Lymphocyte Blastogenic Response Research Articles

Superoxide dismutase activity and blastogenic response of lymphocytes from copper-deficient rats fed diets containing fructose or cornstarch

Male weanling rats were pair-fed diets containing either fructose (F) or starch (S) and either 7 ppm (+) or 0.7 ppm (−) copper (Cu) for 4–5 weeks. The activity of Cu, Zn-superoxide dismutase in splenocytes, cervical lymph node cells (CLNC) and thymocytes and the copper content of thymocytes were lower in rats fed −Cu diets than +Cu diets. Moreover, these indicators of Cu status were lower when rats were fed −Cu diets with F than with S. T-lymphocyte function was assessed by measuring mitogen-stimulated DNA synthesis in cultures of lymphoid cells incubated in medium containing 2% sera pooled from rats fed either the identical diet (homologous system) or the diet with same carbohydrate but different level of Cu (heterologous system) as the tissue donor. In the homologous system, mitogen-induced DNA synthesis was lower in splenocytes, higher in CLNC, and unchanged in thymocytes isolated from rats fed −Cu diets than in the respective cells from +Cu rats. Mitogenic reactivity was inhibited to a greater degree in splenocytes from rats fed −Cu diets containing F instead of S. There was a tendency for unstimulated cells from −Cu rats to incorporate higher levels of 3H-thymidine than cells from copper-adequate rats. 3H-thymidine incorporation in unstimulated cells from rats fed −Cu diets tended to be significantly different in cultures containing heterologous sera than those in cultures containing homologous sera. In contrast, source of sera did not affect mitogen-induced DNA synthesis. These results demonstrate that the impact of dietary copper deficiency on the function of T-lymphocytes is organ specific and can be influenced by the type of dietary carbohydrate.

Colony Forming T Lymphocyte Deficit in the Development of Feline Retrovirus Induced Immunodeficiency Syndrome

The identification and molecular cloning of a feline leukemia virus (FeLV) isolate (FeLV-FAIDS) that consistently produces immunodeficiency syndrome has allowed prospective investigation of events that occur in the prodromal phase of disease. Using a T-lymphocyte colony forming assay (T-CFU-Ic) we have demonstrated that a drastic depletion of circulating T-CFU-Ic prefigures the development of clinical immunodeficiency disease in inoculated cats and correlates with the appearance and replication of the FeLV-FAIDS variant genome in serially collected bone marrow samples. During the same presymptomatic time period, no significant alterations in conventional mitogen-induced lymphocyte blastogenic responses or in circulating lymphocyte numbers were evident. Thus T-CFU-Ic assay but not conventional mitogen-driven blastogenesis identified animals destined to develop immunodeficiency syndrome. The correlation among T-CFU-Ic depletion, the replication of the lymphocytopathic FeLV-FAIDS variant genome in hematopoietic and lymphoid tissues, and the onset of clinical disease, infers that ablation of a colony-forming T lymphocyte progenitor subset is important in the early pathogenesis of feline retrovirus-induced immunodeficiency syndrome.

Colony forming T lymphocyte deficit in the development of feline retrovirus induced immunodeficiency syndrome

The identification and molecular cloning of a feline leukemia virus (FeLV) isolate (FeLV-FAIDS) that consistently produces immunodeficiency syndrome has allowed prospective investigation of events that occur in the prodromal phase of disease. Using a T-lymphocyte colony forming assay (T-CFU-Ic) we have demonstrated that a drastic depletion of circulating T-CFU-Ic prefigures the development of clinical immunodeficiency disease in inoculated cats and correlates with the appearance and replication of the FeLV-FAIDS variant genome in serially collected bone marrow samples. During the same presymptomatic time period, no significant alterations in conventional mitogen-induced lymphocyte blastogenic responses or in circulating lymphocyte numbers were evident. Thus T-CFU-Ic assay but not conventional mitogen-driven blastogenesis identified animals destined to develop immunodeficiency syndrome. The correlation among T-CFU-Ic depletion, the replication of the lymphocytopathic FeLV-FAIDS variant genome in hematopoietic and lymphoid tissues, and the onset of clinical disease, infers that ablation of a colony-forming T lymphocyte progenitor subset is important in the early pathogenesis of feline retrovirus-induced immunodeficiency syndrome.

Virus-Induced Immunosuppression: Infections with Measles Virus and Human Immunodeficiency Virus

Publisher Summary The basic phenomenology of disordered lymphocyte function because of the infection by a lymphotropic virus is neither novel nor unexpected. The problem of measles virus-induced immunosuppression is contemporaneous with modern immunology and virology. This chapter explains that immunosuppression associated with HIV infection currently commands significant attention in biomedical research. Any unexplained disorder of immune response, either heightened or suppressed, might well be investigated with an infectious etiology in mind. Measles virus infects a wide range of epithelial and lymphoblastoid cell lines in vitro . The cell membrane receptor for measles virus is a nonsialated oligosaccharide present on many cells. Productive infection of cells by measles virus is not lytic and constitutive cellular protein synthesis is not altered. Active tuberculosis infection is a common sequela of measles virus infection. Researchers showed that blastogenic response of lymphocytes from the peripheral blood of tuberculosis-immune individuals to tuberculin antigen is significantly reduced during acute measles virus infection. The suppression by measles virus of the proliferative response to mitogen occurs with lymphocytes from both the nonimmune and the measles-immune donors, but serum containing measles antibody abrogates the suppression. During chronic HIV infection in vivo and in vitro , proviral DNA exists in both integrated and nonintegrated forms. Unlike the other lentiviruses and most retroviruses, a cellular receptor for HIV has been well characterized. The immunosuppression in AIDS patients has been characterized by global dysfunction of cellular immunity and polyclonal B cell activation. A significant part of this chapter discusses the immune dysfunction during HIV infection prior to the onset of AIDS and on the effects of HIV infection of lymphocytes and monocytes in vivo .

The inhibitory effects of colchicine, vinblastine and cytochalasin D on lymphocyte blastogenic responses.

The inhibitory effects of colchicine, vinblastine and cytochalasin D on blastogenic responses of equine, bovine and canine peripheral blood lymphocytes (PBL) were investigated. These drugs inhibited blastogenic responses of each PBL. The concentrations for 50% recovery in PBL blastogenic response of colchicine and vinblastine were lower in equine and canine PBL than in bovine PBL. This suggested that microtubules may be more concerned in blastogenic response of equine and canine PBL than of bovine PBL. On the other hand, the concentration for 50% recovery in PBL blastogenic response of cytochalasin D was almost the same in the PBL of each animal. This meant that microfilaments made only a small contribution to the lymphocyte blastogenic response.

Synthesis of a thymosin beta 4-like peptide, deacetyl-thymosin beta Xen4, and its restorative effect on depressed lymphocyte blastogenic response to phytohemagglutinin (PHA) in uremic patients.

An analog of thymosin beta Xen4 isolated from oocytes of Xenopus laevis, deacetyl-thymosin beta Xen4, was synthesized by assembling 6 peptide fragments, followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole (molar ratio, 1:1) in trifluoroacetic acid in the presence of dimethylselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulfoxide on the methionine side chain. The synthetic tritetracontapeptide was found to have a restoring effect on the impaired blastogenic response of T-lymphocytes isolated from uremic patients.

Effect of increased circulating corticosterone in the immature fowl on the blastogenic responses of peripheral blood lymphocytes

Plasma corticosterone concentrations were increased by giving chickens implants containing corticosterone and the effects on lymphocyte numbers and lymphocyte responses to the mitogens, phytohaemag-glutinin (PHA) and concanavalin A (con A), were measured. Raising plasma corticosterone caused a dose-related decrease in numbers of circulating lymphocytes. Blastogenic responses of lymphocytes in whole blood to PHA and con A were reduced and stimulation indices were decreased in those birds with raised circulating corticosterone. When the initial corticosterone-induced lymphocytopenia was taken into account it was evident that corticosterone depressed the responsiveness of the lymphocytes to PHA but not to con A. Additions of corticosterone to cultures of whole blood showed that PHA stimulation was more sensitive than con A stimulation to the inhibitory effects of corticosterone.

An Analysis of in Vitro T Cell Responsiveness in Nontuberculous Mycobacterial Infection

In vitro cell-mediated immunity was examined in patients infected with nontuberculous mycobacteria, Mycobacterium avium-intracellulare complex in Japan. Peripheral blood lymphocytes of patients, as compared with those of tuberculous patients or tuberculin-positive healthy donors, showed depressed in vitro blastogenic responses to purified protein derivative of tuberculin (PPD), not only to PPDs of Mycobacterium tuberculosis but also to PPD-B and PPD-Y of M intracellulare and M kansasii, respectively. Nonspecific lymphocyte blastogenic responses to concanavalin A, phytohemagglutinin and pokeweed mitogen were normal. Analysis of defective in vitro PPD-induced lymphocyte blastogenic responses in these patients revealed that PPD-induced interleukin 2 (IL-2) production was impaired whereas PPD-induced IL-2 responsiveness was normally developed after PPD stimulation. Therefore, addition of exogenous recombinant human IL 2 substantially recovered the in vitro depressed PPD-induced blastogenic responses in these patients with nontuberculous mycobacterial infection.

Immunological abnormalities in children with acute rheumatic carditis and acute post-streptococcal glomerulonephritis

Immunological functions were investigated in 10 children with acute rheumatic fever and 11 children with acute nephritis to try and elucidate the cause of heart damage in acute rheumatic fever. Children with acute rheumatic fever and carditis showed an increase in serum IgG, IgA and antistreptococcal antibodies during the acute stage. Lymphocyte transformation responses to phytohaemagglutinin and streptococcal antigens were reduced but this was due to a serum suppressor effect. After recovering from acute rheumatic fever a lymphocytosis and an increased lymphocyte blastogenic response to streptococcal antigen were found. T-cells, T-helper cells and T-suppressor cells showed some changes in acute rheumatic fever but these were not statistically significant in our study. None of the changes in immunological responses that were seen in acute rheumatic fever were found in acute nephritis. These results support the hypothesis that an abnormal immune response to streptococcal products is involved in the development of carditis and the other phenomena observed in acute rheumatic fever.

Inhibition of normal human natural killer cell activity by human immunodeficiency virus synthetic transmembrane peptides

The inhibitory effect on normal natural killer (NK) cell activity of two synthetic peptides corresponding to amino acid sequences 735–752 and 846–860, respectively, as deduced from the amino acid sequences of HTLV-IIIB gp160, was assessed. Sequences 735–752 and 846–860 correspond to regions located within the HIV transmembrane gp41, the carboxy terminus of HIV gp 160. These two synthetic peptides have been shown previously to suppress the mitogenand alloantigen-induced normal human lymphocyte blastogenic responses. Peptides 735–752 and 846–860 conjugated to protein carriers exerted a significant inhibition on the normal NK cell activity assayed against K562 tumor target cells in an in vitro 51Cr-release cytotoxicity assay. At variance, control peptides similarly conjugated had no effect on NK activity. Addition of exogenous recombinant human interleukin-2 (IL-2) resulted in a partial restoration of the suppression of NK cell activity exerted by both peptides. Binding experiments indicated that peptides 735–752 and 846–860 did not affect the formation of effector cell-target cell conjugates, suggesting inhibitory effect(s) subsequent to the formation of the lytic complex as one potential mechanism of the observed NK suppression. These results suggest that peptides 735–752 and 846–860 homologous to sequences within the HIV transmembrane gp41 may play an important role in the pathogenesis of the defective NK cell activity observed in patients with acquired immunodeficiency syndrome (AIDS).

Effects of antiestrogen and progestin on immune functions in breast cancer patients.

Several immunologic variables were evaluated in 14 patients with untreated primary breast cancer and 20 postmastectomized patients undergoing tamoxifen (TAM) or high-dose medroxyprogesterone acetate (MPA) treatment. Immunologic evaluation in the peripheral blood included lymphocyte count, definition of T-lymphocyte subsets by monoclonal antibodies (OKT3, OKT11, OKT4, and OKT8), and lymphocyte blastogenic response to phytohemagglutinin (PHA) and Concanavalin A (Con A). Moreover, the in vitro effect of TAM and MPA on the blastogenic response of peripheral lymphocytes from normal female subjects was tested. Primary breast cancer patients did not differ from controls in any of the variables tested. Similarly, the immunologic variables of the group treated with TAM were normal, with the exception of a slight reduction of the OKT4+/OKT8+ ratio. In MPA-treated patients, a reduction of the percentage of OKT4+ cells and a decrease of the OKT4+/OKT8+ ratio were observed. Moreover, response to PHA was reduced sharply. However, the addition of interleukin-2 (IL-2) to the culture medium restored PHA response. Likewise, the in vitro addition of MPA to peripheral blood lymphocytes from normal female subjects resulted in a sharp dose-dependent depression of PHA response while TAM was ineffective completely. The inhibitory effect of MPA was not evident when IL-2 was added simultaneously to the culture medium. These results show that the administration of high-dose MPA may alter immunocompetence as defined by T-lymphocyte subsets and response to mitogens. The latter effect may be related to a diminished production of IL-2. In contrast, TAM does not appear to have a significant immunodepressant action either in vitro or in vivo.

Effects of Liposomal Amphotericin B on Human Peripheral Blood Monocytes and Lymphocytes

AbstractThe effects of free amphotericin B (AmpB) and liposome-encapsulated amphotericin B (L-AmpB) on normal human peripheral blood monocytes (HPBM) and lymphocytes were studied. L-AmpB did not reduce HPBM H2O2-generating capacity even at doses of 100 µg/ml. there was no direct induction of H2O2by either AmpB or L-AmpB. L-AmpB (at concentrations of 10µg/ml) did not suppress lymphocyte blastogenic responses (LBRs) to concanavalin A and phyto-hemagglutin. Similar concentrations of AmpB caused a greater than 30% decrease in LBRs related to a direct effect on lymphocytes. Antibody-dependent cell-mediated cytotoxicity and natural killer cell activity were markedly reduced by AmpB at a concentration of 5 µg/ml but not by L-AmpB. L-AmpB was shown to be less toxic to lymphocytes and HPBM than AmpB; however, no potentiation of the immune parameters tested was observed with either drug.

Synthetic peptides homologous to HIV transmembrane glycoprotein suppress normal human lymphocyte blastogenic response

Human immunodeficiency virus (HIV) envelope glycoprotein is synthesized as a polyprotein precursor of 160 kDa (gp 160) and is subsequently cleaved into an amino terminus subunit, gp 120, and a carboxyl terminus transmembrane subunit, gp 41. Two synthetic peptides corresponding to amino acid sequences 735–752 and 846–860, respectively, as deduced from the nucleotide sequence of HTLV-III B gp 160 were synthesized and used to assess their effects on normal human lymphocyte blastogenesis. Peptides 735–752 and 846–860 conjugated to protein carriers, but not free peptides, exerted a pronounced suppression of the normal human lymphocyte proliferative response to concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and alloantigens. A synthetic peptide homologous to a 17 amino acid sequence of the gene product of HIV trans-acting transcriptional (TAT III) gene had no suppressive effects. Peptides 735–752 and 846–860 also inhibited the IL-2-dependent proliferation of the murine CTLL-2 cell line and the PHA-induced proliferation of normal mouse spleen cells. HIV peptide-induced suppression of human blastogenesis required a 2- to 3-day incubation of responder cells with peptides, suggesting that binding of peptides to the cell membrane was not sufficient for suppression. These results suggest that, in addition to the selective cytopathic effects of HIV, the etiological agent of the acquired immunodeficiency syndrome (AIDS), on the T-helper/inducer lymphocyte subset, viral peptide-mediated immunosuppression may also play an important role in the pathogenesis of the disease. Moreover, these data clearly indicate the need to address the potential immunosuppressive property of HIV antigens in the effort to select and develop effective prophylactic means against AIDS.

Effect of erythromycin on the immune response and interferon production

The influence of erythromycin on some aspects of humoral and cell-mediated immunity has been examined employing human as well as murine models, both in vivo and in vitro. No significant differences in antibody synthesis, α- and γ-interferon yield, cutaneous delayed hypersensitivity or lymphocyte blastogenic response to mitogens have been detected between erythromycin-treated subjects and controls. Similarly, in vitro tests on interferon production and blastogenic response to mitogens showed no significant differences when performed with and without erythromycin. Therefore, in contrast with many other antibacterial drugs, erythromycin seems to be devoid of any adverse effects on the immune system.

The clinical and immunologic responses of normal human volunteers to low dose hookworm (Necator americanus) infection.

Five normal human volunteers were exposed to approximately 50 infective larvae of Necator americanus and were observed for the development of clinical signs or symptoms and for changes in blood eosinophil levels, IgG antibody titers, total and parasite-specific IgE, and lymphocyte blastogenic responses for 6-10 weeks. Bronchoalveolar lavage was performed on four subjects prior to infection and at times when larval migration through the pulmonary tree was likely. Eggs were demonstrated in the stools of four volunteers who remained untreated for more than 6 weeks; one volunteer had to be treated at day 40 because of severe gastrointestinal symptoms. All others also complained of abdominal pain and flatulence between days 35-40. All volunteers developed marked blood eosinophilia which peaked between days 38-64 and ranged from 1,350-3,828 eosinophils/mm3. Small increases in total and parasite-specific IgE and IgG were noted in some volunteers. One volunteer showed a significant lymphocyte blastogenic response. With the exception of mucosal erythema, bronchoalveolar lavage results were unremarkable. Our data indicate that a single small inoculum of hookworm larvae is capable of producing significant transient gastrointestinal morbidity and marked blood eosinophilia but does not induce other prominent T cell- and B cell-dependent immune responses.

Macrophage activation as an immune correlate to protective immunity against schistosomiasis in mice immunized with an irradiated, cryopreserved live vaccine.

Immune responses against Schistosoma mansoni were evaluated in C57BL/6 mice injected with one of two populations of irradiated schistosomules, the larval preparations differing only in the degree of freezing-induced damage sustained upon cryopreservation. Mice injected with larvae which successfully withstood cryopreservation showed a significant reduction in worm burden following cercarial challenge. No protection was achieved in mice which received larvae damaged by a suboptimal thawing rate. Parallel comparison of several humoral and cellular responses in mice which received either inoculum revealed that induction of activated macrophages and production of macrophage-activating lymphokine activity were the strongest correlates to development of protective immunity. Protected mice also showed marginal 30-min skin test reactivity and weak but transient 24-h delayed-type hypersensitivity to a soluble adult worm preparation. In contrast, indistinguishable levels of circulating antibodies to soluble and tegumental antigens developed in the two immunization groups, and antigen-stimulated lymphocyte blastogenic responses were strong and essentially equivalent in magnitude. These studies strongly suggested that in this new model for investigating anti-schistosome effector mechanisms, responses contributing to the development of activated macrophages may be essential for induction of protective immunity.

Vitamin E is Immunostimulatory in Calves1,2

Thirty-two Holstein heifer calves, eight per group, were fed 0, 125, 250, or 500 IU/d of supplemental vitamin E/calf, from birth to 24 wk of age, in order to determine the effect on their immune responses. Overall mean lymphocyte blastogenic responses to various T-cell and B-cell mitogens were higher in supplemented calves than in control calves. Mean concentrations of cortisol in serum were lower in all supplemented calves than in control calves. Antibovine herpesvirus type 1 antibody titer (IgG) at 8 and 9 wk, in response to a commercial modified-live intranasal vaccine at 7 wk, was similar in all treatment groups. At 24 wk, in response to a booster at 21 wk, titer was higher in calves given 125 IU of vitamin E/d than in control calves. Based on the concentrations used, it is concluded that supplementation of conventional rations with 125 IU of vitamin E/d may maximize immune responses in calves and may be cost effective.

Application of the fluorometric dna quantitative assay using 4’6-diamidine-2-phenylindole for the evaluation of lymphocyte blastogenic response

A fluorometric DNA microassay using the fluorescent reagent 4'6-diamidine-2-phenylindole dihydrochloride (DAPI) was developed for evaluating a lymphocyte DNA synthesis assay. Triton X-100 (0.05 per cent) was added to the cultured lymphocytes, then DAPI (0.8 micrograms ml-1) was added as a DNA-specific fluorescent intercalating agent. Results are expressed as fluorescence intensity (FI) and stimulation index. FIs measured by DAPI fluorometric assay were highly correlated with [3H]-thymidine incorporation (r = 0.868, P less than 0.01, n = 28). The method is simple, reliable and widely applicable for evaluating blastogenic responses.

EFFECTS OF PYOCYANINE (PYO) AND 1-OH-PHENAZINE (OHP) ON T-LYMPHOCYTE ACTIVATION

Phenazine pigments secreted by P.aeruginosa inhibit lymphocyte blastogenic responses. We studied the mechanism of inhibition of two model compound, PYO and OHP on the T-cell cycle. Peripheral blood mononuclear cells (PBMC) were stimulated with the mitogen, Concanavalin A (Con A) or with the following steps of T-cell activation were studied: 1. Increase in cytosolic-free Ca2+ using Quin-2 fluorescence. 2. Production of Interleukin 2 (IL-2) using the CTLL-20 cell line. 3. Expression of IL-2 receptors (IL-2R) using monoclonal antibodies and flow cytometry. 4). Uptake of 3H-Thymidine (3HTdR). Results with Con A activated PBMC are shown in the table.Lower concentrations of PYO and OHP inhibited Con A-induced 3H TdR uptake synergistically. In ionomycin-activated blastogenesis, 3H TdR uptake was strongly inhibited by PYO (94%), but only to a minor degree by OHP (30%). Therefore, PYO and OHP inhibit T-cell activation at different steps of the signal transduction pathway. OHP blocks the increase in cytosolic Ca2+ and PYO acts at a later step not by-passed by ionomycin. Locally secreted phenazine pigments, even in small amount, may act synergistically to inhibit cellular immune responses necessary to eradicate chronic infections with P.aeruginosa.

Amplification of HTLV-III/LAV infection by antigen-induced activation of T cells and direct suppression by virus of lymphocyte blastogenic responses.

Peripheral blood mononuclear cells (PBMNC) from healthy donors immune to the soluble antigens tetanus toxoid (TT) and/or keyhole limpet hemocyanin (KLH) were exposed to infectious human T lymphotropic virus, type III/lymphadenopathy-associated virus (HTLV-III/LAV) with and without prior activation by TT or KLH. After exposure to the virus, PBMNC that had been activated by antigen were 10 to 100 times more susceptible to viral replication, as estimated by measurement of production of reverse transcriptase and viral antigens, than PBMNC that had been preincubated without antigen. In addition, exposure of PBMNC to HTLV-III/LAV led to a loss of lymphocyte blastogenic responses after 2 to 3 wk in culture. HTLV-III/LAV-induced inhibition of lymphocyte blastogenic responses occurred in the absence of detectable production of RT but required the use of live rather than heat-inactivated virus. These results demonstrate that HTLV-III/LAV infection is amplified by antigen-induced activation of PBMNC, and that low levels of HTLV-III/LAV infection in vitro can suppress lymphocyte blastogenic responses. This study provides an in vitro model for the analysis of HTLV-III/LAV-induced immune defects.