Jan Jansen

Chronic B-cell leukemias: relation between morphological and immunological features

To better understand the heterogeneity of chronic B-cell leukemias we correlated morphological and immunological features by studying the peripheral blood from 80 patients with a panel of anti-immunoglobulin and fourteen monoclonal antibodies, which hitherto were studied separately or with respect to one single morphological entity only. Of these the surface immunoglobulins (sIg) and monoclonal antibodies (McAb) BA-1, BA-2, FMC7, OKM1, and anti-T65 allowed a fair distinction between five cytological subtypes: chronic lymphocytic (CLL), lymphoplasmacytoid (LPL), centrocytic (CL), prolymphocytic (PLL), and hairy cell leukemia (HCL). In that order the sIg showed a decreasing number of cases of mu +/- delta class and an increase of alpha or gamma positivity. The number of BA-1-positive cases was decreased in PLL and HCL. There was a decline of BA-2- and anti-T65-positive cases in the order mentioned, while this was accompanied by an increase of FMC7 and OKM1 positivity. A significant mutual exclusion between anti-T65 and FMC7 was observed and the same was true for FMC7 and BA-2. The antibodies FMC7 and OKM1, and anti-T65 and BA-2 were linked to each other. Also FMC7 positivity was related to sIg of the alpha and gamma classes. On the basis of this unique combination of markers a differentiation scheme of B lymphocytes is proposed, in which prolymphocytic leukemia and hairy cell leukemia seem to represent a maturation arrest at a more advanced stage than chronic lymphocytic or lymphoplasmacytoid leukemia.

Characterization of a new cell line (ESKOL) resembling hairy-cell leukemia: A model for oncogene regulation and late B-cell differentiation

A B-lymphoblastoid cell line ESKOL, composed of differentiated cells resembling hairy-cell leukemia (HCL) has been established from the peripheral blood (PB) of a HCL patient. Morphologically, ESKOL cells share several features with HCL B cells. Flow cytometric analysis revealed that ESKOL cells express HC2, CD21, PCA-1, CD24, FMC7, and CD25. Analysis by Northern-blot hybridization indicated that cultured cells expressed the oncogenes c-myc, H-ras and c-fos. RNA from 3T3 cells transfected with ESKOL DNA hybridized with H-ras and c-fos DNA probes. The ESKOL cells cultured in the presence of increasing concentrations, of alpha interferon demonstrated a decrease in the rate of cellular growth and an increase in the expression of CD21, CD25, FMC7 and PCA-1. Scanning electron microscopy revealed that cells incubated in the presence of alpha interferon underwent membranous changes with a loss of villosity. These observations suggest that IFN tends to drive HC out of their developmental arrest towards maturation.

Infection prevention in severely myelosuppressed patients : a comparison between ciprofloxacin and a regimen of selective antibiotic modulation of the intestinal flora

PURPOSEnTo study whether oral ciprofloxacin would be as effective in preventing bacterial infections in severely myelosuppressed patients as selective antibiotic modulation of the gut flora with neomycin/polymyxin B sulfate/nalidixic acid (NPN).nnnPATIENTS AND METHODSnOne hundred and five patients undergoing allogeneic or autologous bone marrow transplant, or induction therapy for acute leukemia in 1988 and 1989 were studied. Patients were stratified according to the type of therapy, and randomized in a ratio of 2:1 to either oral ciprofloxacin 500 mg BID, or a combination of oral neomycin 250 mg QID, polymyxin-B 100 mg QID, and oral nalidixic acid 1,000 mg BID. Treatment began on admission and continued until the absolute granulocyte count was greater than 500/mm3 for 3 consecutive days.nnnRESULTSnThe 96 evaluable patients were evenly distributed over the 3 treatment groups; 63 patients received ciprofloxacin and 33 received NPN. Fever developed in 92% of patients on ciprofloxacin and in 97% of patients on NPN. (P = 0.66), 6.6 +/- 5.8 and 7.2 +/- 5.3 days from the start of prophylaxis, respectively. Twenty-five patients on ciprofloxacin developed 29 microbiologically documented infections, fewer than the 26 infections in the 22 patients on NPN (P = 0.02). Patients on ciprofloxacin had fewer bacteremias (33%) than did the NPN patients (55%) (P = 0.05). Gram-negative bacteremias were very rare (2 cases; no Enterobacteriaceae), but streptococcal bacteremias were frequent in both arms (27 cases). Side effects were not significantly different, but compliance with ciprofloxacin was better.nnnCONCLUSIONSnCiprofloxacin is at least as effective as the combination of neomycin/polymyxin/nalidixic acid in the prophylaxis of bacterial infections in myelosuppressed patients, and is better tolerated. Additional agents to prevent streptococcal infections are needed.

Characterization of normal human CD3+CD5- and γδ T cell receptor positive T lymphocytes

The functional and phenotypic properties of normal human CD3+ CD5‐ T cells which have a higher frequency of cytotoxic cells than CD3+CD5+ T lymphocytes have been described. Using three‐ and four‐colour immunofluorescence flow cytometric cell sorting, the CD3+CD5‐ and CD3+CD5+ populations were subdivided into αβ or γδ T cell receptor positive cells. The four subsets were examined for the in vitro cytotoxic activity and were also stimulated with mitogens in limiting‐dilution assays to measure the frequencies of proliferating and interleukin‐2 (IL‐2) producing cells. CD3+CD5‐αβ, CD3+CD5‐γδ+ and CD3+CD5+γδ+ cells had lower frequencies of proliferating and IL‐2‐producing cells than did CD3+CD5+αβ+ cells. However, the cytotoxic activity of the different phenotypes was higher in the CD3+CD5‐ subsets, especially when these cells were γδ+ Expression of γδ or lack of expression of CD5 appeared to be associated with the acquisition of cytolytic potentials. CD8 was expressed on 20% of fresh CD3+γδ+ cells. Cultured γδ cells retained the expression of γδ, but quickly lost that of CD8 and with time modulated the expression orCD5, The expression of CD5 was found to be higher on sorted CD3+CD5+γδ‐ than on CD3+CD5+γδ+ cells. These observations indicate that γδ is preferentially expressed on CD5‐negative or weakly positive T lymphocytes and that CD3+CD5‐γδ cells appear to constitute a discrete small subset of mature T lymphocytes which are cytotoxic in nature. However, the exact immunological function of these cells and their place in T cell ontogeny are yet to be elucidated.

Chronic T cell lymphoproliferative disorder and pure red cell aplasia: Further characterization of cell-mediated inhibition of erythropoiesis and clinical response to cytotoxic chemotherapy☆

Two patients with pure red cell aplasia and a T cell lymphoproliferative disorder were studied in order to define the mechanism of suppression of erythropoiesis and the patients response to cytotoxic therapy. In vitro assays demonstrated enhanced formation of both erythroid colonies and bursts following T-cell depletion. Erythroid colony formation was suppressed by the readdition of autologous T cells to a null cell fraction of marrow mononuclear cells. Media conditioned by the patients T cells did not exhibit any inhibitory effect on erythroid colony formation by autologous T cell-depleted marrow cells. These in vitro results suggested that T cell-mediated suppression of erythropoiesis was responsible for the generation of pure red cell aplasia. In both patients, cyclophosphamide therapy resulted in clinical remissions manifested by normalization of the hematocrits associated with a reduction in circulating lymphocytes from more than 10,000/mm3 to under 500/mm3. Maintenance chemotherapy has caused persistent inhibition of lymphocyte counts along with durable remissions with normal hematocrits.

Cytogenetic patterns following bone marrow transplantation for chronic granulocytic leukemia

Allogeneic bone marrow transplantation (BMT) is the only treatment with a potential to cure chronic granulocytic leukemia (CGL). Cytogenetic markers, including the Philadelphia chromosome (Ph), t(9;22)(q34;q11), allow follow-up of these patients after BMT for engraftment and remission status. A variety of cytogenetic patterns have been observed after BMT for CGL. A number of patients experience cytogenetic relapse or mixed chimerism with normal cytogenetics. Furthermore, these types of complications may increase after T-cell depleted BMT. We have closely followed six patients with Ph-positive CGL treated with T-cell depleted bone marrow for 169-720 days after BMT. A number of different cytogenetic patterns were found. The first patient, transplanted while in accelerated phase, relapsed at 155 days with the same clonal abnormalities and died in blast crisis. Four other patients were found to have cytogenetic relapses between days +41 and +233. In every case the Ph reappeared along with other clonal and nonclonal abnormalities, some of which were transient. All four patients presented with hematologic relapse several months after cytogenetic relapse. The final patient has mixed chimerism with Ph-negative cells and continues to be in clinical remission. Close cytogenetic follow-up of CGL patients after BMT can provide important information about early changes in engraftment and remission status.

Hairy cell leukemia

Hairy cell leukemia is a chronic lymphoproliferative disorder that has been recognized as a separate clinical pathologic entity for the last 25 years. After a decade of discussions about the origin of the neoplastic cell, it has now been well established that hairy cells represent a certain, rather mature stage of B-cell differentiation. Evidence for this has been derived from studies using immunophenotyping with monoclonal and polyclonal antibodies, cytochemistry, and immunoglobulin gene rearrangement. For many years, splenectomy was the only therapy of proven value in hairy cell leukemia. For patients who showed insufficient response to the operation, chemotherapy with low-dose alkylating agents was moderately successful, whereas polychemotherapy often resulted in excessive toxicity. More recently, therapy with alpha-interferon has been shown to be very promising, whereas deoxycoformicin may be an attractive alternative. These new advances in immunology and therapy are reviewed.

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes.

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg(2+)-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase D-dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergent availability and cation sensitivity on the apparent distribution of PAPH in neutrophil subcellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzymes was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.