Inhibition of in vitro lymphocyte function by a soluble AKR leukemic suppressor factor.

A soluble suppressor factor(s) found in the serum of AKR mice bearing lymphocytic leukemia (AKR-LSF) has previously been shown to inhibit PHA-induced spleen cell proliferation. In this study we have further characterized the biological activity of the leukemic mouse serum (LMS) in vitro. The LMS inhibits spleen cell proliferation in an MLC as well as generation of functional CTL. The inhibition of CTL generation was not specific since CTL activity resulting from responder and stimulator combinations of various H-2 haplotypes was inhibited. LMS did not inhibit CTL- or NK-mediated cytolytic activity. These results suggested that LMS inhibits lymphocyte proliferative responses but has no effect on cytolytic function. Furthermore, responder cells which had been inhibited by LMS in a primary MLC, washed, and restimulated were able to express CTL activity indicating that the suppression by LMS is reversible. The suppressive effect of LMS is occurring at an early stage of CTL generation since LMS was inhibitory when added on Day 0 or Day 1 but not on Days 2, 3, or 4 of the MLC. Addition of IL-2 did not remove the inhibition by LMS as measured in a PHA proliferative assay. These results suggested that the suppression is not due to a lack of IL-2 but to an inability of the cells to either bind or utilize IL-2.

Normal T cell subsets and lymphocyte activity in multiple sclerosis.

Nine patients with either chronic progressive or relapsing/remitting multiple sclerosis were treated with several courses of lymphopheresis. The lymphocytes obtained by this procedure showed a significantly lower percentage of OKT3+ cells than normal controls at the beginning of therapy. This percentage increased to normal levels after five lymphopheresis treatments and then decreased during the last stages of treatment. Patients had normal levels of OKT4+ and OKT8+ cells compared to controls and these percentages showed no statistically significant changes during the course of treatment. The proliferative response of patient's lymphocytes after stimulation with concanavalin A, PHA and alloantigen was not significantly different from normal controls and these responses were unchanged during the lymphopheresis treatment. Natural killer cell activity was also normal in our patients. The results reported in this study do not suggest a basic T cell abnormality in our patients with multiple sclerosis. Furthermore, the lymphopheresis treatments did not induce any significant change in T cell numbers or functional activity in vitro.

Platelet factor 4 and beta thromboglobulin in Moya-Moya disease.

Increased concentrations of PF4 and beta TG were found in two patients with Moya-Moya disease. Although the pathogenesis of these abnormalities is unknown, the increased concentration of these proteins may result from platelet activation in the abnormal vascular network, leading to platelet aggregation and secretion of these two platelet-derived proteins, or alternatively, the increased concentration of these proteins may be the result of platelet aggregation due to a defect in endothelial prostaglandin production.

Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia.

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [3H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [3H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10(7) BW5147 transplanted leukemia cells had 130 +/- 12 units of AKR-LSF activity/ml of serum compared to 40 +/- 8 units/ml for mice with spontaneous leukemia. Normal mouse serum contained 33 +/- 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.

Chronic granulocytic leukemia and carcinoma of the cervix in situ following renal transplantation.

A 26-year-old white female presented with Philadelphia chromosome positive chronic granulocytic leukemia five years after successful renal transplantation and prolonged therapy with azathioprine and prednisone. This patient represents the seventh case of the chronic granulocytic leukemia reported within the population of immunosuppressed renal transplant recipients. Of further interest in our case is the development of carcinoma in situ of the cervix two years following the transplant. Although multiple malignancies have been recognized in renal homografts, the occurrence of chronic granulocytic leukemia and carcinoma in situ in the same patient has not been previously described. A possible mechanism for the development of chronic granulocytic leukemia is the association of chromosome damage by azathioprine and immunoparalysis of the host, thus setting the stage for the emergency of a malignant clone. The importance of close follow-up of patients on prolonged immunosuppressive therapy is emphasized.

Reversal of cyclophosphamide induced neutropenia by hypertransfusion and androgen therapy.

We injected 300 mg/kg Cyclophosphamide i.p. into female AKR mice and rendered them reversably neutropenic. Group A was hypertransfused; Group B received 5 mg Nandrolone decanoate i.m. q.d X 2; and Group C received both. Group D served as a control. White blood cell counts were followed every other day for 10 days. The nadirs seen on all groups appeared on day 5. At day 10 counts in Groups A, B, and D had returned to normal, while the pattern of granulocyte decline and recovery in Group C was significantly faster than that seen in other groups, returning to normal at day 8. Hypertransfusion and androgen administration together significantly ameliorated drug-induced leukopenia, suggesting synergism. Thus manipulation of the stem-cell compartment may be clinically feasible.

Osteonecrosis in patients with Hodgkin disease following combination chemotherapy.

Three cases of osteonecrosis in patients receiving prednisone as part of multi-drug chemotherapy for lymphoma are presented. These patients are discussed in the context of previously reported series. The etiology of steroid-induced osteonecrosis and its radiologic manifestations are reviewed with regard to the need for biopsy to confirm the diagnosis. In light of this complication, the need for prednisone in lymphoma chemotherapy is considered.

Pure red cell hypoplasia associated with long-arm deletion of chromosome 5.

A further instance of the new hematologic entity of pure red cell hypoplasia with deletion of the the long arm of a chromosome 5(5q-) was seen in a 59-year-old white male. Examination of the bone marrow showed depressed erythroid cells with normal granulocytic and megakaryocytic precursors. Chromosomal analysis showed deletion of a chromosome 5 without any apparent translocation. This karyotype may be associated with acute leukemia.

Physiologic features of hemolysis axxociated with altered cation and 2,3-diphosphoglycerate content.

A hemolytic disorder characterized by altered RBC cation composition (increases Na, decreases K), reduced monovalent cation content (decreased Na + K/liter RBC), and decreased levels of 2,3-diphosphoglycerate (2,3-DPG) is described. The etiology of these RBC abnormalities was not elucidated following extensive laboratory evaluation, although two important physiologic principles were manifested by this case: (1) Hemolysis was relatively well compensated (41% hematocrit) despite a significantly decreased RBC survival (51 Cr t 1/2 = 10.5 days). This effect presumably was due to reduced 2,3-DPG content (1.9 mumol/ml RBC) and the associated increase in whole blood oxygen affinity (P50 = 19.6 mm hg). (2) RBC size and water content were normal in spite of marked cation depletion. This anomaly was thought to reflect the osmotic effects of reduced polyvalent anion (2,3-DPG) content.

Further development of a successful protocol of graft versus leukemia without fatal graft-versus-host disease in AKR mice.

We previously reported a successful model for treatment of BW 5147 leukemia in AKR mice by adoptive immunotherapy using allogeneic spleen cells from C57BL/6 mice. The leukemia cells were given 3 days before initiation of therapy. Graft-versus-host reaction was prevented by treatment with spleen cells from a second allogeneic strain (CBA), followed by cyclophosphamide and syngeneic spleen cells. We now show that it is not necessary to use syngeneic spleen cells in the final transplant since H-2-compatible, allogeneic CBA cells are as effective. In addition, it is possible to initiate successful therapy 5 days after leukemia implantation providing that the initial cyclophosphamide, given in two doses of 100 mg/kg each and spaced 7 days apart, is administered prior to establishment of graft-versus-host reaction. Higher single doses of drugs were followed by fatal graft-versus-host disease.

A simple method for the cryopreservation of human lymphocytes at -80C.

A simple and inexpensive method for the freezing of human blood lymphocytes at -80 C is described. Lymphocytes were frozen in either polystyrene tubes or polyolefin bags. Optimal recovery of cells was obtained when 5% DMSO and 62% plasma were used as cryoprotectants. Viable cell recovery, as assessed by response to phyto hemagglutinin (PHA) and pokeweed mitogen (PWM), was greater than 80% after 60 days of storage. In addition, percentages of T and B lymphocytes were essentially unchanged. The excellent viable cell recovery in polyolefin bags suggests that it is possible to cryopreserve large numbers of lymphocytes in a single container.