidence of or mortality from all forms of interstitial pneumonitis.

Adolescent↗
PubMed1982

Analysis of late infections after human bone marrow transplantation: role of genotypic nonidentity between marrow donor and recipient and of nonspecific suppressor cells in patients with chronic graft-versus-host disease.

Infections occurring 6 mo or later after bone marrow transplantation for severe aplastic anemia or hematologic malignancy were analyzed in 98 long-term survivors. Varicella-zoster (VZ) infections were analyzed separately from all other infections. The factor predisposing most strongly to late VZ infection was genotypic nonidentity for HLA between marrow donor and recipient. There was a suggestion that chronic graft-versus-host disease (GVHD) associated with the presence of nonspecific suppressor cells also predisposed to late VZ infection, while age less than 10 yr was protective against such infections. Chronic GVHD predisposed to late non-VZ infections, but this was not increased by the presence of nonspecific suppressor cells. HLA nonidentify between patient and marrow donor further increased the risk of late non-VZ infections over and above that due to the presence of chronic GVHD. Receipt of a syngeneic transplant appeared protective for late non-VZ infections. These findings suggest that full genotypic identity for HLA between donor and recipient may be required for optimal immune reconstitution after marrow transplantation and may denote a possible biologic role for nonspecific suppressor T cells in humans.

Acute Disease↗
PubMed1981

Characterization of a thrombocytopoietic-stimulating factor from kidney cell culture medium.

The chemical characteristics of a thrombocytopoietic-stimulating factor (TSF or thrombopoietin) found in serum-free kidney cell culture medium were further delineated by subjecting the TSF-rich medium to varying temperatures, different pH, and trypsin digested; the ability of TSF to bind lectins on affinity chromatography was also determined. After treatment, the TSF was assayed in immunothrombocythemic mice by its ability to increase the incorporation of 35S-sodium sulfate into newly formed platelets. TSF appeared to be relatively heat stable; incubation of TSF for 16 h at temperatures of 4, 37, and 56 degrees C showed no loss of TSF activity. However, after incubation at 85 degrees C, TSF was completely inactivated TSF in culture medium was stable of pH 1-8. Above these pH values, the potency of the TSF material decreased sharply. Digestion of TSF with trypsin completely destroyed the thrombocytopoietic-stimulating activity. For TSF purification, two different lectin-agarose derivatives were used; i.e., wheat germ agglutinin (WGA) and concanavalin A (Con A). Both lectins bound TSF, and the hormone was eluted by the sugars specific for the particular lectin. lectins, therefore, can be used to partially purify the hormone; a further 10 to 200-fold purification was achieved by these techniques. Since other workers have shown that TSF from plasma of thrombocytopenic rabbits will bind WGA and Con A, TSF from kidney cell culture medium and TSF from animal sources appear to have similar carbohydrate compositions.

Agglutinins↗
PubMed1979

Assay for thrombopoietin: a comparison of time of isotope incorporation into platelets and the effects of different strains and sexes of mice.

Several workers have used mice for thrombocytopoiesis-stimulating factor (TSF) assays, but the methods have differed. In quest of the optimum TSF assay conditions, we investigated the effects of interval between isotope injection and measurement of 35S incorporation into platelets, different mouse strains and sexes of mice. The 35S incorporation into platelets of mice increased with increase of the interval after isotope injection. However, the greatest difference in radioactivity between control and TSF-injected mice occurred at 16-40 h for normal mice and 24 h for rebound-thrombocytotic mice. When C3H, BALB/c or B6D2F1 mice were injected with platelet specific antisera, similar degrees of thrombocytopenia and rebound-thrombocytosis occurred. After injection of a standard dose of TSF, C3H and B6D2F1 mice showed greater isotopic incorporation levels, compared to suitable controls, than did BALB/c mice. Female and male mice exhibited essentially the same response to a standard dose of TSF. The data show that the mouse strain and isotopic incorporation time influence the sensitivity of the TSF assay but sex of test animals does not.

Animals↗
PubMed1978

Effects of short-term hypoxia on platelet counts of mice.

Recent studies have shown that long-term hypoxia causes decreased platelet counts in mice and short-term hypoxia increased platelet counts. In an attempt to explain the mechanism that increases platelet counts of mice after exposure to short-term hypoxia, we measured platelet counts, total circulating platelet counts (TCPC), total circulating platelet masses (TCPM), percentages of 35S incorporation, and platelet sizes. Platelet counts, as well as TCPC and TCPM of mice, increased after 1-3 days of hypoxia, but these values were decreased after 6-7 days of hypoxia. Although platelet counts were increased in hypoxic mice, the percentage 35S incorporation into platelets and platelet sizes did not show a concurrent increase. After 6 days of hypoxia, average platelet diameters began to increase as platelet counts decreased. Splenic release did not account for the increase in platelet counts of mice after short-term hypoxia. It seems possible, therefore, that megakaryocytes "shed" platelets into the circulation in response to hypoxia. The platelets that enter the circulation in response to short-term hypoxia are smaller and incorporate less 35S than platelets that are produced in response to acute thrombocytopenia.

Animals↗
PubMed1977

Hematologic changes and thrombopoietin production in mice after X-irradiation and platelet-specific antisera.

Sera or plasma thrombopoietin (TSF) levels of mice were determined after: (a) no treatment; (b) induction of thrombocytopenia by injection of rabbit anti-mouse platelet serum (RAMPS); (c) exposure to 750 R or 900 R whole-body x-irradiation; or (d) irradiation and injection with RAMPS. Levels of TSF were assayed in thrombocythemic mice, using Na235SO4 uptake. RAMPS produced an immediate, severe thrombocytopenia without altering RBC or WBC counts of mice. Plasma collected from mice 4 hours after RAMPS injection increased both 35S incorporation into platelets (170% of control, P less than 0.005) and platelet counts (P less than 0.025) of TSF-assay mice. Although severe thrombocytopenia persisted, plasma TSF levels decreased thereafter, i.e., 111% of control after 8 hours and 99% of control after 16 hours. Platelet counts in mice exposed to 750 R and 900 R x-rays progressively decreased to severe thrombocytopenia by day 7, but almost normal RBC counts were maintained. Sera or plasma from animals with x-ray-induced thrombocytopenia caused significant increases in 35S incorporation into platelets of TSF-assay mice (196% of control, P less than 0.005 after 750 R and 141% of control, P less than 0.025 after 900 R). A combination of x-irradiation and RAMPS-injection did not produce greater TSF levels in mice than did x-ray or RAMPS treatment alone.

Animals↗
PubMed1976

A comparison of mice in rebound-thrombocytosis with platelet-hypertransfused mice for the assay of thrombopoietin.

Rebound-thrombocytosis and platelet hypertransfusions were compared as methods of preparing assay animals for the measurement of thrombopoietin (TSF). In immunothrombocythaemic mice, the amount of 35S incorporation into the platelet mass after injections of a standard dose of TSF was related to the length of time after rabbit anti-mouse platelet serum (RAMPS) injection. After 2 platelet transfusions, however, there was no decrease in 35S incorporation values of mice with time after injections of control or TSF-containing substances. When platelet counts were made 3 days after the last platelet transfusion, the counts decreased with the number of transfusions. Mice in rebound-thrombocytosis were responsive to TSF as evidenced by higher platelet counts (P less than 0.05) and increased 35S incorporation into platelets (P less than 0.005), whereas mice made thrombocytotic by platelet transfusions were not. Assuming that increased platelet counts induced by the different techniques affect assay mice only by inhibiting blood cell production by haematopoietic cells, these data are consistent with the hypothesis that sensitivity to TSF depends upon the proliferative state of the megakaryocytic precursor population.

Animals↗
PubMed1976

Mechanism of thrombocytopenia induced in mice by anti-platelet serum.

Although anti-platelet serum is frequently used to produce thrombocytopenia, the mechanism of how platelets are removed from the circulation is poorly understood. In vitro studies indicated that anti-platelet serum caused platelet aggregation, whereas normal rabbit serum did not. The aggregation was dose related; at the lower dose, aggregated platelets were shown to deaggregate and platelets released from the aggregates responded normally to ADP but not to collagen. In mice whose platelets were previously labeled with Na235SO4, anti-platelet serum caused an increase in the amount of 35S mucopolysaccharides (MPS) recovered from the kidneys. No increase in radioactivity was found in the lungs, livers, or spleens of similarly treated mice. The data suggest that platelet specific anti-serum causes platelets to aggregate leading to ADP (and consequently 35S-MPS) release; the accumulation of 35S in kidneys may be due to removal of MPS and/or platelets from the circulation.

Animals↗