Multilocus sequence typing characterizes diversity of Ureaplasma diversum strains, and intra-species variability induces different immune response profiles.

BACKGROUND: Ureaplasma diversum is a pathogen found in the genital tract of cattle and associated with genital disorders such as infertility, placentitis, abortion, birth of weak calves, low sperm motility, seminal vesiculitis and epididymitis. There are few studies evaluating the genetic diversity of U. diversum strains and their influence on the immune response in cattle. Therefore, to better understand genetic relationships of the pathogenicity of U. diversum, a multilocus sequence typing (MLST) scheme was performed to characterize the ATCC 49782 strain and another 40 isolates recovered from different Brazilian states. RESULTS: Primers were designed for housekeeping genes ftsH, polC, rpL22, rpoB, valS and ureA and for virulence genes, phospholipase D (pld), triacylglycerol lipase (tgl), hemolysin (hlyA), MIB-MIP system (mib,mip), MBA (mba), VsA (VsA) and ribose transporter (tABC). PCRs were performed and the targeted gene products were purified and sequenced. Sequence types (STs), and clonal complexes (CCs) were assigned and the phylogenetic relationship was also evaluated. Thus, a total of 19 STs and 4 CCs were studied. Following the molecular analysis, six isolates of U. diversum were selected, inoculated into bovine monocyte/macrophage culture and evaluated for gene expression of the cytokines TNF-α, IL-1, IL-6, IL-10 and IL-17. Differences were detected in the induction of cytokines, especially between isolates 198 and BA78, promoted inflammatory and anti-inflammatory profiles, respectively, and they also differed in virulence factors. CONCLUSION: It was observed that intra-species variability between isolates of U. diversum can induce variations of virulent determinants and, consequently, modulate the expression of the triggered immune response.

Effects of 17ß-Estradiol on Monocyte/Macrophage Response to Staphylococcus aureus: An In Vitro Study.

To describe how 17ß-estradiol (E2) influence in the monocyte/macrophage response induced by S. aureus in in vitro models of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HPBM). MPMs (2 x 105/ml) were isolated from sham (n=3) and ovariectomized (OVX) females (n = 3) and males (n = 3) after induction by thioglycolate. The MPMs obtained from OVX females and males were treated for 24 hours with 17ß-estradiol (E2) (10-7 M), and after that, inoculation with S. aureus was carried out for 6 hours. The macrophages were collected and destined to evaluate the relative gene expression of TNF-α, IL-1ß, IL-6, IL-8 and TLR2. For the in vitro model of HPBMs, six men and six women of childbearing age were selected and HPBMs were isolated from samples of the volunteers' peripheral blood. In women, blood was collected both during menstruation and in the periovulatory period. HPBMs were inoculated with S. aureus for 6 hours and the supernatant was collected for analysis of cytokines by Luminex and the HPBMs were removed for analysis of 84 genes involved in the host's response to bacterial infections by RT-PCR array. Previous treatment with E2 decreased the gene expression and production of proinflammatory cytokines, such as TNF-α, IL-1ß and IL-6 and decreased the expression of TLR2 tanto em MPMs quanto em HPBMs. The analysis of gene expression shows that E2 inhibited the NFκB pathway. It is suggested that 17ß-estradiol acts as an immunoprotective in the monocyte/macrophage response induced by S. aureus.

Pigment production by Fusarium solani BRM054066 and determination of antioxidant and anti-inflammatory properties.

The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1ß, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

Ovarian hormones influence immune response to Staphylococcus aureus infection.

OBJECTIVE: Staphylococcus aureus infections remain associated with considerable morbidity and mortality in both hospitals and the community. There is little information regarding the role of ovarian hormones in infections caused by S. aureus. The aim of this study was to evaluate the effects of ovariectomy in the immune response induced by S. aureus. METHODS: Female mice BALB/c were ovariectomized (OVX) to significantly reduce the level of ovarian hormones. We also used sham-operated animals. The mice were inoculated intraperitoneally with S. aureus. Blood samples were collected for leukocyte count and bacterial quantification. The uterus and spleen were removed and weighed to calculate the uterine and splenic indexes. Lungs were removed and fractionated for immunohistochemical analysis for macrophage detection (anti-CD68) and relative gene expression of IL-6, IL-1ß and TNF-α by RT-PCR. RESULTS: Ovariectomy enlarged spleen size and generally increased circulating lymphocytes. OVX females experienced a continuation of the initial reduction of lymphocytes and a monocyte and neutrophil late response compared to shams (p≥0.05). Moreover, OVX females showed neutropenia after 168h of infection (p≥0.05). Macrophage response in the lungs were less pronounced in OVX females in the initial hours of infection (p≥0.01). OVX females showed a higher relative gene expression of IL-1ß, IL-6 and TNF-α in the lung at the beginning of the infection compared to sham females (p≥0.01). Among the uninfected females, the OVX control females showed a higher expression of IL-6 in the lung compared to the sham control females (p≥0.05). In this model, the lack of ovarian hormones caused a minor increase in circulating leukocytes during the initial stage of infection by S. aureus and increased pulmonary gene expression of IL-1ß, IL-6, and TNF-α. Ovariectomy alone enlarged the spleen and increased circulating lymphocytes. Ovarian hormones acted as immunoprotectors against S. aureus infection.

Ureaplasma diversum and Its Membrane-Associated Lipoproteins Activate Inflammatory Genes Through the NF-κB Pathway via Toll-Like Receptor 4.

Objectives:Ureaplasma diversum is a pathogen of cows that may cause intense inflammatory responses in the reproductive tract and interfere with bovine reproduction. The aims of this study were to evaluate the immune response of bovine blastocysts and macrophages to U. diversum infection and to evaluate the invasion capacity of this microorganism in bovine blastocysts. Methods: Viable and heat-inactivated U. diversum strains ATCC 49782 and CI-GOTA and their extracted membrane lipoproteins were inoculated in macrophages in the presence or absence of signaling blockers of Toll-Like Receptor (TLR) 4, TLR2/4, and Nuclear Factor KB (NF-κB). In addition, the same viable U. diversum strains were used to infect bovine blastocysts. RNA was extracted from infected and lipoprotein-exposed macrophages and infected blastocysts and assayed by qPCR to evaluate the expression of Interleukin 1 beta (IL-1ß), Tumor Necrosis Factor Alpha (TNF-α), TLR2 and TLR4 genes. U. diversum internalization in blastocysts was followed by confocal microscopy. Results: Both Ureaplasma strains and different concentrations of extracted lipoproteins induced a higher gene expression of IL-1ß, TNF-α, TLR2, and TLR4 in macrophages (p < 0.05) when compared to non-infected cells. The used blockers inhibited the expression of IL-1ß and TNF-α in all treatments. Moreover, U. diversum was able to internalize within blastocysts and induce a higher gene expression of IL-1b and TNF- α when compared to non-infected blastocysts (p < 0.05). Conclusion: The obtained results strongly suggest that U. diversum and its lipoproteins interact with TLR4 in a signaling pathway acting via NF-kB signaling to stimulate the inflammatory response. This is the first study to evaluate the in vitro immunological response of macrophages and bovine blastocysts against U. diversum. These results may contribute to a better understanding of the immunomodulatory activity and pathogenicity of this infectious agent.

Ovarian hormones influence immune response to Staphylococcus aureus infection

ABSTRACT Objective: Staphylococcus aureus infections remain associated with considerable morbidity and mortality in both hospitals and the community. There is little information regarding the role of ovarian hormones in infections caused by S. aureus. The aim of this study was to evaluate the effects of ovariectomy in the immune response induced by S. aureus. Methods: Female mice BALB/c were ovariectomized (OVX) to significantly reduce the level of ovarian hormones. We also used sham-operated animals. The mice were inoculated intraperitoneally with S. aureus. Blood samples were collected for leukocyte count and bacterial quantification. The uterus and spleen were removed and weighed to calculate the uterine and splenic indexes. Lungs were removed and fractionated for immunohistochemical analysis for macrophage detection (anti-CD68) and relative gene expression of IL-6, IL-1β and TNF-α by RT-PCR. Results: Ovariectomy enlarged spleen size and generally increased circulating lymphocytes. OVX females experienced a continuation of the initial reduction of lymphocytes and a monocyte and neutrophil late response compared to shams (p ≥ 0.05). Moreover, OVX females showed neutropenia after 168 h of infection (p ≥ 0.05). Macrophage response in the lungs were less pronounced in OVX females in the initial hours of infection (p ≥ 0.01). OVX females showed a higher relative gene expression of IL-1β, IL-6 and TNF-α in the lung at the beginning of the infection compared to sham females (p ≥ 0.01). Among the uninfected females, the OVX control females showed a higher expression of IL-6 in the lung compared to the sham control females (p ≥ 0.05). In this model, the lack of ovarian hormones caused a minor increase in circulating leukocytes during the initial stage of infection by S. aureus and increased pulmonary gene expression of IL-1β, IL-6, and TNF-α. Ovariectomy alone enlarged the spleen and increased circulating lymphocytes. Ovarian hormones acted as immunoprotectors against S. aureus infection.