D- and N-Methyl Amino Acids for Modulating the Therapeutic Properties of Antimicrobial Peptides and Lipopeptides.

Here we designed and synthesized analogs of two antimicrobial peptides, namely C10:0-A2, a lipopeptide, and TA4, a cationic α-helical amphipathic peptide, and used non-proteinogenic amino acids to improve their therapeutic properties. The physicochemical properties of these analogs were analyzed, including their retention time, hydrophobicity, and critical micelle concentration, as well as their antimicrobial activity against gram-positive and gram-negative bacteria and yeast. Our results showed that substitution with D- and N-methyl amino acids could be a useful strategy to modulate the therapeutic properties of antimicrobial peptides and lipopeptides, including enhancing stability against enzymatic degradation. The study provides insights into the design and optimization of antimicrobial peptides to achieve improved stability and therapeutic efficacy. TA4(dK), C10:0-A2(6-NMeLys), and C10:0-A2(9-NMeLys) were identified as the most promising molecules for further studies.

Downregulation of intestinal multidrug resistance transporter 1 in obese mice: Effect on its barrier function and role of TNF-α receptor 1 signaling.

OBJECTIVES: Multidrug resistance transporter 1 (Mdr-1) is a relevant component of the intestinal transcellular barrier that decreases absorption of oral drugs, thus modulating their bioavailability. Obese patients with metabolic disorders take medications that are subjected to intestinal metabolism and the Mdr-1-dependent barrier. This study evaluated the effect of a high-fat diet (HFD; 40% fat for 16 wk) on Mdr-1 expression and transport activity in C57BL/6 (C57) male mice. Comparable studies were performed in tumor necrosis factor α (TNF-α) receptor 1 knockout mice (R1KO) to delineate a possible role of TNF-α signaling. METHODS: mRNA expression was evaluated by real-time polymerase chain reaction and protein levels by western blotting and immunohistochemistry. Mdr-1 activity was assessed using the everted intestinal sac model, with rhodamine 123 as the substrate. Statistical comparisons were made using the Student t test or one-way analysis of variance followed by the post hoc Tukey test. RESULTS: Mdr-1 protein, as well as its corresponding Mdr1a and Mdr1b mRNA, was decreased in C57-HFD mice compared with controls. Immunohistochemical studies confirmed downregulation of Mdr-1 in situ. These results correlated with a 48% decrease in the basolateral to apical transport of rhodamine 123. In contrast, R1KO-HFD modified neither intestinal Mdr-1 mRNA nor its protein expression or activity. In addition, C57-HFD showed elevated intestinal TNF-α mRNA and protein (enzyme-linked immunosorbent assay) levels, whereas R1KO-HFD was undetectable or had a lower increase, respectively. CONCLUSIONS: This study demonstrated an impairment of the Mdr-1 intestinal barrier function induced by HFD as a consequence of downregulation of both Mdr-1 gene homologues, resulting in impaired Mdr-1 protein expression. Inflammatory response mediated by TNF-α receptor 1 signaling was likely involved.

Broadening the spectrum of ivermectin: Its effect on Trypanosoma cruzi and related trypanosomatids.

Chagas disease is an endemic American parasitosis, caused by Trypanosoma cruzi. The current therapies, benznidazole (BZN) and nifurtimox (NFX), show limited efficacy and multiple side effects. Thus, there is a need to develop new trypanocidal strategies. Ivermectin (IVM) is a broad-spectrum antiparasitic drug with low human and veterinary toxicity with effects against T. brucei and Leishmania spp. Considering this and its relatively low cost, we evaluate IVM as a potential repurposed trypanocidal drug on T. cruzi and other trypanosomatids. We found that IVM affected, in a dose-dependent manner, the proliferation of T. cruzi epimastigotes as well as the amastigotes and trypomastigotes survival. The Selectivity Index for the amastigote stage with respect to Vero cells was 12. The IVM effect was also observed in Phytomonas jma 066 and Leishmania mexicana proliferation but not in Crithidia fasciculata. On the epimastigote stage, the IVM effect was trypanostatic at 50 µM but trypanocidal at 100 µM. The assays of the drug combinations of IVM with BNZ or NFX showed mainly additive effects among combinations. In silico studies showed that classical structures belonging to glutamate-gated Cl channels, the most common IVM target, are absent in kinetoplastids. However, we found in the studied trypanosomatid genomes one copy for putative IMPα and IMPß, potential targets for IVM. The putative IMPα genes (with 76% similarity) showed conserved Armadillo domains but lacked the canonical IMPß binding sequence. These results allowed us to propose a novel molecular target in T. cruzi and suggest IVM as a good candidate for drug repurposing in the Chagas disease context.

Essential Bromodomain TcBDF2 as a Drug Target against Chagas Disease.

Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ß-galactosidase.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi. T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission. This paper describes an assay based on measuring the activity of the cytoplasmic ß-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red ß-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a ß-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

Multidrug resistance-associated protein 2 is negatively regulated by oxidative stress in rat intestine via a posttranslational mechanism. Impact on its membrane barrier function.

Oxidative stress (OS) is a key factor in the development of gastrointestinal disorders, in which the intestinal barrier is altered. However, the Multidrug resistance-associated protein 2 (Mrp2) status, an essential component of the intestinal transcellular barrier exhibiting pharmaco-toxicological relevance by limiting the orally ingested toxicants and drugs absorption, has not been investigated. We here evaluated the short-term effect of OS on Mrp2 by treatment of isolated rat intestinal sacs with tert-butyl hydroperoxide (TBH) for 30 min. OS induction by TBH (250 and 500 µM) was confirmed by increased lipid peroxidation end products, decreased reduced glutathione (GSH) content and altered antioxidant enzyme activities. Under this condition, assessment of Mrp2 distribution between brush border (BBM) and intracellular (IM) membrane fractions, showed that Mrp2 protein decreased in BBM and increased in IM, consistent with an internalization process. This was associated with decreased efflux activity and, consequently, impaired barrier function. Subsequent incubation with N-Acetyl-L-Cysteine (NAC, 1 mM) reestablished GSH content and reverted concomitantly the alteration in Mrp2 localization and function induced by TBH. Cotreatment with a specific inhibitor of classic calcium-dependent Protein Kinase C (cPKC) implicated this kinase in TBH-effects. In conclusion, we demonstrated a negative posttranslational regulation of rat intestinal Mrp2 after short-term exposition to OS, a process likely mediated by cPKC and dependent on intracellular GSH content. The concomitant impairment of the Mrp2 barrier function may have implications in xenobiotic absorption and toxicity in a variety of human diseases linked to OS, with notable consequences on the toxicity/safety of therapeutic agents.

Epidemiología de los pacientes pediátricos derivados a un equipo de referencia en violencia doméstica. CASMU- IAMPP, 2016 / Epidemiology of pediatric patients referred to an Expert Reference Team specialized in Domestic Violence. CASMU- IAMPP, 2016 / Epidemiologia dos pacientes pediátricos encaminhados a uma equipe de referência em violência doméstica. CASMU- IAMPP, 2016

Resumen: El maltrato infantil y específicamente el abuso sexual infantil constituyen una violación de los derechos humanos y un importante problema de salud mundial. En Uruguay, los Equipos de Referencia en Violencia Doméstica en las instituciones de salud, asisten a niños víctimas de situaciones de violencia. Este trabajo buscó conocer la incidencia en niños, víctimas de situaciones de maltrato moderado a severo y de probable abuso sexual infantil, asistidos en el equipo de referencia en violencia doméstica de la institución CASMU-IAMPP, y sus características epidemiológicas. Se realizó un estudio observacional, descriptivo, con los usuarios menores de 18 años que fueron derivados en el 2016 a dicho equipo. El total de niños asistidos fue 87, la incidencia anual fue 0,2%. El 38% de los niños fueron víctimas de violencia doméstica, 31% sufrieron probable abuso sexual, 22% violencia física, 24% psicológica y 7% negligencia. En 90% de los casos, las situaciones se detectaron en etapa crónica. El 77% fueron derivados por el sector salud, de ellos un tercio por el pediatra tratante. El 50% convivía con el agresor y en el 38% de las situaciones de abuso sexual, el padre fue el victimario. De las escolares víctimas de abuso sexual, el 65% tenía sobrepeso u obesidad. Se destaca la importancia de conocer la epidemiología de los pacientes atendidos por una patología frecuente y grave para mejorar la calidad de su abordaje.

Summary: Child abuse and specifically child sexual abuse is a violation of Human Rights and a major global health problem. In Uruguay, the expert reference team specialized in domestic violence at health medical institutions assists children who are victims of violence. This paper aims at studying the incidence of children victims of moderate to severe abuse and of probable child sexual abuse assisted at the reference specialized center for domestic violence of the CASMU-IAMPP HMO and its epidemiological characteristics. We carried out an observational, descriptive study with patients under 18 years of age who were referred in 2016. The total number of children assisted was 87, the annual incidence was 0.2%. 38% of the children were victims of domestic violence, 31% suffered probable sexual abuse, 22% physical violence, 24% psychological and 7% negligence. In 90% of the cases, these situations of abuse were detected in the chronic stage. 77% were referred by the health sector; 34% by the treating pediatrician. 50% lived with the aggressor. In 38% of situations of sexual abuse, the father was the abuser. Of the schoolchildren victims of sexual abuse, 65% were overweight or obese. We hereby stress the importance of understanding and knowing the epidemiology of patients treated for a frequent and serious pathology in order to improve their quality of the approach used.

Resumo: O abuso infantil e especificamente o abuso sexual infantil são uma violação dos Direitos Humanos e um grande problema de saúde global. No Uruguai, as equipes de referência em violência doméstica das instituições de saúde assistem a crianças vítimas de situações de violência. Este trabalho procurou conhecer a incidência de crianças vítimas de situações de abuso moderado a grave e provável abuso sexual infantil, nas crianças atendidas pela equipe de referência em violência doméstica da instituição CASMU-IAMPP e suas características epidemiológicas. Realizou-se um estudo descritivo e observacional com crianças menores de 18 anos que foram encaminhados em 2016 à referida equipe. O número total de crianças atendidas foi de 87, a incidência anual foi de 0,2%. 38% das crianças foram vítimas de violência doméstica, 31% sofreram provável abuso sexual, 22% violência física, 24% psicológica e 7% negligência. Em 90% dos casos, detectamos os casos em estágio crônico. 77% foram encaminhados pelo setor de saúde, um terço deles pelo pediatra responsável. 50% moravam com o agressor e em 38% das situações de abuso sexual, o pai era o abusador. Das vítimas escolares de abuso sexual, 65% apresentavam sobrepeso ou obesidade. Destaca-se a importância de conhecer a epidemiologia dos pacientes tratados por uma patologia frequente e séria para melhorar a qualidade de sua abordagem.

Reversion of down-regulation of intestinal multidrug resistance-associated protein 2 in fructose-fed rats by geraniol and vitamin C: Potential role of inflammatory response and oxidative stress.

Intestinal multidrug resistance-associated protein 2 is an ABC transporter that limits the absorption of xenobiotics ingested orally, thus acting as essential component of the intestinal biochemical barrier. Metabolic Syndrome (MetS) is a pathological condition characterized by dyslipidemia, hyperinsulinemia, insulin resistance, chronic inflammation, and oxidative stress (OS). In a previous study we demonstrated that MetS-like conditions induced by fructose in drinking water (10% v/v, during 21 days), significantly reduced the expression and activity of intestinal Mrp2 in rats. We here evaluated the potential beneficial effect of geraniol or vitamin C supplementation, natural compounds with anti-inflammatory and anti-oxidant properties, in reverse fructose-induced Mrp2 alterations. After MetS-like conditions were induced (21 days), animals were cotreated with geraniol or vitamin C or vehicle for another 14 days. Decreased expression of Mrp2 protein and mRNA due to fructose administration was reversed by geraniol and by vitamin C, consistent with restoration of Mrp2 activity evaluated in everted intestinal sacs. Concomitantly, increased intestinal IL-1ß and IL-6 levels induced by fructose were totally and partially counterbalanced, respectively, by geraniol administration. The intestinal redox unbalance generated by fructose was improved by geraniol and vitamin C, as evidenced by decreasing lipid peroxidation products and activity of Superoxide Dismutase and by normalizing glutathione reduced/oxidized glutathione ratio. The restoration effects exhibited by geraniol and vitamin C suggest that local inflammatory response and OS generated under MetS-like conditions represent important mediators of the intestinal Mrp2 down-regulation. Additionally, both agents could be considered of potential therapeutic value to preserve Mrp2 function under MetS conditions.

Glucagon-like peptide 2 prevents down-regulation of intestinal multidrug resistance-associated protein 2 and P-glycoprotein in endotoxemic rats.

Multidrug resistance-associated protein 2 (Mrp2, ABCC2) and P-glycoprotein (P-gp, ABCB1) constitute essential components of the intestinal biochemical barrier that prevent incorporation of food contaminants, drugs or toxic metabolites into the blood stream. Endotoxemia induced in rats by administration of bacterial lipopolysaccharide (LPS) results in elevated intestinal permeability and toxicity of xenobiotics in part associated with down-regulation of expression and activity of Mrp2 and P-gp. We evaluated the protective effect of glucagon-like peptide 2 (GLP-2), a peptide hormone with enterotrophic properties, on Mrp2 and P-gp alterations induced by single i.p. injection of LPS (5mg/kg b.wt.) to rats. Two different protocols of GLP-2 administration, namely prevention and reversion, were examined. The prevention protocol consisted of 7s.c. injections of GLP-2 (125µg/kg b.wt.) administered every 12h, starting 60h before LPS administration. The reversion protocol consisted of 2 doses of GLP-2, starting 3h after LPS injection. Intestinal samples were collected 24h after LPS administration and expression (protein and mRNA) and activity of Mrp2 were evaluated in proximal jejunum whereas those of P-gp were studied in ileum. GLP-2 completely neutralized down-regulation of expression of Mrp2 and P-gp and loss of their respective activities induced by LPS under prevention protocol. GLP-2 was also able to prevent internalization of both transporters from the apical membrane of the enterocyte to intracellular compartments, as detected by confocal microscopy. LPS induced an increase in IL-1ß and oxidized glutathione tissue levels, which were also counterbalanced by GLP-2 administration. In contrast, the reversion protocol failed to attenuate Mrp2 and P-gp down-regulation induced by LPS. We conclude that GLP-2 can prevent down-regulation of intestinal expression and activity of Mrp2 and P-gp in endotoxemic rats and that IL-1ß and oxidative stress constitute potential targets of GLP-2 protective effects.

Osteoma osteoide de cuello femoral: A propósito de un caso / Femoral neck osteoid osteoma: about a case

El osteoma osteoide es un tumor óseo benigno, de pequeño tamaño, sin potencial de crecimiento. Habitualmente se considera a los tumores óseos benignos y malignos como una causa poco frecuente de cojera en apirexia, siendo más frecuentes las patologías inflamatorias inespecíficas como la sinovitis transitoria de cadera, la enfermedad de Perthes y la condropatía conjugal del adolescente. Se presenta el caso clínico de un escolar de 8 años con una cojera dolorosa en apirexia de 4 meses de evolución con sospecha imagenológica de osteoma osteoide de cuello de fémur. Se decide realizar prueba terapéutica con ácido acetilsalicílico. A las 24 horas el niño se encontraba asintomático. Se indicó procedimiento quirúrgico de resección mediante punción bajo tomografía axial computada. El diagnóstico se confirmó mediante anatomía patológica. Se realizó resección completa del tumor con buena evolución. Es importante desde el punto de vista pediátrico realizar un correcto diagnóstico diferencial entre las diferentes causas de cojera dolorosa en apirexia, basándonos en la historia clínica y la imagenología.

Osteoid osteomas are small benign bone tumors which lack growth potential. All bone tumors, whether benign or malign, are considered a rare cause of apyretic limping, being non-specific inflammatory diseases more frequent, such as transient synovitis of the hip, Perthes disease and adolescent conjugal chondropathy. The study presents the clinical case of an 8 year old school boy with a 4 month evolution painful apyretic limp, being there a suspicion of femoral neck osteoid osteoma according to imaging studies. Therapeutic trial of acetylsalicylic acid was performed. Twenty four hours later the boy was asymptomatic. A tomography-guided puncture was indicated. Clinical diagnosis was pathologically confirmed. Subsequently, complete surgical resection of the tumor was performed, the evolution being favorable. From a pediatric perspective it is important to make an accurate differential diagnosis between the different possible causes of painful apyretic limp, based on clinical history and imaging studies.

Trypanosoma cruzi High Mobility Group B (TcHMGB) can act as an inflammatory mediator on mammalian cells.

BACKGROUND: High Mobility Group B (HMGB) proteins are nuclear architectural factors involved in chromatin remodeling and important nuclear events. HMGBs also play key roles outside the cell acting as alarmins or Damage-associated Molecular Patterns (DAMPs). In response to a danger signal these proteins act as immune mediators in the extracellular milieu. Moreover, these molecules play a central role in the pathogenesis of many autoimmune and both infectious and sterile inflammatory chronic diseases. PRINCIPAL FINDINGS: We have previously identified a High mobility group B protein from Trypanosoma cruzi (TcHMGB) and showed that it has architectural properties interacting with DNA like HMGBs from other eukaryotes. Here we show that TcHMGB can be translocated to the cytoplasm and secreted out of the parasite, a process that seems to be stimulated by acetylation. We report that recombinant TcHMGB is able to induce an inflammatory response in vitro and in vivo, evidenced by the production of Nitric Oxide and induction of inflammatory cytokines like TNF-α, IL-1ß and IFN-γ gene expression. Also, TGF-ß and IL-10, which are not inflammatory cytokines but do play key roles in Chagas disease, were induced by rTcHMGB. CONCLUSIONS: These preliminary results suggest that TcHMGB can act as an exogenous immune mediator that may be important for both the control of parasite replication as the pathogenesis of Chagas disease and can be envisioned as a pathogen associated molecular pattern (PAMP) partially overlapping in function with the host DAMPs.

Intestinal multidrug resistance-associated protein 2 is down-regulated in fructose-fed rats.

Expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2) and glutathione-S-transferase (GST) were examined in fructose fed Wistar rats, an experimental model of metabolic syndrome. Animals were fed on (a) control diet or (b) control diet plus 10% w/vol fructose in the drinking water. Mrp2 and the α class of GST proteins as well as their corresponding mRNAs were decreased, suggesting a transcriptional regulation by fructose. Confocal microscopy studies reaffirmed down-regulation of Mrp2. Everted intestinal sacs were incubated with 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl- S-glutathione (DNP-SG; model Mrp2 substrate), was measured in the same compartment to estimate Mrp2 activity. Excretion of DNP-SG was substantially decreased by fructose treatment, consistent with simultaneous down-regulation of Mrp2 and GST. In addition, the effect of fructose on intestinal barrier function exerted by Mrp2 was evaluated in vivo using valsartan, a recognized Mrp2 substrate of therapeutic use. After intraduodenal administration as a bolus, intestinal absorption of valsartan was increased in fructose-drinking animals. Fructose administration also induced oxidative stress in intestinal tissue as demonstrated by significant increases of intestinal lipid peroxidation end products and activity of the antioxidant enzyme superoxide dismutase, by a decreased GSH/GSSG ratio. Moreover, fructose treatment conduced to increased intestinal levels of the proinflammatory cytokines IL-ß1 and IL-6. Collectively, our results demonstrate that metabolic syndrome-like conditions, induced by a fructose-rich diet, result in down-regulation of intestinal Mrp2 expression and activity and consequently in an impairment of its barrier function.

Up-regulation of ATP-binding cassette transporters in the THP-1 human macrophage cell line by the antichagasic benznidazole.

The effect of benznidazole (BZL) on the expression and activity of P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 2 (MRP2, ABCC2), the two major transporters of endogenous and exogenous compounds, was evaluated in differentiated THP-1 cells. BZL induced P-gp and MRP2 proteins in a concentration-dependent manner. The increase in mRNA levels of both transporters suggests transcriptional regulation. P-gp and MRP2 activities correlated with increased protein levels. BZL intracellular accumulation was significantly lower in BZL-pre-treated cells than in control cells. PSC833 (a P-gp inhibitor) increased the intracellular BZL concentration in both pre-treated and control cells, confirming P-gp participation in BZL efflux.

The trypanocidal benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2).

Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only trypanocidal agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200µM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3h of exposure, returning to normality at 24h. Additionally, BZL increased glutathione peroxidase activity at 12h and the oxidized glutathione/total glutathione (GSSG/GSSG+GSH) ratio that reached a peak at 24h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG+GSH returned to control values at 48h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48h, explaining normalization of GSSG/GSSG+GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy.

Tos convulsa: brote epidémico 2011-2012, Hospital Las Piedras: características clínico-evolutivas de los pacientes hospitalizados / Whooping cough: epidemic outbreak 2011-2012, Hospital Las Piedras: clinical-evolutionary characteristics of hospitalized patients

Introducción: la tos convulsa es una enfermedad infecciosa respiratoria aguda debida a Bordetella pertussis y Bordetella parapertussis. Factores de mal pronóstico son edad menor a 6 meses, hiperleucocitosis, coinfección bacteriana. En Uruguay, desde la incorporación de la vacuna antipertussis en 1963, disminuyeron significativamente las notificaciones con comportamiento endémico con brotes. En el 2011, luego del fallecimiento de dos niños con tos convulsa, se registró un aumento de casos en el Hospital Las Piedras (HLP). Objetivo: describir las características clínico-evolutivas de los niños hospitalizados por tos convulsa en el HLP entre el 1º de agosto de 2011 y el 30 de abril de 2012. Material y método: estudio descriptivo, prospectivo, de los niños hospitalizados por tos convulsa. La confirmación se realizó por reacción en cadena de polimerasa (PCR) en secreciones respiratorias. Se analizan variables epidemiológicas, características clínicas y evolutivas. Resultados: se hospitalizaron 41 niños con tos convulsa confirmada (6,4%); 48,8% fueron menores de 6 meses. Diez fueron derivados a centros de tratamiento intensivo (CTI), ocho de ellos menores de 6 meses; dos fallecieron. Tuvieron contacto con casos confirmados 10% y con casos sospechosos 51%. Presentaron leucocitosis > 20.000 cel/mm3, 39%. En tres casos existió coinfección con adenovirus. Discusión y conclusiones: el brote de tos convulsa comenzó en 2011, siendo el HLP el centro que alertó su inicio y registró la tasa más elevada de hospitalización. Las características clínico-evolutivas de los niños afectados son similares a las descritas en la región. Su abordaje requiere accesibilidad a nuevas herramientas diagnósticas de biología molecular. Para el control y la prevención se requiere reforzar las estrategias de vacunación.

Abstract Introduction: whooping cough is an acute respiratory infectious disease caused by a type of bacteria called Bordetella pertussis and Bordetella parapertussis. Being younger than 6 months, hyperleukocytosis and bacterial coinfection are some bad prognosis factors. In Uruguay, since the antipertussis vaccination was included in the vaccination schedule in 1963, notifications with endemic outbreak behavior significantly diminished. In 2011, after two children with whooping cough died, an increase of the cases was recorded at the Las Piedras Hospital. Objective: to describe the clinical characteristics and evolution of children hospitalized for whooping cough at the Las Piedras Hospital from August 1st, 2011 through April 30, 2012. Method: descriptive, prospective study, of the children hospitalized for whooping cough. Confirmation was made by polymerase chain reaction in respiratory secretions. Epidemiological variables, clinical characteristics and evolution are analysed. Results: 41 children were hospitalized with confirmed whooping cough (6.4%); 48.8% were younger than 6 months. Ten of them were referred to intensive care units, eight of them were younger than 6 months and two of them died. Ten per cent were in contact with confirmed cases and 51% were in contact with suspicious cases. Leukocytosis > 20.000 cel/mm3 was seen in 39% of children. There was coinfection with adenovirus in three cases. Discussion and conclusions: the whooping cough outbreak started in 2011, being the Pereira Rossell the health center that issued the warning and had the highest hospitalization rate. Clinical characteristics and evolution of the children affected are the similar to those described in the region. Access to new diagnostic tools of molecular biology is necessary to address this condition. Control and prevention requires strengthening vaccination strategies.

Resumo Introdução: a coqueluche é uma doença infecciosa respiratória aguda devida a Bordetella pertussis e Bordetella parapertussis. Idade menor a 6 meses, hiperleucocitoses e co-infecção bacteriana são fatores de mal prognóstico. No Uruguai, desde a incorporação da vacina contra a coqueluches em 1963, as notificacoes com comportamento endêmico com surtos diminuiram significativamente. Em 2011, depois dos óbitos de duas crianças com coqueluche, registrou-se um aumento de casos no Hospital Las Piedras (HLP). Objetivo: descrever as características clínico-evolutivas das crianças hospitalizadas por coqueluche no HLP no período 1º de agosto de 2011 - 30 de abril de 2012. Material e método: estudo descritivo e prospectivo das crianças hospitalizadas por coqueluche. A confirmação do diagnóstico foi feita por reação em cadeia de polimerase (PCR) em secreções respiratórias. Foram analisadas variáveis epidemiológicas, características clínicas e evolutivas. Resultados: 41 crianças foram hospitalizadas com diagnóstico confirmado de coqueluche (6,4%); 48,8% tinham menos de 6 meses de vida. Dez foram derivadas a unidades de terapia intensiva (UTII) sendo que oito tinham menos de 6 meses; duas faleceram. 10% teve contato com casos confirmados e 51% com casos suspeitos. 39% apresentou leucocitose > 20.000 cel/mm3. Em três casos foi observada co-infecção com adenovirus. Discussão e conclusões: o surto de coqueluche começou em 2011, sendo o HLP o centro que alertou sobre seu inicio e registrou a taxa mais elevada de hospitalização. As características clínico-evolutivas das crianças afetadas foram similares às descritas na região. A abordagem desta patologia requer acesso a novas ferramentas diagnósticas de biologia molecular. Para o controle e a prevenção é necessário reforçar as estratégias de vacinação.

Glomerulonefritis difusa aguda postestreptocócica-psoriasis guttata. A propósito de un caso clínico / A case of acute diffuse poststreptococcal glomerulonephritis: guttate psoriasis

La glomerulonefritis asociada a la infección por estreptococo beta hemolítico del grupo A es la más conocida y es la causa más común de síndrome nefrítico en la edad pediátrica. La psoriasis es una enfermedad cutánea hereditaria eritematodescamativa poco frecuente, representa el 4,1% de las dermatosis que ocurren en niños menores de 16 años. Se presenta el caso de un adolescente de 12 años donde la infección por estreptococo beta hemolítico del grupo A de las vías respiratorias altas ocasionó glomerulonefritis difusa aguda, con expresión concomitante de psoriasis guttata. Se revisan los mecanismos inmunes en ambas patologías.

Glomerulonephritis associated with group A beta-hemolytic streptococcal infection is the best known and most common cause of nephritic syndrome in children. Psoriasis is often an erythematous hereditary skin disease. It represents 4.1% of the dermatoses children under 16 years. The study presents the case of a 12 year old patient where Group A beta-hemolytic streptococcal acute upper respiratory infection caused diffuse glomerulonephritis, with concomitant expression of guttate psoriasis. A review of immune mechanisms of both diseases is added.

Regulation of expression and activity of multidrug resistance proteins MRP2 and MDR1 by estrogenic compounds in Caco-2 cells. Role in prevention of xenobiotic-induced cytotoxicity.

ABC transporters including MRP2, MDR1 and BCRP play a major role in tissue defense. Epidemiological and experimental studies suggest a cytoprotective role of estrogens in intestine, though the mechanism remains poorly understood. We evaluated whether pharmacologic concentrations of ethynylestradiol (EE, 0.05pM to 5nM), or concentrations of genistein (GNT) associated with soy ingestion (0.1-10µM), affect the expression and activity of multidrug resistance proteins MRP2, MDR1 and BCRP using Caco-2 cells, an in vitro model of intestinal epithelium. We found that incubation with 5pM EE and 1µM GNT for 48h increased expression and activity of both MRP2 and MDR1. Estrogens did not affect expression of BCRP protein at any concentration studied. Irrespective of the estrogen tested, up-regulation of MDR1 and MRP2 protein was accompanied by increased levels of MDR1 mRNA, whereas MRP2 mRNA remained unchanged. Cytotoxicity assays demonstrated association of MRP2 and MDR1 up-regulation with increased resistance to cell death induced by 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor, and by paraquat, an MDR1 substrate. Experiments using an estrogen receptor (ER) antagonist implicate ER participation in MRP2 and MDR1 regulation. GNT but not EE increased the expression of ERß, the most abundant form in human intestine and in Caco-2 cells, which could lead in turn to increased sensitivity to estrogens. We conclude that specific concentrations of estrogens can confer resistance against cytotoxicity in Caco-2 cells, due in part to positive modulation of ABC transporters involved in extrusion of their toxic substrates. Although extrapolation of these results to the in vivo situation must be cautiously done, the data could explain tentatively the cytoprotective role of estrogens against chemical injury in intestine.

Modulation of biotransformation systems and ABC transporters by benznidazole in rats.

The effect of antichagasic benznidazole (BZL; 100 mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransformation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltransferase (UGT1A), glutathione S-transferases (alpha glutathione S-transferase [GST-α], GST-µ, and GST-π), multidrug-resistance-associated protein 2 (Mrp2), and P glycoprotein (P-gp) in liver, small intestine, and kidney was estimated by Western blotting. Increases in hepatic CYP3A (30%) and GST-µ (40%) and in intestinal GST-α (72% in jejunum and 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observed without changes in kidney. P-gp and Mrp2 were also increased by BZL in jejunum (170% and 120%, respectively). In ileum, only P-gp was increased by BZL (50%). The activities of GST, P-gp, and Mrp2 correlated well with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5 mg/kg body weight) administered intraduodenally was faster (295%) and the area under the concentration-time curve (AUC) was lower (41%) for BZL-pretreated rats than for controls. The biliary excretion of BZL was higher (60%) in the BZL group, and urinary excretion of BZL did not show differences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransformation enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadministered drugs that are substrates of these systems, including BZL itself.

Regulation of biotransformation systems and ABC transporters by benznidazole in HepG2 cells: involvement of pregnane X-receptor.

BACKGROUND: Benznidazole (BZL) is the only antichagasic drug available in most endemic countries. Its effect on the expression and activity of drug-metabolizing and transporter proteins has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: Expression and activity of P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP2), Cytochrome P450 3A4 (CYP3A4), and Glutathione S-transferase (GST) were evaluated in HepG2 cells after treatment with BZL. Expression was estimated by immunoblotting and real time PCR. P-gp and MRP2 activities were estimated using model substrates rhodamine 123 and dinitrophenyl-S-glutathione (DNP-SG), respectively. CYP3A4 and GST activities were evaluated through their abilities to convert proluciferin into luciferin and 1-chloro-2,4-dinitrobenzene into DNP-SG, respectively. BZL (200 µM) increased the expression (protein and mRNA) of P-gp, MRP2, CYP3A4, and GSTπ class. A concomitant enhancement of activity was observed for all these proteins, except for CYP3A4, which exhibited a decreased activity. To elucidate if pregnane X receptor (PXR) mediates BZL response, its expression was knocked down with a specific siRNA. In this condition, the effect of BZL on P-gp, MRP2, CYP3A4, and GSTπ protein up-regulation was completely abolished. Consistent with this, BZL was able to activate PXR, as detected by reporter gene assay. Additional studies, using transporter inhibitors and P-gp-knock down cells, demonstrated that P-gp is involved in BZL extrusion. Pre-treatment of HepG2 cells with BZL increased its own efflux, as a consequence of P-gp up-regulation. CONCLUSIONS/SIGNIFICANCE: Modifications in the activity of biotransformation and transport systems by BZL may alter the pharmacokinetics and efficiency of drugs that are substrates of these systems, including BZL itself.

Prevención de hepatitis A mediante vacunación en Uruguay (2005-2010) / Preventing hepatitis A through immunization in Uruguay

Introducción: la hepatitis A es un problema sanitario en países en vías de desarrollo. Objetivos: describir estrategias para controlar hepatitis A mediante vacunación. Describir evolución de número de casos, tasas y brotes entre 2005-2010. Material y método: estudio descriptivo retrospectivo 2005-2010. Se analizan:-Estrategias utilizadas por Ministerio de Salud Pública (MSP) para controlar hepatitis A mediante vacunación: bloqueo de brotes; población de riesgo e inclusión universal de vacuna. Descripción: número de casos de hepatitis A en Uruguay y Artigas; número de brotes en ciudades, barrios, familia y otros; número de casos/edad.Resultados: se registraron 21 brotes. Se aplicaron 16.715 primeras dosis y 11.354 segundas dosis. Entre el 10 de setiembre de 2007 y el 26 de noviembre de 2007, el MSP y el Ministerio de Desarrollo Social realizaron una campaña de vacunacióndirigida a niños de entre 1 y 5 años del subsector público. Docentes y estudiantes de Medicina de la Universidad de la República apoyaron y difundieron la campaña. Se aplicaron44.716 primeras dosis y 25.095 segundas dosis. En 2008 se incluye la vacuna en dos dosis en el esquema de vacunación, obligatoria y gratuita. La tasa país disminuyó de 69,6 a 2,7 entre 2005 y 2010 (p<0,05); en el Departamento de Artigas de 780,57/100.000 en 2005 a 0,66 en 2010 (p<0,05). Durante 2005-2008 ocurrieron 22 brotes en ciudades; en 2010 se registraron escasos brotes intrafamiliares y en escuelas. El descenso de número de casos de 2005 a 2010 fue significativo en todas las edades. Comentario: del 2005 al 2010 se produjo una disminución significativay sostenida del número de brotes y tasas de hepatitis A en Uruguay.

Introduction: Hepatitis A is a health problem in developing countries.Objectives: To describe strategies to control Hepatitis A through immunization. To describe the evolutionof the number of cases, rates and outbreaks from 2005 through 2010.Method: Retrospective descriptive study covering the 2005-2010 period of time. The following was analysed:- Strategies used by the Ministry of Public Health to control Hepatitis A through immunization: blocking ofoutbreaks; population at risk; universal immunization. - Description: Number of cases of Hepatitis A in Uruguay, and in the department of Artigas; number ofoutbreaks in cities, neighborhoods, families and other; number of cases/age.Results: 21 outbreaks were recorded. 16.715 first doses and 11.354 second doses were applied. From 10 September, 2007 through 26 November, 2007 the Ministry of Public Health and the Ministry of Social Developmentconducted an immunization campaign aimed at children in the public sub-sector who were between 1 and 5 years old. Professors at the School of Medicine of the University of the Republic and medical students supported and contributed to the dissemination of the campaign. 44.716 first doses and 25.095 second doses were applied. In 2008 the two dose vaccine is included in the mandatory and free immunization plan. The national rate dropped from 69.6 to 2.7 from 2005 through 2010 (p<0,05); and in the department ofArtigas from 780.57/100.000 in 2005 to 0.66 in 2010 (p<0,05). Between 2005 and 2008 there were 22 outbreaks in cities, in 2010 few intra-family outbreaks and in schools were registered. The decrease in the number of cases from 2005 through 2010 was significant for all ages. Comment: A significant and sustained decrease ofHepatitis A outbreaks and rates was evidenced in Uruguay from 2005 through 2010.

Introdução: a hepatite A é um problema sanitário nos países em vias de desenvolvimento. Objetivos: Descrever estratégias para controlar hepatiteAporvacinação. Descrever a evolução do número de casos, taxas e surtos entre 2005-2010. Material e método: estudo descritivo retrospectivo 2005-2010. Foram analisadas: - Estratégias utilizadas pelo Ministério de Salud Pública (MSP) para controlar a hepatite A por vacinação: bloqueio de surtos; população de risco; inclusão universal da vacina. - Descrição: número de casos de hepatite A no Uruguai e especificamente no departamento de Artigas; número de surtos en cidades, bairros, famílias e outros; número de casos/idade. Resultados: foram registrados 21 surtos. Foram aplicadas16.715 primeiras doses e 11.354 segundas doses. Entre o dia 10 de setembro de 2007 e o dia 26 de novembro de 2007, o MSP e o Ministério de Desenvolvimento Social realizaram una campanha de vacinação dirigida acrianças entre 1 e 5 anos do setor público. Docentes e estudantesde Medicina da Universidad de la República apoiaram e difundiram a campanha. Foram aplicadas 44.716 primeiras doses e 25.095 segundas doses. Em 2008 a vacina em duas doses foi incluída ao esquema de vacinação, obrigatória e gratuita. La taxa país diminuiu de 69,6 a 2,7 entre 2005 y 2010 (p<0,05); no departamento de Artigas passou de 780,57/100.000 em 2005 a 0,66 em 2010 (p<0,05). Durante 2005-2008 foram registrados 22 surtos em cidades; em 2010, foram registrados poucos surtos intrafamiliarese em escolas. A redução do número de casos de 2005 a 2010 foi significativa em todas as idades. Comentário: de 2005 a 2010 observou-se uma diminuição significativa e mantida do número de surtos e das taxas de hepatite A no Uruguai.