Oropouche virus experimental infection in the golden hamster (Mesocrisetus auratus)

Oropouche virus (OROV), of the family Bunyaviridae, is the second most frequent arbovirus causing febrile disease in Brazil. In spite of this, little is known about pathogenesis of OROV infection. This report describes an experimental model of OROV in golden hamster (Mesocricetus auratus). Following subcutaneous inoculation of OROV, over 50% of the animals developed disease characterized by lethargy, ruffled fur, shivering, paralysis, and approximately one third died. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation to collect tissue samples from brain, heart, liver, lung, spleen, muscle and blood for virus titration, histology and OROV immunohistochemistry. OROV was detected in high titers in blood, liver and brain, but not in the other organs. Histopathology revealed meningoencephalitis and hepatitis, with abundant OROV antigen detected in liver and brain. Diffuse galectin-3 immunostaining in brain and liver supports microglial and Kupfer cells activation. This is the first description of an experimental model for OROV infection and should be helpful to study pathogenesis and possibly to test antiviral interventions such as drugs and vaccine candidates. (C) 2010 Elsevier B.V. All rights reserved.

Assessment of the Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta in a Rat Model of Intrauterine Growth Restriction

Objective: To investigate glomerular development and expression of insulin and insulin-like growth factor receptors in an experimental model of intrauterine growth restriction (IUGR). Material and Methods: We studied three groups of Sprague-Dawley fetuses: IUGR - restricted by ligation of the right uterine artery; C-IUGR - left horn controls, and EC - external controls (non-manipulated). Body and organs were weighed, and glomerular number and volume were analyzed. Expression of IR beta, IRS-1, IRS-2 and IGF-IR beta was analyzed in liver, intestine and kidneys by immunoblotting. Results: Organ/body weight ratios were similar. In IUGR, glomerular number and volume were increased compared to C-IUGR and EC (p < 0.001). In the IUGR liver, increases were found in IGF-IR beta compared to C-IUGR and EC; IR beta compared to EC, and IRS-2 compared to C-IUGR. However, decreases in IR beta were noted in IUGR compared to C-IUGR; IRS-1 compared to C-IUGR and EC, and IRS-2 compared to EC. In IUGR intestine, increases were detected in IR beta, IRS-1 and IGF-IR beta compared to C-IUGR and EC. In IUGR kidneys, increases were observed in IR beta and IGF-IR beta compared to C-IUGR and EC, and IRS-1 compared to EC. Decreased IRS-2 in the intestine and kidney were noticed in IUGR compared to C-IUGR and EC. Conclusion: IUGR fetuses had less glomeruli and alterations in insulin receptors, which may be associated with an increased risk of disease occurrence in adulthood. Copyright (C) 2010 S. Karger AG, Basel

CXCL12 rs1801157 polymorphism and expression in peripheral blood from breast cancer patients

The role of chemokines has been extensively analyzed both in cancer risk and tumor progression. Among different cytokines, CXCR4 and its ligand CXCL12 have been recently subjected to a closer examination. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/SDF1-3`A) in the CXCL12 gene and the relative expression of mRNA CXCL12 in peripheral blood were assessed in breast cancer patients, since the chemokine CXCL12 and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using MspI restriction enzyme and the expression analyses by quantitative RT-PCR. No difference in GG genotype and allele A carrier frequencies were observed between breast cancer patients and healthy blood donors and nor when CXCL12 mRNA expression was assessed among patients with different tumor stages. However a significant difference was observed when CXCL12 mRNA relative expression was analyzed in breast cancer patients in accordance to the presence or absence of the CXCL12 rs1801157 allele A. Allele A breast cancer patients presented a mRNA CXCL12 expression about 2.1-fold smaller than GG breast cancer patients. Estrogen positive patients presenting CXCL12 allele A presented a significantly lower expression of CXCL12 in peripheral blood (p = 0.039) than GG hormone positive patients. Our findings demonstrated that allele A is associated with low expression of CXCL12 in the peripheral blood from ER-positive breast cancer patients, which suggests implications on breast cancer clinical outcome. (C) 2011 Elsevier Ltd. All rights reserved.

Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15: 17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer ""stem"" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), Fc gamma RIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) possibly through a methylation-dependent mechanism, indicating that C/EBP alpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease. (Blood. 2009; 114: 5415-5425)

CXCL12 rs1801157 Polymorphism in Patients with Breast Cancer, Hodgkin`s Lymphoma, and Non-Hodgkin`s Lymphoma

Chemokines and their receptors regulate the trafficking of immune cells during their development, inflammation, and tissue repair. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/ stromal cell-derived factor-1 (SDF1)-3`A) in CXCL12/SDF1 gene was assessed in breast cancer, Hodgkin`s lymphoma (HL), and non-Hodgkin`s lymphoma (NHL), since the chemokine CXCL12, previously known as SDF1, and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis, and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using a restriction enzyme Hpall cleavage. No significant difference was observed in genotype distribution between breast cancer patients (GG: 57.3%; GA: 39.8%; AA: 2.9%) and healthy female controls (GG: 62.9%; GA: 33%; AA: 4.1%) nor between HL patients (GG: 61.1%; GA:27.8%; AA: 11.1%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%), whereas a significant difference was observed in genotype distribution between NHL patients (GG: 51.4%; GA: 47.1%; AA: 1.5%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%). Further studies will be necessary to elucidate the cancer chemokine network. However, this study suggests that CXCL12 rs1801157 polymorphism may have important implications in the pathogenesis of NHL. J. Clin. Lab. Anal. 23:387-393, 2009. (C) 2009 Wiley-Liss, Inc.

Essential Role of CCR2 in Neutrophil Tissue Infiltration and Multiple Organ Dysfunction in Sepsis

Rationale Sepsis is defined as a systemic inflammatory response to infection, which in its severe form is associated with multiple organ dysfunction syndrome (MODS). The precise mechanisms by Which MODS develops remain unclear. Neutrophils have a pivotal role in the defense against infections; however, overwhelming activation of neutrophils is known to elicit tissue damage. Objectives: We investigated the role of the chemokine receptor CCR2 in driving neutrophil infiltration and eliciting tissue damage in remote organs during sepsis. Methods: Sepsis was induced in wild-type mice treated with CCR2 antagonist (RS504393) or CCR2(-/-) mice by cecal ligation and puncture (CLP) model. Neutrophil infiltration into the organs was measured by myeloperoxidase activity and fluorescence-activated cell sorter. CCR2 expression and chemotaxis were determined in neutrophils stimulated with Toll-like receptor agonists or isolated from septic mice and patients. Measurements and Main Results: CCR2 expression and responsiveness to its ligands was induced in circulating neutrophils during CLP-induced sepsis by a mechanism dependent on Toll-like receptor/nuclear factor-kappa B pathway. Genetic or pharmacologic inhibition of CCR2 protected mice from CLP-induced mortality. This protection was associated with lower infiltration of neutrophils into the lungs, heart, and kidneys and reduced serum biochemical indicators of organ injury and dysfunction. Importantly, neutrophils from septic patients express high levels of CCR2, and the severity of patient illness correlated positively with increasing neutrophil chemotaxis to CCR2 ligands. Conclusions: Collectively, these data identify CCR2 as a key receptor that drives the inappropriate infiltration of neutrophils into remote organs during sepsis. Therefore, CCR2 blockade is a novel potential therapeutic target for treatment of sepsis-induced MODS.

Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR

Aims We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. Methods and results Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-beta-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. Conclusion We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

Treatment with cannabidiol reverses oxidative stress parameters, cognitive impairment and mortality in rats submitted to sepsis by cecal ligation and puncture

Oxidative stress plays an important role in the development of cognitive impairment in sepsis. Here we assess the effects of acute and extended administration of cannabidiol (CBD) on oxidative stress parameters in peripheral organs and in the brain, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and perforation (CLP). To this aim, male Wistar rats underwent either sham operation or CLP. Rats subjected to CLP were treated by intraperitoneal injection with ""basic support"" and CBD (at 2.5, 5, or 10 mg/kg once or daily for 9 days after CLP) or vehicle. Six hours after CLP (early times), the rats were killed and samples from lung, liver, kidney, heart, spleen, and brain (hippocampus, striatum, and cortex) were obtained and assayed for thiobarbituric acid reactive species (TBARS) formation and protein carbonyls. On the 10th day (late times), the rats were submitted to the inhibitory avoidance task. After the test, the animals were killed and samples from lung, liver, kidney, heart, spleen, and brain (hippocampus) were obtained and assayed for TBARS formation and protein carbonyls. The acute and extended administration of CBD at different doses reduced TBARS and carbonyl levels in some organs and had no effects in others, ameliorated cognitive impairment, and significantly reduced mortality in rats submitted to CLP. Our data provide the first experimental demonstration that CBD reduces the consequences of sepsis induced by CLP in rats, by decreasing oxidative stress in peripheral organs and in the brain, improving impaired cognitive function, and decreasing mortality. (C) 2010 Elsevier B.V. All rights reserved.

Vitamin E and tannic acid improve DNA damage in rats submitted chronic ethanol administration

Purpose - Chronic ethanol consumption induces lipid peroxidation by increasing free radicals or reducing antioxidants and may increase damage to hepatic DNA. Tannins are polyphenolic metabolites present in various plants and one of their effects is antioxidant activity that reduces lipoperoxidation, as is the case for vitamin E. This paper aims to assess the role of tannic acid and vitamin E in lipid peroxidation and in DNA damage in rats receiving ethanol. Design/methodology/approach - A total of 60 Wistar rats were divided into six groups: control + ethanol (0-24hs), tannic acid + ethanol (0-24 hs), and vitamin E + ethanol (0-24 hs). The animals were sacrificed immediately (0 hour) or 24 hours after a period of four weeks of ethanol administration and the following measurements were made: plasma vitamin E and liver glutathione, thiobarbituric acid resistant substances, and a-tocopherol. The comet test was also applied to hepatocytes. Findings - Ethanol administration led to an increase in DNA damage (148.67 +/- 15.45 versus 172.63 +/- 18.94) during a period of 24 hours which was not detected in the groups receiving tannic acid or vitamin E. Steatosis was lower in the groups receiving tannic acid. Originality/value - The paper highlights that antioxidant role of vitamin E and of tannic acid in biological systems submitted to oxidative stress should be reevaluated, especially regarding the protective role of tannic acid against hepatic steatosis.

Postembryonic Development of Rectal Pads in Bees (Hymenoptera, Apidae)

The morphology and development of the digestive tract of insects has been extensively studied, but little attention has been given to the development of the rectal pads. These organs are responsible for absorption of water and salts. In insects where they occur, there are usually six ovoid rectal pads located in the medial-anterior portion of the rectum. The rectal pad has three types of cells: principal, basal, and junctional. The arrangement of these three cell types delimits an intrapapillary lumen. The aim of the current study is to describe the development of the rectal pads during postembryonic development of Melipona quadrifasciata anthidioides and Melipona scutellaris. Specimens were analyzed at the following developmental stages: white-, pink-, brown-, and black-eyed pupae, and adult workers. The development of the rectal pad begins as a thickening of the epithelium in white-eyed pupae at 54 hr. At this stage, there is neither a basal cell layer nor intrapapillary lumen. The basal layers begin to form in the pink-eyed pupa and are completely formed at the end of the development of the brown-eyed pupa. In the brown-eyed pupal stage, the intrapapillary lumen is formed and the junctional cells are positioned and completely differentiated. Necrotic and apoptotic cell death were detected along with cell proliferation in the whole rectum during pupal development, suggesting that the development of the rectal pads involves cell proliferation, death, and differentiation. The rectal pads originate only from the ectoderm. Anat Rec, 292:1602-1611, 2009. (C) 2009 Wiley-Liss, Inc.

NO EVIDENCE OF PATHOLOGICAL AUTOIMMUNITY FOLLOWING MYCOBACTERIUM LEPRAE HEAT-SHOCK PROTEIN 65-DNA VACCINATION IN MICE

Heat-shock proteins (HSPs) are currently one of the most promising targets for the development of immunotherapy against tumours and autoimmune disorders. This protein family has the capacity to activate or modulate the function of different immune system cells. They induce the activation of monocytes, macrophages and dendritic cells, and contribute to cross-priming, an important mechanism of presentation of exogenous antigen in the context of MHC class I molecules, These various immunological properties of HSP have encouraged their use in several clinical trials. Nevertheless, an important issue regarding these proteins is whether the high homology among HSPs across different species may trigger the breakdown of immune tolerance and induce autoimmune diseases. We have developed a DNA vaccine codifying the Mycobacterium leprae Hsp65 (DNAhsp65), which showed to be highly immunogenic and protective against experimental tuberculosis. Here, we address the question of whether DNAhsp65 immunization could induce pathological autoimmunity in mice. Our results show that DNAhsp65 vaccination induced antibodies that can recognize the human Hsp60 but did not induce harmful effects in 16 different organs analysed by histopathology up to 210 days after vaccination. We also showed that anti-DNA antibodies were not elicited after DNA vaccination. The results are important for the development of both HSP and DNA-based immunomodulatory agents.

Chemokines expression during Leptospira interrogans serovar Copenhageni infection in resistant BALB/c and susceptible C3H/HeJ mice

The role of innate immune responses in protection against leptospirosis remains unclear. We examined the expression of the chemokines CCL2/JE (MCP-1), CCL3/MIP-1 alpha (MIP-1 alpha) and CXCL1/KC (IL-8) regarding resistance and susceptibility to leptospirosis in experimental mice models BALB/c and C3H/HeJ, respectively. A virulent strain of Leptospira interrogans serovar Copenhageni was used in this study. Twenty-five animals of each mouse strain of C3H/HeJ and BALB/c, were infected intraperitoneally with 106 cells. Five un-infected animals of each strain were kept as control. Mortality of C3H/HeJ mouse was observed while BALB/c mice were asymptomatic. The presence of leptospire DNA in tissues of infected animals was demonstrated by PCR. Chemokines were measured in serum, spleen, liver, kidney and lung of both strains of animals using immunoenzymatic assay (ELISA). Elevations in the levels of chemokines MCP-1 and IL-8 occurred in all organs and sera of C3H/HeJ and BALB/c infected mice. The levels of MIP-1 alpha were lower when compared to MCP-1 and IL-8 in all analyzed organs, with a slight increase in liver and kidney. Our results indicate that the expression of inflammatory mediators can vary greatly, depending on the tissue and mouse strains. It is possible that the resistance to Leptospira can be partially correlated to the increase of MIP-1 alpha observed in BALB/c mice, while an increasing and a sustained expression of MCP-1 and IL-8 in the lungs of C3H/HeJ mice can be correlated to the severity and progression of leptospirosis. (C) 2009 Elsevier Ltd. All rights reserved.