Evaluation of immunostimulatory activity of Chyawanprash using in vitro assays.

Chyawanprash is an ayurvedic formulation used in Indian traditional medicinal system for its beneficial effect on human health. We investigated the immunostimulatory effects of Chyawanprash (CHY) using in vitro assays evaluating the secretion of cytokines such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1beta (IL-1β) and Macrophage Inflammatory Protein-1-alpha (MIP-1-α) from murine bone marrow derived Dendritic Cells (DC) which play pivotal role in immunostimulation. The effects of CHY on phagocytosis in murine macrophages (RAW264.7) and Natural Killer (NK) cell activity were also investigated. At non-cytotoxic concentrations (20–500 µg/ml), CHY enhanced the secretion of all the three cytokines from DC. CHY also stimulated both, macrophage (RAW264.7) as well as NK cell activity, in vitro. In conclusion, the data substantiates the immunoprotective role of CHY at cellular level mediated by immunostimulation in key immune cells viz. dendritic Cells, macrophages and NK cells.

Activation of macrophages with N-formyl-methionyl-leucyl-phenylalanine: involvement of protein kinase C and tyrosine kinase.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) a potent chemotactic peptide stimulates immune responses by activating macrophages and other cells of the immune system. The present study reports inhibition of fMLP-induced activation of murine peritoneal and P388D-1 macrophage cell line by protein kinase C (PKC) inhibitors, H-7 and chelerythrine chloride. Similarly, tumoricidal activity was also downregulated by protein tyrosine kinase (PTK) inhibitors genestein and lavendustin A. Further, fMLP increased tyrosine phosphorylation of several proteins in murine macrophages, which were inhibited in presence of genestein and lavendustin A. These findings suggest the involvement of PKC and PTK in the activation of murine macrophages with fMLP.

Protective action of an anti-oxidant (L-Ascorbic acid) against genotoxicity and cytotoxicity in mice during p-DAB-induced hepatocarcinogenesis.

BACKGROUND: DNA damage from micronutrient deficiencies has been suggested as one major cause of cancer. Therefore studies involving vitamin supplementation, particularly with those with anti-oxidant activity, in combating cancer have routinely been carried out in both in vivo and in vitro systems, but relatively much less in mice. AIMS: The present study examines if L-Ascorbic acid (AA; vitamin C) administration has any protective abilities in combating p-DAB induced hepatocarcinogenesis in mice at cytogenetical, biochemical, histological and ultra-structural levels. SETTINGS AND DESIGN: To test if AA had a protective action against genotoxicity, cytotoxicity and tissue damage in liver during p-dimethylaminoazobenezene (p-DAB) induced hepatocarcinogenesis in mice, a group of mice were chronically fed 0.06% p-DAB and 0.05% phenobarbital (PB) for a varying period of time (7, 15, 30, 60, 90 and 120 days). A sub-group of the p-DAB plus PB fed mice were also fed 1% L-ascorbic acid. Several assays were periodically conducted (at the six intervals of fixation) for determination of genotoxic (based on chromosomal, nuclear and sperm head anomalies), cytotoxic (based on the marker enzymes aspartate transaminase; AST, alanine aminotransferase; ALT; acid phosphatase; ACP; alkaline phosphatase; ALKP; lipid peroxidation; LPO); and tissue damaging (based on optical and electron microscopic studies of liver at day 60 only) effects in these different groups of mice as compared to normal healthy control. METHODS AND MATERIAL: Adult healthy mice of Swiss Albino strain, reared and maintained in the animal house of the Department of Zoology, Kalyani University, under supervision of Animal Welfare Committee (which oversees ethical issues), served as materials for the present study. Widely practiced standard technique has been followed for each protocol. STATISTICAL ANALYSIS USED: The significance test between different series of data was conducted by student's t-test. RESULTS AND CONCLUSIONS: The results of all these studies indicated that AA had protective action against p-DAB induced hepatocarcinogenesis in mice.

Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.

Cholangiocarcinoma is known to be relatively resistant to chemotherapy. One alternative approach is to use a combination of an immunomodulating agent with an anticancer drug. Here we studied the synergistic actions of TNF-alpha and triptolide (a diterpene epoxide prepared from Tripterygium wilfordii), previously shown to have antitumor activity against hamster cholangiocarcinoma (CCA) cells. Three human CCA cell lines (HuCCA-1, HubCCA-1, KKU-100 cell lines) were subjected to a combined treatment of TNF-alpha (0.1-10 ng/ml) and triptolide (5-50 ng/ml) for 24 hours in microculture plates. The combination of TNF-alpha and triptolide had a significantly increased cytotoxic activity over that of triptolide alone (p < 0.05). Under the same conditions, TNF-alpha by itself was not cytotoxic to these cell lines. Similarly, the combined treatment could also accelerate apoptotic cell death in all three human cholangiocarcinoma cell lines. The combined treatment of TNF-alpha at 10 ng/ml and triptolide at 50 ng/ml for 6-10 hours achieved a percentage of apoptotic cells shown by DAPI staining of 18-65%, compared to only 6-20% apoptotic cells for triptolide alone. Analyzing the possible mechanisms of the combined treatment, we found by Western blot that at 6 hours, there was a poly (ADP-ribose) polymerase (PARP) cleavage which was not detectable by the treatment of either TNF-alpha or triptolide alone. The cleavage of PARP was inhibited when the cells were pretreated with the enzyme inhibitor AC-DEVD-CMK, suggesting that apoptosis induced by the combination of TNF-alpha and triptolide involved activation of caspase 3. These results indicate that apoptosis of human cholangiocarcinoma cell lines as induced by a combination of TNF-alpha and triptolide is mediated through caspase 3 activation.

Cell proliferation and natural killer cell activity by polyherbal formulation, Immu-21 in mice.

Immunomodulatory activity of an Ayurvedic polyherbal formulation, Immu-21 containing extracts of Ocimum sanctum, Withania somnifera, Emblica officinalis and Tinospora cordifolia was studied on proliferative response of splenic leukocytes to T cell mitogens, concanavalin (Con)-A and phytohemagglutinin (PHA) and B cell mitogen, lipopolysaccharide (LPS) in vitro by [3H]-thymidine uptake assay in mice. The cytotoxic activity of Immu-21 was tested by measuring the splenic leukocyte natural killer (NK) cell activity against K 562 cells. Intraperitoneal (i.p.) treatment with Immu-21 (30 mg/kg) once a day for 14 and 21 days did not cause change in body weight and spleen weight, where as splenocytes/spleen count was increased. Treatment of Immu-21 (30 mg/kg, i.p.) for 14 days and 1 mg/kg for 21 days significantly increased LPS induced leukocyte proliferation. NK cell activity was significantly increased when mice were pretreated with Immu-21 (10 and 30 mg/kg, i.p.) once a day for 7 days. The results indicate that pretreatment with Immu-21 selectively increased the proliferation of splenic leukocyte to B cell mitogen, LPS and cytotoxic activity against K 562 cells in mice.

Cyclins D1, E, A expression and synergistic cytotoxicity to a cholangiocarcinoma cell line from recombinant TNF-alpha and PHA supernate.

We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.

Cell-mediated immunosuppression in mice by street rabies virus not restored by calcium ionophore or PMA.

It is known that rabies virus can suppress the host immune system. In this study we demonstrate a depression of cell-mediated immunity in mice, peripherally infected with Thai street rabies virus. The cell-mediated cytolysis of spleen cells from mice increased transiently on day 5 after infection and declined rapidly thereafter until death. The proliferation of spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin or concanavalin A, was significantly suppressed during the course of infection. There was also a marked suppression of IL-2 secretion in parallel with a decrease of the T-cell proliferative response to mitogen. The suppression of T-cell proliferation was not restored by treatment with a calcium ionophore (A 23187) or phorbol 12-myristate-13 acetate (PMA).

Influencia del coralán sobre la respuesta in vitro de células inmunocompetentes / Coralan influence on the in vitro response of inmunocompetent cells

El coralán, aislado de la Pseudopterogorgia americana, un coral blando, es una glicoproteìna sulfatada que contiene 1% de sulfato, se ha informado que tienen influencia en el rechazo del trasplante del tumor ascìtico de Ehrlich. Diferentes concentraciones de esta sustancia se probaron en células mononucleares de sangre periférica (CMN) frente a la PHA, se observó un incremento significativo de la respuesta, lo que indica que una de las vías por las que este producto ajerce su actividad es por la vía de los linfocitos T. Tambièn se produce un ligero incremento de la actividad de las células NK frente a las células K 562 cuando las CMN son preincubadas con el producto 2 horas antes de realizarse el experimento, datos que en nuestras condiciones fueron comparables al procucido por el IFN, que se ha descrito que aumenta esta actividad.

The immunomodulatory and antitumor activities of trichosanthin-an abortifacient protein isolated from tian-hua-fen (Trichosanthes kirilowii).

Trichosanthin, a basic protein purified from the root tuber of Trichosanthes kirilowii, has been used effectively in China to induce midterm abortion in humans. In this paper, we show that trichosanthin at non-cytotoxic concentrations markedly inhibited the mitogen-induced lymphoproliferative response and the generation of a primary alloreactive CTL response in vitro. Similarly, the production of IL-2 by Con A activated splenocytes and the in vitro effector functions of macrophages were also significantly suppressed. In contrast, the cytolytic activity of CTL and NK cells was unimpaired. Moreover, the in vivo activation of NK cells was not significantly altered by a single injection of a non-toxic microgram amount of trichosanthin into mice. However, other immune reactivities such as the induction of a DTH response and the humoral antibody formation to SRBC were markedly depressed. Our data suggest that trichosanthin is a potent immunosuppressive protein that could affect humoral immunity and a variety of cell-mediated processes. In addition, our preliminary results show that this abortifacient protein could also inhibit the growth of a murine malignant tumour (MBL-2), both in vivo and in vitro.