Salmonella enterica serovar typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes

The pattern of global gene expression in Salmonella enterica serovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell division in vivo were lower than those in vitro. S. Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed to D-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial and Salmonella pathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization. S. Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source. © 2011, American Society for Microbiology.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Chronic Ethanol Consumption Alters All-Trans-Retinoic Acid Concentration and Expression of Their Receptors on the Prostate: A Possible Link Between Alcoholism and Prostate Damage

Background: Ethanol (EtOH) alters the all-trans-retinoic acid (ATRA) levels in some tissues. Retinol and ATRA are essential for cell proliferation, differentiation, and maintenance of prostate homeostasis. It has been suggested that disturbances in retinol/ATRA concentration as well as in the expression of retinoic acid receptors (RARs) contribute to benign prostate hyperplasia and prostate cancer. This study aimed to evaluate whether EtOH consumption is able to alter retinol and ATRA levels in the plasma and prostate tissue as well as the expression of RARs, cell proliferation, and apoptosis index. Methods: All animals were divided into 4 groups (n = 10/group). UChA: rats fed 10% (v/v) EtOH ad libitum; UChACo: EtOH-naïve rats without access to EtOH; UChB: rats fed 10% (v/v) EtOH ad libitum; UChBCo: EtOH-naïve rats without access to EtOH. Animals were euthanized by decapitation after 60 days of EtOH consumption for high-performance liquid chromatography and light microscopy analysis. Results: EtOH reduced plasma retinol concentration in both UChA and UChB groups, while the retinol concentration was not significantly different in prostate tissue. Conversely, plasma and prostate ATRA levels increased in UChB group compared with controls, beyond the up-regulation of RARβ and -γ in dorsal prostate lobe. Additionally, no alteration was found in cell proliferation and apoptosis index involving dorsal and lateral prostate lobe. Conclusions: We conclude that EtOH alters the plasma retinol concentrations proportionally to the amount of EtOH consumed. Moreover, high EtOH consumption increases the concentration of ATRA in plasma/prostate tissue and especially induces the RARβ and RARγ in the dorsal prostate lobe. EtOH consumption and increased ATRA levels were not associated with cell proliferation and apoptosis in the prostate. © 2012 by the Research Society on Alcoholism.

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R- B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30. dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1β and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1β and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R- B. ovis infection. During the chronic phase of R- B. ovis infection (120 and 240. dpi), cytokine production was down-regulated in the epididymus (IL-1β and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1β and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R- B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation. © 2012 Elsevier B.V.

Infliximab prevents increased systolic blood pressure and upregulates the AKT/eNOS pathway in the aorta of spontaneously hypertensive rats

High systolic blood pressure caused by endothelial dysfunction is a comorbidity of metabolic syndrome that is mediated by local inflammatory signals. Insulin-induced vasorelaxation due to endothelial nitric oxide synthase (eNOS) activation is highly dependent on the activation of the upstream insulin-stimulated serine/threonine kinase (AKT) and is severely impaired in obese, hypertensive rodents and humans. Neutralisation of circulating tumor necrosis factor-α (TNFα) with infliximab improves glucose homeostasis, but the consequences of this pharmacological strategy on systolic blood pressure and eNOS activation are unknown. To address this issue, we assessed the temporal changes in the systolic pressure of spontaneously hypertensive rats (SHR) treated with infliximab. We also assessed the activation of critical proteins that mediate insulin activity and TNFα-mediated insulin resistance in the aorta and cardiac left ventricle. Our data demonstrate that infliximab prevents the upregulation of both systolic pressure and left ventricle hypertrophy in SHR. These effects paralleled an increase in AKT/eNOS phosphorylation and a reduction in the phosphorylation of inhibitor of nuclear factor-κB (Iκβ) and c-Jun N-terminal kinase (JNK) in the aorta. Overall, our study revealed the cardiovascular benefits of infliximab in SHR. In addition, the present findings further suggested that the reduction of systolic pressure and left ventricle hypertrophy by infliximab are secondary effects to the reduction of endothelial inflammation and the recovery of AKT/eNOS pathway activation. © 2012 Elsevier B.V.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.

Sialidase activity in aerobic vaginitis is equal to levels during bacterial vaginosis

Objective: To evaluate levels of proinflammatory cytokines and sialidase activity in aerobic vaginitis (AV) in relation to normal vaginal flora and bacterial vaginosis (BV). Study design: In this cross-sectional study, a total of 682 consecutive non-pregnant women attending the gynecology service were assessed and 408 women were included. Vaginal rinsing samples were collected from 223 women with microscopic finding of BV (n = 98), aerobic vaginitis (n = 25) and normal flora (n = 100). Samples were tested for interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, and sialidase activity. Results: Compared to women with normal flora, vaginal levels of IL-1β were highly increased in both BV and AV (p < 0.0001). Significantly higher vaginal IL-6 was detected in AV (p < 0.0001) but not in BV, in relation to normal flora. Women with AV also presented increased IL-8 levels (p < 0.001), while those with BV presented levels similar to normal flora. Sialidase was increased in BV and AV compared with the normal group (p < 0.0001) but no difference in sialidase activity was observed between BV and AV. Conclusion: A more intense inflammatory host response occurs for AV than for BV when compared with normal flora. Furthermore, the increased sialidase activity in AV and BV indicates that both abnormal vaginal flora types can be harmful to the maintenance of a healthy vaginal environment. © 2012 Elsevier B.V.

Osteogenesis-inducing calcium phosphate nanoparticle precursors applied to titanium surfaces

This study investigated the effects of the morphology and physicochemical properties of calcium phosphate (CaP) nanoparticles on osteogenesis. Two types of CaP nanoparticles were compared, namely amorphous calcium phosphate (ACP) nano-spheres (diameter: 9-13 nm) and poorly crystalline apatite (PCA) nano-needles (30-50 nm x 2-4 nm) that closely resemble bone apatite. CaP particles were spin-coated onto titanium discs and implants; they were evaluated in cultured mouse calvarial osteoblasts, as well as after implantation in rabbit femurs. A significant dependence of CaP coatings was observed in osteoblast-related gene expression (Runx2, Col1a1 and Spp1). Specifically, the PCA group presented an up-regulation of the osteospecific genes, while the ACP group suppressed the Runx2 and Col1a1 expression when compared to blank titanium substrates. Both the ACP and PCA groups presented a more than three-fold increase of calcium deposition, as suggested by Alizarin red staining. The removal torque results implied a slight tendency in favour of the PCA group. Different forms of CaP nanostructures presented different biologic differences; the obtained information can be used to optimize surface coatings on biomaterials. © 2013 IOP Publishing Ltd.

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.

The renin-angiotensin system plays a major role in voiding dysfunction of ovariectomized rats

Aims: The renin-angiotensin system (RAS) plays a major role in cardiovascular diseases in postmenopausal women, but little is known about its importance to lower urinary tract symptoms. In this study we have used the model of ovariectomized (OVX) estrogen-deficient rats to investigate the role of RAS in functional and molecular alterations in the urethra and bladder. Main methods: Responses to contractile and relaxant agents in isolated urethra and bladder, as well as cystometry were evaluated in 4-month OVX Sprague-Dawley rats. Angiotensin-converting enzyme activity and Western blotting for AT1/AT2 receptors were examined. Key findings: Cystometric evaluations in OVX rats showed increases in basal pressure, capacity and micturition frequency, as well as decreased voiding pressure. Angiotensin II and phenylephrine produced greater urethral contractions in OVX compared with Sham group. Carbachol-induced bladder contractions were significantly reduced in OVX group. Relaxations of urethra and bladder to sodium nitroprusside and BAY 41-2272 were unaffected by OVX. Angiotensin-converting enzyme activity was 2.6-fold greater (p < 0.05) in urethral tissue of OVX group, whereas enzyme activity in plasma and bladder remained unchanged. Expressions of AT1 and AT2 receptors in the urethra were markedly higher in OVX group. In bladder, AT1 receptors were not detected, whereas AT2 receptor expression was unchanged between groups. 17β-Estradiol replacement (0.1 mg/kg, weekly) or losartan (30 mg/kg/day) largely attenuated most of the alterations seen in OVX group. Significance: Prolonged estrogen deprivation leads to voiding dysfunction and urethral hypercontractility that are associated with increased ACE activity and up-regulation of angiotensin AT1/AT2 receptor in the urethral tissue. © 2013 Elsevier Inc. All rights reserved.

Avaliação da expressão gênica em leucócitos de eqüinos: análise pela técnica do microarray em modelo ex vivo de endotoxemia

Pós-graduação em Ciência Animal - FMVA

Expressão gênica de citocinas no encéfalo de cães com leishmaniose visceral

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterações neuroquímicas no tecido retiniano murino em modelo de malária cerebral induzida pela infecção por Plasmodium berghei (ANKA)

A Malária Cerebral (MC) apresenta-se como uma severa complicação resultante da infecção por Plasmodium falciparum. Esta condição encontra-se comumente associada a disfunções cognitivas, comportamentais e motoras, sendo a retinopatia uma das mais graves conseqüências da doença. Diversos modelos experimentais já foram descritos no intuito de elucidar os mecanismos fisiopatológicos relacionados a esta síndrome, no entanto, estes ainda permanecem pouco compreendidos. Dentro deste contexto, o presente trabalho procurou investigar as alterações neuroquímicas envolvidas na patologia da MC. Os camundongos C57Bl/6 (fêmeas e machos) inoculados com ≈106 eritrócitos parasitados (PbA) apresentaram baixa parasitemia (15-20%) com sinais clínicos evidentes como: deficiência respiratória, ataxia, hemiplegia e coma seguido de morte, condizentes com o quadro de MC. A análise no tecido retiniano demonstrou uma diminuição nos níveis de GSH com 2 dias após a inoculação. Entretanto, essa diminuição não foi tão evidente com o decorrer da infecção (4º e 6º dias após infecção). Concomitante a este aumento durante o processo infeccioso, observamos um progressivo aumento na captação de 3H-glutamato (4º e 6º dia após infecção) por um sistema independente de Na+, sugerindo que o quadro de MC é responsável por um aumento na atividade de uma proteína transportadora. Dados obtidos com a imunofluorescência demonstram que além de aumentar a atividade do sistema de transporte, o quadro de MC também estimula o aumento na expressão do sistema xCG - no tecido retiniano. O presente trabalho demonstra ainda que estes eventos neuroquímicos no tecido retiniano são independentes de ativação inflamatória, visto que os níveis de TNF-α e expressão de NOS-2, apresentam-se alterados somente no tecido retiniano.

Expressão gênica e viabilidade de folículos pré-antrais inclusos em fragmentos de córtex ovarianos cultivados in vitro de macaco-prego (Sapajus apella)

O objetivo desse trabalho foi analisar a ativação e o crescimento de folículos préantrais de Sapajus apella, submetidos a um sistema de cultivo in vitro em curto-prazo. Nesse sentido, dois experimentos foram realizados neste trabalho. Experimento I: exposição à fresco e à crioprotetores de biopsias de fragmentos do córtex ovariano. Ambos os fragmentos ovarianos foram submetidos à extração total de RNA e síntese de cDNA. Após a amplificação do cDNA por PCR em tempo-real (RT-PCR), os software GeNorm, bestkeeper e Normfinder foram usados para avaliar a estabilidade dos genes GAPDH (glyceraldehyde-2-phosphate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyltransferase 1) e TBP (TATA-binding protein). Experimento II: o tecido do córtex ovariano de quatro fêmeas foi oletado e dividido em nove pedaços de 1 mm³. Um fragmento ovariano (grupo controle) foi imediatamente dividido em dois pedaços, os quais foram destinados para análise da viabilidade ou por RT-PCR. Os 8 fragmentos restantes foram individualmente cultivados in vitro em um meio constituído de TCM suplementado com 100 ng/mL EGF (T1), com adição de 10 μM de BME (T2), 100 ng/mL de BMP4 (T3), 25 IU de PMSG (T4), 10 μM de BME e 100 ng/mL de BMP4 (T5), 10 μM de BME, 25 IU de PMSG (T6), 100 ng/mL de BMP4, 25 IU de PMSG (T7) ou 10 μM de BME, 100 ng/mL de BMP4, 25 IU de PMSG (T8). Os resultados demonstraram que no tecido do córtex ovariano de S. apella, os genes HPRT1 e TBP foram os mais apropriados como genes de referência, podendo ser usados como parâmetro para normalizar dados em estudos futuros. Ao contrário, o GAPDH se apresentou como menos estável dos genes de referencia testados. Após o cultivo in vitro, todos os tratamentos alcançaram percentuais similares da viabilidade de folículos pré-antrais. O tecido ovariano cultivado na presença de GF+BME/BMP4/PMSG resultou no aumento da taxa de ativação e crescimento folicular, assim como no aumento da expressão de AMH, BMP15 e GDF9, genes conhecidos como indicadores específicos de desenvolvimento folicular. Dessa forma, o HPRT1 e TBP são os genes de referência mais estáveis, na exposição à crioprotetores, a fresco e no o cultivo de tecidos de córtex ovariano de S. apella. Os folículos pré-antrais são capazes de desenvolverem-se in vitro quando cultivados em meio suplementado com PMSG, BME e BMP4. A viabilidade folicular, entretanto, permaneceu independentemente do meio de cultivo in vitro e o uso de fatores de crescimento, como marcadores de desenvolvimento folicular, foi crucial para identificar o melhor meio de cultivo in vitro.

Células binucleadas trofoblásticas: efeito dos seus produtos de secreção na produção de Prostaglandina F2'alfa' e avaliação da expressão gênica relativa de lactogênio placentário e fator de crescimento endotélio vascular em placentas bovinas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)