Effects of recombinant interferon-alpha and -gamma on B-CLL cells in serum-free medium: expression of activation, differentiation, and CALLA antigens.

Chronic B-lymphocytic leukemia (B-CLL) cells from 10 patients were cultured serum-free with recombinant interferon (rIFN)-alpha 2, rIFN-gamma, or phorbol ester (TPA) for 5 days. All three agents induced functional differentiation, as evidenced by IgM secretion, without concomitant proliferation. A panel of monoclonal antibodies was used to detect changes in cell surface antigens defining pre-B cells (CALLA), resting B cells (HH1), early (4F2, MHM6) and late (anti-Tac, OKT9) B cell activation, and terminally differentiated B cells (OKT10). The activation markers 4F2, MHM6, and anti-Tac and the plasma cell marker T10 were all significantly induced with TPA, rIFN-alpha 2, an rIFN-gamma, whereas the expression of HH1 decreased. CALLA was detected on substantial proportions of differentiated (4-38%) but not resting (0-4%) B-CLL cells. The CALLA-positive B-CLL cells were negative for nuclear terminal deoxynucleotidyl transferase (TdT). The T9 antigen was expressed on TPA-treated cells (1-16%) only. The present findings indicate novel properties of IFN-alpha and IFN-gamma in inducing terminal differentiation of human monoclonal B cells without prior activation.

TPA-induced differentiation of chronic lymphocytic leukemia cells: studies on mu-chain expression.

The regulation of IgM synthesis and secretion was studied in chronic lymphocytic leukemia cells, with a phenotype roughly similar to peripheral resting B cells, during phorbol ester (12-O-tetradecanoylphorbol-13-acetate)-induced differentiation. TPA treatment caused a 20 times increase in total RNA synthesis and 20 to 50 times increase in the protein synthesis as compared to control cells. Morphologically, 70-90% of the cells reached the lympho- or plasmablast stage of differentiation. In control culture cells, approximately equal amounts of mRNA coding for secretory (s) and membrane (m) mu-chains were found. The micron message was translated as surface IgM expression was detected. A posttranscriptional regulation of microsecond synthesis appears to exist, since only low amounts of cytoplasmic mu-chains were detected by immunoprecipitation and SDS-PAGE, and no secretion of pentameric IgM was detected as measured by an RIA. TPA induction caused a relative increase in the microseconds to microns mRNA ratio, demonstrating differentiation associated control mechanisms operating at the level of mRNA processing. The high levels of cytoplasmic microsecond-chain precursor and the efficient secretion of pentameric IgM in TPA-induced chronic lymphocytic leukemia cells indicated the presence also of posttranscriptional controls.

Treatment of the Guillain-Barré syndrome by plasmapheresis.

Treatment by plasmapheresis was performed in eight adult patients with the Guillain-Barré syndrome. One patient with a chronic relapsing form underwent four separate courses of plasmapheresis and her condition improved rapidly each time. Of seven patients with acute Guillain-Barré syndrome, the condition of three improved markedly, in one partially, and in three it did not improve in association with treatment. There were no apparent differences concerning clinical and neurophysiologic parameters between those whose conditions improved and those whose conditions did not.

Selenium protection against toxicity from cadmium and mercury studied at the cellular level.

Interaction between selenium and the heavy metals cadmium and mercury was studied in an experimental rat model (Sprague-Dawley). The rats were administered either one single trace element or combinations of selenium and cadmium as well as selenium and mercury. Salts of these trace elements were administered intraperitoneally daily during thirty days. Thereafter the animals were sacrificed and kidneys and livers excised rapidly. Thin sections were produced by a cryotome and subsequently freeze-dried. Nuclear microscopy of the sections showed that in the combination groups there was a co-localization of selenium and the heavy metals. None of the expected pathological signs of cadmium and mercury toxicity were observed. The conclusion was that selenium exerted a protective effect against the toxicity of cadmium and mercury through mechanisms still to be unveiled.

Cyclosporin-A is selectively cytotoxic to human leukemic T cells in vitro.

We have studied the antileukemic effects of cyclosporin-A (CyA) on the cells of a number of human hematopoietic biopsies and established cell lines in vitro. Cya killed the tumor cells but not the nonmalignant control cells from three of four patients with T-lymphocytic leukemia/lymphoma at concentrations comparable to those recommended in clinical long-term immunosuppressive therapy. The leukemic cells from 7 patients with B-type chronic lymphocytic leukemia were insensitive to CyA. The drug was cytostatic and cytolytic to three of five T-lymphoblastic leukemia cell lines tested. With the possible exception of a plasma cell line, all other control cell lines (B-lymphocytic lymphoma, histiocytic lymphoma, fibroblast, and glia cell lines) were resistant to CyA. These observations indicate that CyA or its derivatives may be useful as a highly selective antitumor agent in different T-cell malignancies.

Preoperative autologous donation of 6 units of blood during rh-EPO treatment.

PURPOSE: To determine if donation of six units of blood in three weeks is possible with self-administered subcutaneous recombinant human erythropoietin (rhEPO) injections and oral iron treatment. METHODS: A prospective trial where a total of 32 otherwise healthy patients were phlebotomised before revision hip arthroplasty during rhEPO and oral iron treatment (ferrofumarate). Adverse events were noted and compliance was controlled. Routine laboratory tests were performed at each visit including reticulocytes and 2,3-DPG. The relative oxygen releasing capacity (RORC) and the oxygen releasing capacity (ORC) were calculated. Blood donation was postponed until the next visit if haemoglobin concentrations was < 115 g.l-1 (men) or < 105 g.l-1 (women). RESULTS: All but two patients were able to donate six units of blood with an acceptable haemoglobin concentration on the day of operation. One serious adverse event occurred when the Hb was 119 g.l-1, compared with 149 g.l-1 before treatment. During the first two weeks before phlebotomy there was no increase in Hb, the mean nadir was reached after six phlebotomies (31 g.l-1 below pre-study level), while at operation it was 19 g.l-1 below pre-study level. There was an increase in 2,3-DPG and oxygen releasing capacity after the initiation of rhEPO therapy, before the first phlebotomy. CONCLUSION: It is possible to donate six units of blood in a three week period before surgery during self-administered subcutaneous rhEPO treatment and oral iron therapy at a rhEPO dose of 60 U.kg-1 BW three times a week. It is suggested that rhEPO per se initiates a right-shift of the oxygen dissociation curve via an increased 2,3-DPG level, which could explain that some patients report subjective benefit of rhEPO in spite of no change in Hb concentration.

Production of beta 2-microglobulin by chronic lymphocytic leukaemia cells in vitro.

The in vitro production of beta 2-microglobulin (beta 2m) by leukaemic cells was studied in 22 patients with chronic B-lymphocytic leukaemia (CLL). In addition, the concentration of beta 2m in serum (S-beta 2m) was determined and expressed as percent of the upper normal limit, after a correction for elevated S-Creatinine values. Patients with progressive disease usually had CLL cells with a high rate of in vitro synthesis and an increased S-beta 2m. This was not found in patients with non-progressive disease. The in vitro synthesis of beta 2m X the lymphocyte count correlated with S-beta 2m in the total material (r = 0.65). The increased S-beta 2m frequently observed in CLL may therefore originate from the tumour cells. Hence, S-beta 2m is promising as a clinically useful tumour cell-associated marker in CLL.

Treatment of therapy-resistant Sézary syndrome with Cyclosporin-A: suppression of pruritus, leukaemic T cell activation markers and tumour mass.

A patient with Sézary's syndrome resistant to conventional therapy was successfully treated with Cyclosporin-A (CyA) for 14 months. Pre-treatment in vitro studies showed that the leukaemic T cells were sensitive to therapeutic concentrations of CyA. The patient's pruritus disappeared promptly after 2 d of treatment. The positive effect of CyA on the pruritus was related to the dose and plasma concentrations of the drug. Analysis of leukaemic cell surface markers using monoclonal antibodies showed that episodes of pruritus were correlated with the appearance of cells expressing the T cell "activation" markers interferon- gamma (IFN-gamma) and HLA-DR. This indicated that CyA may control pruritus by suppressing the production/release of lymphokines. Immunohistochemical staining of repeated skin biopsies demonstrated a reduction of infiltrating T cells. Clinically, this was correlated with an improved skin status and a partial regress of palpable lymph node size. Apart from an initial reversible drop in the circulating helper/suppressor T cell ratio, the number of circulating Sézary cells was unaltered during CyA treatment. After 5 months, higher doses of CyA were needed to control symptoms, and this was correlated wit with partial drug resistance also in vitro.

Phorbol ester-induced differentiation of chronic B lymphocytic leukaemia cells--regulatory impact of autologous and allogeneic accessory cells.

Phorbol ester (TPA) induction of chronic lymphocytic leukaemia (CLL) cells can be used as a model system for the study of human B cell differentiation. We have analysed the role of accessory cells and the correlation to target cell surface Ig phenotype in TPA-induced morphological and functional differentiation of 12 CLL populations. A more than three-fold increase in secreted IgM was seen in 10 of 12 cases, with the strongest responses in patients having monoclonal serum IgM. CLL populations negative for or having only weak surface mu chain expression were less inducible. The impact of autologous and allogeneic accessory cells on TPA induction was studied in cell enrichment/depletion experiments using both physical and cytotoxic antibody techniques. CLL cells physically depleted of autologous E+ and monocytic (light density fraction) cells still responded to TPA. This response could be enhanced by allogeneic E- light fraction cells. Further depletion of autologous accessory cells by treatment of the E- high density fraction CLL cells with a panel of monoclonal antibodies plus complement demonstrated the permissive role of one or two populations of autologous cells expressing low avidity E receptors and the T3, T4 and T8 antigens. Augmenting T cells of similar phenotype were found among allogeneic cells from normal individuals. Thus, TPA-induced IgM secretion in biopsy B-CLL cells is regulated by minute numbers of autologous helper T cells. Furthermore, the Ig secretory response of CLL populations seems to be correlated with the surface Ig the surface immunoglobulin phenotype of the leukaemic cells.

Physiological response to phlebotomies for autologous transfusion at elective hip-joint surgery.

In order to study the physiological response to phlebotomies for autotransfusion, an autotransfusion program was designed for 10 patients undergoing hip-joint replacement surgery for arthrosis. 4 phlebotomies of 450 ml each were performed within 12 days. Blood samples were taken immediately before phlebotomy for blood hemoglobin (Hb), serum erythropoietin (Epo), reticulocyte count (ret) and erythrocyte 2,3-diphosphoglycerate (DPG). All 4 phlebotomies could be performed in 9/10 patients, and only 1 patient had significant symptoms (fatigue). The operation was performed 2 weeks after the last phlebotomy. None of the patients had recovered the initial Hb level at operation (24.8 +/- 9 per liter lower than initially), and they were all even more anemic after the operation (36.8 +/- 16.9 g/l lower than initially). Serum Epo increased from 13.6 +/- 7.2 IU to 30.6 +/- 12.2 (SD) IU per liter, and reticulocyte counts increased to a maximum of 3.68 +/- 1.69%. DPG increased in all patients except the one who had significant fatigue. It is concluded that the patients tolerated the phlebotomy program well but that a significant anemia developed. The compensatory increase in erythropoietin and reticulocyte count, adequate for this degree of anemia, was small compared to the increase seen at more severe anemia, indicating that there may be a role for pharmacological stimulation of erythropoiesis in blood predeposit programs.