G1-4A, a Polysaccharide from Tinospora cordifolia Inhibits the Survival of Mycobacterium tuberculosis by Modulating Host Immune Responses in TLR4 Dependent Manner.

Rapid emergence of drug resistance in Mycobacterium tuberculosis (MTB) is a major health concern and demands the development of novel adjunct immunotherapeutic agents capable of modulating the host immune responses in order to control the pathogen. In the present study, we sought to investigate the immunomodulatory effects of G1-4A, a polysaccharide derived from the Indian medicinal plant Tinospora cordifolia, in in-vitro and aerosol mouse models of MTB infection. G1-4A treatment of MTB infected RAW264.7 macrophages significantly induced the surface expression of MHC-II and CD-86 molecules, secretion of proinflammatory cytokines (TNF-α, IL-ß, IL-6, IL-12, IFN-γ) and nitric oxide leading to reduced intracellular survival of both drug sensitive (H37Rv) as well as multi drug resistant strains (Beijing and LAM) of MTB, which was partially attributed to G1-4A induced NO production in TLR4-MyD88 dependent manner. Similarly, bacillary burden was significantly reduced in the lungs of MTB infected BALB/c mice treated with G1-4A, with simultaneous up-regulation of the expression of TNF-α, INF-γ and NOS2 in the mouse lung along with increased levels of Th1 cytokines like IFN-γ, IL-12 and decreased levels of Th2 cytokine like IL-4 in the serum. Furthermore, combination of G1-4A with Isoniazid (INH) exhibited better protection against MTB compared to that due to INH or G1-4A alone, suggesting its potential as adjunct therapy. Our results demonstrate that modulation of host immune responses by G1-4A might improve the therapeutic efficacy of existing anti-tubercular drugs and provide an attractive strategy for the development of alternative therapies to control tuberculosis.

Amelioration of radiation-induced hematopoietic syndrome by an antioxidant chlorophyllin through increased stem cell activity and modulation of hematopoiesis.

Hematopoietic stem cells and progenitor cells (HSPC) are low in abundance and exhibit high radiosensitivity and their ability to divide dramatically decreases following exposure to ionizing radiation. Our earlier studies have shown antiapoptotic, immune-stimulatory, and antioxidant effects of chlorophyllin, a constituent of the over the counter drug derifil. Here we describe the beneficial effects of chlorophyllin against radiation-induced hematopoietic syndrome. Chlorophyllin administration significantly enhanced the abundance of HSPC in vivo. It induced a transient cell cycle arrest in lineage-negative cells in the bone marrow. However, the chlorophyllin-treated mice exposed to whole body irradiation (WBI) had a significantly higher proportion of actively dividing HSPC in the bone marrow as compared to only WBI-exposed mice. It significantly increased the number of colony forming units (CFUs) by bone marrow cells in vitro and spleen CFUs in irradiated mice in vivo. Pharmacokinetic study showed that chlorophyllin had a serum half-life of 141.8 min in mice. Chlorophyllin upregulated antiapoptotic genes and antioxidant machinery via activation of prosurvival transcription factors Nrf-2 and NF-κB and increased the survival and recovery of bone marrow cells in mice exposed to WBI. Chlorophyllin stimulated granulocyte production in bone marrow and increased the abundance of peripheral blood neutrophils by enhancing serum levels of granulocyte-colony stimulation factor (GCSF). Most importantly, prophylactic treatment of mice with chlorophyllin significantly abrogated radiation-induced mortality. Chlorophyllin mitigates radiation-induced hematopoietic syndrome by increasing the abundance of hematopoietic stem cells, enhancing granulopoiesis, and stimulating prosurvival pathways in bone marrow cells and lymphocytes.

TGF-ß1-ROS-ATM-CREB signaling axis in macrophage mediated migration of human breast cancer MCF7 cells.

Macrophages in the tumor microenvironment play an important role in tumor cell survival. They influence the tumor cell to proliferate, invade into surrounding normal tissues and metastasize to local and distant sites. In this study, we evaluated the effect of conditioned medium from monocytes and macrophages on growth and migration of breast cancer cells. Macrophage conditioned medium (MϕCM) containing elevated levels of cytokines TNF-α, IL-1ß and IL-6 had a differential effect on non-invasive (MCF7) and highly invasive (MDA-MB-231) breast cancer cell lines. MϕCM induced the secretion of TGF-ß1 in MCF7 cells. This was associated with apoptosis in a fraction of cells and generation of reactive oxygen and nitrogen species (ROS and RNS) and DNA damage in the remaining cells. This, in turn, increased expression of cAMP response element binding protein (CREB) and vimentin resulting in migration of cells. These effects were inhibited by neutralization of TNF-α, IL-1ß and IL-6, inhibition of ROS and RNS, DNA damage and siRNA mediated knockdown of ATM. In contrast, MDA-MB-231 cells which had higher basal levels of pCREB were not affected by MϕCM. In summary, we have found that pro-inflammatory cytokines secreted by macrophages induce TGF-ß1 in tumor cells, which activate pCREB signaling, epithelial-mesenchymal-transition (EMT) responses and enhanced migration.

Plumbagin, a vitamin K3 analogue, abrogates lipopolysaccharide-induced oxidative stress, inflammation and endotoxic shock via NF-κB suppression.

Plumbagin has been reported to modulate cellular redox status and suppress NF-κB. In the present study, we investigated the effect of plumbagin on lipopolysaccharide (LPS)-induced endotoxic shock, oxidative stress and inflammatory parameters in vitro and in vivo. Plumbagin inhibited LPS-induced nitric oxide, TNF-α, IL-6 and prostaglandin-E2 production in a concentration-dependent manner in RAW 264.7 cells without inducing any cell death. Plumbagin modulated cellular redox status in RAW cells. Plumbagin treatment significantly reduced MAPkinase and NF-κB activation in macrophages. Plumbagin prevented mice from endotoxic shock-associated mortality and decreased serum levels of pro-inflammatory markers. Plumbagin administration ameliorated LPS-induced oxidative stress in peritoneal macrophages and splenocytes. Plumbagin also attenuated endotoxic shock-associated changes in liver and lung histopathology and decreased the activation of ERK and NF-κB in liver. These findings demonstrate the efficacy of plumbagin in preventing LPS-induced endotoxemia and also provide mechanistic insights into the anti-inflammatory effects of plumbagin.

Cytokines from the tumor microenvironment modulate sirtinol cytotoxicity in A549 lung carcinoma cells.

Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor ß (TGF-ß) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs - cisplatin and taxol and inhibitors of epidermal growth factor receptor - AG658, Janus kinase - AG490 and SIRT1 - sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K-AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.

Involvement of ERK-Nrf-2 signaling in ionizing radiation induced cell death in normal and tumor cells.

Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s) in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.

G1-4 A, an arabinogalactan polysaccharide from Tinospora cordifolia increases dendritic cell immunogenicity in a murine lymphoma model.

The immunogenicity of dendritic cells (DC) is known to increase with their maturation state and both are induced by microbial products like LPS. In this study, we have investigated the effect of G1-4A, a polysaccharide isolated from Indian medicinal plant, Tinospora cordifolia on phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDC) and its ability to be used as an adjuvant in immunotherapy. G1-4A, enhanced surface expression of CD40, CD80, CD86, MHCII by BMDC in vitro and splenic DC in vivo. T cell allostimulatory activity and secretion of IL-12 and TNFα by BMDC were also increased. Treatment with G1-4A resulted in decreased phagocytosis and increased antigen processing that are characteristic of mature DC. G1-4A treated DC cross presented exogenous antigens on a MHC I background which resulted in the activation of cytotoxic T cells. These cells thus activated could cause lysis of target tumor cells in vitro. Administration of tumor lysate pulsed G1-4A treated DC resulted in decreased tumor burden in preventive as well as therapeutic tumor challenge experiments in a murine lymphoma model. These results thus confirm that G1-4A could be a promising nontoxic maturation agent to be potentially used in DC based immunotherapy of tumor.

Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-κB, AP-1 and NF-AT.

BACKGROUND: Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. METHODOLOGY/PRINCIPAL FINDINGS: The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. CONCLUSIONS/SIGNIFICANCE: The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders.

Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner.

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.

Pro-oxidants ameliorate radiation-induced apoptosis through activation of the calcium-ERK1/2-Nrf2 pathway.

There are no reports describing the ability of pro-oxidants to protect against radiation-induced apoptosis. Activation of the redox-sensitive transcription factor Nrf2 by low levels of ROS is known to protect against oxidative stress-induced cell death. In this study, hydrogen peroxide, diethylmaleate, and 1,4-naphthoquinone (NQ) exhibited complete protection against radiation-induced cell death in lymphocytes as estimated by propidium iodide staining. Radioprotection by NQ was demonstrated by inhibition of caspase activation, decrease in cell size, DNA fragmentation, nuclear blebbing, and clonogenic assay. Interestingly, NQ offered protection to lymphocytes even when added to cells postirradiation. NQ increased intracellular ROS levels and decreased GSH levels. NQ activated Nrf2 and increased the expression of the cytoprotective gene heme oxygenase-1 in lymphocytes. NQ increased ERK phosphorylation, which is upstream of Nrf2, and this ERK activation was through increased intracellular calcium levels. Administration of NQ to mice offered protection against whole-body irradiation (WBI)-induced apoptosis in splenic lymphocytes and loss of viability of spleen and bone marrow cells. It restored WBI-mediated changes in hematological parameters and functional responses of lymphocytes. Importantly, NQ rescued mice against WBI-induced mortality. These results demonstrated that a pro-oxidant such as NQ can protect against radiation-induced apoptosis by activation of multiple prosurvival mechanisms including activation of the calcium-ERK1/2-Nrf2 pathway.

Immune responses to novel allergens and modulation of inflammation by vitamin K3 analogue: a ROS dependent mechanism.

The possibility of newer allergens being responsible for atopy needs to be explored at regional level due to environmental variables. Current studies were undertaken to identify common environmental allergens causing atopy in a defined population of India and to correlate the presence of various risk factors with the clinical presentation of allergy. Newer allergens like human dander and rice grain dust were identified and reported as the most common cause of atopy in this region. Atopy, elevated serum total IgE and familial tendency, was observed in 88%, 69% and 58% of allergic patients respectively. Further, allergen-specific immune responses like lymphocyte proliferation and cytokine secretion were studied in vitro using peripheral blood mononuclear cells (PBMC) isolated from both allergic and non-allergic individuals. Although, some allergens induced significant lymphocyte proliferation in vitro, allergen-induced cytokine secretion except that of TNF-α was not seen. Significantly higher ratio of secreted IL-4/IFN-γ cytokines was observed in PBMC isolated from allergic subjects in response to PHA. Plumbagin (vitamin K3 analogue) completely inhibited PHA-induced cytokine production in PBMC, in both allergic and non-allergic individuals. Plumbagin modulated the levels of intracellular reactive oxygen species and glutathione and suppressed PHA induced activation of NF-κB in human PBMC. The results thus show in human PMBC, for the first time, the anti-allergic and anti-inflammatory effects of plumbagin and underscore its therapeutic potential.

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ).

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2-5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.

A novel N-alkylated prodigiosin analogue induced death in tumour cell through apoptosis or necrosis depending upon the cell type.

PURPOSE: To investigate the mechanism of cell death induced by the N-alkylated prodigiosin analogue, 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrryl-methene (MAMPDM) in S-180 and EL-4 tumour cell lines. METHODS: Effect of MAMPDM on cell viability was assessed by MTT dye conversion. Induction of apoptosis was assessed by monitoring caspase 3 activity using a fluorogenic substrate, fragmentation of DNA by gel electrophoresis and sub-diploid DNA containing cells by flowcytometry. Necrosis was estimated by flowcytometric analysis of the uptake of propidium iodide. RESULTS: MAMPDM inhibited the proliferation of murine fibrosarcoma, S-180 cells and induced cell death. Investigations into the mechanism of cell death by MAMPDM in S-180 cells showed absence of hallmarks of apoptotic cell death such as activation of caspase 3, DNA fragmentation and presence of cells with sub-diploid DNA content. However, there was a rapid loss of membrane integrity as assessed by uptake of propidium iodide, which is characteristic of necrosis. In contrast to induction of necrosis in S-180 cells, MAMPDM induced apoptotic cell death in EL-4 cells as evident by activation of caspase 3, fragmentation of DNA and sub-diploid DNA containing cells. CONCLUSIONS: MAMPDM could induce cell death by either apoptosis or necrosis depending upon the cell type. This would be of advantage in elimination of tumor cells defective in apoptotic pathway and therefore, refractory to the conventional therapies.

Role of interleukin-2 regulated proteins in immunosuppression by the N-alkylated prodigiosin analogue.

Prodigiosins have emerged as a novel family of tripyrrole compounds with significant T cell specific immunosuppressive potential. We had previously reported the immunosuppressive activity of a novel N-alkylated prodigiosin analogue, 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrryl-methene (MAMPDM) in mitogen stimulated spleen cells. There was an increase in the accumulation of IL-2 and induction of apoptotic cell death. Since IL-2 regulates both cellular proliferation and activation induced cell death (AICD), we have investigated the effect of MAMPDM on the expression of IL-2 regulated genes that are involved in these two opposite processes. The mitogen stimulated mouse spleen cells did not undergo a single division in presence of proliferation inhibitory concentrations of MAMPDM. An increase in the percentage of apoptotic cells was observed in the undivided cell population. The cells were arrested in G1 phase independent of the p53 expression. Expression of IL-2 regulated genes such as CD71, proliferating cell nuclear antigen (PCNA) and cyclin D was suppressed while that of Fas was not. Thus, in the presence of MAMPDM there was selective inhibition of only the pro-mitogenic signaling and not that of pro-apoptotic signaling by IL-2. The induction of apoptosis in presence of MAMPDM was the effect rather than cause for the anti-proliferative activity.

Identification of a red-pigmented bacterium producing a potent anti-tumor N-alkylated prodigiosin as Serratia marcescens.

A bacterial strain producing a novel prodigiosin analogue 2,2'-[3-methoxy-1'amyl-5'-methyl-4-(1''-pyrryl)] dipyrrylmethene (MAMPDM) possessing potent cytotoxic activity towards cancer cells was isolated and identified. The bacterial cells were spherical and occurred singly, and some of the biochemical tests matched with Micrococcus. Therefore, the isolate was earlier tentatively reported to be Micrococcus sp. In the present studies, analytical profile index (API) suggested this organism to be Klebsiella. However, Klebsiella is not known to produce the red pigment prodigiosin, which is produced by Serratia species and some other bacteria. Based on other biochemical characteristics, particularly DNase, gelatinase, lipase, ornithine decarboxylase, presence of a cell-associated N-alkylated prodigiosin (MAMPDM) and organic solvent tolerance, the strain has now been identified as a variant of Serratia marcescens. 16S rRNA gene analysis conclusively established this organism as S. marcescens ost3. The red pigment (MAMPDM) of this organism showed selective cytotoxic activity in cancer cell lines of different origin (LS-A and U937) and reduced toxicity to non-malignant cells. The LC50 of MAMPDM was 1.59 microM and 0.176 microM for U937 and LS-A cells, respectively, while there was no effect on the viability of L929, a non-malignant cell line, at these concentrations. Thus, S. marcescens ost3 may serve as a source of a new anti-cancer compound.

Prodigiosins: a novel family of immunosuppressants with anti-cancer activity.

Prodigiosins (PrGs) are a family of promising therapeutic molecules, isolated mostly from Gram-negative bacteria and characterized by a common pyrryldipyrrylmethene structure with varying side chains. They show a broad spectrum of activities such as anti-microbial, anti-malarial, anti-cancer and immunosuppressive. PrGs are attracting increasing attention due to the ongoing research for less toxic, but effective agents for cancer chemotherapy and immunosuppression for preventing allograft rejection and autoimmunity. Different analogues have been synthesized and evaluated. This review discusses the immunosuppressive and anti-cancer activities of this class of compounds, as both involve inhibition of cell proliferation. The main focus is on the in vitro and in vivo immunosuppressive activity of the different PrGs and the mechanisms involved. PrGs primarily target the T cells, though some effects are observed on other cell types also. Unlike the well-known immunosuppressant cyclosporin A, PrGs do not inhibit the secretion of IL-2 but inhibit the mitogenic signaling from IL-2, suggesting a different mechanism of action. Janus tyrosine kinase 3 (Jak3) that associates with IL-2R upon activation is considered as the molecular target for PrGs. Its restricted expression makes Jak3 as an attractive target for immunosuppressive therapy. However, the available literature suggests that some other pathways are also influenced by the PrGs. These may be important for the anti-cancer activity, as well as immunosuppressive action. Therefore, PrGs appear to be potential candidates for pharmaceutical development as immunosuppressants and also as anti-cancer agents.

Antiapoptotic and immunomodulatory effects of chlorophyllin.

Chlorophyllin (CHL) was earlier shown to reduce the level of intracellular ROS and apoptosis induced by ionizing radiation and 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH). In the present studies, the effect of CHL on radiation-induced immunosuppression and modulation of immune responses in mice was examined. Chlorophyllin inhibited the in vitro lymphocyte proliferation induced by concanavalin A (Con A) in a dose dependent manner at doses>or=50 microM. At lower doses (10 microM) CHL significantly inhibited activation induced cell death (AICD) in Con A stimulated spleen cells. Spleen cells obtained from CHL treated mice showed an inhibition of response to Con A depending on dose of CHL and the time after its administration. Spleen cells obtained from CHL treated mice (24 h) showed lower inhibition of response to Con A following in vitro (5 Gy) as well as whole body irradiation (2 Gy). The expression of antiapoptotic genes bcl-2 and bcl-xL was up-regulated in these cells. Chlorophyllin treatment of mice led to splenomegaly and increase in the number of peritoneal exudate cells (PEC). The numbers of T cells, B cells and macrophages in the spleen were also increased. Increased phagocytic activity was seen in PEC obtained from CHL treated mice. Most importantly, CHL administration to mice immunized with sheep red blood cells (SRBC) augmented both humoral and cell-mediated immune responses.

Cell cycle regulators modulating con A mitogenesis and apoptosis in low-dose radiation-exposed mice.

Low doses of ionizing radiation (LDR) are reported to induce transient changes in mouse lymphocytes, such as enhanced response to polyclonal T cell mitogens, increased expression of heat shock proteins, and p53. We evaluated the role of cell cycle proteins and apoptosis in lymphocytes of C57BL/6 mice exposed to 20 cGy fractionated LDR. We found an enhanced cell proliferation in response to the mitogen concanavalin A (con A). The expression of several cell cycle and apoptosis-related intracellular and extracellular proteins was analyzed by flowcytometry following labeling with specific antibodies. An increased response to con A was accompanied by an increase in the expression of proliferating cell nuclear antigen (PCNA) and cyclins D and A. The expression of cyclin B did not change significantly. In 20 cGy-exposed C57BL/6 mice, the caspase activity and apoptosis were reduced in con A-stimulated spleen cells as compared to sham controls. The expression of Fas and Fas ligand was analyzed by labeling with specific antibodies followed by flowcytometry. There was no change in the expression of Fas and Fas ligand. The change in the mitochondrial transmembrane potential was followed by labeling the cells with the dye 5,5',6,6',-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (JC-I) and analyzing by flowcytometry. Mitochondrial stability was increased in spleen cells of LDR-treated mice. These data suggest that LDR induces augmentation of mitogenic response by modulation of expression of cyclins and the mitochondrial membrane potential leading to reduced apoptosis.

Low dose radiation induced immunomodulation: effect on macrophages and CD8+ T cells.

PURPOSE: The aim of the present investigation was to study the effect of fractionated whole body low dose ionizing radiation (LDR) on the functional responses of T lymphocytes, their subpopulations and macrophages. MATERIALS AND METHODS: C57BL/6 mice were exposed to 4 cGy from a (60)Co source, at 0.31 cGy/min, at 24 h intervals for 5 days (total dose 20 cGy). Phagocytic activity was measured by flow cytometry using Bioparticles and nitric oxide generation was estimated by spectrophotometry. Proliferation of lymphocytes in response to concanavalin A (con A) and alloantigens was measured by (3)H thymidine incorporation. Expression of cell surface markers was assessed by flow cytometric analysis of antibody labeled cells. Target cell killing by cytotoxic T cells (CTL) generated against allogenic cells was assessed by flow cytometry using PKH26 labeled target cells. Cytokines were estimated by enzyme linked immunosorbent assay. RESULTS: Exposure to LDR enhanced nitric oxide secretion and phagocytosis. The expression of early activation antigen, CD69, was enhanced in CD8(+) T lymphocytes concomitant with enhanced proliferation in response to con A. In addition, mixed lymphocyte reaction (MLR) and CTL response were augmented and secretion of interferon gamma (IFN-gamma) was suppressed following LDR exposure. CONCLUSIONS: LDR exposure enhanced the function of macrophages and responses of CD8(+) T cells in C57BL/6 mice.

Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo.

Chlorophyllin (CHL) has been examined as an antioxidant/radioprotector in splenic lymphocytes from BALB/c mice. CHL inhibited lipid peroxidation induced by 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic stimulation of lymphocytes in vitro. Generation of intracellular reactive oxygen species (ROS) by radiation or AAPH was measured as oxidation of dichlorodihydrofluorescein diacetate (H(2)DCF-DA) using flow cytometry. Addition of CHL to lymphocytes in vitro significantly inhibited the increase in intracellular ROS. Further, lymphocytes from mice treated with CHL (100-400 microg/gbw i. p.) showed varying levels of ROS depending on the dose and the time (24 to 72 h) after injection. The extent of radiation-induced apoptosis and suppression of concanavalin A (con A)-induced mitogenesis ex vivo corresponded with changes in ROS levels in CHL-administered mice. Antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were also estimated in lymphocytes from CHL-treated mice. CHL offered protection against whole body irradiation (WBI)-induced lipid peroxidation and apoptosis in lymphocytes at all the time points studied. These results demonstrate antioxidant effect of CHL in vivo.