Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi

"Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patterns and in the parasitic peptides recognition patterns of experimentally infected Wistar rats. The rats experimentally infected with T. evansi isolates obtained from coatis, dogs and horses were submitted to indirect immunofluorescence test (IgM e IgG) and Western blotting. The serological titers for IgM and IgG ranged between 1:40 and 1:160. The most recognized polypeptide profiles were in a range of 17 and 74 kDa. Our data suggest that the humoral immune response in Wistar rats is not sufficient for granting an effective control of T. evansi infections.

Molecular pathways involved in the antineoplastic effects of calcitriol on insulinoma cells.

We have previously reported that in tumorigenic pancreatic beta-cells, calcitriol exerts a potent antitumorigenic effect by inducing apoptosis, cell growth inhibition, and reduction of solid beta-cell tumors. Here we have studied the molecular pathways involved in the antineoplastic activity of calcitriol on mouse insulinoma beta TC(3) cells, mouse insulinoma beta TC expressing or not expressing the oncogene p53, and beta TC-tet cells overexpressing or not the antiapoptotic gene Bcl2. Our results indicate that calcitriol-induced apoptosis was dependent on the function of p53 and was associated with a biphasic increase in protein levels of transcription factor nuclear factor-kappa B. Calcitriol decreased cell viability by about 40% in p53-retaining beta TC and in beta TC(3) cells; in contrast, beta TC p53(-/-) cells were only minimally affected. Calcitriol-induced cell death was regulated by members of the Bcl-2 family of apoptosis regulatory proteins, as shown by calcitriol-induced up-regulation of proapoptotic Bax and Bak and the lack of calcitriol-induced cytotoxicity in Bcl-2-overexpressing insulinoma cells. Moreover, calcitriol-mediated arrest of beta TC(3) cells in the G(1) phase of the cell cycle was associated with the abnormal expression of p21 and G(2)/M-specific cyclin B2 genes and involved the DNA damage-inducible factor GADD45. Finally, in beta TC(3) cells, calcitriol modulated the expression of IGF-I and IGF-II genes. In conclusion, these findings contribute to the understanding of the antitumorigenic effects of calcitriol on tumorigenic pancreatic beta-cells and further support the rationale of its utilization in the treatment of patients with malignant insulinomas.

Promoter and transcription analysis of penicillin-binding protein genes in Streptococcus gordonii.

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

Serum and CSF levels of neuron-specific enolase (NSE) in cardiac surgery with cardiopulmonary bypass: a marker of brain injury?

We investigated whether neuron-specific enolase (NSE) in serum or cerebrospinal fluid (CSF) reflects subtle or manifest brain injury in children undergoing cardiac surgery using cardiopulmonary bypass (CPB). NSE was measured in serum (s-NSE) before, and up to, 102 h after surgery in 27 children undergoing cardiac surgery with CPB. In 11 children, CSF-NSE was also measured 48 or 66 h post-surgery. As erythrocytes contain NSE, hemoglobin concentration in the samples was determined spectrophotometrically at 550 nm (cut-off limit: absorbance 0.4 = 560 mg/l) in 14 children and in a further 13 children by spectroscopic multicomponent analysis (cut-off limit 5 micromol/l = 80 mg/l). One hundred and one of 214 post-operative serum samples (47%) had to be discarded because of hemolysis (18% spectrophotometrically at 550 nm and 88% with spectroscopic multicomponent analysis). On the first and second post-operative day, the median s-NSE values were significantly higher when compared with samples taken after 54 h or longer (P = 0.008 and P = 0.002). All CSF-NSE levels were within the normal range and below the s-NSE measured in the same patient. Although in our study elevated s-NSE seems to indicate brain injury in CPB-surgery, the low concentration of NSE in the post-operative CSF of 11 children puts the neuronal origin of s-NSE in question. NSE from other non-neuronal tissues probably contributes to the elevated s-NSE. Additionally, normal post-operative CSF-NSE values in two children with post-operative neurological sequelae might question the predictive value of CSF-NSE with regard to brain injury.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Report of the Working Group to examine the development of appropriate systems to determine nursing and midwifery staffing levels

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Augmented epithelial multidrug resistance-associated protein 4 expression in peritoneal endometriosis: regulation by lipoxin A(4).

OBJECTIVE: To compare the expression of the prostaglandin (PG) E(2) transporter multidrug resistance-associated protein 4 (MRP4) in eutopic and ectopic endometrial tissue from endometriosis patients with that of control subjects and to examine whether MRP4 is regulated by the antiinflammatory lipid lipoxin A(4) (LXA(4)) in endometriotic epithelial cells. DESIGN: Molecular analysis in human samples and a cell line. SETTING: Two university hospitals and a private clinic. PATIENT(S): A total of 59 endometriosis patients and 32 age- and body mass index-matched control subjects undergoing laparoscopy or hysterectomy. INTERVENTION(S): Normal, eutopic, and ectopic endometrial biopsies as well as peritoneal fluid were obtained during surgery performed during the proliferative phase of the menstrual cycle. 12Z endometriotic epithelial cells were used for in vitro mechanistic studies. MAIN OUTCOME MEASURE(S): Tissue MRP4 mRNA levels were quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and localization was analyzed with the use of immunohistochemistry. Cellular MRP4 mRNA and protein were quantified by qRT-PCR and Western blot, respectively. PGE(2) was measured in peritoneal fluid and cell supernatants using an enzyme immunoassay (EIA). RESULT(S): MRP4 was expressed in eutopic and ectopic endometrium, where it was overexpressed in peritoneal lesions and localized in the cytoplasm of glandular epithelial cells. LXA(4) attenuated MRP4 mRNA and protein levels in endometriotic epithelial cells in a dose-dependent manner, while not affecting the expression of enzymes involved in PGE(2) metabolism. Investigations employing receptor antagonists and small interfering RNA revealed that this occurred through estrogen receptor α. Accordingly, LXA(4) treatment inhibited extracellular PGE(2) release. CONCLUSION(S): We report for the first time that MRP4 is expressed in human endometrium, elevated in peritoneal endometriosis, and modulated by LXA(4) in endometriotic epithelial cells.

Molecular mechanisms for the transcriptional regulation of the Connexin36 gene expression in insulin-secreting cells

Résumé Régulation de l'expression de la Connexin36 dans les cellules sécrétrices d'insuline La communication intercellulaire est en partie assurée via des jonctions communicantes de type "gap". Dans la cellule ß pancréatique, plusieurs observations indiquent que le couplage assuré par des jonctions gap formées parla Connexine36 (Cx36) est impliqué dans le contrôle de la sécrétion de l'insuline. De plus, nous avons récemment démontré qu'un niveau précis d'expression de la Cx36 est nécessaire pour maintenir une bonne coordination de l'ensemble des cellules ß, et permettre ainsi une sécrétion synchrone et contrôlée d'insuline. Le développement du diabète et du syndrome métabolique est partiellement dû à une altération de la capacité des cellules ß à sécréter de l'insuline en réponse à une augmentation de la glycémie. Cette altération est en partie causée par l'augmentation prolongée des taux circulant de glucose, mais aussi de lipides, sous la forme d'acides gras libres, et de LDL (Low Density Lipoproteins), particules assurant le transport des acides gras et du cholestérol dans le sang. Nous avons étudié la régulation de l'expression de la Cx36 dans différentes conditions reflétant la physiopathologie du diabète de type 2 et du syndrome métabolique et démontré qu'une exposition prolongée à des concentrations élevées de glucose, de LDL, ainsi que de palmitate (acide gras saturé le plus abondant dans l'organisme), inhibent l'expression de la Cx36 dans les cellules ß. Cette inhibition implique l'activation de la PKA (Proteine Kinase A), qui stimule à son tour l'expression du facteur de transcription ICER-1 (Inductible cAMP Early Repressor-1). Ce puissant répresseur se fixe spécifiquement sur un motif CRE (cAMP Response Element), situé dans le promoteur du gène de la Cx36, inhibant ainsi son expression. Nous avons de plus démontré que des cytokines pro-inflammatoires, qui pourraient contribuer au développement du diabète, inhibent également l'expression de la Cx36. Cependant, les cytokines agissent indépendamment du répresseur ICER-1, mais selon un mécanisme requérant l'activation de l'AMPK (AMP dependant protein kinase). Sachant qu'un contrôle précis des niveaux d'expression de la Cx36 est un élément déterminant pour une sécrétion optimale de l'insuline, nos résultats suggèrent que la Cx36 pourrait être impliquée dans l'altération de la sécrétion de l'insuline contribuant à l'apparition du diabète de type 2. Summary A particular way by which cells communicate with each other is mediated by gap junctions, transmembrane structures providing a direct pathway for the diffusion of small molecules between adjacent cells. Gap junctional communication is required to maintain a proper functioning of insulin-secreting ß-cells. Moreover, the expression levels of connexin36 (Cx36), the sole gap junction protein expressed in ß-cells, are critical in maintaining glucose-stimulated insulin secretion. Chronic hyperglycemia and hyperlipidemia exert deleterious effects on insulin secretion and may contribute to the progressive ß-cell failure linked to the development of type 2 diabetes and metabolic syndrome. Since modulations of the Cx36 levels might impair ß-cell function, the general aim of this work was to elucidate wether elevated levels of glucose and lipids affect Cx36 expression. The first part of this work was dedicated to the study of the effect of high glucose concentrations on Cx36 expression. We demonstrated that glucose transcriptionally down-regulates the expression of Cx36 in insulin-secreting cells through activation of the protein kinase A (PKA), which in turn stimulates the expression of the inducible cAMP early repressor-1 (ICER-1). This repressor binds to a highly conserved cAMP response element (CRE) located in the Cx36 promoter, thereby inhibiting Cx36 expression. The second part of this thesis consisted in studying the effects of sustained exposure to free fatty acids (FFA) and human lipoproteins on Cx36 levels. The experiments revealed that the most abundant FFA, palmitate, as well as the atherogenic low density lipoproteins (LDL), also stimulate ICER-1 expression, resulting in Cx36 down-regulation. Finally, the third part of the work focused on the consequences of long-term exposure to proinflammatory cytokines on Cx36 content. Interleukin-1 ß (IL-1 ß) inhibits Cx36 expression and its effect is potentialized by tumor necrosis factor α (TNFα) and interferon γ (IFNγ). We further unveiled that the cytokines effect on Cx36 levels requires activation of the AMP dependent protein kinase (AMPK). Prolonged exposures to glucose, palmitate, LDL, and pro-inflammatory cytokines have all been proposed to contribute to the development of diabetes and metabolic syndrome. Since Cx36 expression levels are critical to maintain ß-cell function, Cx36 down-regulation by glucose, lipids, and cytokines might participate to the ß-cell failure associated with diabetes development.

Astrocyte dense-core vesicle gliosecretion is governed by rest/nrsf

Astrocytes are the brain non-nerve cells competent for the expression of clear and dense-core vesicles (DCVs) and for their regulated exocytosis. This process, called gliosecretion, nearly resembles the neurosecretion occurring in neurons and neurosecretory cells. REST/NRSF is a transcription repressor known to orchestrate nerve-cell differentiation, governing the expression of hundreds of neuron-specific genes through their repression in the non-nerve and their fine modulation in the nerve cells. Our previous studies in neurosecretory rat PC12 cells identified REST as the critical factor for the expression not only of individual genes, but also of the whole neurosecretory process via multiple, direct and indirect mechanisms (D'Alessandro et al., J. Neurochem., 2008; Klajn et al., J. Neurosci., 2009). Therefore we wondered whether gliosecretion was governed by REST. We investigated rat astrocyte primary cultures: they exhibited high REST, which directly represses the transcription of at least one target gene, and expressed neither DCVs nor their markers (granins, peptides, membrane proteins). Transfection of a dominant-negative construct of REST (REST/ DBD-GFP) induced the appearance of DCVs filled with secretogranin2 and NPY that are distinct from other intracellular organelles. TIRF analysis of astrocytes co-transfected with REST/DBD-GFP and NPY-mRFP constructs revealed NPY-mRFP-positive DCVs undergoing Ca2þ-dependent exocytosis, largely prevented by BoNT/B. Immunohistochemistry of the I-II layers of the human temporal brain cortex showed all neurons and microglia exhibiting the expected inappreciable and high levels of REST, respectively. In contrast astrocyte RESTwas variable, going from inappreciable to high, accompanied by variable expression of DCVs. In this work it has been demonstrated that astrocyte DCV expression and gliosecretion are governed by REST (Prada et al., 2011 in press). The variable in situ REST levels may contribute to the well known structural/functional heterogeneity of astrocytes and this new observation might be of great interest for the understanding of both astrocyte physiology and pathology.

Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation.

Lymphatic valves are essential for efficient lymphatic transport, but the mechanisms of early lymphatic-valve morphogenesis and the role of biomechanical forces are not well understood. We found that the transcription factors PROX1 and FOXC2, highly expressed from the onset of valve formation, mediate segregation of lymphatic-valve-forming cells and cell mechanosensory responses to shear stress in vitro. Mechanistically, PROX1, FOXC2, and flow coordinately control expression of the gap junction protein connexin37 and activation of calcineurin/NFAT signaling. Connexin37 and calcineurin are required for the assembly and delimitation of lymphatic valve territory during development and for its postnatal maintenance. We propose a model in which regionally increased levels/activation states of transcription factors cooperate with mechanotransduction to induce a discrete cell-signaling pattern and morphogenetic event, such as formation of lymphatic valves. Our results also provide molecular insights into the role of endothelial cell identity in the regulation of vascular mechanotransduction.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Increased plasma levels of tumor necrosis factor-alpha in asymptomatic/"indeterminate" and Chagas disease cardiomyopathy patients

We compared plasma tumor necrosis factor-alpha (TNF-alpha) levels among asymptomatic/"indeterminate" Chagas disease patients (ASY) and patients across the clinical spectrum of chronic Chagas disease cardiomyopathy (CCC). Idiopathic dilated cardiomyopathy (DCM) patients and normal controls (NC) were included as controls. ASY Chagas disease patients had significantly higher plasma TNF-alpha levels than NC. TNF-alpha levels among severe CCC patients with significant left ventricular (LV) dysfunction were similar to those of DCM patients, showing average 2-fold higher levels than CCC patients without LV dysfunction and ASY patients, and 8-fold higher levels than NC. In Chagas disease, chronic TNF-a production prior to heart failure may play a role in CCC progression.

Variants in MTNR1B influence fasting glucose levels.

To identify previously unknown genetic loci associated with fasting glucose concentrations, we examined the leading association signals in ten genome-wide association scans involving a total of 36,610 individuals of European descent. Variants in the gene encoding melatonin receptor 1B (MTNR1B) were consistently associated with fasting glucose across all ten studies. The strongest signal was observed at rs10830963, where each G allele (frequency 0.30 in HapMap CEU) was associated with an increase of 0.07 (95% CI = 0.06-0.08) mmol/l in fasting glucose levels (P = 3.2 x 10(-50)) and reduced beta-cell function as measured by homeostasis model assessment (HOMA-B, P = 1.1 x 10(-15)). The same allele was associated with an increased risk of type 2 diabetes (odds ratio = 1.09 (1.05-1.12), per G allele P = 3.3 x 10(-7)) in a meta-analysis of 13 case-control studies totaling 18,236 cases and 64,453 controls. Our analyses also confirm previous associations of fasting glucose with variants at the G6PC2 (rs560887, P = 1.1 x 10(-57)) and GCK (rs4607517, P = 1.0 x 10(-25)) loci.