Inhibitory effect of the Pseudobrickellia brasiliensis (Spreng) R.M. King & H. Rob. aqueous extract on human lymphocyte proliferation and IFN-γ and TNF-α production in vitro

Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Co-administration of CpG oilgonucleotides and Chenopodium album extract reverse IgG2a/IgH1 ratios and increase IFN-gamma and IL-10 prpductions in a murine model of asthma

Asthma is a disorder of increasing severity and prevalence. Recent knowledge about the pathogenesis of asthma emphasizes its inflammatory nature. CpG oligonucleotides are a class of compounds containing motifs based on the cytosine-guanine dinucleotides [CpG-ODNs]. These motifs are suppressed in mammalian DNA. They induce inflammation in mammals characterized by the induction of T helper type 1 and regulatory responses. In this paper, the effect of CpG DNA co-administration with a homemade Chenopodium album [Ch.a] extract in a murine model of asthma is reported for the first time. Balb/C mice were sensitized using Ch.a. pollen allergenic extract plus CpG-ODNs intraperitoneally and were challenged with aerosolized allergen. Results measured included IL-10 and IFN-gamma cytokines as well as IgG subclasses. For this, splenocytes from mice treated with CpG/Ag or Ag alone, were cultured in the presence of antigen. The results showed that CpG ODN administered at the time of Ch.a sensitization, effectively increased cytokines and IgG2a/IgG1 ratios compared with those in mice treated with antigen or with PBS alone[P

Baicalin reduces the severity of experimental autoimmune encephalomyelitis

Scutellaria baicalensis Georgi is one of the important medicinal herbs widely used for the treatment of various inflammatory diseases in Asia. Baicalin (BA) is a bioactive anti-inflammatory flavone found abundantly in Scutellaria baicalensis Georgi. To explore the therapeutic potential of BA, we examined the effects of systemic administration of the flavone (5 and 10 mg/kg, ip) on relapsing/remitting experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein 139-151 in SJL/J mice, an experimental model of multiple sclerosis. The mice treated with PBS or BA at day -1 and for 3 consecutive days were observed daily for clinical signs of disease up to 60 days after immunization. In the PBS-EAE group, neurological scores were: incidence (100 percent), mean day of onset (8.0 ± 0.73), peak clinical score (3.0 ± 0.4), and cumulative disease index (141.8 ± 19.4). In the BA-EAE group (5 or 10 mg kg-1 day-1, respectively), incidence (95 or 90 percent), mean day of onset (9.0 ± 0.80 or 9.2 ± 0.75; P = 0.000), peak clinical score (2.2 ± 0.3 or 2.0 ± 0.3; P = 0.000), and cumulative disease index (75.9 ± 10.1 or 62.9 ± 8.4; P = 0.000) decreased, accompanied by the histopathological findings (decrease of dense mononuclear infiltration surrounding vascellum) for the spinal cord. Additionally, the in vitro effects of BA (5, 10, and 25 µM) on mononuclear cells collected from popliteal and inguinal lymph nodes of day-10 EAE mice were evaluated using an MTT reduction assay for cell proliferation, and ELISA to measure IFN-g and IL-4 cytokines. Compared with the control group, BA caused an increase in IL-4 (EAE-DMSO: 3.56 ± 0.42 pg/mL vs EAE-BA (5, 10, and 25 µM): 6.03 ± 1.1, 7.83 ± 0.65, 10.54 ± 1.13 pg/mL, respectively; P < 0.001); but inhibited IFN-g (EAE-DMSO: 485.76 ± 25.13 pg/mL vs EAE-BA (5, 10, and 25 µM): 87.08 ± 9.24, 36.27 ± 5.44, 19.18 ± 2.93 pg/mL, respectively; P < 0.001) and the proliferation of mononuclear cells (EAE-DMSO:...

Effects of Saussurea lapp a roots extract in ethanol on leukocyte phagocytic activity, lymphocyte proliferation and interferon - gamma [IFN-gamma]

Effects of Saussurea lappa root extracts prepared in ethanol according to the homeopathic principles were ssessed on leukocyte phagocytic activity, lymphocyte transformation and mitogen-induced interferon-gamma [INF-gamma] in the cultures of peripheral blood mononuclear cells of goats [PBMC] in vitro. Leukocyte phagocytic activity was measured by flow cytometry, lymphocyte proliferation by MTT and IFN-gamma level in cell culture supernatants was determined by ELISA. The results obtained demonstrated that all tests dilutions [D4, D6, D8] of Saussurea lappa in ethanol have exerted a stimulating effect on leukocyte phagocytic activity in dose-dependent manner. A 10 micro liters dose of Saussurea lappa of each dilution markedly enhanced phagocytic activity, while other does tested made only a feeble stimulating effect. The increase with 10 micro liter dose were found significantly [p<0.01] different between each dilution, maximal stimulationwas observed by D8 dilution. Different does [10 micro liters, 2 micro liters, 0.5 micro liters] of all test dilutions [D4, D6, D8] of Saussurea lappa in sterile 0.9% NaCl solution inhibited lymphocyte proliferation. Maximal inhibitory effect was observed with the 2 micro liter dose. Similarly, Saussurea lappa suppressed the secretion of IFN-gamma by mitoren-activated [PHA; 2.5 micro g/ml] of peripheral mononuclear cells in dose-dependent manner. In conclusion these findings suggest that enhanced leukocyte phagocytic activity may be helpful to clear the soluble immune complexes produced during a sustained immune response against self antigens which causes chronic inflammatory injury of tissue. On the other hand, inhibition of lymphocyte proliferation and IFN-gamma by Saussurea lappa may contribute to suppress immune-mediated inflammatory reactions possibly through a cell-mediated cytokine pathway. Thus it is conceivable that ethanolic extracts of Saussurea lappa roots in homeopathetic dilutions may be considered as a potential candidate for therapeutic support in autoimmune and chronic inflammatory disorders

Tuberculin specific T cell responses in BCG vaccinated children.

The effector mechanisms of BCG protection were examined 5-7 years after vaccination. The in vitro lymphoproliferation, following stimulation with tuberculin, in normal, (A) vaccinated and (B) unvaccinated children and children with tuberculosis (C), were assayed. The mean stimulation index (SI) of lymphocyte transformation in normal subjects were significantly (P < 0.05) higher than those with tuberculosis. The ratio of tuberculin-specific CD4 to CD8 cells in short-term cultures were significantly (P less than 0.05) higher in the vaccinees. In group (A), 70 % had positive ratios as against 20 %and 0 %in groups (B) and (C), respectively. Secretion of IL-2 by the cells was significantly (P < 0.05) high in the vaccinated. None of the unvaccinated children had positive levels of IL-2. The vaccinees also had highly significant (P < 0.01) levels of IFN-)in the supernatants of cell-cultures, following tuberculin stimulation. In majority of the BCG vaccinated children, the stimulation of specific TH1 cells seem to be considerably high, in short-term in vitro cultures. While these responses were not so marked in the unvaccinated, they were almost totally absent in the patients.

Interferon-gamma (IFN-gamma) response to different hepatitis B virus antigens in hepatitis B virus infection.

The IFN-gamma levels in serum and cultured supernatant of peripheral blood mononuclear cells (PBMCs) were compared after stimulation by HBsAg ad, HBsAg ay and HBcAg among 3 groups of subjects i.e. patients with acute HBV infection, patients with chronic HBV infection and subjects recovered from HBV infection. Uninfected vaccinated group was taken as control. Serum and PBMCs were obtained from 38 individuals between 18-50 years of age. PBMCs were separated from heparinised blood by Ficoll-Hypaque density gradient centrifugation technique and cultured in CO2 incubator after stimulation by HBV surface and core antigens. IFN-gamma concentration was measured in serum and culture supernatant of PBMCs by an in-house ELISA technique. The mean serum IFN-gamma levels in acute, chronic, recovered and control groups were 88 pg/ml, 96.6 pg/ml, 155 pg/ml and 205 pg/ml respectively. On stimulation by HBsAg ad, IFN-gamma levels in cultured PBMCs of the above mentioned groups were 282.50 pg/ml, 307.45 pg/ml, 915.62 pg/ml and 511.67 pg/ml respectively, while in the same group on HBsAg ay stimulation, IFN-gamma levels were 246.25 pg/ml, 374.70 pg/ml, 1040 pg/ml and 465.83 pg/ml respectively. On stimulation by HBcAg, the IFN-gamma levels were 875 pg/ml, 128.50 pg/ml, 905 pg/ml and 235.33 pg/ml respectively in the acute, chronic, recovered and control groups. When compared with serum, significantly higher levels of IFN-gamma in cultured supernatant of PBMCs were observed after stimulation by HBsAg ad and HBsAg ay subtype in cases of chronic (p<0.05) and recovered groups (p<0.01 and p<0.001 respectively). However, no statistically significant difference of IFN-gamma level was observed between serum and PBMCs amongst the acute and control groups when stimulated by either of the HBsAg subtypes or HBcAg. In the recovered group, IFN-gamma levels produced by PBMCs after stimulation by HBcAg were significantly higher than that of serum (p<0.01). The study concludes that on subsequent exposure, PBMCs of the recovered group produces higher levels of IFN-gamma in response to different hepatitis B antigens. This response perhaps is able to protect individuals who are unable to develop anti-HBs.

A study on interferon-gamma (IFN-gamma) response by T cells stimulated by hepatitis B virus core antigen.

Cell-mediated immune response by lymphocyte induced through recognition of HBV core antigen during acute HBV infection, chronic HBV infection and in subjects recovered from HBV infection was investigated in the present study by assessing the competence of IFN-gamma secretion by cultured PBMCs on stimulation by HBV nucleocapsid antigen (HBcAg). Fresh blood was collected in heparin from acute, chronic and recovered groups of HBV infected patients and uninfected vaccinated healthy controls aged between 18-50 years. PBMCs were separated by Ficoll-Hypaque density gradient centrifugation technique and were stimulated with hepatitis B virus core antigen (HBcAg) and mitogen (lectin). Stimulated PBMCs were cultured in CO2 and IFN-gamma levels were measured from the culture supernatant by an in-house ELISA technique. The mean+/-SE levels of IFN-gamma in HBcAg stimulated PBMCs in acute, chronic, recovered and controls groups were 875 pg/ml+/-297.56, 128.50 pg/ml+/-33.66, 905 pg/ml+/-172.51 and 235.33 pg/ml+/-111.28 respectively. IFN-gamma levels produced by HBcAg stimulated PBMCs of acute and recovered groups were significantly higher than that of chronic and control group (p<0.001). All groups responded strongly to lectin stimulation. Thus, it may be concluded that patients with acute HBV infection and those who had recovered from HBV infection show vigorous response to HBcAg stimulation in contrast to patients with chronic HBV infection.

Incremento en la concentración de IFN-ç y TNF-Ó en el plasma de sujetos sanos a los que se administró un RNAt de origen fúngico. Reporte preliminar / Increase in the concentrations of IFN-ç and TNF-Ó in the plasm of healthy subjects treated with fungal tRNA. Preliminary report

Antecedentes: Es importante el uso con fines terapéuticos de inmunomoduladores que funcionen como inductores de la sintesis de interferón natural (IFN-n), principalmente si se considera el costo y toxicidad del interferón recombinante. En este trabajo, se estudia el efecto que la aplicación de uno de estos inductores, el ácido ribonucleico de transferencia (RNAt), tuvo sobre los niveles séricos del interferón gamma (IFN-ç) y del factor de necrosis tumoral alfa (TNF-Ó). Objetivos: Determinar la concentración del INF-ç y el TNF-Ó en el plasma de sujetos a quienes se les aplicó un RNAt de origen fúngico como inductor de la síntesis de IFN-n. Material y Métodos: A nueve voluntarios sanos se les administró por vía IM 200 mg del RNAt, y se valoró en plasma de sangre venosa antes y dos horas después de la apliación del RNAt, la concentración del IFN-ç y del TNF-Ó por la técnica de ELISA. Resultados: Se observó que los niveles de IFN-ç y del TNF-Ó se incrementaron de 267.83 ñ 208.03 a 2230 ñ 2088.73 pg/mL, y de 48.92 ñ 88.07 a 261.65 ñ 80.61 pg/mL respectivamante (Prom. ñ D.S, p < 0.05), a las dos horas de la administración de RNAt. Conclusión: Con la administración del RNAt se observa una tendencia de incremento en la concentración de IFN-ç y del TNF-Ó, en el plasma de sujetos normales