The effect of linoleic acid on the sensitivity of human lymphocytes to cortisol and their capacity to catabolize the steroid

We have shown previously that cortisol-sensitive lymphocytes (thymocytes) have a much lower capacity than cortisol-resistant cells to catabolize cortisol. In the present study, we attempt to demonstrate that inhibition of cortisol catabolism may make cortisol-resistant lymphocytes vulnerable to the steroid. Linoleic acid, which has the capacity to inhibit the catabolism of cortisol by lymphocytes, was used for this purpose. By using various concentrations of linoleic acid (20-60 micrograms/mL) we showed an inverse linear relationship between linoleic acid concentration and the rate of cortisol catabolism by lymphocytes. During this experiment which took 17 h the viability of cells did not change significantly (minimum viability 95%), even at the highest concentration of linoleic acid. Keeping the metabolism of cortisol at a level of 40% of that obtained by the control, by adding linoleic acid to lymphocyte cultures (50 micrograms/mL) and measuring the viability of the cells for a period of 3 days in the presence or absence of cortisol, we were able to show a rise in the death rate of the cells which started after 24 h of incubation owing to the presence of the steroid.     

Phosphatidylethanolamine methylase and cyclic nucleotide phosphodiesterase activities in human B lymphoid hemopathies

Phospholipid methylase and cyclic nucleotide phosphodiesterase activities were studied in human B lympho?d hemopathies (51 patients: acute lymphoblastic leukemia, B lymphoma, chronic lymphocytic leukemia, hairy cell leukemia) and compared with activities in lymphoblast?d and Burkitt lymphoma cell lines and with normal B lymphocytes: methylase activity proved to be lower in ALL and high grade lymphoma and inversely related to the percent of cells in S phase state; the A/G ratio of phosphodiesterases was low in ALL and CLL and high in hairy cell leukemia and it was related to the percent of cells in S phase state.     

Cyclic AMP phosphodiesterase in human lymphocytes and lymphoblasts.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.     

Interaction of blood lymphocyte Fc receptors with IgG during in vitro cultivation

Bovine blood lymphocytes taken from normal cows and those suffering from chronic lymphocytic leukemia were cultured in complete medium 199 with 10% of heat-inactivated fetal bovine serum. After a 48 hour culturing an enhanced quantity of the Fc-receptor bound fluoresceinated immunoglobulin G (IgG) was established. When lymphocyte fractions enriched with T- or B-cells were cultured, the binding capacity of Fc-receptors for IgG (48 hours after culture) increased in both the cell populations. A study of the kinetics of interactions of Fc-receptors with IgG showed that the increased number of Fc-receptors after culturing was followed by an enhanced affinity of Fc-receptors towards IgG. The affinity of Fc-receptors of blood lymphocytes of cows with chronic lymphocytic leukemia was lower than that of normal lymphocytes.     

Electrophoretic mobility of lymphocytes in chronic lymphatic leukemia (author's transl)]

Lymphocytes and other blood cells can be separated by means of free flow cell electrophoresis. Immunofluorescence of the separated lymphocytes of four healthy volunteers with antiimmunoglobulins IgD and IgM produced different distribution profiles for each immunoglobulin class, the IgD positive cells migrating faster than the IgM positive ones. Amongst five patients with chronic lymphocytic leukemia four with IgD positive lymphocytes (greater than 80%) showed an identical electrophoretic distribution. The IgM positive lymphocytes (greater than 80%) of the fifth patient migrated much more slowly. The weighted mean of each distribution profile of either the IgD or IgM positive lymphocytes in CLL is similar to that of normal subjects.     

In vitro culture studies of granulocyte/macrophage and erythroid progenitor cells in lymphoproliferative disorders

Granulocyte/macrophage progenitor cells (CFU-GM) and erythroid progenitor cells (BFU-E) have been assayed in peripheral blood (PB) and/or bone marrow (BM) from 12 patients with acute lymphocytic leukemia (ALL), 16 patients with chronic lymphocytic leukemia (CLL) and 31 patients with various forms of non-Hodgkin lymphoma (NHL) without BM involvement. Progenitor cell growth in PB and BM from the NHL patients did not differ statistically from controls (p greater than 0.1). CFU-GM and BFU-E per ml PB were markedly increased in ALL and CLL patients (p less than 0.001) while CFU-GM and BFU-E per plated BM cells from these patients were severely depressed (p less than 0.001). Lymphoblasts from one ALL patient failed to inhibit CFU-GM and BFU-E-derived colony growth from control PB mononuclear cells. The high levels of circulating progenitor cells in ALL and CLL patients clearly distinguish them from other cytopenic hematological malignancies, in which decreased progenitor cell levels have been demonstrated previously (acute myeloid leukemia, hairy cell leukemia). The cause of this finding and its pathophysiological implication still remains to be established.     

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 specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.     

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.     

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.     

Lymphoma immunophenotyping: "borderline" lymphomas

Immunophenotyping of B-cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(-) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as "borderline" CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.     

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.     

Enhancement of 5'-nucleotidase activity of human leukemic cells after fractionation: implications for cancer and aging

Previous studies reported that 5'-nucleotidase activity was undetectable or at much lower levels in the homogenate of human chronic lymphocytic leukemic (CCL) cells than in normal lymphocytes. In the present study, 5'-nucleotidase specific activity in acute myelocytic leukemia (AML), which varied in a range from undetectable to 1.4 (nmoles/min.mg protein), was enhanced by cell fractionation, from undetectable in the homogenate, up to 18.8 +/- 1.2, 6.4 +/- 0.7 and 0.68 +/- 0.12 in plasma membranes, microsomes, and cytosol fraction, respectively. In a further fractionation of the cytosol of various leukemic cells with ammonium sulfate, 5'-nucleotidase specific activity increased up to 14-fold in the 60% (NH4)2SO4 fraction, with a recovery of 1266 +/- 115%. These data suggest that 5'-nucleotidase activity in fractionated leukemic cells is higher than reported previously and that the sum of 5'-nucleotidase activity in subcellular compartments is higher than that detected in the homogenate. Furthermore, even when 5'-nucleotidase was undetectable in a homogenate, it became detectable in the plasma membranes, suggesting that its ecto-enzyme function is still active in leukemic cells. The undetectable or low 5'-nucleotidase in the homogenate is indicative of (1) the enzyme itself being in an inactive form but becoming active after the fractionations, or (2) the presence of a factor(s) that prevents the enzyme from being detected but that is separated from the enzyme by the fractionations. In both cases, the rate of nucleotide catabolism by inactive 5'-nucleotidase in rapidly proliferating leukemic cells should be slower than when the enzyme is active. The present finding is consistent with our previous findings that during normal cell aging the high 5'-nucleotidase activity is associated with senescent non-proliferating cells but low or undetectable activity with rapidly proliferating immortal cells. The implications of 5'-nucleotidase for DNA synthesis in aging and cancer are discussed.     

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.     

Effect of cortisol on the ultrastructure of normal, leukemic, and cultured human lymphocytes.

The effect of cortisol on the ultrastructure of normal, leukemic, and cultured human lymphocytes during a 2-hr incubation was investigated. The presence of 10(-5) M cortisol in the incubation medium produced in normal lymphocytes a variety of alterations in cytoplasmic organelles. Mitochondria were most affected and showed evidence of irreversible deterioration (formation of myelin figures). Occasional cells demonstrated an overt rearrangement of their cytoplasmic membranes resulting in a bizarre array of parallel cisternae-like structures. More commonly, the usually underdeveloped Golgi of normal lymphocytes became very pronounced in structure. All of these alterations were produced within 2 hr of incubation, but only in normal human lymphocytes. Under identical conditions, no evidence of ultrastructural changes were produced by cortisol in either lymphocytes from chronic lymphocytic leukemic patients, or those from the RPMI 1788 cell line.     

The pathology and homing of a transplantable murine B cell leukemia (BCL1).

The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to kappa- and lambda-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 lambda-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.     

New antigenic determinant (Sa) on human lymphocytes and phagocytes

Presence of antigenic determinants reacting with homogeneous IgM/kappa cold agglutinin (CA) of a new specificity, tentatively called Sa, was investigated by bithermic cytotoxicity assay and by immunofluorescence. CA Sa killed on average 38% allogeneic peripheral blood lymphocytes (PBL) and up to 74% of autologous PBL. There was preferential kill of B-PBL compared to T-PBL. Some preference toward B cells was also noted using tonsillary B and T lymphocytes. Cytotoxic activity of CA Sa against chronic lymphocytic leukemia cells of B-type was almost equal to that of potent anti-I CA and much stronger than anti-i CA. Presence of additional B-cytotoxic factor in the serum was excluded by the use of red blood cell eluate composed solely of homogeneous CA. Thymocytes and helper-type T cells from a patient with T cell chronic lymphocytic leukemia were very susceptible to the cytotoxic action of Sa. CA Sa killed 39% of monocytes, but there was almost no kill of polymorphonuclear leukocytes. Lymphocytotoxicity of CA Sa was abolished by sialyllactose and was not influenced by I-active glycoproteins. Comparison of CA Sa to CA of other specificities showed marked differences, supporting the view that Sa has new, previously unrecognized specificity.     

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     

Lymphocyte cholesterol synthesis in chronic lymphocytic leukemia

Studies in B and T lymphocytes from two patients with B cell chronic lymphocytic leukemia (CLL) showed that the rate of cholesterol synthesis and hydroxymethylglutaryl (HMG)-CoA reductase activity in the malignant cell--the B lymphocyte--did not differ from that in normals, before or after the treatment. Cholesterol synthesis and HMG-CoA reductase activity in T cells, on the other hand, from one patient (H.E.) before therapy, were only one-fifth of that in normals and in the other patient (J.R.) receiving treatment. Following chemotherapy and treatment with prednisone, T lymphocytes from H.E. exhibited normal cholesterol metabolism.