Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F2 alpha (PGF2 alpha) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10(6) cells, and PGF 2 alpha increased from a normal value of 31 pgrams to 482 pgrams/10(6) cells. In CLL lymphocytes, levels of PGE and PGF2 alpha were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF2 alpha fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3':5'-adenosine monophosphate (c-AMP) level was constantly low, and the initial level of PGF2 alpha fluctuated in relation to similar oscillations of cyclic 3':5'-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF2 alpha, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

The immunomodulating properties of staphylotoxin]

The action of native staphylotoxin has been tested on T lymphocytes obtained from 21 healthy human donors. As revealed in this investigation, toxic action affects Fc-receptors to IgG, IgM and IgA. The dose-dependent effect of the preparation suggests its direct contacts with Fc-receptors of lymphocytic membranes. High doses of the toxin suppress the expression of Fc-receptors 6- to 12-fold, low doses of the toxin are capable of stimulating the expression of Fc gamma-receptors.     

Transient deficiency of peripheral blood accessory cells in supporting T cell mitogenesis in patients suffering from chronic idiopathic thrombocytopenic purpura after intravenous gammaglobulin treatment

Mitogenic response of blood lymphocytes to phytohemagglutinin (PHA) and to OKT3 monoclonal antibodies was investigated in 7 patients suffering from chronic idiopathic thrombocytopenic purpura (ITP) before, during and after high-dose intravenous (i.v.) immunogammaglobulin (IgG) infusion. The platelet count rose above the pre-treatment values during infusion therapy in all patients but one. Five out of seven patients presented elevated platelet-associated IgG (PA-IgG) levels at the time of the first infusion; four of these showed an increase in platelet count and a transient reduction or normalization of PA-IgG after IgG infusion. Five out of seven patients showed an impairment of T lymphocyte mitogenic response to PHA and OKT3 before therapy. All patients responded to IgG therapy with a transient deficiency of FcR mediated monocytes (Mo) in supporting T cell mitogenesis induced by both mitogens during and after IgG infusion. This reduced cooperative capability of Mo disappeared at various times after the end of therapy (range 3-12 days). The transient alteration of Mo function, possibly due to a modification in the surface number or in the affinity of Fc-receptors, can explain in part, the increase in platelet count during and after IgSRK infusion.     

Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F₂ (PGF₂) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10⁶ cells, and PGF₂ increased from a normal value of 31 pgrams to 482 pgrams/10⁶ cells. In CLL lymphocytes, levels of PGE and PGF₂ were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF₂ fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3′:5′-adenosine monophosphate (c-AMP) level was constantly low, and the initial high level of PGF₂ fluctuated in relation to similar oscillations of cyclic 3′:5′-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF₂, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

DNA-synthesizing T and non-T cells in chronic lymphocytic leukemia.

In 21 patients with chronic lymphocytic leukemia (CLL) and in 8 hematologically normal persons the number of DNA-synthesizing peripheral blood lymphocytes was investigated by autoradiographic techniques. The lymphocytes were differentiated by EN-rosette tests into T and non-T lymphoid cells. The results show a normal number of proliferating T lymphoid cells and an increased number of proliferating non-T lymphoid cells in clinical stages O-I. Stages III-IV demonstrate a significant increase of the proliferation rate of both T and non-T lymphoid cells. The possible pathogenetic factors and the prognostic value of these results are discussed.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

Cortisol catabolism by lymphocytes of patients with chronic lymphocytic leukemia

A low rate of catabolism of cortisol by lymphocytes correlates with high sensitivity of the cells to the steroid and causes them to die at a greater rate than control samples. Since lymphocytes of patients with chronic lymphocytic leukemia respond to treatment with glucocorticosteroids and are cortisol sensitive, we attempted to see whether their capability to catabolize cortisol differs from that of normal lymphocytes. No difference was found between the two groups of cells with regard to the pattern of cortisol metabolites. However, the lymphocytes of the chronic lymphocytic leukemia groups showed a total cortisol catabolism per cell that was significantly lower than that of the control group. Patients with low lymphocyte count in peripheral blood showed a relatively higher cortisol metabolism by lymphocytes per cell than those with high counts.     

Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes,and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.Abbreviations dCMP deaminase deoxycytidylate deaminase - PHA Phytohemagglutinin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - AMOL acute monocytic leukemia - WBC white blood cells     

Xenoantisera to human DR antigens: Serological and immunochemical characterization

Antibodies to DR antigens were detected using serological and immunochemical tests in sera from rabbits and goats immunized with cultured human B-lymphoid cells mixed with an anti-T-cell xenoantiserum or with partially purified DR antigens. After absorption with human red blood cells, cultured melanoma cells, and/or T-lymphoid cells, DR xenoantisera become specifically cytotoxic to B lymphocytes. Three out of nine sera tested with a panel of T-depleted peripheral lymphocytes and chronic lymphocytic leukemia cells showed correlation with DR alloantisera submitted to the Seventh International Histocompatibility Workshop. Although the correlation coefficients were lower than those obtained with DR alloantisera, the results obtained suggest that DR xenoantisera may recognize allotypic specificities.     

Identification and purification of cytolytic antibodies directed against O-acetylated sialic acid in childhood acute lymphoblastic leukemia

Sialic acids typically present as terminal sugars of oligo-saccharides are reported to be modified by O-acetylation at the C-9 position on lymphoblasts of childhood acute lymphoblastic leukemia (ALL) patients (Sinha et al., 1999a, Leukaemia, 13, 119-125). We now report high titers of IgG antibodies directed against O-acetylated derivatives of sialic acids (O-AcSA) in serum of ALL patients. These antibodies were purified using bovine submaxillary mucin (BSM) and the IgG distribution was confined to IgG(1)and IgG(2)subclasses; their binding was totally abolished with de-O-acetylation confirming their specificity towards O-AcSA determinants. Flow cytometry demonstrated binding of these antibody fractions to peripheral blood mononuclear cells (PBMC) of both T- and B-ALL patients having increased cell surface 9-O-AcSA determinants. Western blotting of membranes derived from PBMC of ALL patients confirmed binding of the antibody to O-acetylated sialoglycoconjugates corresponding to 144, 135, 120, 90, and 36 kDa whereas binding to PBMC from normal individuals corresponded to 144 and 36 kDa. Specificity of the antibody fraction towards 9-O-AcSA was substantiated by hemagglutination and hemagglutination-inhibition assays. The antibody purified from ALL serum selectively mediates complement dependent cytolysis of lymphoblasts expressing O-AcSAs and thereby possibly confers passive protection. The enhanced anti O-AcSA antibody levels allowed for development of a serodiagnostic assay (BSM-ELISA) specific for ALL. Minimal crossreactivity was observed with other hematological disorders like acute myeloid leukemia (n = 16), chronic myeloid leukemia (n = 6), chronic lymphocytic leukemia (n = 7) and non-Hodgkin's lymphoma (n = 3) as well as normal healthy individuals (n = 28). The BSM-ELISA therefore provides a simple, noninvasive alternative diagnostic approach for ALL and merits clinical consideration.     

Acid phosphatase cytochemistry of mitogen-transformed normal and chronic lymphocytic leukemia lymphocytes

Acid phosphatase cytochemistry was performed on lymphocytes stimulated in vitro with phytohemagglutinin, pokeweed mitogen, or concanavalin A. These electron microscopic studies demonstrated that activated lymphocytes from both normals and patients with chronic lymphocytic leukemia (CLL) had an increased number of lysosomes relative to resting cells. At the time of maximum thymidine incorporation, a reduced number of lysosomes was present in many transformed CLL lymphocytes, mainly medium-sized blast cells, in comparison to transformed normal cells. The findings demonstrate a lysosomal abnormality in phytomitogen transformed CLL lymphocytes which may be related to functional defects of these cells or to an incomplete transformation of a residual population of normal lymphocytes.     

A unique antigen (sigma) on sézary cells

Summary Lymphapheresis was performed on a patient with Sézary syndrome. The Sézary cells were purified by removing E-rosette-forming and Fc receptor-bearing cells. Antiserum against these purified Sézary cells was raised in rabbits. This antiserum had cytotoxicity against Sézary cells as well as against normal peripheral blood lymphocytes. Absorption was carried out with chronic lymphocytic leukemia (CLL) and normal lymphocytes. The absorbed antiserum maintained cytotoxicity against Sézary cells but lost cytotoxicity against CLL and normal peripheral blood lymphocytes.Indirect immunofluorescence assay showed that the antiserum reacted against purified Sézary cells and a high percentage (66%) of peripheral blood mononuclear cells from five patients with Sézary syndrome. It also reacted against 5.7% of normal lymphocytes, 8% of CLL cells, 5% of the lymphocytes from a patient who had undergone splenectomy, 2% of lymphocytes from a patient with multiple myeloma, 5% of lymphocytes from a hairy cell leukemia patient, and 1% of acute lymphocytic leukemia cells (T cell). The antiserum did not react against thymocytes but reacted against 34.6% of the bone marrow lymphocytes. This unique marker was designated as sigma () antigen. It was suggested that Sézary syndrome may represent proliferation or malignant transformation of normally present antigen-positive lymphocytes.     

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with established leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50 000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.     

A new human T-cell differentiation antigen: Unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with extablished leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.PJM is a Junior Faculty Clinical Fellow of the American Cancer Society.     

Cap formation on lymphocytes from patients with leukemic diseases induced by four different lectins.

When ficoll purified peripheral blood lymphocytes were treated with fluorescein conjugated lectins from lentils (LCH), castor beans (RCA) and phaseolus coccineus beans (L-and E-PHA) for 15 min and the percentages of the cap forming cells were examined, the values of leukemic lymphocytes were reduced compared to the values obtained with normal lymphocytes. The reduction was more than half in patients with acute and chronic myelogenous leukemia and immunoblastoma, it was only one quarter in patients with chronic lymphocytic leukemia, Hodgkin's disease and lymphosarcoma. The lowest number of cap forming cells was found in lymphoblasts of established lymphoblastoid cell lines. The four different lectins showed nearly the same capacity in the induction of caps. After successive binding, the different lectins showed cocapping on the lymphocyte surface.     

A human B lymphocyte antigen (P-76) shared by B-cell chronic lymphocytic leukemia cells and hairy cell leukemia cells

A membrane antigen with an apparent specificity to B lymphocytes was detected with immunochemical techniques and its properties were analyzed. Anti-B-CLL serum was raised in a rabbit by immunization with B-cell chronic lymphocytic leukemia (B-CLL) cells. This anti-B-CLL serum was absorbed with erythrocytes, liver homogenate and insolubilized immunoglobulins. After further absorption with T-CLL cells, chronic myelocytic leukemia (CML) cells and acute myelocytic leukemia (AML) cells, the anti-B-CLL serum still reacted with peripheral blood B lymphocytes, B-CLL cells and hairy cell leukemia (HCL) cells. In contrast, no reactivity was seen with peripheral blood T lymphocyte or monocytes, or leukemia cells of non-B cell origin. An immunoprecipitation of radiolabeled cell surface proteins was attempted using the anti-B-CLL serum in the presence of Staphylococcus Aureus Cowan 1 (SaCl), and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A membrane antigen with an apparent molecular weight of 76,000 daltons (P-76) was immunoprecipitated with the anti-B-CLL serum from the lysates of normal B lymphocyte, B-CLL cells and HCL cells. The antigen (P-76) is not composed of disulfide-linked subunits and has no structural relationship with HLA-DR (Ia-like) antigens or other known antigens. These results suggest that this antigen is B-lymphocyte specific, and favour the B-lymphocyte nature of HCL cells.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Osmotic fragility in subfractions of leucocytes in hematological diseases (author's transl)]

Leucocytes of normal persons and patients with acute and chronic granulocytic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma were separated into subfractions by centrifugation in discontinuous Ficoll density gradient. Osmotic resistance was examined in hypotonic NaCl solutions with decreasing concentration and by determining LDH activity in the supernatant. Suspensions of myelocytes, polymorphnuclear granulocytes, and lymphocytes of normal persons and patients with chronic lymphocytic leukemia demonstrated the same osmotic resistance. Only myeloblasts were osmotically less fragile, and tumor cells of non-Hodgkin lymphoma more fragile.     

Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay

The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV-induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases.