Covariates of vaginal microbiota and pro-inflammatory cytokine levels in women of reproductive age.

This study aimed to assess the correlation between covariates of the vaginal microbiota and local levels of proinflammatory cytokines in women of reproductive age presenting four molecularly defined bacterial community-state types (CSTs). We enrolled 133 non-pregnant women who attended primary care health clinics for routine Pap-testing. Molecular profiling of vaginal microbiota was performed by V3-V4 16S rRNA sequencing. The covariates of vaginal microbiota included were: vaginal pH, total bacterial cell count, diversity (Shannon index), -richness and dominant taxa abundances. Levels of interleukin (IL)-1ß, IL-6, IL-8, and tumour necrosis factor (TNF-α) were measured by enzyme-linked immunosorbent assays in supernatants of cervicovaginal fluids. Nonparametric Kruskal-Wallis test was used to compare microbiota covariates and cytokines among different CSTs. Spearman's tests were performed to assess correlations across the measured parameters. A total of 96 (72.2%) participants had CSTs dominated by Lactobacillus spp. (Lactobacillus crispatus CST I, n=38; Lactobacillus gasseri CST II, n=20; and Lactobacillus iners CST III, n=38). A total of 37 (27.8%) presented the Lactobacillus-depleted CST IV. Total bacterial count was higher in CST II (1.29E+05, 3.40E+04-6.69E+05) compared to other Lactobacillus-dominated CSTs (p=0.0003). The highest values of microbiota diversity (1.85; 0.23-2.68) and richness (27.0; 5.0-37.0) were observed in CST IV (P<0.0001). Lower levels of IL-1ß were observed in CST I (5.4; 0.0-3,256) when compared to CST III (51.7; 0.0-2,616) and to CST IV (56.2; 0.0-3,407) (P=0.008). Levels of IL-6 were higher in CST II (4.13; 0-131.4) than in CST IV (0.0-58.27) (P=0.02). Correlation tests showed an overall distinct profile of CST II when compared to other Lactobacillusdominated CSTs, particularly regarding the correlation between total bacterial load and cytokines (r>0.39). In conclusion, this study provides evidence of a single pro-inflammatory signature of L. gasseri-dominated microbiota in response to bacterial load. Further studies evaluating a broader range of inflammation markers are warranted.

Effect of propolis on Th2 and Th17 cells: interplay with EtxB- and LPS-treated dendritic cells.

Dendritic cells (DCs) are antigen-presenting cells that drive the differentiation of T CD4+ cells into different profiles according to the nature of the antigen or immunomodulator. Propolis is a resinous product made by bees that has numerous pharmacological properties, including an immunomodulatory action. To assess whether propolis can modulate the activation of CD4+ T cells by stimulating DCs with heat-labile enterotoxin B subunit (EtxB) or lipopolysaccharide (LPS), we aimed to elucidate the mechanisms affected by propolis in the differential activation of T lymphocytes. Cell viability, lymphocyte proliferation, gene expression (GATA-3 and RORc), and cytokine production (interleukin (IL)-4 and IL-17A) were analyzed. Propolis, EtxB, and LPS induced a higher lymphoproliferation compared with the control. Propolis induced GATA-3 expression and, in combination with EtxB, maintained the baseline levels. Propolis alone or in combination with LPS inhibited RORc expression. EtxB alone and in combination with propolis increased IL-4 production. Propolis in combination with LPS prevented LPS-induced IL-17A production. These results opened perspectives for the study of biological events that may be favored by propolis by promoting Th2 activation or helping in the treatment of inflammatory conditions mediated by Th17 cells.

Effect of propolis on Th2 and Th17 cells: interplay with EtxB- and LPS-treated dendritic cells

Dendritic cells (DCs) are antigen-presenting cells that drive the differentiation of T CD4+ cells into different profiles according to the nature of the antigen or immunomodulator. Propolis is a resinous product made by bees that has numerous pharmacological properties, including an immunomodulatory action. To assess whether propolis can modulate the activation of CD4+ T cells by stimulating DCs with heat-labile enterotoxin B subunit (EtxB) or lipopolysaccharide (LPS), we aimed to elucidate the mechanisms affected by propolis in the differential activation of T lymphocytes. Cell viability, lymphocyte proliferation, gene expression (GATA-3 and RORc), and cytokine production (interleukin (IL)-4 and IL-17A) were analyzed. Propolis, EtxB, and LPS induced a higher lymphoproliferation compared with the control. Propolis induced GATA-3 expression and, in combination with EtxB, maintained the baseline levels. Propolis alone or in combination with LPS inhibited RORc expression. EtxB alone and in combination with propolis increased IL-4 production. Propolis in combination with LPS prevented LPS-induced IL-17A production. These results opened perspectives for the study of biological events that may be favored by propolis by promoting Th2 activation or helping in the treatment of inflammatory conditions mediated by Th17 cells.

Evaluation of renal protection from high doses of melatonin in an experimental model of renal ischemia and reperfusion in hyperglycemic rats.

BACKGROUND: Melatonin is a free radical scavenger with important actions in the study of renal ischemia and reperfusion (I/R). This study evaluated possible renal protection of high doses of melatonin in an experimental model of I/R in which rats were submitted to acute hyperglycemia under anesthesia with isoflurane. METHOD: Forty-four male Wistar rats, weighing more than 300 g, were randomly divided into 5 groups: G1, sham (n = 10); G2, melatonin (n = 10; 50 mg.kg(-1)); G3, hyperglycemia (n = 9; glucose 2.5 g.kg(-1)); G4, hyperglycemia/melatonin (n = 10; 2.5 g.kg(-1) glucose + melatonin 50 mg.kg(-1)); and G5, I/R (n = 5). In all groups, anesthesia was induced with 4% isoflurane and maintained with 1.5% to 2.0% isoflurane. Intraperitoneal injection of melatonin (G1, G4), glucose (G3, G4), or saline (G1, G5) was performed 40 minutes before left renal ischemia. Serum plasma values for creatinine and glucose were determined at baseline (M1), immediately following reperfusion (M2), and 24 hours after completion of the experiment (M3). Histological analysis was performed to evaluate tubular necrosis (0-5). RESULTS: Serum glucose was higher at M2 in the groups supplemented with glucose, hyperglycemia (356.00 ± 107.83), and hyperglycemia/melatonin (445.3 ± 148.32). Creatinine values were higher at T3 (P = .0001) for I/R (3.6 ± 0.37), hyperglycemia/melatonin (3.9 ± 0.46), and hyperglycemia (3.71 ± 0.69) and lower in the sham (0.79 ± 0.16) and melatonin (2.01 ± 1.01) groups, P < .05. Histology showed no necrosis injury in the G1, lesion grade 2 in the G2, and severe acute tubular necrosis in the G3: (grade 4), G4: (grade 5) and G5: (grade 4) groups (P < .0001). DISCUSSION: Melatonin protected the kidneys submitted to I/R in rats without hyperglycemia; however, this did not occur when the I/R lesion was associated with hyperglycemia. CONCLUSIONS: Due to its antioxidant and antiapoptotic action, melatonin was able to mitigate, but not prevent acute tubular necrosis in rats with hyperglycemia under anesthesia by isoflurane.

Evaluation of mesenchymal stem cell migration after equine tendonitis therapy.

REASONS FOR PERFORMING STUDY: Mesenchymal stem cells (MSCs) have been used to treat equine tendonitis with promising results; however, little is known about the potential migration of these cells. OBJECTIVES: To assess the possible migration of MSCs from an implantation site in the superficial digital flexor tendon (SDFT) to a lesion in the SDFT of the contralateral limb. STUDY DESIGN: In vivo experimental study. METHODS: Adipose-derived MSCs were isolated from 4 healthy horses. Lesions were induced in the SDFTs of both forelimbs, followed by intralesional implantation of autologous adipose-derived MSCs labelled with nanocrystals into one of the limbs. Flow cytometry of the peripheral blood mononuclear cells and fluorescence microscopy of biopsies of the SDFT lesions were used to search for the labelled cells. RESULTS: Labelled cells were detected among the peripheral blood mononuclear cells in all animals, but labelled cells were present only in the SDFTs that were treated with the intralesional implants. CONCLUSION: Nanocrystals were a valuable in vivo marker of MSCs to be used for tendonitis treatment. Although migration of MSCs to the bloodstream was observed, it was not possible to identify the labelled cells in the untreated tendons.

Characterization of mesenchymal stem cells derived from equine adipose tissue / Caracterização das células tronco mesenquimais derivadas do tecido adiposo equino

Stem cell therapy has shown promising results in tendinitis and osteoarthritis in equine medicine. The purpose of this work was to characterize the adipose-derived mesenchymal stem cells (AdMSCs) in horses through (1) the assessment of the capacity of progenitor cells to perform adipogenic, osteogenic and chondrogenic differentiation; and (2) flow cytometry analysis using the stemness related markers: CD44, CD90, CD105 and MHC Class II. Five mixed-breed horses, aged 2-4 years-old were used to collect adipose tissue from the base of the tail. After isolation and culture of AdMSCs, immunophenotypic characterization was performed through flow cytometry. There was a high expression of CD44, CD90 and CD105, and no expression of MHC Class II markers. The tri-lineage differentiation was confirmed by specific staining: adipogenic (Oil Red O), osteogenic (Alizarin Red), and chondrogenic (Alcian Blue). The equine AdMSCs are a promising type of adult progenitor cell for tissue engineering in veterinary medicine.

O uso de células tronco tem demonstrado resultados promissores na terapia da tendinite e osteoartrite na medicina equina. O objetivo deste trabalho foi caracterizar as células tronco mesenquimais derivadas do tecido adiposo (AdCTMs) em cavalos através da (1) avaliação da capacidade das células progenitoras para realizar a diferenciação adipogênica, osteogênica e condrogênica; e (2) através da análise por citometria de fluxo, utilizando os marcadores stemness relacionados: CD44, CD90, CD105 e MHC de Classe II. Cinco cavalos sem raça definida, de 2 a 4 anos de idade foram utilizados para a coleta do tecido adiposo da base da cauda. Após o isolamento e cultivo das AdCTMs, a caracterização imunofenotípica foi realizada pela citometria de fluxo. Houve alta expressão dos marcadores CD44, CD90 e CD105, e não houve expressão do MHC Classe II. A diferenciação foi confirmada pela coloração específica: adipogênica (Oil Red O), osteogênico (Alizarin Red), e condrogênico (Alcian Blue). As AdCTMs são um tipo promissor de células progenitoras adulta para a engenharia tecidual na medicina veterinária.

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets.

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.

PP063. TLR-4 expression and pro-inflammatory cytokine production by peripheral blood monocytes from preeclamptic women.

INTRODUCTION: Toll-like receptor (TLR)-4 and TLR-2 are involved in inflammatory response of monocytes. These cells are activated in pregnant women with preeclampsia (PE), and over-produce inflammatory cytokines. TLR4 may recognize endogenous ligands, the so-called danger signals released by damaged cells, leading to production of pro-inflammatory cytokines. OBJECTIVES: This study investigated TLR2 and TLR4 expression and cytokine production by monocytes from women with PE before and after stimulation with TLR ligands. METHODS: Monocytes (5×10(5)cell/mL) were obtained from 32 preeclamptic (PE) and 20 normotensive (NT) pregnant women in the last trimester of pregnancy. TLR2 and TLR4 expression on monocyte surface was determined by flow cytometry in non-stimulated cells, and after 18h of culture with lipopysaccharide (LPS) and peptidoglycan (PG). TNF-α, IL-10 and IL-12p70 production by these cells stimulated or not with LPS or PG was evaluated by enzyme immunoassay. Results were analyzed by non-parametric tests with significance level set at 5%. RESULTS: In the absence of stimulation, the basal TLR4 expression by monocytes detected by the median fluorescence intensity (MFI) was significantly higher in the PE group than in the NT group while no significant differences were observed between groups in relation to endogenous TLR2 expression. An increase in TLR4 MFIs was detected after monocytes from NT pregnant women were stimulated with LPS while TLR2 expression was increased after PG-stimulation. No alterations in TLR expression was detected after LPS or PG-stimulation in monocytes from patients with PE. Evaluation of endogenous cytokine levels in supernatant culture of monocytes showed higher concentrations of TNF-α and IL-12p70 in preeclamptic women in comparison with the NT group, whereas IL-10 values were significantly higher in NT pregnant women than in the PE group. In contrast, when monocytes were stimulated with the TLRs ligands LPS and PG, the release of TNF-α was significantly reduced, while IL-12p70 levels were significantly higher in women with PE compared to NT group. IL-10 production was similar in both groups studied. CONCLUSION: The basal up-regulation of TLR4 expression associated with endogenous high TNF-α and IL-12p70 production by monocytes from preeclamptic women confirms the activated profile of these cells by the disease process. These findings provide new insights into possible roles for TLRs in the pathogenesis of systemic inflammation detected in PE. FINANCIAL SUPPORT: FAPESP 2009/11924-3 and 2010/20207-0.

PP064. M1 Monocyte subpopulation is associated with pro-inflammatory cytokineproduction in pregnant women with preeclampsia.

INTRODUCTION: Monocytes from peripheral blood of pregnant women with preeclampsia are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. OBJECTIVES: This study aimed at evaluating whether the inflammatory state of monocytes observed in preeclampsia is associated with the polarization of monocyte to M1 profile in peripheral blood, correlating the expression of surface receptors CD64, TLR2, TLR4, and CD163 and CD206 with cytokine production. METHODS: We studied 90 pregnant women, 30 normotensive and 60 with preeclampsia, matched for gestational age. Peripheral blood monocytes obtained from normotensive pregnant or preeclamptic pregnant women were cultured for 18h, and the expression of surface receptors on M1 inflammatory monocyte subpopulation (TLR2, TLR4 and CD64) and M2 suppressor monocyte subpopulation (CD163 and CD206) were evaluated by flow cytometry, using specific monoclonal antibodies, labeled with fluorochromes. The values were expressed as ??the mean fluorescence intensity. Moreover, the production of proinflammatory cytokines associated with M1 profile (TNF-α, IL-12p70 and IL-23) and the anti-inflammatory cytokine associated with M2 profile (IL-10) were evaluated in the monocyte supernatant of culture by enzyme immunoassay. Results were analyzed using nonparametric tests with significance level set at 5%. RESULTS: The expression of CD4 and TLR4 on monocyte surface, from women with preeclampsia was significantly higher, while the expression of CD163 and CD206 was significantly decreased compared with normotensive pregnant women, suggesting the predominance of monocyte M1 profile. Endogenous production of TNF-α, IL-12p70 and IL-23 by monocytes was increased, while synthesis of IL-10 was lower in women with preeclampsia compared with normotensive pregnant women. Positive correlations between TLR4 and CD64 (r=0.5849), TLR4 and TNF-α (r=0.5126) or TLR4 and IL-23 (r=0.8095), as well as between CD64 and TNF-α (r = 0.7133) or CD64 and IL-23 (r = 0.6051) were observed in the preeclamptic group. The results confirm the activated state of monocytes from women with preeclampsia by increased production of proinflammatory cytokines and the expression of receptors characteristic of the M1 subpopulation. CONCLUSION: This study provides evidence that monocytes from women with preeclampsia are classically activated and the systemic inflammatory environment may differentiate and polarize these cells to the M1 profile. FINANCIAL SUPPORT: CNPq, FAPESP 2009/11924-3 and 2010/20207-0.

Erythropoietin attenuates apoptosis after ischemia-reperfusion-induced renal injury in transiently hyperglycemic Wister rats.

BACKGROUND: Hyperglycemia is associated with a decreased tolerance to ischemia and an increased severity of renal ischemia reperfusion (I/R) injury. It has been suggested that erythropoietin (EPO) attenuates this effect in normoglycemic animals. This study sought to examine the effects of EPO on treatment renal I/R injury (IRI) in transiently hyperglycemic rats. MATERIAL AND METHODS: Twenty-eight male Wister rats anesthetized with isoflurane received glucose (2.5 g.kg(-1) intraperitoneally) before right nephrectomy. They were randomly assigned to four groups: sham operation (S); IRI (ISO); IRI+EPO, (600 UI kg(-1) low-dose EPO [EL]); and IRI+EPO 5000 UI kg(-1) (high-dose EPO [EH]). IRI was induced by a 25-minute period of left renal ischemia followed by reperfusion for 24 hours. Serum creatinine and glucose levels were measure at baseline (M1), immediately after the ischemic period (M2), and at 24 hours after reperfusion (M3). After sacrificing the animals, left kidney specimens were submitted for histological analysis including flow cytometry to estimate tubular necrosis and the percentages of apoptotic, dead or intact cells. RESULTS: Scr in the ISO group was significantly higher at M3 than among the other groups. Percentages of early apoptotic cells in ISO group were significantly higher than the other groups. Percentages of late apoptotic cells in S and ISO groups were significantly greater than EL and EH groups. However, no significant intergroup differences were observed regarding the incidence of tubular necrosis. CONCLUSIONS: Our results suggested that, although not preventing the occurrence of tubular necrosis, EPO attenuated apoptosis and glomerular functional impairment among transiently hyperglycemic rats undergoing an ischemia/reperfusion insult.

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm.

Tritrichomonas fetus causes infertility and abortion in cattle; however, there is scarce information regarding the susceptibility of bovine sperm to this parasite. The objective of this study was to analyze in vitro the interaction between T. fetus and bovine sperm and to evaluate the effect of extracellular products secreted by the parasite on these reproductive cells. Sperm from five fertile bulls (Bos taurus taurus, Holstein-Friesian), selected through a Percoll gradient, adhered to T. fetus after 30min of interaction, resulting in agglutination between the two kinds of cells. Based on reverse transcription-polymerase chain reaction (RT-PCR), T. fetus continuously expressed its gene for cysteine peptidase in the presence or absence of sperm. Computer-assisted semen analysis (CASA) revealed that, after 1h incubation of sperm in T. fetus culture extract, the extracellular products secreted by the parasite decreased sperm progressive motility (P<0.05). Although T. fetus extracellular products did not lead to loss of sperm viability (P<0.05) based on the Annexin-V/propidium iodide assay, the percentage of Annexin-V fluorescein isothiocyanate-positive and propidium iodide-positive cells increased (P<0.05) during incubation of sperm in T. fetus culture extract, consistent with cellular damage. In conclusion, extracellular products secreted by T. fetus were cytotoxic to bovine sperm, as they decreased sperm progressive motility; perhaps this contributes to the pathogenesis of T. fetus-induced infertility.

Viability and cell cycle analysis of equine fibroblasts cultured in vitro.

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD((R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: role of interleukin-8.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production.

Assessment of cytokine values in serum by RT-PCR in HIV-1 infected individuals with and without highly active anti-retroviral therapy (HAART)

A cross-sectional study was performed on HIV-1 infected individuals with or without antiretroviral treatment (ARV) in the AIDS Day Hospital, Botucatu Medical School, UNESP. Between August 2004 and October 2005, 73 HIV-1 infected individuals were divided into three groups: infected individuals with or without AIDS who had never received ARV (G1 = 15); patients on HAART that had had plasma HIV-1 RNA viral load (VL) equal to or greater than 50 copies/mL (G2 = 27); and patients on HAART with undetectable VL for at least the past six months (G3 = 31). There was also an additional group that comprised blood donors without any sign of the disease and with negative HIV serum tests (G4 = 20), which was the control group. Serum cytokine levels (values in pg/mL) were measured by enzyme-linked immunosorbent assay (ELISA) and specific mRNA expression by reverse transcription polymerase chain reaction (RT-PCR). Both techniques were performed on the four groups for TNF-á, IL-2, INF-ã, IL-4 and IL-10. All patients were submitted to VL determination and CD4+ and CD8+T lymphocyte counts. The analysis of the results revealed a significant comparison among groups for both methods and an association between the latter (> 80% r² > 0.80). There was only one exception, in control individuals for IL-2 by ELISA. The cytokine profiles, in both methods, for the three patient groups, were mature Th-0. The behaviors of IL-2 and INF-ã required emphasis due to consequent expression of dominant Th profile. Both methods showed low IL-2 and high mean values of INF-ã in the three groups. Several authors have recently drawn attention to the substantial apoptosis of infected and non-infected CD4+T cells, mainly during primary infection, persisting only in those with INF-ã phenotype producer and not IL-2. HIV infected individuals submitted to HAART are expected to produce IL-2 in an attempt to present Th-1 profile, but in most cases this did not occur.(AU)