Rat Splenic Cells Research Articles

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs.

Juniperus procera and Majra honey are well-known as a folk medicine in many countries. This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells' growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

Silver Nanoparticle Production by Ruta graveolens and Testing Its Safety, Bioactivity, Immune Modulation, Anticancer, and Insecticidal Potentials.

Ruta graveolens, a plant belonging to the family Rutaceae, is traditionally used as a medicinal plant and a flavoring agent in food. This work aimed to prepare silver nanoparticles (AgNPs) using the ethanol extract from R. graveolens leaves and test different biological activities as well as insecticidal potentials in the extract and extract prepared AgNPs. Dried and powdered R. graveolens leaves were subjected to extraction using ethanol, and this extract was used to synthesize AgNPs. AgNP synthesis was monitored by the change in color, UV spectrophotometry, and electron microscopy (scanning). Fourier transform infrared (FT-IR) spectroscopy was used to monitor the functional groups in the extracts. Immunological, physiological, anticancer, antibacterial, and insecticidal potentials of the extract and its prepared AgNPs were tested. Results showed the ability of the leaf extract to synthesize. SEM examination revealed a spherical shape of AgNPs with a size of 40–45 nm. The extract contained many functional groups as indicated by FT-IR. The extract alone inhibited the growth of normal rat splenic cells, while the extract containing AgNPs stimulated its growth. Extract alone stimulated HeLa cell proliferation and inhibited HepG2 growth, while both cell line growth was inhibited by the extract containing AgNPs. Both the extract and extract with AgNPs were safe on RBCs and did not cause any severe elevation in liver enzymes. The extract alone and with AgNPs showed insecticidal activity against Culex pipiens. Our findings suggest that the R. graveolens leaf extract, alone or with AgNPs, is biologically safe on animal cells and has antibacterial, insecticidal, and immunomodulation potentials.

Phyto-Synthesis, Characterization and Biological Applications of Gold Nanoparticles (AuNPs) Using Toxicosis Mediated Plant Nicotiana glauca Graham

Nanotechnology is a newer branch of material science that thrives in the interdisciplinary fields including biomedical, therapeutics, diagnostics etc. Among the metallic nanoparticles, gold nanoparticles (AuNPs) in particular are known for their versatile biological applications in several scientific fields in general and specifically the biomedical field. The current article accounts for an environmentally safe method for AuNPs synthesis using the chloroauric acid and ethanolic leaf extract of Nicotiana glauca (NG), for the first time. Various analytical techniques identified properties of synthesized AuNP to include UV-Vis absorption spectroscopy, Fourier-Transform Infrared Radiation (FT-IR), and scanning electron microscopy (SEM) for surface morphology. The average diameter of synthesized AuNP was about 100 nm. Antimicrobial activity of AuNPs was investigated on Grampositive ( Staphylococcus aureus ), Gram-negative (Escherichia coli , Proteus mirabilis, and Shigella flexneri) and fungi (Candida albicans) using well diffusion method. Cytotoxic action on Hela/HepG2 tumor cell lines and normal rat splenic cells were tested through cell viability. Furthermore, Larvicidal toxicity was performed on 4th instar larvae of Culex pipens . NG extract (NGEx) alone exerted significantly lower (6–7 mm) antimicrobial activity than that of NG extract with AuNPs (14–15 mm). Cytotoxicity screening revealed that both preparations were growth inhibitory on both cell lines and splenic cells. For larvicidal properties, the extract and gold nanoparticles (NGExAu) showed larvicidal effect against 4th instar larvae of Culex pipiens. NGExAu was more lethal (LC50 = 164.4072 ppm; LC90 = 613.366 ppm) against Cx. pipiens under exposure for 24 h with lesser lethal content com- pared to NGEx of only (LC50 = 752.865 ppm; LC90 = 4455.726 ppm).

Age and sex determine CD4+ T cell stimulatory and polarizing capacity of rat splenic dendritic cells.

The study investigated influence of sex and age on splenic myeloid dendritic cells (DCs) from Dark Agouti rats. Freshly isolated DCs from young males exhibited less mature phenotype and greater endocytic capacity compared with those from age-matched females. Upon LPS stimulation in vitro they were less potent in stimulating allogeneic CD4+ cells in mixed leukocyte reaction (MLR), due to lower expression of MHC II, and greater NO and IL-10 production. In accordance with higher TGF-β production, young male rat DCs were less potent in stimulating IL-17 production in MLR than those from young females. Irrespective of sex, endocytic capacity and responsiveness of DCs to LPS stimulation in culture, judging by their allostimulatory capacity in MLR decreased with age, reflecting decline in MHC II surface density followed by their greater NO production; the effects more prominent in females. Additionally, compared with LPS-stimulated DCs from young rats, those from sex-matched aged rats were more potent in stimulating IL-10 production in MLR, whereas capacity of DCs from aged female and male rats to stimulate IL-17 production remained unaltered and decreased, respectively. This reflected age-related shift in IL-6/TGF-β production level ratio in LPS-stimulated DC cultures towards TGF-β, and sex-specific age-related remodeling CD4+ cell cytokine pathways. Additionally, compared with LPS-stimulated DCs from young rats, those cells from sex-matched aged rats were less potent in stimulating IFN-γ production in MLR, the effect particularly prominent in MLRs encompassing male rat DCs. The study showed that stimulatory and polarizing capacity of DCs depends on rat sex and age.

Green Synthesis, Structural, In Vitro and In Vivo Bioactivity Properties of ZnO Nanoparticles for Biomedical Applications

Owing to the fascinating applications of ZnO in modern devices, it is interesting to explore its more aspects for future devices. Hence, herein, we have synthesized the high purity spherical ZnO nanoparticles (SNPs) through a facile green synthesis route and robust structural and biomedical studies are carried out. Hexagonal phase with 93.2% crystallinity was confirmed through XRD analysis. ZnO nanoparticles were tested for their bioactivities both in vivo (acute cytotoxicity test) and in vitro (Anti-cancer activities on the liver (HepG2) and cervical (Hela) cancer cell lines, stimulatory/inhibitory effects on normal rat splenic cells, hemolytic effects on red blood cells and antimicrobial activity). Results showed that ZnO SNPs has no cytotoxic effects on the vital organ like the liver and has no hemolytic action on red blood cells. ZnO SNPs showed inhibitory consequence on normal rat splenic cells growth at all tested concentrations. The ZnO nanoparticles showed a stimulatory effect on Hela cell line. Moreover, ZnO nanoparticles showed an inhibitory effect on HepG2 cell line and microbial cells.

Cellular proliferation/cytotoxicity and antimicrobial potentials of green synthesized silver nanoparticles (AgNPs) using Juniperus procera.

Juniperus spp. are used as medicinal plants in many countries like Bosnia, Lebanon, and Turkey. In folk medicines, these plants have been used for treating skin and respiratory tract diseases, urinary problems, rheumatism and gall bladder stones. The objectives of this work were to synthesize silver nanoparticles (AgNPs) using a coniferous tree, Juniperus procera leaf extract and testing the synthesized AgNPs for its antimicrobial potentials, hemolytic activity, toxicity and the proliferative effects against normal and activated rat splenic cells. Leaf extract was prepared using acetone and ethanol as solvents. AgNPs were prepared using the acetone extract. AgNPs were validated using UV-Vis spectroscopy and scanning electron microscopy (SEM). Functional groups in the extract were identified using Fourier Transform Infrared (FT-IR) spectroscopy. SEM images of AgNPs showed spherical and cubic shapes with a uniform size distribution with an average size of 30-90 nm. FT-IR spectroscopy showed the presence of many functional groups in the plant extract. AgNPs showed promising antimicrobial activity against tested bacteria and fungus. AgNPs also expressed a stimulating activity towards the rat splenic cells in a dose dependent manner. Acetone as solvent was safer on cells than ethanol. Green synthesized AgNPs using J. procera might be used as a broad-spectrum therapeutic agent against microorganisms and as an immunostimulant agent.

Effects of chelidonic acid, a secondary plant metabolite, on mast cell degranulation and adaptive immunity in rats

The present study evaluated the immunomodulatory effects of chelidonic acid, a secondary plant metabolite, with therapeutic potential in allergic disorders, in experimental animals. In mast cell degranulation studies, ovalbumin immunized and challenged rats, chelidonic acid (1, 3 and 10mg/kg, i.p.) dose relatedly prevented ovalbumin challenge induced mast cell degranulation by differing degrees when compared with vehicle treated group, and these effects were comparable with prednisolone (10mg/kg). A reduction in post-challenge mortality was also observed in all treated groups. Further, there were reductions in the blood eosinophil counts and serum IgE levels after chelidonic acid treatment. Chelidonic acid also inhibited histamine release from rat peritoneal mast cells (RPMC) in vitro, in a dose related manner. In tests for adaptive immunity, in rats immunized with sheep RBC, chelidonic acid differentially suppressed the (a) plaque forming cell (PFC) count in rat splenic cells, (b) anti-SRBC antibody titre and serum IgG levels and (c) increases in foot pad thickness in the DTH assay — all of which were comparable with prednisolone. These experimental results are discussed in light of the possible therapeutic potential of chelidonic acid in allergic disorders.

Study on effects and mechanism of traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis in rats

To study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA). Wister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated. Wuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity. The anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.

A Novel NKR-P1Bbright NK Cell Subset Expresses an Activated CD25+CX3CR1+CD62L−CD11b−CD27− Phenotype and Is Prevalent in Blood, Liver, and Gut-Associated Lymphoid Organs of Rats

The inhibitory NKR-P1B receptor identifies a subset of rat splenic NK cells that is low in Ly49 receptors but enriched for CD94/NKG2 receptors. We report in this study a novel NKR-P1B(bright) NK subpopulation that is prevalent in peripheral blood, liver, and gut-associated lymphoid organs and scarce in the spleen, peripheral lymph nodes, bone marrow, and lungs. This NKR-P1B(bright) NK subset displays an activated phenotype, expressing CD25, CD93, CX(3)CR1 and near absence of CD62-L, CD11b, and CD27. Functionally, NKR-P1B(bright) NK cells are highly responsive in terms of IFN-γ production and exert potent cytolytic activity. They show little spontaneous proliferation, are reduced in numbers upon in vivo activation with polyinosinic:polycytidylic acid, and have poor survival in ex vivo cytokine cultures. Our findings suggest that NKR-P1B(bright) NK cells are fully differentiated effector cells that rapidly die upon further activation. The identification of this novel rat NK cell subset may facilitate future translational research of the role of distinct NK cell subsets under normal physiological conditions and during ongoing immune responses.

Systemic Delivery of Nanoliposomal Ceramide Displays Anti-Leukemic Activity in a Rat Model of Large Granular Lymphocytic Leukemia

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.

Immunosuppressive effects of the heat shock protein 90-binding antibiotic geldanamycin.

Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamycin depleted cellular Raf-1 of rat splenic cells without affecting the steady state levels of hsp90 and downstream mitogen activated protein (MAP) kinases, ERK1 and ERK2. In parallel, it inhibited mitogen-induced nuclear factor-kappa B (NF-kappa B) activation in these cells. Geldanamycin also had a potent suppressive effect on recall antigen-induced T cell proliferation, with an IC50 value of 1 nM. In vivo, geldanamycin suppressed the progression of adjuvant-induced arthritis dose-dependently. These results suggest that geldanamycin exerts an immunosuppressive effect partly through destabilizing Raf-1, and raise a new strategy for the prevention of inflammatory diseases.

Interleukin-10 Reduces Natural Killer Sensitivity and Downregulates MHC Class I Expression on H-ras-Transformed Cells

We examined the effect of IL-10 on sensitivity to NK-cell-mediated cytotoxicity of the H-ras-induced transformants, W14 and W31. Incubation of cells with recombinant human (rh) IL-10 resulted in a dose-dependent decrease in the expression of MHC class I antigens, but not in the ICAM-1 expression. However, prior incubation of W31 cells with rhIL-10 markedly decreased their susceptibility to cytolysis by rat splenic NK cells. This fact suggested that the IL-10-mediated decrease in MHC class I expression might not dominate the regulation of the NK sensitivity. This was true when rat IL-10 cDNA-introduced W31 cells were used as an endogenous IL-10 producer. The NK sensitivityin vitroof W31T-H, a high IL-10-producer clone, was suppressed downward to the equivalent level of W31 cells pretreated with exogenous rhIL-10. The decreased NK-sensitivity of W31T-H cells was further confirmed byin vivoWinn assay, in which nude mice challenged with W31T-H cells and rat NK cells together developed tumors, whereas nude mice challenged with W31T-L, a minimal-IL-10 producer clone, and NK cells did not. Since neither exogenous nor endogenous IL-10 affected the proliferation of W31 cells, the data indicated that W31T-H cells could evade the NK-cell-mediated immune responsein vivo.Taken together, our data reveal a novel mechanism for an IL-10-mediated escape of tumor cells from host immune system by NK cells.

Intracellular melatonin distribution in cultured cell lines.

A specific antibody combined with a fluorescein-labeled immunoglobulin was used to investigate the topographic distribution of melatonin in a variety of cells of different origins. Positive identification of both nuclear and cytosolic melatonin was confirmed in all the tested cells: Swiss 3T3 mouse fibroblasts, BCG1 bovine granulosa, NB41A3 mouse neuroblastoma, F9 mouse teratocarcinoma, MDCK normal canine kidney derived and human HeLa cell lines, as well as in human peripheral blood mononuclear leukocytes and rat splenic cells. In 3T3 mouse fibroblasts melatonin immunofluorescence partially colocalized with actin and serotonin immunostaining, but not with tubulin or actin stress fibers. Several distinct patterns of subcellular melatonin distribution, different from the bromodeoxyuridine-labeled replication profiles, have been discerned throughout the cell cycle of synchronized 3T3 cells. In addition, synchronized 3T3 mouse fibroblasts cultured in the presence of 10(-3) M melatonin progressed more slowly through the cell cycle than control cells. These results suggest that melatonin may interact directly with nuclear and cytoskeletal structures probably affecting different cell functions such as cell cycle control, subcellular organization, and genome stability.

Carbon monoxide induces murine thymocyte apoptosis by a free radical-mediated mechanism.

Carbon monoxide (CO) induces acute or chronic toxicity, according to the level and duration of the exposure. Since chronic CO exposure was shown to have immunosuppressive effects (as it decreases the frequency of rat splenic immunocompetent cells and immunoglobulin production), we investigated the effect of CO on thymocytes, since these are the most sensitive cells to oxidative damage from the lymphoid lineage. We exposed thymocytes to CO, then determined their apoptotic index after 6 h of incubation at 37 degrees C using the fluorochrome Hoechst 33342 and electron microscopy and found an increase of apoptosis in CO-exposed thymocytes. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), an antioxidant vitamin E analog, decreased CO-induced thymocyte apoptosis unlike methylene blue, L-nitroarginine methyl ester or pyrrolidine dithiocarbamate. We also observed that lipid peroxidation was increased in the CO-exposed thymocytes and that it was inhibited by Trolox. Our results suggest that CO induces thymocyte apoptosis by a free radical-mediated mechanism which can be inhibited by Trolox but which does not involve the activation of the guanylyl cyclase-cGMP pathway.

Caffeine-DNA interactions: biochemical investigations comprising DNA-repair enzymes and nucleic acid synthesis.

Chicken embryo cells were treated with caffeine (0.5-8.0 mM) alone or combined with various chemical and physical DNA-and/or chromatin-interactive agents. Analytical procedures comprised scheduled (SDS) and unscheduled (UDS) DNA synthesis, RNA synthesis (RNS), the activities of O6-alkylguanine-DNA alkyltransferase (AT) and poly (ADP-ribose) polymerase (PARP) as well as nucleoid sedimentation. Additional investigations were done in rat thymic and splenic cells. The effect of caffeine on DNase-I activity served as an in vitro-model system. When present in the PARP-, SDS-, UDS- and RNS-assays, caffeine inhibited the corresponding tracer (14C-NAD, dT-3H, 3H-U) incorporation in a dose-dependent manner. The AT activity was slightly stimulated. At concentrations of 0.06-0.3 mM, caffeine inhibited DNase-I activity by excess substrate. No specific effects of caffeine could be shown by nucleoid sedimentation. Besides the reduced permeability of the cells to nucleic acid precursors, the results obtained with the PARP- and DNase-I assays give evidence for the formation of a DNA-caffeine adduct as a prominent mechanism of cellular caffeine effects including DNA repair inhibition.

Assessment of immunotoxicity by multiparameter flow cytometry.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Assessment of Immunotoxicity by Multiparameter Flow Cytometry

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+ -dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Retinoic acid induces translocation of protein kinase C (PKC) and activation of nuclear PKC (nPKC) in rat splenocytes

Retinoic acid (RA), a vitamin A metabolite, has marked effects on growth of normal and malignant cells; however, the exact mechanism of action remains unclear. The effect of two RA analogs, 13- cis-RA and all- trans-RA, on transmembrane signalling processes was investigated in rat splenocytes. Treatment of rat splenic cells with these retinoic acid analogs resulted in translocation of protein kinase C (PKC) from the cytosol to the membrane. Previous studies have described nuclear RA receptors (RARs and RXRs) for several species and the biologic activity of RA has been shown to be mediated by specific interaction with these nuclear receptors. Thus, activation of nuclear pool(s) of protein kinase C (nPKC) by RA analogs was also studied. Rat splenocyte nuclei pure by enzymatic and electron microscope criteria demonstrated a biphasic pattern of bell-shaped curves for both cis- and trans-RA with maximum statistically significant peak of phosphate incorporation into endogenous substrates at 10 −16 M cis-RA and 10 −16-10 −17 M trans-RA. A monoclonal antibody to PKC and the PKC inhibitors, H-7, sphingosine, and staurosporine, blocked the RA-stimulated nuclear phosphorylation. The ability of RA to activate cell membrane PKC resulting in an increase in particulate PKC activity correlates well with the activation of nPKC since the particulate fraction would include nuclear enzyme systems. This ability of RA to activate nPKC and possibly affect the growth status of a cell may provide a missing link to our understanding of the cellular sites of action for this vitamin.

Generation of Lymphokine-Activated Killer Activity in Rodents but Not in Humans Is Nitric Oxide Dependent

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG -monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO-2), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+ cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.

Prenatal diazepam induced persisting downregulation of peripheral (ω3) benzodiazepine receptors on rat splenic macrophages

Prenatal treatment with a low dose of diazepam (1.25mg/kg/d, gestational day 14–20) has previously been found to cause longterm impairment of cellular immune responses in rat offspring. A possible site of action of diazepam, i.e., the peripheral-type (ω3) benzodiazepine receptor, was characterized on splenic macrophages as well as on a rat splenic cell preparation containing mainly lymphocytes. In membranes of both preparations, [3H]PK 11195 bound to a single site which in competition experiments exhibited characteristics of the (ω3) site. Prenatal exposure to diazepam was followed, at 8 weeks of life, by a marked decrease in maximal binding capacity (Bmax) of spleen macrophage membranes in offspring of both sexes, while membranes of the splenic cell preparation exhibited an increase of the dissociation constant of [3H]PK 11195 at 2 and 8 weeks of life. Both types of delayed effects may reduce the metabolic capacity of these immune cells, in which we have also observed deficits of cytokine release.