Effect of D2O on phytohemagglutinin-induced lymphocyte proliferation.

The effect of D2O on phytohemagglutinin (PHA)-induced proliferation of human lymphocytes in vitro was examined. A significant dose-dependent inhibition of the lymphocyte reactivity by D2O was observed. This inhibition did not require the simultaneous presence of D2O and PHA in the cultures. Both cytotoxic and cytostatic mechanisms for this inhibition are implicated.     

Lack of effect of azathioprine on phytohemagglutinin-induced lymphocyte transformation and established delayed cutaneous hypersensitivity.

The effect of experimental long-term azathioprine administration on pre-existing in vitro and in vivo manifestations of delayed hypersensitivity was examined. Adult ewes were administered 3 mg/kg/day azathioprine for periods of up to 28 days. Neither phytohemagglutinin (PHA)-induced nor tuberculin-induced lymphocyte transformation was affected. Similarly, pre-existing skin test positivity to tuberculin showed no observable change following drug therapy. These results agree with the majority of those involving clinical situations and indicate that immunosuppression with azathiprine cannot be effectively monitored by either the lymphocyte transformation response to PHA or by changes in the in vivo or in vitro manifestations of pre-existing delayed cutaneous hypersensitivity.     

Effect of normal mouse serum on mouse lymphocyte transformation in vitro

Mouse spleen cells were cultured in the presence or absence of one of the following mitogens: phytohemagglutinin (PHA), concanavalin A, allogeneic spleen cells or bacterial lipopolysaccharide. The addition of normal mouse serum (NMS) to the cultures usually depressed mitogen-induced lymphocyte transformation as measured by DNA, RNA or protein synthesis, whether calculated as gross or net synthesis. The degree of depression was increased as the concentration of NMS serum increased. DNA and RNA synthesis in unstimulated cultures were also usually depressed by NMS. NMS added 24 hours or more after the start of cultures with PHA or allogeneic lymphocytes was less depressive than serum present from the start and in some cases, actually increased DNA synthesis. The presence of NMS in cultures containing supra-optimal doses of concanavalin A could also increase DNA synthesis. Cells incubated with NMS for 24 hours survived as well as cells incubated without NMS, but subsequently responded less well to PHA. Cells passed through a column of glass wool had a lower baseline DNA synthesis, which was not inhibited by NMS; they responded less well to mitogens and this response was further depressed by NMS. It is suggested that NMS contains a factor which damps the proliferation of mouse T and B cells, without exerting an overt cytotoxic effect, and that it acts directly on lymphocytes.     

Isoprinosine augmentation of phytohemagglutinin-induced lymphocyte proliferation.

The need for agents designed to modify immune response in the treatment of patients with viral infection, immunodeficiency, or cancer prompted the present study on the mechanisms of action of isoprinosine, a compound developed for antiviral use and whose therapeutic activity may involve the immune system. The effect of isoprinosine on in vitro proliferation of human peripheral blood lymphocytes stimulated by phytohemagglutinin (PHA) and on lymphocyte levels of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate was analyzed. Over a concentration range from 0.2 to 250 mug/ml, isoprinosine augmented PHA-induced proliferation; maximal stimulation was observed between 25 to 50 mug/ml. Isoprinosine in the absence of PHA had no effect on proliferation. The relative lack of effect of isoprinosine during a 90-min exposure and the lack of effect on lymphocyte cyclic nucleotide levels indicate that isoprinosine potentiates the PHA response by a mechanism different than a number of hormonal agents and such immunopotentiators as levamisole, polyadenylic-acid, and endotoxin. Further evaluation of isoprinosine as an immunopotentiator is indicated.     

Clearance of Cryptococcus neoformans from immunologically suppressed mice.

To assess the effects of cryptococcal antigen-induced immunosuppression on a Cryptococcus neoformans infection, CBA/J mice were injected intravenously with saline or suppressive doses of cryptococcal antigen (CneF) at weekly intervals and were then infected with viable C. neoformans cells. By the second week after infection, the cryptococcal antigen-injected mice had suppressed anticryptococcal delayed-type hypersensitivity (DTH) responses compared with the responses of the saline-treated, infected control mice. In addition, the immunosuppressed mice had higher numbers of cryptococcal CFU cultured from their lungs, livers, spleens, lymph nodes, and brains than did the control animals. A direct correlation of suppression of the anticryptococcal DTH response and reduced clearance of cryptococci from tissues was also observed after mice were given a single intravenous injection of CneF and infected. To determine whether or not the cryptococcal antigen was specifically reducing the clearance of C. neoformans or had a more generalized effect, mice were injected with saline or suppressive doses of CneF, infected with Listeria monocytogenes, and then followed daily for 7 days for the clearance of L. monocytogenes from spleens and on day 7 for DTH reactivity to Listeria antigen. There were no differences between the saline- and CneF-treated mice with respect to anti-Listeria DTH responses or clearance of L. monocytogenes from spleens, indicating that CneF was not altering natural resistance mechanisms responsible for early clearance of L. monocytogenes, nor was the CneF influencing the induction of the acquired immune response which was responsible for the late clearance of the bacteria. Together, these data indicate that the specific suppression of this cell-mediated immune response induced by cryptococcal antigen reduces the ability of the animals to eliminate the homologous organism (C. neoformans) but not a heterologous infectious agent, such as L. monocytogenes.     

Some properties of a phytohemagglutinin-induced lymphocyte mitogenic factor

The principles governing production and the effect on lymphocytes of the mitogenic factor (MF), secreted by human lymphocytes in vitro under the influence of phytohemagglutinin (PHA) and described previously by the author, were investigated. PHA was inactivated by anti-PHA antiserum and immunosorbents. Lymphocytes stimulated by PHA secrete MF in the G₁-period and at the beginning of the S-period of the cell cycle. During cultivation of the cells in a protein-free medium DNA and protein synthesis was sharply inhibited but MF production was 1.6 times higher than in media containing serum. The kinetics of the reaction of lymphocytes to MF is described: DNA synthesis (reflected in thymidine-H³ incorporation) began on the 5th–6th day and reached an maximum on the 6th–7th day.Belorussian Research Institute of Hematology and Blood Transfusion, Minsk. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 9, pp. 76–79, September, 1975.     

Effect of uremia on lymphocyte transformation and chemiluminescence by spleen cells of normal and Cryptococcus neoformans-infected mice.

The effect of uremia on immune incompetence was studied. BALB/c mice were infected with a minimally virulent strain of Cryptococcus neoformans 6 weeks before immune assay. Uremia was induced by intramuscular injection of 0.15 ml of glycerol. Pooled spleen cells from four experimental groups (normal, uremic, infected, and infected and uremic) were assayed by lymphocyte transformation (LT) and luminol-dependent chemiluminescence (CL) 24 h after induction of uremia. A greater response to phytohemagglutinin and concanavalin A stimulation in tests of LT and CL was exhibited by uremic cells than by nonuremic cells; however, the presence of BALB/c uremic serum resulted in lower responses by both LT and CL. Infected mice showed a greater response to mitogens than did noninfected mice, but no significant stimulation in response to heat-killed whole cells of C. neoformans. Spleen cell populations of uremic mice had a lower viability and a different composition of spleen cell subpopulations than did cell preparations from nonuremic mice.     

Effect of sport stress on lymphocyte transformation and antibody formation.

The effect of a heavy (marathon, 2.5 hr) and moderate (35 min of running) sport stress on the number and function of lymphocytes, and on the plasma cortisol and leucocyte levels was investigated. Marathon running had a profound effect on the lymphocytes. Though the total number of lymphocytes did not change, their responsiveness to PHA and Con A, especially to PPD, was clearly depressed. The suppression of lymphocyte transformation was transient, the recovery occurring in 24 hr. The marathon running had no effect on antibody-forming capacity when the antigen was given immediately after the performance of the marathon, i.e. at the time when the response of lymphocytes to PHA, Con A and PPD stimulation was impaired. A clear-cut granulocytosis and elevation of plasma cortisol was seen in all the marathon runners. The 35 min of running also resulted in granulocytosis and an increase of plasma cortisol, but it did not cause any impairment of the lymphocyte function.     

Immunoglobulin synthesis and lymphocyte transformation by anti-immunoglobulin sera in bursectomized chickens

It was found in bursectomized chickens that selective inhibition of IgM or IgG immunoglobulin synthesis occurs with equal frequency. Alterations in the immunoelectrophoretic pattern of immunoglobulins were described. Lymphocyte transformation induced by rabbit anti-IgM serum was inhibited in chickens lacking either IgM or IgG. Spleen lymphocytes from bursectomized chickens with a normal pattern of immunoglobulins exhibited transformation induced by anti-globulin serum like controls, despite a decreased humoral antibody response.     

肺癌组织培养液和病人血清对自身T细胞集落形成能力的影响

本文观察了10例肺癌病人围术期的肺癌组织、正常肺组织培养液和血清对自身外周血T细胞集落形成能力的影响,并以14例正常人的血清作为对照。结果显示:肺癌人病在去肿瘤负荷前T细胞集落产率为160.73±124.02/10~5,故低于正常人的397.81±133.89/10~5(P<0.001),也低于去肿瘤负荷后的(P<0.001)306.53±79.86/10~5(P<0.001)。术前病人的血清对T细胞集落的抑制率为46.97±21.42%,术后则下降至27.63±23.25%(P<0.01)。加进肺癌组织培养液时,病人的T细胞集落产率为224.83±104.05/10~5,正常肺组织培养液则为323.12±125.27/10~5(P<0.001),肺癌组织培养液和病人血清两者对T细胞集落产率的抑制性呈正相关关系(r=0.817,P<0.005)。本研究结果认为,肺癌组织能产生细胞免疫抑制物质进入血循环,导致病人T细胞集落形成能力明显下降。手术根治性切除肺癌组织,能有效地解除T细胞集落形成能力受抑制的情况。     

Tuberculous anergic sera or purified protein derivative treatment induces modification in lymphocyte transformation of cells from patients with tuberculosis.

Peripheral blood mononuclear cells from 20 purified protein derivative (PPD)-reactive (PPD+) tuberculous patients were cultured in autologous, tuberculous anergic, or normal serum. After 12 h of incubation, the serum was eliminated and lymphocyte transformation with PPD was performed. Transformation was inhibited only in cells incubated with anergic serum. In contrast, cells from 11 anergic tuberculous (PPD-) individuals recovered the ability to respond to an optimal PPD dose after treatment with high PPD concentrations followed by several washings. The cells which recovered returned to their initial anergic state when incubated with sera from anergic patients. Under both conditions, incubation with sera did not abolish the response to the mitogen phytohemagglutinin. Cells from healthy PPD+ or PPD- individuals were used as controls. The most important finding derived from serum analysis was the increased levels of specific immunoglobulins G and A in anergic patients.     

In vitro spleen leucocyte migration and phytohaemagglutinin-stimulated lymphocyte transformation of immunosuppressed mice cells. I. Cells from mice treated by thymectomy and/or rabbit anti-mouse lymphocyte serum

An in vitro study of spleen leucocyte migration ability and phytohaemagglutinin (PHA) stimulated peripheral lymphocyte transformation response was made on cells derived from mice treated by thymectomy, thymectomy and/or rabbit anti-mouse lymphocyte serum (RAMLS). These responses were compared with those shown by normal mice and found to be considerably depressed. Thymectomy combined with RAMLS treatment produced the greatest change. This decreased activity persisted for up to 100 days. The spleen leucocyte migration results changed in parallel with the PHA-stimulated lymphocyte response and were considered to reflect a T-cell function.     

Effects of monocytes from human neonates on lymphocyte transformation

Adherent cells (approximately 75% monocytes, 25% lymphocytes) obtained from neonates and from adults were studied to compare their effects on mitogen-induced lymphocyte transformation. The response of autologous lymphocytes to concanavalin A (Con A) was enhanced significantly by adherent cells from neonates and from adults. Whereas the addition of 2-mercaptoethanol (2-ME) did not enhance the response of unseparated peripheral blood mononuclear cells or fully restore the response of adherent cell-depleted lymphocytes from neonates, both of these effects were observed when 2-ME was added to cell preparations from adults. In fact, the response to Con A of lymphocytes from adults was significantly greater in the presence of 2-ME than in the presence of autologous adherent cells. Equivalent enhancement of the response to Con A was observed when adherent cells from neonates were added to lymphocytes from adults or when adherent cells from adults were added to lymphocytes from neonates.

Adherent cells from neonates consistently inhibited the autologous lymphocyte response to the specific B-cell mitogen, NWSM, a water-soluble extract of Nocardia opaca. Lymphocytes from five out of nine neonates failed to respond to NWSM unless adherent cells were depleted. The presence of adherent cells did not prevent the response of lymphocytes from any of the eight adults tested. This difference in response to NWSM between lymphocytes from neonates and adults was significant. Inhibition of the response of autologous lymphocytes to NWSM by adherent cells from adults was of lesser magnitude and could be demonstrated consistently only when 2-ME was added to adherent cell-depleted lymphocyte preparations. We conclude that the effects of adherent cells which were observed were due to monocytes. The enhancing effect of monocytes from adults on lymphocyte response to Con A could be replaced by 2-ME, whereas this was not true for neonates. In contrast to their effects on response to Con A, monocytes from neonates inhibited the response to NWSM more consistently and to a greater degree than did monocytes from adults.

     

Influence of growth hormone-releasing hormone (GHRH) on phytohemagglutinin-induced lymphocyte activation: comparison of two synthetic forms. GHRH and PHA-induced lymphocyte activation.

The in vitro effect of synthetic human growth hormone-releasing hormone (GHRH) on mitogen-induced lymphocyte proliferation and lymphokine secretion was investigated. Peripheral blood mononuclear cells (PBMC) of healthy adults were incubated in the presence and absence of increasing concentrations (from 0.006 to 50 micrograms/ml) of two forms of GHRH differing in amino-acid sequence (GHRH 1-44 and GHRH 1-29) or of increasing concentrations (from 0.0012 to 20 U/ml) of recombinant human insulin (rh-insulin). Low concentrations of GHRH 1-29 increased phytoemoagglutinin (PHA)-induced lymphoproliferation, while high concentrations inhibited lymphocyte response, interleukin-2 (IL-2) secretion and IL-2 receptor expression on activated cells. A toxic effect was excluded since no differences in cell viability were observed between cells cultured with and without hormone. GHRH 1-44 did not affect PHA-induced lymphoproliferation, IL-2 production and IL-2 receptor expression. Low concentrations of rh-insulin increased PHA-elicited lymphoproliferation, while high concentrations did not decrease lymphocyte response. The present study suggests that GHRH modulates in vitro human T lymphocyte functions.     

Inhibition of phytohemagglutinin-induced lymphocyte proliferation by dexamethasone: mechanisms of individual susceptibility

Direct positive correlation has been shown between the level of stimulation of peripheral blood lymphocytes and of the inhibition degree of PHA stimulation by dexamethasone. On the basis of this correlation we subdivided all the examined individuals into three groups of susceptibility: resistant, sensitive, and intermediate. The studies carried out in these groups showed that the reception effectiveness of glucocorticoids (GC) was much higher in the sensitive group than in the group of resistant individuals. In this group interleukin 2 (IL-2) production was relatively high; in the intermediate group this parameter was lower, and in the group of sensitive individuals the mean indices of IL-2 production were the lowest. However, this latter group unlike the two others was heterogeneous and may be divided into two subgroups: high and low IL-2 producers. HLA antigens B7 and DR2 were also shown to be associated with a decrease in sensitivity to GC inhibition of PHA-induced lymphocyte proliferation. Our results indicate that the method we used is adequate enough for dividing individuals into sensitivity groups.     

Characterization of lymphocyte E rosette inhibitory factor in sera from tuberculous patients.

Rosette inhibitory factor (RIF) in serum of patients suffering from active pulmonary tuberculosis, after in vitro contact with lymphocytes from healthy subjects, inhibits spontaneous rosette formation of these lymphocytes with sheep red blood cells (SRBC). RIF is not a C-reactive protein. By the use of chromatography on Sephadex G-200 and single analytic flotation, RIF was found to be contained in the beta-lipoprotein fraction. The presumable mechanism of formation of this factor and suggestions concerning its action on T cells are discussed.