A synthetic lethality screen reveals ING5 as a genetic dependency of catalytically dead Set1A/COMPASS in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/complex of proteins associated with SET1 histone methyltransferase (Set1A/COMPASS) in mouse ESCs does not impair their viability or ability to self-renew; however, it leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF (monocytic leukaemia zinc finger protein/monocytic leukaemia zinc finger protein-related factor) and HBO1 (HAT bound to ORC1) acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mouse ESCs decreases the fitness of these cells, and the simultaneous loss of ING5 and in Set1AΔSET leads to up-regulation of differentiation-associated genes. Taken together, our results point toward Set1A/COMPASS and ING5 as potential coregulators of the self-renewal and differentiation status of ESCs.

Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb and DNA methylation.

The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.

JNK activation and translocation to mitochondria mediates mitochondrial dysfunction and cell death induced by VDAC opening and sorafenib in hepatocarcinoma cells.

The multikinase inhibitor sorafenib, and opening of voltage dependent anion channels (VDAC) by the erastin-like compound X1 promotes oxidative stress and mitochondrial dysfunction in hepatocarcinoma cells. Here, we hypothesized that X1 and sorafenib induce mitochondrial dysfunction by increasing reactive oxygen species (ROS) formation and activating c-Jun N-terminal kinases (JNKs), leading to translocation of activated JNK to mitochondria. Both X1 and sorafenib increased production of ROS and activated JNK. X1 and sorafenib caused a drop in mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, after 60 min. Mitochondrial depolarization after X1 and sorafenib occurred in parallel with JNK activation, increased superoxide (O2•-) production, decreased basal and oligomycin sensitive respiration, and decreased maximal respiratory capacity. Increased production of O2•- after X1 or sorafenib was abrogated by JNK inhibition and antioxidants. S3QEL 2, a specific inhibitor of site IIIQo, at Complex III, prevented depolarization induced by X1. JNK inhibition by JNK inhibitors VIII and SP600125 also prevented mitochondrial depolarization. After X1, activated JNK translocated to mitochondria as assessed by proximity ligation assays. Tat-Sab KIM1, a peptide selectively preventing the binding of JNK to the outer mitochondrial membrane protein Sab, blocked the depolarization induced by X1 and sorafenib. X1 promoted cell death mostly by necroptosis that was partially prevented by JNK inhibition. These results indicate that JNK activation and translocation to mitochondria is a common mechanism of mitochondrial dysfunction induced by both VDAC opening and sorafenib.

Pediatric Logistic Organ Dysfunction-2 (Pelod-2) score as a model for predicting mortality in pediatric burn injury.

Multiple organ dysfunction syndrome (MODS) is an important cause of mortality in burn injury. Pediatric Organ Logistic Dysfunction (PELOD)-2 score as a descriptive scoring system for organ dysfunction has been highly predictive of mortality in children with suspected infection, but its usefulness for burn patients is unknown. All pediatric burn patients hospitalized in Cipto Mangunkusumo Hospital (CMH) in Jakarta, Indonesia, from January 2012 to January 2017 were studied. Gender, age, nutritional status, burn characteristics, total body surface area burned (%TBSA), depth of burn, inhalation injury, time interval to resuscitation and surgery, day one ABSI and PELOD-2 score, and mortality as outcome were recorded. Bivariate and multivariate analysis logistic regressions were done to generate a mortality prediction model. Mortality rate among subjects was 20.3%. Bivariate analysis showed that extensive %TBSA, depth of burn, presence of inhalation injury, PELOD-2 score and ABSI score in pediatric burn patients were significantly associated with mortality (p<0.001). In multivariate analysis, only PELOD-2 score was independently associated with mortality. PELOD-2 score mortality prediction rate was far lower than actual mortality. Mortality rate by the new model was close to the actual mortality rate. Our new combined model could be used to calculate probability of death based on day 1 PELOD-2 score in pediatric burn patients.

La mort après brûlure est fréquemment due à une défaillance multiviscérale. Le score PELOD 2 s'est révélé efficace dans la prédiction de mortalité de l'enfant septique mais n'a pas été évalué chez l'enfant brûlé. Tous les enfants brûlés hospitalisés dans l'hôpital Cipto Mangunkusumo de Djakarta (Indonésie) entre janvier 2012 et janvier 2017 ont été évalués. L'âge, le sexe, l'état nutritionnel, la surface brûlée, sa profondeur, l'existence d'une inhalation de fumées, les délais jusqu'à la réanimation et la chirurgie, ABSI et PELOD 2 à J1 et mortalité (20,3%) ont été colligés. Des analyse bivariée puis multivariée ont été réalisées afin de construire un modèle prédictif de mortalité. PELOD 2 comme ABSI étaient de bons prédicteurs de mortalité, les prédictions de PELOD 2 s'avérant très optimistes. Toutefois, seul PELOD 2 apparaissait comme un prédicteur indépendant de mortalité. Un modèle combinant les mortalités prédites par ABSI et PELOD 2 s'est avéré mieux corrélé à la mortalité observée. Il pourrait être utilisé chez les enfants brûlés.

Impact of smoking on imiquimod response in patients with vulval intraepithelial neoplasia.

BACKGROUND: Vulval intraepithelial neoplasia (VIN) is a precancerous condition that may progress to invasive malignancy. VIN is associated with human papillomavirus (HPV) infection in most cases, and with inflammatory skin disorders in a smaller proportion of patients. Treatment of VIN has traditionally been surgical excision; however, topical treatments, including imiquimod cream, are becoming increasingly used. Patient factors influencing response to imiquimod therapy, in particular smoking, have not yet been published. AIM: To assess the impact of smoking and other patient characteristics that may influence the treatment response to topical imiquimod for VIN. METHODS: This was a retrospective cohort study of 46 women treated with topical imiquimod for VIN in a single centre dermatology unit from January 2011 to July 2017. RESULTS: Complete clinical resolution of VIN was observed in 28 of 46 patients (61%), but was significantly reduced in the smoking cohort. CONCLUSIONS: Smoking may impair response to imiquimod for VIN, and should be considered when discussing VIN treatment options with patients.

Burden of Paediatric Sepsis in a Tertiary Centre from a Developing Country / Carga de sepsis pediátrica en un centro terciario de un país en desarrollo

ABSTRACT Background: Sepsis causes significant paediatric morbidity and mortality in developing countries. This paper describes the outcome of paediatric sepsis in the University Hospital of the West Indies (UHWI), Jamaica, using administrative data from hospital records from 2010 to 2014. Objective: To provide baseline data on the burden and outcome of paediatric sepsis in a tertiary institution in Jamaica between 2010 and 2014 so as to embark on initiatives to build capacity to provide care in order to decrease the burden and improve the outcomes from sepsis in children. Methods: Data were abstracted from electronic discharge summaries of children hospitalized at the UHWI, a tertiary centre that accepts referrals for newborns and children with complex disorders. The medical records of children aged 0-16 years who were hospitalized with a diagnosis of sepsis, septicaemia and neonatal sepsis were reviewed, using the World Health Organization's International Classification of Diseases, 10th Revision (ICD-10). Demographic and outcome data were extracted. Results: Among 7011 children aged 0-16 years who were admitted, sepsis accounted for 801 hospitalizations in 782 children with a male to female ratio of 1.27 to 1. Neonates comprised 86% (n = 691), of which 36.2% (n = 250) were preterm. The median duration of hospitalization was 10 days (range: 0-366 days; interquartile range: 7-19 days). The sepsis-attributable mortality rate was 10.1% (n = 70) in neonates and was higher in preterm versus term neonates (18.4%, n = 46/250 versus 6.0%, n = 26/434, respectively). The annual crude mortality rate for paediatric sepsis was 9-11% (12 per 1000 paediatric hospitalizations). Conclusion: Sepsis accounted for a high number of admissions and consumed significant resources as evidenced by the long duration of hospitalization. The mortality rate for paediatric sepsis was high, especially in newborns. Targeted interventions are needed to reduce the sepsis-attributable burden and improve outcomes established by the Global Sepsis Alliance and United Nations' Sustainable Development Goals.

RESUMEN Anteceentes: La sepsis causa morbilidad y mortalidad pediátricas significativas en los países en desarrollo. Este artículo describe el resultado de la sepsis pediátrica en el Hospital Universitario de West Indies (HUWI), Jamaica, utilizando datos administrativos de registros hospitalarios de 2010 a 2014. Objetivo: Proporcionar datos de referencia sobre la carga y el resultado de la sepsis pediátrica en una institución terciaria en Jamaica entre 2010 y 2014, a fin de emprender iniciativas para aumentar la capacidad de prestar atención a disminuir la carga y mejorar los resultados de la sepsis en los niños. Métodos: Se obtuvieron datos de resúmenes de descargas electrónicas de niños hospitalizados en el HUWI, un centro terciario que acepta remisiones de recién nacidos y niños con trastornos complejos. Se realizó una revisión de las historias clínicas de niños de 0 a 16 años de edad que fueron hospitalizados con un diagnóstico de sepsis, septicemia y sepsis neonatal, utilizando la Clasificación Internacional de Enfermedades de la Organización Mundial de la Salud, 10th Revisión (ICD-10). Se obtuvieron datos en relación con la demografía y los resultados clínicos. Resultados: Entre los 7011 niños de 0 a 16 años de edad que fueron ingresados, la sepsis fue la causa de 801 hospitalizaciones en 782 niños, en una proporción varón-mujer de 1.27 a 1. Los neonatos abarcaron el 86% (n = 691), el 36.2% de los cuales (n = 250) eran prematuros. La duración promedio de la hospitalización fue de 10 días (rango: 0-366 días; rango intercuartil: 7-19 días). La tasa de mortalidad atribuible a la sepsis fue de 10.1% (n = 70) en los neonatos, y fue mayor entre los prematuros en comparación con los neonatos a término (18.4%, n = 46/250 frente a 6.0%, n = 26/434, respectivamente). La tasa de mortalidad bruta anual para la sepsis pediátrica fue de 9 a 11% (12 por cada 1000 hospitalizaciones pediátricas). Conclusión: La sepsis representó un elevado número de ingresos y consumió recursos significativos como se evidenció en la larga duración de la hospitalización. La tasa de mortalidad por sepsis pediátrica fue alta, especialmente en recién nacidos. Se necesitan intervenciones específicas para reducir la carga atribuible a la sepsis y mejorar los resultados establecidos por la Alianza Mundial contra la Sepsis y los Objetivos del Desarrollo Sostenible de las Naciones Unidas.

A cytoplasmic COMPASS is necessary for cell survival and triple-negative breast cancer pathogenesis by regulating metabolism.

Mutations and translocations within the COMPASS (complex of proteins associated with Set1) family of histone lysine methyltransferases are associated with a large number of human diseases, including cancer. Here we report that SET1B/COMPASS, which is essential for cell survival, surprisingly has a cytoplasmic variant. SET1B, but not its SET domain, is critical for maintaining cell viability, indicating a novel catalytic-independent role of SET1B/COMPASS. Loss of SET1B or its unique cytoplasmic-interacting protein, BOD1, leads to up-regulation of expression of numerous genes modulating fatty acid metabolism, including ADIPOR1 (adiponectin receptor 1), COX7C, SDC4, and COQ7 Our detailed molecular studies identify ADIPOR1 signaling, which is inactivated in both obesity and human cancers, as a key target of SET1B/COMPASS. Collectively, our study reveals a cytoplasmic function for a member of the COMPASS family, which could be harnessed for therapeutic regulation of signaling in human diseases, including cancer.

Histone H3K4 methylation-dependent and -independent functions of Set1A/COMPASS in embryonic stem cell self-renewal and differentiation.

Of the six members of the COMPASS (complex of proteins associated with Set1) family of histone H3 Lys4 (H3K4) methyltransferases identified in mammals, Set1A has been shown to be essential for early embryonic development and the maintenance of embryonic stem cell (ESC) self-renewal. Like its familial relatives, Set1A possesses a catalytic SET domain responsible for histone H3K4 methylation. Whether H3K4 methylation by Set1A/COMPASS is required for ESC maintenance and during differentiation has not yet been addressed. Here, we generated ESCs harboring the deletion of the SET domain of Set1A (Set1AΔSET); surprisingly, the Set1A SET domain is dispensable for ESC proliferation and self-renewal. The removal of the Set1A SET domain does not diminish bulk H3K4 methylation in ESCs; instead, only a subset of genomic loci exhibited reduction in H3K4me3 in Set1AΔSET cells, suggesting a role for Set1A independent of its catalytic domain in ESC self-renewal. However, Set1AΔSET ESCs are unable to undergo normal differentiation, indicating the importance of Set1A-dependent H3K4 methylation during differentiation. Our data also indicate that during differentiation, Set1A but not Mll2 functions as the H3K4 methylase on bivalent genes and is required for their expression, supporting a model for transcriptional switch between Mll2 and Set1A during the self-renewing-to-differentiation transition. Together, our study implicates a critical role for Set1A catalytic methyltransferase activity in regulating ESC differentiation but not self-renewal and suggests the existence of context-specific H3K4 methylation that regulates transcriptional outputs during ESC pluripotency.

SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.

MLL3/MLL4/COMPASS Family on Epigenetic Regulation of Enhancer Function and Cancer.

During development, precise spatiotemporal patterns of gene expression are coordinately controlled by cis-regulatory modules known as enhancers. Their crucial role in development helped spur numerous studies aiming to elucidate the functional properties of enhancers within their physiological and disease contexts. In recent years, the role of enhancer malfunction in tissue-specific tumorigenesis is increasingly investigated. Here, we direct our focus to two primary players in enhancer regulation and their role in cancer pathogenesis: MLL3 and MLL4, members of the COMPASS family of histone H3 lysine 4 (H3K4) methyltransferases, and their complex-specific subunit UTX, a histone H3 lysine 27 (H3K27) demethylase. We review the most recent evidence on the underlying roles of MLL3/MLL4 and UTX in cancer and highlight key outstanding questions to help drive future research and contribute to our fundamental understanding of cancer and facilitate identification of therapeutic opportunities.

Chikungunya in Jamaica: Public health effects and clinical features in children / Chikungunya en Jamaica ­ Efectos en la Salud Pública y Características Clínicas en Niños

Chikungunya virus (CHIKV) entered the Caribbean for the first time in 2013 and Jamaica experienced its maiden epidemic with Chikungunya Fever in 2014. We aimed to describe the public health effects and describe the clinical features in children and adolescents in Jamaica. METHODS: This study reviewed the public health effects of the illness in Jamaica by reviewing available data sources and the clinical features in 210 children and adolescents meeting the case definition at two hospitals, Bustamante Hospital for Children and University Hospital of the West Indies between August 23 and October 31, 2014 by chart review. Descriptive analyses and comparisons between groups using the Mann-Whitney U test were performed with SPSS version 22. RESULTS: The majority of households were affected by the illness which caused widespread absenteeism from school and work, loss of productivity and economic losses estimated at 60 billion dollars. The health sector was impacted by increased numbers seen in clinics and emergency departments, increased need for bed space and pharmaceuticals. Ninety-nine per cent of the children were febrile with a median maximal temperature of 102.4 F. Ninety-three per cent had household contacts of 0­20 persons. In addition to fever, maculopapular rash and joint pains, infants six months and younger presented with irritability and groaning (p = 0.00) and those between six months and six years presented with febrile seizures (p = 0.00). Neurologic involvement was noted in 24%. Apart from anaemia, few had other laboratory derangements. Few had severe organ dysfunction and there were no deaths. CONCLUSION: The Chikungunya Fever epidemic had significant public health and economic impact in Jamaica. In children, there were characteristic presentations in neonates and young infants and in children six months to six years. Neurologic involvement was common but other organ dysfunction was rare. These findings underscore the need to prevent further epidemics and the quest for a vaccine.(AU)

Antecedentes: El virus de Chikungunya (CHIKV) entró en el Caribe por primera vez en 2013, y Jamaica experimentó su primera epidemia de fiebre de Chikungunya en 2014. Nos propusimos como objetivo describir sus efectos en la salud pública y describir sus características clínicas en niños y adolescentes en Jamaica. Métodos: Este estudio examinó los efectos de la enfermedad en la salud pública en Jamaica. El examen se realizó mediante la revisión de fuentes de datos disponibles y las características clínicas en 210 niños y adolescentes que cumplían con la definición del caso en dos hospitales ­ Hospital Pediátrico Bustamante y el Hospital Universitario de West Indies ­ entre el 23 de agosto y 31 de octubre de 201, según las historias clínicas. Se realizaron análisis descriptivos y comparaciones entre los grupos usando la prueba U de Mann-Whitney y la versión 22 de SPSS Resultados: La mayoría de los hogares fueron afectados por la enfermedad, que causó un ausentismo generalizado en escuelas y trabajos, pérdida de productividad, y pérdidas económicas estimadas en 60 billones de dólares. El sector de la salud fue afectado por un aumento del número de personas atendidas en clínicas y departamentos de urgencias, y una mayor necesidad de camas en los hospitales y productos farmacéuticos. Noventa y nueve por ciento de los niños presentaron un estado febril con una temperatura mediana máxima de 102.4 F. Un noventa y tres por ciento tuvo contactos domésticos de personas de 0­20. Además de fiebre, erupciones maculopapulares y dolores en las articulaciones, los niños de seis meses o menos edad, presentaron irritabilidad y quejidos (p = 0.00), y aquellos entre seis meses y seis años de edad presentaron convulsiones febriles (p = 0.00). Se observó compromiso neurológico en el 24%. Aparte de anemia, algunos tenían otros trastornos de laboratorio. Otros presentaban una disfunción orgánica severa y no hubo muertes. Conclusión: La epidemia de fiebre de Chikungunya tuvo un impacto significativo tanto en la salud pública como en la economía de Jamaica. Los niños presentaron manifestaciones características, observadas tanto en recién nacidos y bebés pequeños como en niños de seis meses a seis años. El compromiso neurológico fue común, pero cualquiera otra disfunción orgánica fue rara. Estos hallazgos subrayan la necesidad de hacer más por evitar las epidemias y buscar la solución de una vacuna.(AU)

Transient Increased Calcium and Calcitriol Requirements After Discontinuation of Human Synthetic Parathyroid Hormone 1-34 (hPTH 1-34) Replacement Therapy in Hypoparathyroidism.

Synthetic human PTH 1-34 (hPTH 1-34) replacement therapy in hypoparathyroidism maintains eucalcemia and converts quiescent bone to high-turnover bone. However, the skeletal and metabolic effects of drug discontinuation have not been reported. Nine subjects with hypoparathyroidism received subcutaneous injections of hPTH 1-34 two to three times daily for 19.8 to 61.3 months and then transitioned back to calcium and calcitriol. Biochemistries and bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) were assessed at baseline, while on treatment, and at follow-up 3 to 12 months after drug discontinuation. Two subjects developed hypocalcemia when hPTH 1-34 was abruptly discontinued. Thus, to avoid hypocalcemia, subjects were slowly weaned from hPTH 1-34 over several weeks. When hPTH 1-34 was stopped, subjects were requiring two to three times pretreatment doses of calcitriol and calcium to maintain blood calcium levels. Doses were gradually reduced over many weeks until calcium levels were stable on doses similar to baseline. Bone-specific alkaline phosphatase (BSAP), N-telopeptide (NTX), and osteocalcin (OC) increased significantly with hPTH 1-34; at follow-up, BSAP and NTX had returned to baseline while OC was still slightly elevated. During treatment, BMD was unchanged at the hip and lateral spine but declined at the anterior-posterior (AP) spine, radius, and total body. During weaning, BMD increased, with the hip and lateral spine exceeding pre-hPTH 1-34 values and the whole body returning to baseline. AP spine was increased non-significantly compared to baseline at follow-up. hPTH 1-34 must be gradually weaned in hypoparathyroid patients with high doses of oral medications given to avoid hypocalcemia. The transient increased requirements accompanied by increased BMD after long-term hPTH 1-34 therapy suggest a reversal of the expanded remodeling space favoring bone formation as the skeleton returns to a low-turnover state, reminiscent of the hungry bone syndrome. Further study and close monitoring is required to ensure safe transition to conventional therapy and to elucidate the physiological mechanism of this phenomenon.

Evaluation of Ginkgo biloba extract as an activator of human glucocorticoid receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba, which is one of the most frequently used herbal medicines, is commonly used in the management of several conditions, including memory impairment. Previously, it was reported to decrease the expression of peripheral benzodiazepine receptor and the biosynthesis of glucocorticoids, thereby regulating glucocorticoid levels. However, it is not known whether Ginkgo biloba extract regulates the function of the glucocorticoid receptor. AIM OF THE STUDY: We determined whether Ginkgo biloba extract and several of its chemical constituents affect the activity of human glucocorticoid receptor (hGR). MATERIALS AND METHODS: A hGR-dependent reporter gene assay was conducted in HepG2 human hepatocellular carcinoma cells and hGR target gene expression assays were performed in primary cultures of human hepatocytes. RESULTS: Multiple lots and concentrations of the extract and several of its chemical constituents (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide) did not increase hGR activity, as assessed by a cell-based luciferase reporter gene assay. The extract did not influence the expression of hGR target genes, including tyrosine aminotransferase (hTAT), constitutive androstane receptor (hCAR), or pregnane X receptor (hPXR), in primary cultures of human hepatocytes. Moreover, hGR antagonism by mifepristone (also known as RU486) did not attenuate the extent of induction of hCAR- and hPXR-regulated target genes CYP2B6 and CYP3A4 by Ginkgo biloba extract. CONCLUSION: Ginkgo biloba extract, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide are not activators of hGR. Furthermore, the extract does not influence the hGR-hCAR or the hGR-hPXR signaling pathway in primary cultures of human hepatocytes.

Species-dependent and receptor-selective action of bilobalide on the function of constitutive androstane receptor and pregnane X receptor.

Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6ß-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16α-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

Differential effect of meclizine on the activity of human pregnane X receptor and constitutive androstane receptor.

Conflicting data exist as to whether meclizine is an activator of human pregnane X receptor (hPXR). Therefore, we conducted a detailed, systematic investigation to determine whether meclizine affects hPXR activity by performing a cell-based reporter gene assay, a time-resolved fluorescence resonance energy transfer competitive ligand-binding assay, a mammalian two-hybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. In pregnane X receptor (PXR)-transfected HepG2 cells, meclizine activated hPXR to a greater extent than rat PXR. It bound to hPXR ligand-binding domain and recruited steroid receptor coactivator-1 to the receptor. Consistent with its hPXR agonism, meclizine increased hPXR target gene expression (CYP3A4) in human hepatocytes. However, it did not increase but decreased testosterone 6ß-hydroxylation, suggesting inhibition of CYP3A catalytic activity. Meclizine has also been reported to be an inverse agonist and antagonist of human constitutive androstane receptor (hCAR). Therefore, given that certain tissues (e.g., liver) express both hPXR and hCAR and that various genes are cross-regulated by them, we quantified the expression of a hCAR- and hPXR-regulated gene (CYP2B6) in cultured human hepatocytes treated with meclizine. This drug did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist-mediated increase in CYP2B6 mRNA and CYP2B6-catalyzed bupropion hydroxylation levels. These observations reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a hCAR reporter gene assay and mammalian two-hybrid assay. In conclusion, meclizine is a hPXR agonist, and it does not act as a hCAR inverse agonist or antagonist in cultured human hepatocytes.

Human pregnane X receptor agonism by Ginkgo biloba extract: assessment of the role of individual ginkgolides.

Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6ß-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.

Antibiotic resistance among pathogens causing disease in Jamaican children with HIV/AIDS.

OBJECTIVE: There are limited data regarding the antimicrobial resistance patterns of pathogens in children with HIV/AIDS from developing countries. We aimed to determine the prevalence and antibiotic susceptibility patterns of bacterial pathogens causing urinary tract infections (UTIs) and sepsis in a cohort of 219 HIV-infected Jamaican children. METHODS: This cross-sectional study examined clinical and microbiological data for children enrolled in the Kingston Paediatric/Perinatal HIV/AIDS programme from September 1, 2002 to May 31, 2007. Cases were defined as physician-diagnosed, laboratory confirmed UTIs and sepsis based on Centers for Disease Control and Prevention (CDC) criteria. Only isolates from urine, blood and sterile sites were considered. RESULTS: Forty-four patients (20.1%) accounted for 74 episodes of UTIs and sepsis. Mean number of infections was 1.7 +/- 1.3 per patient. There were 31 males (70.5%) and mean age at time of infection was 5.6 +/- 4.7 years. Bacterial infections comprised cystitis (n = 52, 70.3%), bacterial pneumonia (n = 15, 20.3%), meningitis (n = 4, 5.4%), septicaemia (n = .2, 2.7%) and bone infection (n = 1, 1.4%). Among 52 UTIs, 39 were caused by a single organism. The most common UTI isolates included Escherichia coli (n = 21, 53.8%) and Enterobacter spp (n = 5, 12.8%). Among 22 cases of sepsis, isolates included Streptococcus pneumoniae (n = 8, 36.4%) and coagulase negative Staphylococcus (n = 6, 27.3%). All E coli isolates at two of three clinical sites were resistant to cotrimoxazole. There were 79.7% (n = 51) of infectious episodes with a cotrimoxazole-resistant organism occurring among those on cotrimoxazole prophylaxis. CONCLUSIONS: Escherichia coli was the most frequent bacterial isolate. Cotrimoxazole is a poor choice for empiric treatment of sepsis and UTIs in this clinical setting.

Effectiveness of antiretroviral therapy in treating paediatric HIV/AIDS in Jamaica.

BACKGROUND AND PURPOSE: Paediatric HIV/AIDS remains a significant challenge in developing countries. We describe the effectiveness of interventions in HIV-infected children attending Paediatric Infectious Diseases Clinics in Jamaica. METHODS: One hundred and ninety-seven HIV-infected children were followed prospectively in multicentre ambulatory clinics between September 1, 2002 and August 31, 2005, in the Kingston Paediatric and Perinatal HIV/AIDS Programme, Jamaica, and their outcomes described. RESULTS: Median follow-up was 23 child-months (interquartile range [IQR] 12-31) with 12 children (6.0%) lost to follow-up and deaths (n=13) occurred at 4.64 per 100 child-years of follow-up. Median age was 5.0 years (IQR 2.2-8.1) and 32.1% had Centers for Disease Control and Prevention (CDC) category C disease at enrollment; 62% were ever on antiretroviral therapy (ART) with median duration of 15.4 months (IQR 5.5-25.5); 85% initiated ART with zidovudine/lamivudine/nevirapine. Mean weight-for-height 0.13 +/- 1.02 (mean difference -1.71 [95% Confidence interval (CI) -2.73, -0.69]; p = 0.001) and body mass index-for-age 0.05 +/- 1.11 (mean difference -1.11, [CI -1.79, -0.43]; p = 0.002); z scores increased after 24 months on ART; however, children remained stunted. Reductions in the incidence of hospitalizations (mean diff 30.95, [CI 3.12, 58.78]; p = 0.03) and in episodes of pneumonia, culture-positive sepsis and tuberculosis occurred in those on ART. CONCLUSIONS: A successfully implemented ambulatory model for paediatric HIV care in Jamaica has improved the quality of life and survival of HIV-infected children.

Acute lead poisoning associated with backyard lead smelting in Jamaica / Envenenamiento agudo con plomo asociado con la fundición casera de plomo en Jamaica

Long-term backyard smelting of lead in a district known as Mona Commons, Kingston, Jamaica, has produced lead burdens as high as 30 000 mg/kg in soils near to the smelter, and indoor dust loadings of 373 microg/f2 in the residents' home. The blood lead levels (BPb) of 107 children from the district were in the range 2.2-202 microg/dL. Fifty-nine per cent of these had BPb levels above 10 microg/dL and the population mean was an unacceptably high 25.1 microg/dL. The highest levels were observed for five siblings, two of whom presented with lead encephalopathy. This severe chronic exposure to lead was exacerbated by a significant history of pica, and chronic nutritional anaemia. Chelation therapy significantly reduced the BPb levels but due to lead storage in other organs, the values after several months were still higher than desirable. This study emphasizes the importance of reducing the exposure of children to lead.