Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

Background: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. Methods: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. Results: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. Conclusions: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background: Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy. Methods: Mif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days. Results: During the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009). Conclusions: The up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.

Cellular Renewal and Improvement of Local Cell Effector Activity in Peritoneal Cavity in Response to Infectious Stimuli

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (Mempty set) subsets, namely small peritoneal Mempty set (SPM) and large peritoneal Mempty set (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for beta-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-gamma stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal Mempty set subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

Syzygium jambolanum treatment improves survival in lethal sepsis induced in mice

Background: The leaves and the fruits from Syzygium jambolanum DC.(Myrtaceae), a plant known in Brazil as sweet olive or 'jambolao', have been used by native people to treat infectious diseases, diabetes, and stomachache. Since the bactericidal activity of S. jambolanum has been confirmed in vitro, the aim of this work was to evaluate the effect of the prophylactic treatment with S. jambolanum on the in vivo polymicrobial infection induced by cecal ligation and puncture (CLP) in mice. Methods: C57BI/6 mice were treated by the subcutaneous route with a hydroalcoholic extract from fresh leaves of S. jambolanum (HCE). After 6 h, a bacterial infection was induced in the peritoneum using the lethal CLP model. The mice were killed 12 h after the CLP induction to evaluate the cellular influx and local and systemic inflammatory mediators' production. Some animals were maintained alive to evaluate the survival rate. Results: The prophylactic HCE treatment increased the mice survival, the neutrophil migration to infectious site, the spreading ability and the hydrogen peroxide release, but decreased the serum TNF and nitrite. Despite the increased migration and activation of peritoneal cells the HCE treatment did not decrease the number of CFU. The HCE treatment induced a significant decrease on the bone marrow cells number but did not alter the cell number of the spleen and lymph node. Conclusion: We conclude that the treatment with S. jambolanum has a potent prophylactic antiseptic effect that is not associated to a direct microbicidal effect but it is associated to a recruitment of activated neutrophils to the infectious site and to a diminished systemic inflammatory response.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Hemoplasma Infection in HIV-positive Patient, Brazil

Hemotrophic mycoplasmas infect a variety of mammals. Although infection in humans is rarely reported, an association with an immunocompromised state has been suggested. We report a case of a Mycoplasma haemofelis-like infection in an HIV-positive patient co-infected with Bartonella henselae.

High Prevalence of bla(CTX-M) Extended Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae Isolates from a Tertiary Care Hospital: First report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil

The aim of this study was to investigate the presence and prevalence of bla(TEM), bla(SHV), and bla(CTX-M) and bla(GES)-like genes, responsible for extended spectrum beta-lactamases (ESBLs) production in clinical isolates of Klebsiella pneumoniae collected from a Brazilian tertiary care hospital. Sixty-five ESBL producing K. pneumoniae isolates, collected between 2005 and 2007, were screened by polymerase chain reaction (PCR). Identification of bla genes was achieved by sequencing. Genotyping of ESBL producing K. pneumoniae was performed by the enterobacterial repetitive intergenic consensus-PCR with cluster analysis by the Dice coefficient. The presence of genes encoding ESBLs was confirmed in 59/65 (90.8%) isolates, comprising 20 bla(CTX-M-2), 14 bla(CTX-M-59), 12 bla(CTX-M-15), 9 bla(SHV-12), 1 bla(SHV-2), 1 bla(SHV-2a), 1 bla(SHV-5), and 1 bla(SHV-31) genes. The ESBL genes bla(SHV-12), bla(SHV-31), and bla(CTX-M-15), and the chromosome-encoded SHV-type beta-lactamase capable of hydrolyzing imipenem were detected in Brazil for the first time. The analysis of the enterobacterial repetitive intergenic consensus-PCR band patterns revealed a high rate of multiclonal bla(CTX-M) carrying K. pneumoniae isolates (70.8%), suggesting that dissemination of encoding plasmids is likely to be the major cause of the high prevalence of these genes among the K. pneumoniae isolates considered in this study.

Impaired Innate Immunity in Tlr4(-/-) Mice but Preserved CD8(+) T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8(+) T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-gamma secreting CD8(+) T cells specific for H-2K(b)-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2(-/-), Tlr4(-/-), Tlr9(-/-) or Myd88(-/-) mice generated both specific cytotoxic responses and IFN-gamma secreting CD8(+) T cells at levels comparable to WT mice, although the frequency of IFN-gamma(+)CD4(+) cells was diminished in infected Myd88(-/-) mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-gamma, TNF-alpha and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4(-/-) mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.

The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5610 6 trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+) CD8(+), CD69(+) CD4(+) and CD25(+) CD122(+) CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.

Aspergillus nidulans as a biological system to detect the genotoxic effects of mercury fumes on eukaryotes

Mercury (Hg) pollution is one of the most serious environmental problems. Due to public concern prompted by the symptoms displayed by people who consumed contaminated fish in Minamata, Japan in 1956, Hg pollution has since been kept under constant surveillance. However, despite considerable accumulation of knowledge on the noxious effects of ingested or inhaled Hg, especially for humans, there is virtually nothing known about the genotoxic effects of Hg. Because increased mitotic crossing over is assumed to be the first step leading to carcinogenesis, we used a sensitive short-term test (homozygotization index) to look for DNA alterations induced by Hg fumes. In one Aspergillus nidulans diploid strain (UT448//UT184), the effects of the Hg fumes appeared scattered all over the DNA, causing 3.05 times more recombination frequencies than the mean for other strains. Another diploid (Dp II- I//UT184) was little affected by Hg. This led us to hypothesize that a genetic factor present in the UT184 master strain genome, close to the nicB8 genetic marker, is responsible for this behavior. These findings corroborate our previous findings that the homozygotization index can be used as a bioassay for rapid and efficient assessment of ecotoxicological hazards.

Identification and Phylogenetic Analysis of Heme Synthesis Genes in Trypanosomatids and Their Bacterial Endosymbionts

It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellate's cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.

Hyperbaric Oxygen Prevents Early Death Caused by Experimental Cerebral Malaria

Background: Cerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis. Methodology/Principal Findings: C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O(2), 3.0 ATA, 1-2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-alpha, IFN-gamma and IL-10 mRNA levels and percentage of gamma delta and alpha beta CD4(+) and CD8(+) T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB)dysfunction and hypothermia. Conclusions/Significance: The data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.

Effect of oat bran on time to exhaustion, glycogen content and serum cytokine profile following exhaustive exercise

The aim of this study was to evaluate the effect of oat bran supplementation on time to exhaustion, glycogen stores and cytokines in rats submitted to training. The animals were divided into 3 groups: sedentary control group (C), an exercise group that received a control chow (EX) and an exercise group that received a chow supplemented with oat bran (EX-O). Exercised groups were submitted to an eight weeks swimming training protocol. In the last training session, the animals performed exercise to exhaustion, (e.g. incapable to continue the exercise). After the euthanasia of the animals, blood, muscle and hepatic tissue were collected. Plasma cytokines and corticosterone were evaluated. Glycogen concentrations was measured in the soleus and gastrocnemius muscles, and liver. Glycogen synthetase-alpha gene expression was evaluated in the soleus muscle. Statistical analysis was performed using a factorial ANOVA. Time to exhaustion of the EX-O group was 20% higher (515 +/- 3 minutes) when compared with EX group (425 +/- 3 minutes) (p = 0.034). For hepatic glycogen, the EX-O group had a 67% higher concentrations when compared with EX (p = 0.022). In the soleus muscle, EX-O group presented a 59.4% higher glycogen concentrations when compared with EX group (p = 0.021). TNF-alpha was decreased, IL-6, IL-10 and corticosterone increased after exercise, and EX-O presented lower levels of IL-6, IL-10 and corticosterone levels in comparison with EX group. It was concluded that the chow rich in oat bran increase muscle and hepatic glycogen concentrations. The higher glycogen storage may improve endurance performance during training and competitions, and a lower post-exercise inflammatory response can accelerate recovery.