Targeting nuclear receptor corepressors for reversible male contraception.

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

Corepressor SMRT is required to maintain Hox transcriptional memory during somitogenesis.

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRTmRID), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRTmRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRTmRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci.

Identification and structure activity relationships of quinoline tertiary alcohol modulators of RORγt.

A high-throughput screen of the ligand binding domain of the nuclear receptor retinoic acid-related orphan receptor gamma t (RORγt) employing a thermal shift assay yielded a quinoline tertiary alcohol hit. Optimization of the 2-, 3- and 4-positions of the quinoline core using structure-activity relationships and structure-based drug design methods led to the discovery of a series of modulators with improved RORγt inhibitory potency and inverse agonism properties.

6-Substituted quinolines as RORγt inverse agonists.

We identified 6-substituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). The synthesis of this class of RORγt modulators is reported, and optimization of the substituents at the quinoline 6-position that produced compounds with high affinity for the receptor is detailed. This effort identified molecules that act as potent, full inverse agonists in a RORγt-driven cell-based reporter assay. The X-ray crystal structures of two full inverse agonists from this chemical series bound to the RORγt ligand binding domain are disclosed, and we highlight the interaction of a hydrogen-bond acceptor on the 6-position substituent of the inverse agonist with Glu379:NH as a conserved binding contact.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Rescue of a primary myelofibrosis model by retinoid-antagonist therapy.

Molecular targeting of the two receptor interaction domains of the epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors (SMRT(mRID)) produced a transplantable skeletal syndrome that reduced radial bone growth, increased numbers of bone-resorbing periosteal osteoclasts, and increased bone fracture risk. Furthermore, SMRT(mRID) mice develop spontaneous primary myelofibrosis, a chronic, usually idiopathic disorder characterized by progressive bone marrow fibrosis. Frequently linked to polycythemia vera and chronic myeloid leukemia, myelofibrosis displays high patient morbidity and mortality, and current treatment is mostly palliative. To decipher the etiology of this disease, we identified the thrombopoietin (Tpo) gene as a target of the SMRT-retinoic acid receptor signaling pathway in bone marrow stromal cells. Chronic induction of Tpo in SMRT(mRID) mice results in up-regulation of TGF-ß and PDGF in megakaryocytes, uncontrolled proliferation of bone marrow reticular cells, and fibrosis of the marrow compartment. Of therapeutic relevance, we show that this syndrome can be rescued by retinoid antagonists, demonstrating that the physical interface between SMRT and retinoic acid receptor can be a potential therapeutic target to block primary myelofibrosis disease progression.

PI3Kγ kinase activity is required for optimal T-cell activation and differentiation.

Phosphatidylinositol-3-kinase gamma (PI3Kγ) is a leukocyte-specific lipid kinase with signaling function downstream of G protein-coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T-cell function, we studied T cells from PI3Kγ kinase-dead knock-in (PI3Kγ(KD/KD)) mice expressing the kinase-inactive PI3Kγ protein. We show that CD4(+) and CD8(+) T cells from PI3Kγ(KD/KD) mice exhibit impaired TCR/CD28-mediated activation that could not be rescued by exogenous IL-2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ(KD/KD) T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ(KD/KD) CD4(+) T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ(KD/KD) mice also exhibited an impaired response to immunization and a reduced delayed-type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T-cell activation and differentiation, as well as for mounting an efficient T cell-mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.

PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization.

Although peroxisome proliferator-activated receptor gamma (PPARgamma) agonists such as thiazolidinediones (TZDs) are widely used to treat type 2 diabetes, how its activation in individual tissues contributes to TZD's therapeutic action remains controversial. As TZDs are known to have receptor-independent effects, we sought to establish gain-of-function animal models to delineate the receptor's insulin-sensitizing actions. Unexpectedly, we find that selective activation of PPARgamma in adipocytes, but not in macrophages, is sufficient for whole-body insulin sensitization equivalent to systemic TZD treatment. In addition to improved adipokine, inflammatory, and lipid profiles, PPARgamma activation in mature adipocytes normalizes serum insulin without increased adipogenesis. Co-culture studies indicated that PPARgamma-activated adipocytes broadly suppress induction of inflammatory cytokines and C-X-C family chemokines in macrophages. Collectively, these data describe an "adipocentric" model in which adipose activation of PPARgamma is sufficient for complete insulin sensitization and suggest a specific application for fat selective PPARgamma modulators in diabetic therapy.

Preclinical and clinical characterization of the RORγt inhibitor JNJ-61803534.

The nuclear receptor retinoid-related orphan receptor gamma t (RORγt) plays a critical role in driving Th17 cell differentiation and expansion, as well as IL-17 production in innate and adaptive immune cells. The IL-23/IL-17 axis is implicated in several autoimmune and inflammatory diseases, and biologics targeting IL-23 and IL-17 have shown significant clinical efficacy in treating psoriasis and psoriatic arthritis. JNJ-61803534 is a potent RORγt inverse agonist, selectively inhibiting RORγt-driven transcription versus closely-related family members, RORα and RORß. JNJ-61803534 inhibited IL-17A production in human CD4+ T cells under Th17 differentiation conditions, but did not inhibit IFNγ production under Th1 differentiation conditions, and had no impact on in vitro differentiation of regulatory T cells (Treg), nor on the suppressive activity of natural Tregs. In the mouse collagen-induced arthritis model, JNJ-61803534 dose-dependently attenuated inflammation, achieving ~ 90% maximum inhibition of clinical score. JNJ-61803534 significantly inhibited disease score in the imiquimod-induced mouse skin inflammation model, and dose-dependently inhibited the expression of RORγt-regulated genes, including IL-17A, IL-17F, IL-22 and IL-23R. Preclinical 1-month toxicity studies in rats and dogs identified doses that were well tolerated supporting progression into first-in-human studies. An oral formulation of JNJ-61803534 was studied in a phase 1 randomized double-blind study in healthy human volunteers to assess safety, pharmacokinetics, and pharmacodynamics. The compound was well tolerated in single ascending doses (SAD) up to 200 mg, and exhibited dose-dependent increases in exposure upon oral dosing, with a plasma half-life of 164 to 170 h. In addition, dose-dependent inhibition of ex vivo stimulated IL-17A production in whole blood was observed, demonstrating in vivo target engagement. In conclusion, JNJ-61803534 is a potent and selective RORγt inhibitor that exhibited acceptable preclinical safety and efficacy, as well as an acceptable safety profile in a healthy volunteer SAD study, with clear evidence of a pharmacodynamic effect in humans.

Immunological characterization of HM5023507, an orally active PI3Kδ/γ inhibitor.

Phosphoinositide 3-kinases, delta (PI3Kδ) and gamma (PI3Kγ) are enriched in immune cells and regulate the development and function of innate and adaptive immunity. Dual PI3Kδγ inhibitors are considered high value targets for their potential to treat a variety of immune-mediated diseases, but their discovery has been challenging. Here we describe the preclinical pharmacology of HM5023507, an orally active dual inhibitor of δγ isoforms in immune signaling. HM5023507 inhibited PI3Kδ and PI3Kγ isoforms with greater than 100-fold selectivity against PI3Kα and PI3Kß in recombinant enzymatic assays and in primary human immune cells with an exquisite selectivity against other targets. HM5023507 attenuated the PI3Kδ/γ signaling in human basophils (IC50: 42/340 nmol/L; selectivity ratio ~1:8). HM5023507 attenuated the activation and function of human B and T cells, Th17 differentiation of CD4 T cells in the blood of healthy donors and rheumatoid arthritis patients, and cytokine and IgG production in human T and B cell cocultures, in vitro. Orally dosed HM5023507 attenuated PI3K δ/γ-mediated immune signaling in the rat in a dose-related manner. In addition, HM5023507 inhibited semiestablished collagen-induced arthritic inflammation in the rats (ED50 of 0.25mg/kg, p.o. BID or 0.5 mg/kg, QD, AUC: 1422 ng/mL*h), improved histopathology- and micro-computed tomography (µCT)-based indices of joint damage, bone destruction, and attenuated the levels of anti-collagen antibody, with an overall anti-inflammatory profile matching that of a TNFα neutralizing antibody. The PI3K δγ inhibitory profile of HM5023507 and its selectivity make it a useful tool to further delineate immunobiology of dual PI3K δγ targeting.

T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers.

Kv1.3 is a voltage-gated potassium channel expressed on T cells that plays an important role in T cell activation. Previous studies have shown that blocking Kv1.3 channels in human T cells during activation results in reduced calcium entry, cytokine production, and proliferation. The aim of the present study was to further explore the effects of Kv1.3 blockers on the response of different human T cell subsets under various stimulation conditions. Our studies show that, unlike the immune suppressor cyclosporine A, the inhibitory effect of Kv1.3 blockers was partial and stimulation strength dependent, with reduced inhibitory efficacy on T cells under strengthened anti-CD3/CD28 stimulations. T cell responses to allergens including house dust mites and ragweed were partially reduced by Kv1.3 blockers. The effect of Kv1.3 inhibition was dependent on T cell subsets, with stronger effects on CCR7- effector memory compared to CCR7+ central memory CD4 T cells. Calcium entry studies also revealed a population of CD4 T cells resistant to Kv1.3 blockade. Activation of CD4 T cells was accompanied with an increase in Kv1.3 currents but Kv1.3 transcripts were found to be reduced, suggesting a posttranscriptional mechanism in the regulation of Kv1.3 activities. In summary, Kv1.3 blockers inhibit T cell activation in a manner that is highly dependent on the T cell identity and stimulation strength, These findings suggest that Kv1.3 blockers inhibit T cells in a unique, conditional manner, further refining our understanding of the therapeutic potential of Kv1.3 blockers.

RORγt and RORα signature genes in human Th17 cells.

RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.

A Prospective Virtual Screening Study: Enriching Hit Rates and Designing Focus Libraries To Find Inhibitors of PI3Kδ and PI3Kγ.

Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process.

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis.

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.

Curva epidemiológica de Bordetella pertussis en el Ecuador referente a los años 1999 al 2014 / Bordetella pertussis epidemiological curve in Ecuador referring to the years 1999 to 2014

Bordetella pertussises un cocobacilo Gram negativo aerobio estricto agente causal de la enfermedad infectocontagiosa conocida como tosferina que afecta predominantemente a lactantes, produciendo un alto índice de mortalidad. Mediante la elaboración de una curva epidemiológica del número de casos de la patología por Bordetella pertussis en un periodo epidémico dispuesto según etapas del tiempo, se evidenciará la evolución de los brotes en el Ecuador como aporte a la epidemiología nacional. Como objetivo específico se determina la frecuencia de aparición de B. pertussis, mediante el aislamiento de la cepa se procede a describir el comportamiento epidemiológico según la frecuencia de aparicióncomparando los datos epidemiológicos obtenidos en Ecuador con datos en el mundo, para poder destacar la importancia de implementar nuevas técnicas de laboratorio para el diagnóstico de la tosferina. El método aplicado en la investigación es descriptivo de cohorte transversal, siendo analizada la curva epidemiológica con los datos obtenidos de los aislamientos durante los años 1999 al 2014; como resultados del comportamiento epidemiológico de la Bordetella pertussisen el Ecuador se pueden visualizar picos en las gráficas que indican el inicio y el final de un brote, esto ocurre cada 3 a 5 años con la presencia de 0, 1 a 2 casos esporádicos entre cada una, tal como lo describen los reportes epidemiológicos de la OMS.Los resultados obtenidos en el estudio revelan la categoría en cuanto los recursos humanos, educación y comunicación, sus dimensiones, procedimientos clínicos, técnicos para la vigilancia de enfermedades transmisibles, las técnicas de cultivo, serología, y la unidad de análisis de las muestras clínicas recibidas de las diferentes zonas del Ecua

Bordetellapertussis is a strict aerobic Gram negative coccobacillus, causative agent of the infectious disease known as pertussis, which predominantly affects infants, producing a high mortality rate. By means of the elaboration of an epidemiological curve of the number of cases of the pathology by Bordetella Pertussis in an epidemic period arranged according to time stages, the evolution of the outbreaks in Ecuador as a contribution to the National Epidemiology will be evidenced.As a specific objective, the frequency of appearance of B. pertussis is determined by means of the isolation of the strain, we proceed to describe the epidemiological behavior according to the frequency of appearance, comparing the epidemiological data obtained in Ecuador with data obtainedin the world, in order to highlight the importance of implementing new laboratory techniques for the diagnosis of whooping cough.The method applied in the research is descriptive of a transversal cohort, the epidemiological curve being analyzed with the data obtained from the isolations during the years 1999 to 2014, as results of the epidemiological behavior of the Bordetella pertussis in Ecuador can be seen peaks in the graphs that indicate the beginning and end of an outbreak, this occurs every 3 to 5 years with the presence of 0, 1 to 2 sporadic cases between each, as described in the WHO epidemiological reports.The results obtained in the study reveal the category in terms of human resources, education and communication, its dimensions, clinical, technical procedures for the surveillance of communicable diseases, culture techniques, serology, and the unit of analysis of the clinical samples received of the different zones of Ecuador.

Tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining.

Previous attempts to define dengue virus (DENV) tropism in human autopsy tissues have detected DENV antigens that are abundant in circulation during severe dengue, and thus may be present in uninfected cells. To better define DENV tropism, we performed immunostaining for the DENV2 nonstructural protein 3 (NS3) in humans and in a mouse model of DENV infection. In mice, NS3 was detected in phagocytes of the spleen and lymph node, hepatocytes in liver, and myeloid cells in bone marrow. In human autopsy tissues, NS3 was present in phagocytes in lymph node and spleen, alveolar macrophages in lung, and perivascular cells in brain. This protein was also found in hepatocytes in liver and endothelial cells in spleen, although NS3 was not present in endothelium in any other tissue. Thus, NS3-specific immunostaining supports roles for infected phagocytes, hepatocytes, and, to a limited degree, endothelial cells in the pathogenesis of severe dengue.

Aspectos comparativos da localização, distribuição e irrigação do nó atrioventricular em suínos (Sus domesticus) da raça Landrace / Comparatives aspects of localization, distribution and irrigation of the atrioventricular node in pigs (Sus domesticus) of the Landrace race

A análise morfológica buscou verificar a localização do nó e do fascículo atrioventriculares, bem como sua distribuição sob o endocárdio dos ventrículos e descrever as artérias responsáveis pela nutrição do nó, comparando tais aspectos com características já descritas em seres humanos. Foram coletados 30 corações de suínos Landrace, sendo fêmeas com idade entre 2,5 a 3 anos. Inicialmente, 10 corações foram dissecados a fresco, e outros 20 corações foram injetados com solução de neoprene látex, posteriormente fixados em formaldeído a 10% por 72 horas, para então serem dissecados. Foi observado que o nó atrioventricular localiza-se na região caudoventral do septo interatrial, próximo à abertura do seio coronário. Também foram observadas quatro tipos de irrigações, sendo que a principal irrigação encontrada nos suínos trata-se do primeiro ramo septal caudal, proveniente da artéria coronária direita. O fascículo atrioventricular atravessou o esqueleto fibroso em direção ao septo interventricular e se dividiu em dois ramos. O ramo direito desceu em direção ao ápice do coração sob o endocárdio, se distribuindo na parede lateral do ventrículo direito e o ramo esquerdo permaneceu único ou se dividiu em dois e três ramos, que se distribuíram na parede do ventrículo esquerdo. A localização e irrigação do nó atrioventricular, assim como a divisão e a distribuição do fascículo atrioventricular são semelhantes à anatomia humana, podendo ser viável a utilização de suínos como modelo experimental em estudos comparativos que investiguem esse sistema.

Morphological analysis aimed to verify the location of the node and the atrioventricular fasciculus, and its distribution in the endocardium of the ventricles and describe the arteries responsible for nourishing the node, comparing these aspects with features in humans. Were collected 30 Landrace pig hearts, females, 2.5 to 3 years. Initially, 10 hearts were fresh dissected, and in 20 others hearts were injected with neoprene latex solution, later fixed in 10% formaldehyde for 72 hours, then were dissected. It was observed that the atrioventricular node was located in the caudoventral region of the atrial septum, near the opening of the coronary sinus. It was also observed four types of irrigation, and the main irrigation found in pigs was the first septal branch caudal coming from the right coronary artery. The atrioventricular fascicle crossed the fibrous skeleton into the interventricular septum and split into two branches. The right branch came down towards the apex of the heart under the endocardium, up by distributing itself in the lateral wall of the right ventricle, and the left branch remained single or divided in two and three branches, which are distributed in the wall of the left ventricle. Location and irrigation of the atrioventricular node, as well as the division and distribution of fascicle, are similar to Human Anatomy, being viable the use of pigs as an experimental model in comparative studies to investigate this system.

Tinta china en orina como método de diagnóstico en criptococosis diseminada asociado a VIH/SIDA. Estudio transversal realizado en el hospital de infectología José Rodríguez M. durante el año 2009 / China ink in the urine as a diagnostic tool in disseminated cryptococcosis associated with HIV/AIDS. Sectional study of infectious diseases in the José Rodríguez M. Hospital in 2009

Tipo estudio: transversal y descriptivo. Objetivos: determinar la utilidad de la aplicación de tinta china en orina como método de diagnóstico en los pacientes con sospecha de criptococosis diseminada asociada a VIH/SIDA. Métodos: el universo del estudio está establecido por los pacientes VIH/SIDA que ingresaron durante el año 2009 con sospecha de esta enfermedad al hospital de Infectología “José Rodríguez Maridueña”. Resultados: la sensibilidad del test de tinta en orina fue de un 71,79% con un valor predictivo positivo del 90,32%. Del universo total de pacientes, se obtuvo una media de CD4 de 97,5 +/- 90,6 ul; no hubo diferencia estadísticamente significativa entre el test de tinta china y las variables independientes. Conclusión: el test de tinta china en orina se establece como un método práctico, no invasivo y económico en los pacientes VIH/SIDA que ingresan con alta presunción de criptococosis en este nosocomio. El test de tinta china en orina nos propicia una detección de casos de forma anticipada, ante la expectativa de la convalidación de los cultivos o de métodos más específicos que nos permitan su confirmación o corrección.

Studio: transverse and descriptive. Objectives: to determine the usefulness of applying ink in urine as a diagnostic method in patients with suspected disseminated cryptococcosis associated with HIV / AIDS. Methods: the universe of study is established by the HIV / AIDS patients admitted during 2009 with suspicion of this disease at the "José Rodríguez Maridueña" Infectious Diseases Hospital Results: the sensitivity of the ink test in urine was 71.79% with a positive predictive value of 90.32%. Out of the total sample of patients, we obtained a CD4 mean of 97.5 +/- 90.6 ul; there was no statistically significant difference between the china ink test and the independent variables. Conclusion: the china ink test in urine was established as a practical, noninvasive and economical test in HIV / AIDS patients admitted with high presumption of cryptococcosis. The China ink test in urine allows the early detection of cases, before the validation of the culture or more specific methods that allow us confirmation or correction.

Interação comunicativa em contexto lúdico de duas crianças com Síndrome de Down, comportamentos autísticos e privação de estímulos / Communicative interaction in a playful context of two children with Down Syndrome, autistic behavior and deprivation of stimuli

Esta pesquisa qualitativa teve como objetivo analisar o processo de interação comunicativa entre duas crianças que apresentam comportamentos autísticos associados à síndrome de Down e entre estas e a terapeuta, enfocando modalidades de comunicação verbal e não-verbal estabelecidas durante brincadeiras de faz-de-conta. As duas crianças têm histórias de vida com privação de estímulos. Os dados foram coletados em sessões fonoaudiológicas semanais durante 6 meses. Após a transcrição dos episódios, a partir da vídeo-gravação das sessões realizadas, as análises evidenciaram desenvolvimento qualitativo na interação comunicativa entre as crianças e a terapeuta, possibilitando o compartilhamento de significados e inserção destes sujeitos nas situações sociais apresentadas.

The objective of this qualitative research was to analyze the communicative interaction process between two children who show autistic behavior associated with Down syndrome, as well as the process between them and their therapist. Different modalities of verbal and non-verbal communication demonstrated during make-believe activities were also observed. Both children have life history of deprivation of stimuli. Data were collected during weekly sessions with a speech therapist over 6 months. The sessions were video-taped and then the episodes were transcribed. The analyses displayed a qualitative development in the communicative interaction between the children and the therapist which favored the subjects to share meanings and insert themselves in the social situations presented.