Gastroprotective potential of hydro-alcoholic extract of Pattanga (Caesalpinia sappan Linn.).

ETHNO-PHARMACOLOGICAL RELEVANCE: Pattanga is botanically equated as Caesalpinia sappan Linn. (Family: Caesalpiniaceae) and is used in Ayurveda system of medicine since ages. According to Ayurveda, useful part is Heartwood, which is bitter, astringent and acrid and is useful in vitiated conditions of vata and pitta, burning sensation, wounds, ulcers, leprosy, skin diseases, menorrhagia, leucorrhea, and diabetes. It is used as a major ingredient in Ayurvedic formulations and preparations like Patrangasava, Chandanadya Thalia, and Karpuradyarka. AIM OF THE STUDY: The present study is planned to evaluate the gastroprotective activity of the selected Ayurvedic drug using three different in vivo gastric ulcer models, so as to provide scientific evidence for the Ayurvedic claims. MATERIALS AND METHODS: For this study, Wistar albino rats fasted overnight were selected. The hydroalcoholic extract of Caesalpinia sappan heartwood at the dose level 250 and 500mg/kg body weight was selected and administered orally before necrotizing agents. Antioxidant and antiulcer parameters were evaluated and the stomach samples were subjected for histopathological studies. In addition, PGE2 estimation and protein expressions of COX-1, COX-2 and iNOS were analyzed by Western blot. The plant extract was subjected to LCMS/MS analysis. In addition, Cytoprotective effect in isolated gastric mucosal cells, TUNEL Assay, Acid neutralizing capacity assay, H+/K+ ATPase inhibitory assay were performed. RESULTS: The ulcer protection was found to be 92%, 86% and 64% against ethanol, NSAID and pylorus ligation induced ulcer respectively. The hydro-alcoholic extract of C. sappan heartwood exhibited cytoprotective effect with 76.82% reduction against indomethacin-induced cytotoxicity at the concentration of 25µg/ml. C. sappan showed 63.91% inhibition in H+/K+ ATPase inhibitory assay at the concentration 500µg/ml. CONCLUSIONS: Our results depict that Caesalpinia sappan heartwood possesses gastroprotective activity, possibly mediated through cytoprotection and antioxidant mechanisms. The data obtained in the present study provides scientific support for the traditional use of Caesalpinia sappan in the management of peptic ulcer.

Comparison between C57Bl/6 and C57Bl/10 mycobacterial mouse pleurisy with respect to cellular migration and nitric oxide production.

Mycobacterium bovis-BCG (BCG) and Mycobacterium leprae (ML) have opposite inflammatory properties. Mycobacteria-induced pleurisy in C57Bl/6 and C57Bl/10 mice was evaluated to establish if their innate responses could be comparable, verifying cellular migration and nitrite production. Kinetic responses after ML or BCG intrathoracic injection were compared in those mice, sharing the H-2(b) MHC haplotype. BCG led to acute eosinophilia and late neutrophilia in both mice. In C57Bl/6 late pleurisy, monocytes and neutrophil recruitment was dose- and iNOS-dependent, inhibited by methotrexate but not by indomethacin. Pleural macrophages released nitrites ex vivo after 7 days of BCG stimulus, without "priming" and blocked by the nitrite inhibitor L-N5-(1-iminoethyl)-ornithine (L-NIO). ML did not induce cellular migration or nitrite production, independent of the mouse strain, timing, or number of bacilli. Although these mycobacteria have high homology, there was no effect of ML on BCG-evoked secondary cellular recruitment. Both C57Black mice trigger similar onset of inflammatory responses to these mycobacteria, so far can alternatively be used in experimental studies.

Inhibition of IL-12 production by thalidomide.

The immunomodulatory properties of thalidomide are currently being exploited therapeutically in conditions as diverse as erythema nodosum leprosum, chronic graft-vs-host disease, rheumatoid arthritis, and sarcoidosis. The relevant mechanism of action of thalidomide in these diseases remains unclear. The important role recently ascribed to IL-12, a cytokine critical to the development of cellular immune responses, in the pathogenesis of several of these conditions led us to examine whether thalidomide affects the production of IL-12. Thalidomide potently suppressed the production of IL-12 from human PBMC and primary human monocytes in a concentration-dependent manner. Thalidomide-induced inhibition of IL-12 production was additive to that induced by suboptimal inhibiting doses of dexamethasone, and occurred by a mechanism independent of known endogenous inhibitors of IL-12 production. These results suggest that thalidomide may have therapeutic utility in a wide range of immunologic disorders that are characterized by inappropriate cellular immune responses.

Monocyte derived IL 10 and PGE2 are associated with the absence of Th 1 cells and in vitro T cell suppression in lepromatous leprosy.

Our previous studies had shown that the clinicopathological spectrum in leprosy was associated with discrete T cell subsets in circulation, with tuberculoid patients having antigen-induced Th 1, whereas lepromatous leprosy patients with antigen-specific T cell anergy possessed Th 2 cells. The present study shows that infected monocytes from lepromatous but not tuberculoid leprosy patients released soluble factors (MoF(s)) containing IL-10 and PGE2 which inhibited M. leprae induced in vitro lymphoproliferation of previously sensitised healthy or tuberculoid leprosy subjects. A strong negative correlation was observed between adherent cell derived IL-10 and IL-2 at the level of both the product and cytokine mRNA. Moreover, anti-IL-10 antibodies and indomethacin partially reversed the suppressor effects of MoF(s). Taken together these studies indicate that infected monocytes contribute to the development of T cell anergy by releasing factors that affect regulatory cytokines and T cell subset differentiation in lepromatous leprosy.

Regulation of murine macrophage effector functions by lipoarabinomannan from mycobacterial strains with different degrees of virulence.

Lipoarabinomannan (LAM) is the major arabinose- and mannose-containing phosphorylated lipopolysaccharide (LPS) in mycobacterial cell walls. LAM preparations from a virulent strain (Erdman) (LAM(Erdman)) and an attenuated strain (H37Ra) (LAMH37Ra) of Mycobacterium tuberculosis, as well as from M. leprae (a virulent mycobacterium), were analyzed for their effects on various macrophage (M phi) effector functions. LAMH37Ra, like gram-negative LPS, exhibited a dose-dependent ability to induce tumor necrosis factor alpha (TNF-alpha) production in normal M phi, and gamma interferon (IFN-gamma) priming of the M phi greatly augmented the levels of TNF-alpha. However, the effects of LAMH37Ra were unaffected by polymyxin B, which totally abrogated the effects of LPS. LAM(Erdman) and LAM from M. leprae, on the other hand, induced virtually no TNF-alpha production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of TNF-alpha mRNA induced by the various preparations correlated with the levels of TNF-alpha protein detected. Interestingly, both LAMH37Ra and LAM(Erdman) could block subsequent IFN-gamma- and LPS-induced M phi activation, a previously reported measure of the potent ability of LAM to down-regulate M phi effector functions. Two lines of evidence suggested, however, that M phi cyclooxygenase products did not play a role in this down-regulation. LAMH37Ra and LPS could induce the production of NO2- in both normal and IFN-gamma-primed M phi, whereas LAM(Erdman) could stimulate NO2- production only in primed M phi. Both LAMH37Ra and LAM(Erdman) could substitute for LPS as a triggering signal for IFN-gamma-primed M phi in a toxoplasma killing assay. The triggering ability of LAM(Erdman), however, was abrogated by an anti-TNF-alpha antibody, suggesting that sufficient TNF-alpha production was stimulated by LAM(Erdman) to drive a M phi function relevant in host resistance. Thus, mycobacterial LAM is a potent regulator of M phi functions, a fact that may have important consequences in mycobacterial disease.

Rat Schwann cells produce interleukin-1.

There is increasing evidence that Schwann cells of peripheral nerves may be able to function as accessory cells, interacting with the immune system in T cell-mediated immune responses, by expression of the major histocompatibility complex (MHC) class II molecules. In addition to MHC class II-associated presentation of antigen to T lymphocytes, the release of a co-stimulatory factor, interleukin-1 (IL-1), is an essential function of accessory cells for T cell activation. In this study, we investigated if Schwann cells were able to produce IL-1. Purified cultures of neonatal and adult rat Schwann cells were incubated with various stimulatory agents. Supernatants and cell lysates were collected from these cultures and IL-1 activity was assayed. Both neonatal and adult rat Schwann cells produced IL-1 activity in response to bacterial antigens and the IL-1 activity was often higher in the cell lysate than in the supernatant. When stimulated neonatal or adult rat Schwann cells were examined with antibody against IL-1, strong immunolabelling was seen intracellularly, but no IL-1 was detected on the cell surface. Since IL-1 plays an important role in the initiation of immune responses, these observations support the view that Schwann cells may function as antigen-presenting cells, thereby taking part in neuroimmunological responses within peripheral nerves.

Route-related variation in immunogenicity of mycobacteria.

The route of immunization was observed to play a significant role in deciding the T-cell response to immunization with killed mycobacterial vaccines. Slow-growing mycobacteria were found to be immunogenic by both the intraperitoneal (i.p.) and intradermal (i.d.) routes; rapid-growing mycobacteria were immunogenic by the i.d. route only. The nonresponder state following i.p. immunization with Mycobacterium vaccae could be corrected by treatment of the mice with poly I:poly C or indomethacin prior to immunization. Both poly I:poly C, an interferon inducer, and indomethacin, a prostaglandin inhibitor, are known to enhance the expression of major histocompatibility complex glycoproteins. Since they are so important in antigen preparation, it was concluded that the inability of mice to respond to M. vaccae by the i.p. route is likely due to defective presentation of the bacterial antigens by the antigen-presenting cells at the site, namely, the peritoneal macrophages. These findings are significant because M. leprae has been reported to be antigenically similar to M. vaccae, and the response of mice to i.p. immunization with both of these mycobacteria is very similar.

Induction of unresponsiveness to gamma interferon in macrophages infected with Mycobacterium leprae.

We have previously demonstrated that Mycobacterium leprae-burdened granuloma macrophages isolated from infected nude mice are refractory to activation by gamma interferon (IFN-gamma). To explore further both the afferent and efferent functional capacity of M. leprae-infected macrophages, we examined the IFN-gamma-mediated activation of resident mouse peritoneal macrophages infected in vitro with live or dead M. leprae. When IFN-gamma was administered within 24 h of M. leprae infection, macrophages were fully activated. However, defective activation was evident at 3 to 5 days postinfection in macrophages that were heavily burdened with viable M. leprae. This defect was evident by four parameters of activation in which IFN-gamma failed to stimulate the enhancement of microbicidal activity, cytotoxicity for tumor target cells, O2- production, and surface Ia antigen expression. The development of defective activation closely followed an increase in macrophage production of prostaglandin E2. Defective activation of M. leprae-burdened macrophages was reversible by indomethacin, and a similar block in IFN-gamma activation was observed in three of these four parameters in normal macrophages treated with exogenous prostaglandin E2. Thus, infection of mouse macrophages with M. leprae appears to restrict IFN-gamma-mediated activation at least in part by induction of inhibitory levels of prostaglandin E2.

Interleukin 1 and prostaglandin production by cells of the mononuclear phagocyte system isolated from mycobacterial granulomas.

A study has been made of the activity of interleukin 1 (IL-1) and prostaglandins (PGs) in the culture supernatants from unstimulated and lipopolysaccharide (LPS)-stimulated mycobacteria-induced granuloma cells. Both epithelioid cells from bacillus Calmette-Guerin (BCG)-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas, separated on a fluorescence-activated cell sorter using monoclonal antibody specific to guinea pig macrophages, spontaneously secreted low levels of IL-1 (assayed by thymocyte comitogenic and fibroblast mitogenic activities) into culture supernatants. However, culture supernatants from LPS-stimulated epithelioid cells showed significantly higher IL-1 activity than those from unstimulated cells. In contrast, LPS stimulation of M. leprae granuloma macrophages failed to enhance IL-1 production. Nevertheless, IL-1 activity in the culture supernatants from stimulated mycobacterial granuloma cells of both types was much lower than that from LPS-stimulated peritoneal exudate macrophage culture supernatants. There was no detectable amount of prostaglandin E2 (PGE2) in the culture supernatants from both unstimulated and LPS-stimulated BCG- and M. leprae-induced granuloma cells in comparison to much higher levels of PGE2 produced by unstimulated (0.28-6.2 ng/ml) or LPS-stimulated (greater than 15 ng/ml) peritoneal exudate macrophages. However, BCG granuloma cells either secreted prostaglandin F2 alpha (PGF2 alpha) spontaneously or produced comparable levels of PGF2 alpha to those from peritoneal exudate macrophages on stimulation, while M. leprae granuloma macrophages produced much lower levels of PGF2 alpha.

Interleukin-1 released by blood-monocyte-derived macrophages from patients with leprosy.

In highly purified blood-monocyte-derived macrophages collected from patients with leprosy and from healthy individuals and cultured in vitro with mycobacterial antigens such as Mycobacterium bovis BCG or Mycobacterium leprae, we nonspecifically induced the synthesis of interleukin-1. Normally, all supernatants from cultured macrophages of all subjects tested produced similar amounts of interleukin-1. However, only in patients with lepromatous leprosy, M. leprae, but not BCG, induced high-level synthesis of prostaglandin E2, which acted as a suppressor factor in the mouse thymocyte proliferative assay used to measure the interleukin-1 content of the supernatants. Normal interleukin-1 content of those supernatants was demonstrated by blocking the prostaglandin E2 synthesis by the addition of indomethacin to the medium throughout the experimental procedure. We also tested the efficiency of a combination of BCG and M. leprae in reducing the prostaglandin E2 synthesis, but with the methodology used, we did not observe any beneficial effect of such a combination. These results demonstrate the possible role of M. leprae in the induction of at least one of the suppressive monokines and are additional arguments for the involvement of macrophages in the suppression of the specific cell-mediated immunity to M. leprae observed in lepromatous leprosy.

T-cell-dependent polyclonal activation by soluble mycobacterial extracts of B cells in peripheral blood mononuclear cell populations from leprosy patients and normal donors.

Soluble extracts of ultrasonically disrupted mycobacteria (including Mycobacterium leprae) cause strong T-cell-dependent, Cyclosporin A-sensitive, polyclonal B-cell activation, in human peripheral blood mononuclear cell preparations. It is not enhanced by cortisol. Cells from cases of lepromatous leprosy show no polyclonal B-cell activation in the presence of extracts of M. leprae, although they respond normally to other mycobacterial species. The polyclonal B-cell activation response to the M. leprae extract, of a small group of normal leprosy contacts, was reduced relative to that of non-contacts.

Prostaglandin-dependent regulation of the in vitro proliferative response to mycobacterial antigens of peripheral blood lymphocytes from normal donors and from patients with tuberculosis or leprosy.

The response to soluble mycobacterial antigens of peripheral blood mononuclear cells, from most normal donors, tuberculosis patients or cases of tuberculoid leprosy (TT/BT) was enhanced by the addition of indomethacin. In contrast, indomethacin caused no enhancement of the response of cells from lepromatous leprosy (BL/LL) cases. Moreover the addition of 10-5 M prostaglandin E2 (PGE2) failed to inhibit the proliferative responses of cells from the BL/LL patients, although it markedly inhibited the responses of peripheral blood mononuclear cells from the other groups. The addition of PGE2 or indomethacin to cells which had been precultured for 48 hr had no significant effect on the proliferative responses of cells from any of the groups of donors. These results suggest that a normal, prostaglandin-dependent, indomethacin-sensitive regulatory mechanism is absent from the peripheral blood mononuclear cells of BL/LL patients.