James K. Brennan

Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro

SummaryWe have compared several methods for reducing calcium and magnesium concentrations in tissue culture medium, with the objective of producing selective deficiency effects on the growth of mouse (L5178Y) and human (P1R) lymphoblasts. In experiments in which calcium- and magnesium-“free” McCoy’s medium was supplemented with 15% horse or fetal calf serum, enough calcium and magnesium was provided by serum to support normal lymphoblast growth rate. Either dialysis or chelating-resin treatment of horse or fetal calf serum reduced calcium and magnesium contents approximately 100-fold. Use of dialyzed sera resulted in reduced growth rate, although in most cases the reduction in growth could be attributed to other effects of dialysis on serum, inasmuch as growth in those experiments was not restored to normal by the addition of calcium and magnesium to the medium.In contrast, the reduction of lymphoblast growth rate that occurred when resin-treated serum was used was always attributable to removal of calcium and magnesium, as normal growth always occurred in cultures to which calcium and magnesium were added.To demonstrate a growth-inhibiting effect on either mouse or human lymphoblasts by severe reduction of either calcium or magnesium in the presence of normal amounts of the alternative cation, it was necessary to (a) expose McCoy’s Ca−Mg-“free” medium to chelating-resin to reduce further the residual cation concentrations; (b) wash cells from stock cultures in a medium devoid of calcium and magnesium prior to inoculation into experimental cultures; (c) reduce the proportion of serum in the final medium from 15 to 5%; and (d) add 100 μM EGTA to cultures. Under these conditions, growth of both cell types was completely abolished in the presence of normal magnesium but in the absence of added calcium, and markedly reduced in the presence of normal calcium but in the absence of magnesium. These modifications did not compromise growth in cultures containing normal concentrations of both ions.

Treatment of C4D-positive acute humoral rejection with plasmapheresis and rabbit polyclonal antithymocyte globulin

Background. Alloantibody-mediated acute rejection is a major cause of renal allograft loss despite aggressive therapy. Patients with humoral rejection can be identified with high sensitivity and specificity by the presence of peritubular capillary C4d staining on renal biopsy and donor-specific anti-human leukocyte antigen antibodies. Standard therapy for acute humoral rejection (AHR) has been removal of donor-specific antibodies by plasmapheresis (PPH) in conjunction with intravenous immunoglobulin therapy. We describe a series of seven patients with C4d positive AHR who received combined therapy with PPH and polyclonal rabbit antithymocyte globulin (rATG). Methods. PPH (1.4 volume exchange) was initiated on diagnosis of AHR on an alternate day basis for a mean number of 6.8 treatments, in conjunction with rATG (0.75 mg/kg/day 5–10 days) until the serum creatinine returned to 120% of nadir. Results. The nadir posttreatment creatinine was significantly lower than pretreatment creatinine (1.0±1.2 vs. 2±1.4, P <0.007) with only one episode of graft loss. On follow-up there was no difference in renal allograft survival between the AHR group and the 60 patients without AHR who underwent transplantation during the same period. We describe the ability of rATG to induce apoptosis in vitro peripheral blood and activated B cells. Conclusion. Combination therapy using PPH and rATG is an effective means of reversing AHR in renal allografts.

Relationship between plasma adriamycin levels and the outcome of remission induction therapy for acute nonlymphocytic leukemia

SummaryPlasma adriamycin and adriamycinol levels were masured in 45 patients with acute nonlymphocytic leukemia 3 h after the drug was administered. A wide range of levels was found. Plasma levels increased after the administration of each of three daily doses of the drug. High plasma levels were associated with both death during remission induction therapy and, for patients who entered remission, long remissions.

Prediction of response of patients with acute nonlymphocytic leukaemia to remission induction therapy: use of clinical measurements

Two hundred patients with acute nonlymphocytic leukaemia received remission induction therapy consisting of cytosine arabinoside and an anthracycline antibiotic. Analysis of the pretherapy characteristics of the patients demonstrated that patient age was the most important factor in determining whether or not the patient would survive remission induction therapy. Assessment of the characteristics of the bone marrow after 6 d of therapy permitted the recognition of patients who were likely to fail to enter remission because of persistent leukaemia. Taken together, these observations demonstrate that it is possible to identify patients for whom conventional chemotherapy is not likely to be of benefit either because it is too intensive or because it is not intensive enough to produce a complete remission.

Inhibition of DNA synthesis by cytosine arabinoside: Relation to response of acute non-lymphocytic leukemia to remission induction therapy and to stage of the disease

The sensitivity of leukemic marrow cell DNA synthesis to cytosine arabinoside (araC) exposure in vitro was studied in specimens obtained from patients with acute non-lymphocytic leukemia. Cells from patients who had been treated with araC in the past were more likely to be resistant to the effect of araC on DNA synthesis than cells obtained from patients who had not been so-treated previously. Resistance to the effects of araC on DNA synthesis was associated with the failure of high-dose araC therapy to induce remissions in relapsed patients, whereas remission induction failure in previously untreated patients was not associated with araC resistance.

Secretion of plasminogen activator by the human macrophage‐like cell line, GCT: separation from colony‐stimulating and erythropoiesis‐enhancing activities

Summary. The human macrophage‐like cell line, GCT, elaborates monokines such as colony‐stimulating activity (CSA) and erythropoiesis‐enhancing activity (EEA) which stimulate the growth of primitive blood progenitors in culture. These cells also secrete a fibrinolysis activator (FA), which can be identified if cells are cultured in serum‐free medium. FA was found to have a similar molecular weight to CSA and EEA by gel filtration but could be separated from them by ion exchange chromatography. Subcellular fractionation of GCT cells indicated that fibrinolytic activity was present in the cell membranes and cytosol, whereas CSA and EEA were present only in the cytosol. FA resembled urokinase in molecular weight and its strict requirement for plasminogen as a substrate. Double immunodiffusion of GCT activator and urokinase against anti‐urokinase antiserum resulted in a line of identity, and incubation of activator with antiserum resulted in loss of its fibrinolytic activity. Thus, GCT activator was similar, if not identical to the plasminogen activator, urokinase.