Preventive and curative glycoside kaempferol treatments attenuate the TH2-driven allergic airway disease.

Asthma is a chronic respiratory disease characterized by airway inflammation and airway hyperresponsiveness (AHR). One strategy to treat allergic diseases is the development of new drugs. Flavonoids are compounds derived from plants and are known to have antiallergic, anti-inflammatory, and antioxidant properties. To investigate whether the flavonoid kaempferol glycoside 3-O-[beta-d-glycopiranosil-(1-->6)-alpha-l-ramnopiranosil]-7-O-alpha-l-ramnopiranosil-kaempferol (GRRK) would be capable of modulating allergic airway disease (AAD) either as a preventive (GRRK P) or curative (GRRK C) treatment in an experimental model of asthma. At weekly intervals, BALB/c mice were subcutaneously (sc) sensitized twice with ovalbumin (OVA)/alum and challenged twice with OVA administered intranasally. To evaluate any preventive effect, GRRK was administered 1h (hour) before each OVA-sensitization and challenge, while to analyze the curative effect, mice were first sensitized with OVA, followed by GRRK given at day 18 through 21. The onset of AAD was evaluated 24h after the last OVA challenge. Both treatments resulted in a dose-dependent reduction in total leukocyte and eosinophil counts in the bronchoalveolar lavage fluid (BAL). GRRK also decreased CD4(+), B220(+), MHC class II and CD40 molecule expressions in BAL cells. Histology and lung mechanic showed that GRRK suppressed mucus production and ameliorated the AHR induced by OVA challenge. Furthermore, GRRK impaired Th2 cytokine production (IL-5 and IL-13) and did not induce a Th1 pattern of inflammation. These findings demonstrate that GRRK treatment before or after established allergic lung disease down-regulates key asthmatic features. Therefore, GRRK has a potential clinical use for the treatment of allergic asthma.

Anti-allergic effect of bee pollen phenolic extract and myricetin in ovalbumin-sensitized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The bee pollen is used in folk medicine to alleviate allergic reactions. The bee pollen phenolic extract (BPPE) consists in phenolic compounds (flavonoids) from plants picked by Apis mellifera bee. AIM OF THIS STUDY: Here we evaluated the anti-allergic property of the BPPE and the flavonoid myricetin (MYR) in murine model of ovalbumin (OVA)-induced allergy. MATERIALS AND METHODS: The study focused on the BPPE or myricetin treatment of OVA-sensitized BALB/c mice and their effects on the IgE and IgG1 production, pulmonary cell migration, eosinophil peroxidase (EPO) activity and anaphylactic shock reaction. RESULTS: The BPPE treatment (200mg/kg) showed inhibition of the paw edema, IgE and IgG(1) OVA-specific production, leukocyte migration to the bronchoalveolar lavage (BAL) and EPO activity in lungs. In addition, BPPE treatment showed partial protection on the anaphylactic shock reaction induced by OVA. Treatment with myricetin (5 mg/kg) also inhibited pulmonary cell migration and IgE and IgG(1) OVA-specific production. CONCLUSIONS: These results support the hypothesis the myricetin is one of the flavonoids of BPPE responsible for the anti-allergic effect and a potential tool to treat allergies.

Warifteine, a bisbenzylisoquinoline alkaloid, decreases immediate allergic and thermal hyperalgesic reactions in sensitized animals.

Warifteine is a bisbenzylisoquinoline alkaloid isolated from the Cissampelos sympodialis Eichl (Menispermaceae). This plant is used in the folk medicine for the treatment of airway respiratory diseases. A murine model of immediate allergic reaction was used to evaluate warifteine treatment in the IgE production, leukocyte activation, thermal hyperalgesia, mast cell degranulation and scratching behavior. BALB/c mice treated with warifteine (0.4-10 mg/Kg) 1 h before OVA sensitization reduced OVA induced paw edema as well as the OVA-specific IgE serum titers as compared with non-treated and OVA-sensitized animals. Warifteine also reduced the mice death evoked by IgE-dependent anaphylactic shock reaction at 30 min after intravenous OVA challenge. To assess the effect of warifteine treatment on T cell proliferative response, spleen cells from warifteine treated or non-treated and OVA-sensitized animals were evaluated. Spleen cells from warifteine treated animals (2.0 mg/kg) did not proliferate following OVA stimulation as compared with spleen cell cultures from non-treated animals. This response may be related with the increase of NO production as observed in peritoneal macrophage cultures treated with warifteine. Thermal hyperalgesia evoked by IgE or histamine/5-hydroxytryptamine challenge was inhibited on rats at dose of 4.0 mg/kg. Warifteine treatment (0.6 or 6.0 microg/ml) also decreased the IgEalphaDNP-BSA sensitized mast degranulation after DNP-BSA challenge measured by histamine release. In addition, compound 48/80-induced scratching behavior was also sensitive to warifteine treatment. These results demonstrate for the first time that warifteine treatment reduced the allergy-associated responses.

Cissampelos sympodialis Eichl. (Menispermaceae) inhibits anaphylactic shock reaction in murine allergic model

The murine model of OVA-induced immediate allergic reaction was used to evaluate the effectiveness of intraperitoneal sub-acute treatment with the leaf hydroalcoholic extract of Cissampelos sympodialis (AFL) in the anaphylactic shock reaction, IgE production and the background proliferative response. BALB/c mice treated with AFL ranging from 200 to 400 mg/kg/day for 5 days before and during OVA-sensitization strongly reduced the animal death and promoted reduction in total and OVA-specific serum IgE level. Spleen cells from AFL-treated sensitized animals showed a decreased proliferative background response when compared with non-sensitized animals. These results demonstrated that sub-acute intraperitoneal treatment with Cissampelos sympodialis extract has an anti-allergic effect.

Cissampelos sympodialis Eichl. (Menispermaceae): oral treatment decreases IgE levels and induces a Th1-skewed cytokine production in ovalbumin-sensitized mice.

The murine model of ovalbumin (OVA)-induced allergy was used to evaluate the effectiveness of oral treatment with the leaf extract of Cissampelos sympodialis Eichl. (Menispermaceae) (CS) in the modulation of immunoglobulin E (IgE) production and T cell activation. CS treatment with doses ranging from 200 to 600 mg/Kg/day for 15 days before and during OVA-sensitization promoted reduction in total and OVA-specific serum IgE. CS at 400 or 600 mg/Kg/day also reduced paw edema induced by local OVA challenge. Daily intake of up to 600 mg/Kg of oral CS by BALB/c mice did not reduce weight gain, which is indicative of a lack of systemic toxicity. To assess the effect of CS treatment on T cell proliferative response to stimuli in vitro, the mitogenic response of spleen cells of treated and control animals were evaluated. Cells from CS-treated animals showed an elevated background proliferative response to concanavalin-A (Con-A) when compared to those from control animals. Oral intake of CS increased the in vitro production of IFN-gamma and IL-10 by Con-A stimulated cells. Mice treated with 200 mg/Kg/day CS showed increasing levels of IFN-gamma. These results show that oral treatment with Cissampelos sympodialis extract has an immunomodulatory effect, reducing allergy-associated responses possibly by a preferential activation of Th1-type cytokines.

Modulation of B lymphocyte function by an aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl (Menispermaceae)

Cissampelos sympodialis Eichl species are used in folk medicine for the treatment of asthma, arthritis and rheumatism. In the present study, we investigated the immunomodulatory effect of an aqueous fraction of a 70 percent (v/v) ethanol extract of C. sympodialis leaves on B lymphocyte function. The hydroalcoholic extract inhibited the in vitro proliferative response of resting B cells induced by LPS (IC50 = 17.2 æg/ml), anti-delta-dextran (IC50 = 13.9 æg/ml) and anti-IgM (IC50 = 24.3 æg/ml) but did not affect the anti-MHC class II antibody-stimulated proliferative response of B cell blasts obtained by stimulation with IL-4 and anti-IgM. Incubation with the hydroalcoholic extract used at 50 æg/ml induced a 700 percent increase in intracellular cAMP levels. IgM secretion by resting B cells (obtained from normal mice) and polyclonally activated B cells (obtained from Trypanosoma cruzi-infected animals) was inhibited by the hydroalcoholic extract. The latter were more sensitive to the hydroalcoholic extract since 6.5 æg/ml induced a 20 percent inhibition in the response of cells from normal mice while it inhibited the response of B cells from infected animals by 75 percent. The present data indicate that the alcoholic extract of C. sympodialis inhibited B cell function through an increase in intracellular cAMP levels. The finding that the hydroalcoholic extract inhibited immunoglobulin secretion suggests a therapeutic use for the extract from C. sympodialis in conditions associated with unregulated B cell function and enhanced immunoglobulin secretion. Finally, the inhibitory effect of the hydroalcoholic extract on B cells may indicate an anti-inflammatory effect of this extract.

Modulation of B lymphocyte function by an aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl (Menispermaceae).

Cissampelos sympodialis Eichl species are used in folk medicine for the treatment of asthma, arthritis and rheumatism. In the present study, we investigated the immunomodulatory effect of an aqueous fraction of a 70% (v/v) ethanol extract of C. sympodialis leaves on B lymphocyte function. The hydroalcoholic extract inhibited the in vitro proliferative response of resting B cells induced by LPS (IC50 = 17.2 g/ml), anti-delta-dextran (IC50 = 13.9 g/ml) and anti-IgM (IC50 = 24.3 g/ml) but did not affect the anti-MHC class II antibody-stimulated proliferative response of B cell blasts obtained by stimulation with IL-4 and anti-IgM. Incubation with the hydroalcoholic extract used at 50 g/ml induced a 700% increase in intracellular cAMP levels. IgM secretion by resting B cells (obtained from normal mice) and polyclonally activated B cells (obtained from Trypanosoma cruzi-infected animals) was inhibited by the hydroalcoholic extract. The latter were more sensitive to the hydroalcoholic extract since 6.5 g/ml induced a 20% inhibition in the response of cells from normal mice while it inhibited the response of B cells from infected animals by 75%. The present data indicate that the alcoholic extract of C. sympodialis inhibited B cell function through an increase in intracellular cAMP levels. The finding that the hydroalcoholic extract inhibited immunoglobulin secretion suggests a therapeutic use for the extract from C. sympodialis in conditions associated with unregulated B cell function and enhanced immunoglobulin secretion. Finally, the inhibitory effect of the hydroalcoholic extract on B cells may indicate an anti-inflammatory effect of this extract.

Cissampelos sympodialis Eichl (Menispermaceae) leaf extract induces interleukin-10-dependent inhibition of Trypanosoma cruzi killing by macrophages.

The aqueous fraction of the ethanolic extract (AFL) of Cissampelos sympodialis Eichl (Menispermaceae), popularly known as milona, has been shown to have both immunosuppressive and anti-inflammatory effects. In the present study we investigated the modulation of macrophage antimicrobicidal activity by in vitro treatment with the extract from C. sympodialis. Normal and thioglycolate-elicited mouse peritoneal macrophages were infected in vitro with the protozoan Trypanosoma cruzi DM28c clone. We observed that the AFL (used at doses ranging from 13 to 100 microg/ml) increased T. cruzi growth and induced a 75% reduction in nitric oxide production. This inhibition could be mediated by the stimulation of macrophage interleukin-10 (IL-10) secretion since the in vitro treatment with the AFL stimulated IL-10 production by T. cruzi-infected macrophages. These results suggest that the anti-inflammatory effect of the AFL from C. sympodialis could be, at least in part, mediated by the inhibition of macrophage functions and that the inhibition of macrophage microbicidal activity induced by the C. sympodialis extract may be mediated by the decrease in macrophage function mediated by interleukin-10 production.

Cissampelos sympodialis Eichl (Menispermaceae) leaf extract induces interleukin-10-dependent inhibition of Trypanosoma cruzi killing by macrophages

The aqueous fraction of the ethanolic extract (AFL) of Cissampelos sympodialis Eichl (Menispermaceae), popularly known as milona, has been shown to have both immunosuppressive and anti-inflammatory effects. In the present study we investigated the modulation of macrophage antimicrobicidal activity by in vitro treatment with the extract from C. sympodialis. Normal and thioglycolate-elicited mouse peritoneal macrophages were infected in vitro with the protozoan Trypanosoma cruzi DM28c clone. We observed that the AFL (used at doses ranging from 13 to 100 æg/ml) increased T. cruzi growth and induced a 75 percent reduction in nitric oxide production. This inhibition could be mediated by the stimulation of macrophage interleukin-10 (IL-10) secretion since the in vitro treatment with the AFL stimulated IL-10 production by T. cruzi-infected macrophages. These results suggest that the anti-inflammatory effect of the AFL from C. sympodialis could be, at least in part, mediated by the inhibition of macrophage functions and that the inhibition of macrophage microbicidal activity induced by the C. sympodialis extract may be mediated by the decrease in macrophage function mediated by interleukin-10 production

Studies on the anti-inflammatory and analgesic activity of Curatella americana L.

Curatella americana L. (Dilleneaceae) popularly known as 'cajueiro-bravo' and 'sambaiba' is used in Brazilian folk medicine for the treatment of inflammation and ulcer. The anti-inflammatory and analgesic tests were conducted with the hydroalcoholic extract (HAE) of the bark of the plant. The HAE inhibited mouse ear oedema induced by o-tetradecanoyl phorbol acetate (TPA) and by capsaicin. While the ID50 values obtained for the HAE against these two irritants were 40.8 +/- 1.7 and 30 +/- 1.2 mg/kg i.p. (mean +/- S.E.M., n = 6), respectively, the corresponding value for carrageenan induced paw oedema (3 h) was 21.8 +/- 2.1 mg/kg, i.p., n = 6. In the established adjuvant-induced arthritis model, the HAE significantly inhibited the oedema in daily doses of 50 mg/kg, i.p. (n = 10). The HAE also inhibited acetic acid-induced writhing (ID50 23.2 +/- 0.8 mg/kg, i.p., n = 6) and the formalin-induced late phase paw licking response (ID50 11.9 +/- 1.2 mg/kg, i.p., n = 10) in the mice. However, the HAE was inactive in the formalin-induced initial paw licking response in mice or heat induced tail flick response in rats. The HAE has shown both anti-inflammatory and peripheral analgesic activities when administrated in the mouse by the intraperitoneal route in doses which are at least 12 times lower than its LD50 dose of 647 mg/kg, i.p.

Cissampelos sympodialis Eichl. leaf extract increases the production of IL-10 by concanavalin-A-treated BALB/c spleen cells.

The effect of the aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl. (Menispermaceae) leaves (AFL) on Concanavalin-A (Con-A)-activated spleen cell proliferation and cytokine (IL-2, IL-4, IL-10 and IFN-gamma) secretion were analyzed. BALB/c spleen cells were treated, in vitro, with different concentrations, ranging from 6.25 to 400 microg/ml of AFL either in the presence or the absence of 5 microg/ml of the mitogen Con-A. It was observed that concentrations of the AFL higher then 50 microg/ml were toxic to the cells and concentrations ranging from 6.25 to 50 microg/ml decreased the lymphocyte proliferative response in the presence of the mitogen. This inhibition of mitogen induced proliferation was not reverted by the addition of exogenous recombinant IL-2. The AFL did not significantly inhibit IL-2 secretion. In the presence of AFL there was a reduction in the levels of secreted IFN-gamma while the production of both IL-10 and IL-4 were increased. AFL did not induce the production of any of the cytokines analyzed, in the absence of Con-A. It is suggested that increased IL-10 production down regulates IFN-gamma secretion and T cell proliferative responses.

Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase.

Human infection with Trypanosoma cruzi (Chagas' disease) is usually accompanied by humoral and cellular immune responses to GP57/51, a major antigen that was recently identified as a prominent cysteinyl proteinase (cruzipain). The PBMC responses of 11 chronic chagasic patients and the properties of anti-cruzipain T cell lines are reported herein. GP57/51, isolated from Y strain epimastigotes (n-cruzipain) or the recombinant protein expressed in E. coli (r-cruzipain), elicited proliferative responses of variable intensity from the patient's PBMC. T cell lines were then generated using each of these antigens. These lines, which always carried the CD4+ phenotype, were reciprocally stimulated by n-cruzipain or r-cruzipain, the responses to the former being usually stronger. The analysis of cytokine production suggested that Th1-like subsets dominate the patient's responses: IFN-gamma was consistently induced on stimulation with either n-cruzipain or r-cruzipain. In contrast, IL-4 was present in very small concentrations or was undetectable. We then sought to define T cell epitopes of cruzipain using synthetic peptides spanning portions of the central (catalytic) domain and COOH-terminal extension. From a panel of 11 peptides, only one 33 mer peptide (P214) elicited a strong proliferative response on anti-cruzipain T cell lines, the intensity being comparable to that induced by r-cruzipain. Conversely, T cell lines started with P214 were responsive to either n-cruzipain or r-cruzipain, the proliferative responses again being accompanied by IFN-gamma production, but not IL-4. Interestingly, P214 is located in a conserved region of the catalytic domain of cruzipain, hence may propitiate opportunities for cross-recognition of other members of the papain superfamily. Fine epitope mapping should reveal whether structurally similar regions of host thiol-cathepsins can be potential targets for cross-reactive T cell responses during chronic human infection.

Rapid competitive enzyme-linked immunosorbent assay using a monoclonal antibody reacting with a 15-kilodalton tegumental antigen of Schistosoma mansoni for serodiagnosis of schistosomiasis.

A competitive enzyme-linked immunosorbent assay (CELISA) for antibody detection was developed by using a monoclonal antibody which reacts with a 15-kDa tegumental antigen of the adult worm of Schistosoma mansoni. This monoclonal antibody was not able to react with antigens of Schistosoma japonicum or Schistosoma haematobium in enzyme-linked immunoelectrotransfer blot (EITB) and indirect immunofluorescence tests. The assay was performed in a period of 1 h using an adult worm crude extract antigen. To evaluate the CELISA, a total of 73 serum samples was analyzed: 35 were from S. mansoni-infected patients, 23 were from individuals with parasitic infections other than schistosomiasis, and 14 were from healthy individuals. All serum samples from healthy individuals and from patients infected with other parasites were negative, as were two (6%) samples from patients infected with S. mansoni. EITB analysis showed that 32 of 33 CELISA-positive samples were positive in the EITB but with different patterns of reactivity. A 15-kDa protein reacted with 60% of serum samples, and a 60-kDa protein showed the highest level of reactivity (85%). The two samples from patients infected with S. mansoni that were negative in the CELISA reacted with 70-, 60-, 50-, 47-, and 38-kDa proteins. One sample, positive in CELISA, did not react with proteins of the antigenic extract.

Interleukin 10 production correlates with pathology in human Leishmania donovani infections.

We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.

Characterization of responses of normal human T cells to Trypanosoma cruzi antigens.

The stimulation of normal human PBMC by Trypanosoma cruzi Ag was analyzed. PBMC showed significant in vitro proliferation in response to parasite lysate (Tct), with stimulation indices ranging from 10 to 400, peaking at 6 to 7 days. The cells stimulated with Tct produced significant levels of IL-2. To determine which cells proliferated in response to Tct, PBMC were separated into T- and B-enriched cell populations. Purified T cells, but not B cells, proliferated strongly to Tct. The T cell response required APC and was processing dependent. T cell lines generated against Tct proliferated in response to parasite lysate only in the presence of autologous APC and produced IL-2, IL-6, and IFN-gamma but not IL-4 in response to PMA plus ionomycin. Although there were a significant number of CD45Ra+ cells, the majority of the cells in these T cell lines were CD45Ro+. The V beta usage of Tct-responding T cells was heterogeneous, with most V beta genes represented among the responding cells. An immunodominant repeat Ag (TcD) and a ribosomal phosphoprotein (P0) of T. cruzi elicited strong proliferative responses in all subjects tested. These data indicate the presence of T cell-stimulatory Ag in Tct, characterized by nonpreferential usage of the V beta gene families. The strong stimulation of normal human PBMC by Tct may contribute to immunologic alterations seen in T. cruzi infection.

Cyclophosphamide blocks both antigen-specific and polyclonal immunoglobulin responses in experimental visceral leishmaniasis.

Cyclophosphamide (Cy) has been shown to modulate antibody responses in a wide range of diseases both in humans and experimental animals. Our results in Syrian hamsters infected with Leishmania donovani have shown that Cy blocks specific and polyclonal antibody production both in vivo and in vitro. This effect was achieved by weekly 100 mg/kg doses and also by a 300 mg/kg single dose. Although Cy provokes a significant decrease in B-cell numbers in infected animals, this cannot explain the suppression of antibody production since a 50% decrease in B-cells of only-infected hamsters did not reproduce the same effect in in vitro assays. Also, this suppression was not reversed either by elimination of adherent cells or by the presence of indomethacin. These data suggest that Cy affects T-cell populations involved in the control of antibody production by B-cells.