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Abnormal pain has recently been estimated to affect â¼50 million adults each year within the United States. With many treatment options for abnormal pain, such as opioid analgesics, carrying numerous deleterious side effects, research into safer and more effective treatment options is crucial. To help elucidate the mechanisms controlling nociceptive sensitivity, the Drosophila melanogaster larval nociception model has been used to characterize well-conserved pathways through the use of genetic modification and/or injury to alter the sensitivity of experimental animals. Mammalian models have provided evidence of ß-catenin signaling involvement in neuropathic pain development. By capitalizing on the conserved nature of ß-catenin functions in the fruit fly, here we describe a role for Armadillo, the fly homolog to mammalian ß-catenin, in regulating baseline sensitivity in the primary nociceptor of the fly, in the absence of injury, using under- and over-expression of Armadillo in a cell-specific manner. Underexpression of Armadillo resulted in hyposensitivity, while overexpression of wild-type Armadillo or expression of a degradation-resistant Armadillo resulted in hypersensitivity. Neither underexpression nor overexpression of Armadillo resulted in observed dendritic morphological changes that could contribute to behavioral phenotypes observed. These results showed that focused manipulation of Armadillo expression within the nociceptors is sufficient to modulate baseline response in the nociceptors to a noxious stimulus and that these changes are not shown to be associated with a morphogenetic effect.
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Proteínas del Dominio Armadillo , Proteínas de Drosophila , Nocicepción , Factores de Transcripción , beta Catenina , Animales , Analgésicos Opioides , beta Catenina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Dolor , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismoRESUMEN
Chronic pain is a debilitating condition affecting millions of people worldwide, and an improved understanding of the pathophysiology of chronic pain is urgently needed. Nociceptors are the sensory neurons that alert the nervous system to potentially harmful stimuli such as mechanical pressure or noxious thermal temperature. When an injury occurs, the nociceptive threshold for pain is reduced and an increased pain signal is produced. This process is called nociceptive sensitization. This sensitization normally subsides after the injury is healed. However, dysregulation can occur which results in sensitization that persists after the injury has healed. This process is thought to perpetuate chronic pain. The Hedgehog (Hh) signaling pathway has been previously implicated in nociceptive sensitization in response to injury in Drosophila melanogaster. Downstream of Hh signaling, the Bone Morphogenetic Protein (BMP) pathway has also been shown to be necessary for this process. Here, we describe a role for nuclear components of BMP's signaling pathway in the formation of injury-induced nociceptive sensitization. Brinker (Brk), and Schnurri (Shn) were suppressed in nociceptors using an RNA-interference (RNAi) "knockdown" approach. Knockdown of Brk resulted in hypersensitivity in the absence of injury, indicating that it normally acts to suppress nociceptive sensitivity. Animals in which transcriptional activator Shn was knocked down in nociceptors failed to develop normal allodynia after ultraviolet irradiation injury, indicating that Shn normally acts to promote hypersensitivity after injury. These results indicate that Brk-related transcription regulators play a crucial role in the formation of nociceptive sensitization and may therefore represent valuable new targets for pain-relieving medications.
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Drosophila melanogaster/metabolismo , Nocicepción/fisiología , Dolor/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Proteínas Morfogenéticas Óseas/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/genética , Proteínas Hedgehog/metabolismo , Nociceptores/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismoRESUMEN
Recognition of influenza A virus (IAV) by the innate immune system triggers pathways that restrict viral replication, activate innate immune cells, and regulate adaptive immunity. However, excessive innate immune activation can exaggerate disease. The pathways promoting excessive activation are incompletely understood, with limited experimental models to investigate the mechanisms driving influenza virus-induced inflammation in humans. Interferon regulatory factor 5 (IRF5) is a transcription factor that plays important roles in the induction of cytokines after viral sensing. In an in vivo model of IAV infection, IRF5 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine production, specifically reducing cytokine-producing myeloid cell populations in Irf5-/- mice but not impacting type 1 interferon (IFN) production or virus replication. Using cytometry by time of flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses by myeloid cells to IAV, we employed human-induced pluripotent stem cells (hIPSCs) with biallelic mutations in IRF5, demonstrating for the first time that induced pluripotent stem cell-derived dendritic cells (iPS-DCs) with biallelic mutations can be used to investigate the regulation of human virus-induced immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or macrophages, corresponded with reduced virus-induced inflammatory cytokine production, with IRF5 acting downstream of Toll-like receptor 7 (TLR7) and, possibly, retinoic acid-inducible gene I (RIG-I) after viral sensing. Thus, IRF5 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in mice and humans and drives immune-mediated viral pathogenesis independently of type 1 IFN and virus replication.IMPORTANCE The inflammatory response to influenza A virus (IAV) participates in infection control but contributes to disease severity. After viral detection, intracellular pathways are activated, initiating cytokine production, but these pathways are incompletely understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN and virus replication, implying that IRF5 could be specifically targeted to treat influenza virus-induced inflammation. We show for the first time that human iPSC technology can be exploited in genetic studies of virus-induced immune responses. Using this technology, we deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired influenza virus-induced cytokine production and revealed that IRF5 acts downstream of Toll-like receptor 7 and possibly retinoic acid-inducible gene I. Our data demonstrate the importance of IRF5 in influenza virus-induced inflammation, suggesting that genetic variation in the IRF5 gene may influence host susceptibility to viral diseases.
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Células Madre Pluripotentes Inducidas/inmunología , Virus de la Influenza A/inmunología , Factores Reguladores del Interferón/metabolismo , Inmunidad Adaptativa/fisiología , Animales , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/fisiología , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Factores Reguladores del Interferón/inmunología , Interferón Tipo I/metabolismo , Pulmón/virología , Macrófagos/virología , Ratones , Infecciones por Orthomyxoviridae/virología , Replicación Viral/fisiologíaRESUMEN
Although great progress has been made to advance the scientific understanding of oil spills, tools for integrated assessment modeling of the long-term impacts on ecosystems, socioeconomics and human health are lacking. The objective of this study was to develop a conceptual framework that could be used to answer stakeholder questions about oil spill impacts and to identify knowledge gaps and future integration priorities. The framework was initially separated into four knowledge domains (ocean environment, biological ecosystems, socioeconomics, and human health) whose interactions were explored by gathering stakeholder questions through public engagement, assimilating expert input about existing models, and consolidating information through a system dynamics approach. This synthesis resulted in a causal loop diagram from which the interconnectivity of the system could be visualized. Results of this analysis indicate that the system naturally separates into two tiers, ocean environment and biological ecosystems versus socioeconomics and human health. As a result, ocean environment and ecosystem models could be used to provide input to explore human health and socioeconomic variables in hypothetical scenarios. At decadal-plus time scales, the analysis emphasized that human domains influence the natural domains through changes in oil-spill related laws and regulations. Although data gaps were identified in all four model domains, the socioeconomics and human health domains are the least established. Considerable future work is needed to address research gaps and to create fully coupled quantitative integrative assessment models that can be used in strategic decision-making that will optimize recoveries from future large oil spills.
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Brucellosis is a zoonosis caused by bacteria of the Brucella genus. In ruminants, brucellosis causes abortion, followed by chronic infection and secretion of bacteria in milk. In humans, it usually presents as flu-like symptoms, with serious complications if untreated. Epidemiological studies have only recently established that brucellosis can also cause pregnancy complications in women, but the pathogenic mechanisms are unknown. Pioneering studies in ruminants showed that Brucella infect trophoblasts and then colonise the placenta where they grow to high density. A recent study showed that the main zoonotic Brucella species can infect human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT). In this work, we show that Brucella papionis (associated with stillbirth in primates) also infects human trophoblasts. However, it replicates actively in CTB, whereas its replication is very restricted within EVT. We also observed alteration of several trophoblastic functions upon infection by B. papionis or Brucella melitensis (the most prevalent species in human brucellosis). Infection altered the production of hormones, the ability of CTB to form syncytiotrophoblasts, and the invasion capacity of EVT. We also found that infection can spread between different types of trophoblasts. These findings constitute a new step in understanding how Brucella infection causes adverse pregnancy outcomes.
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Brucella melitensis/patogenicidad , Brucella/patogenicidad , Brucelosis/microbiología , Trofoblastos/microbiología , Brucelosis/patología , Femenino , Humanos , Embarazo , Trofoblastos/patologíaRESUMEN
1. Quizartinib absorption, metabolism and excretion were characterized in six healthy men receiving a single oral dose of 60 mg (≈100 µCi) of [14C]-quizartinib. Blood, plasma, urine and faeces were collected ≤336 h postdose. 2. Four hours postdose, maximum mean ± SD blood radioactivity concentrations were 296 ± 67.4 ng equivalents/g. A mean ± SD of 1.64 ± 0.482% and 76.3 ± 6.23% of the dose was recovered in urine and faeces, respectively, within 336 h postdose. 3. Radio-detector high-performance liquid chromatography (radio-HPLC) and liquid chromatography-mass spectrometry (LC-MS) showed two main radioactive peaks in plasma, unchanged quizartinib and mono-oxidative metabolite, AC886. Five additional metabolites in plasma were identified by LC-MS, but low levels prevented radio-HPLC detection. Although unchanged quizartinib was the main radioactive component in faeces (mean, 4.0% of administered dose), 15 metabolites representing a mean of 1.0-3.5% of administered dose were found. Quizartinib was predominantly metabolized by phase I biotransformations (oxidation, reduction, dealkylation, deamination, hydrolysis and combinations thereof). 4. This study indicated that quizartinib was rapidly and orally bioavailable, extensively metabolized, with AC886 as the major circulating metabolite, and predominantly eliminated in faeces. Quizartinib was well tolerated in the subjects.
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Benzotiazoles/metabolismo , Inhibidores Enzimáticos/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Compuestos de Fenilurea/metabolismo , Benzotiazoles/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Humanos , Masculino , Compuestos de Fenilurea/uso terapéuticoRESUMEN
The intestinal mucosa forms the first line of defense against infections mediated by enteric pathogens such as salmonellae. Here we exploited intestinal "organoids" (iHOs) generated from human induced pluripotent stem cells (hIPSCs) to explore the interaction of Salmonella enterica serovar Typhimurium with iHOs. Imaging and RNA sequencing were used to analyze these interactions, and clear changes in transcriptional signatures were detected, including altered patterns of cytokine expression after the exposure of iHOs to bacteria. S. Typhimurium microinjected into the lumen of iHOs was able to invade the epithelial barrier, with many bacteria residing within Salmonella-containing vacuoles. An S. Typhimurium invA mutant defective in the Salmonella pathogenicity island 1 invasion apparatus was less capable of invading the iHO epithelium. Hence, we provide evidence that hIPSC-derived organoids are a promising model of the intestinal epithelium for assessing interactions with enteric pathogens.
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Interacciones Huésped-Patógeno , Células Madre Pluripotentes Inducidas/microbiología , Células Madre Pluripotentes Inducidas/fisiología , Organoides/microbiología , Organoides/fisiología , Salmonella typhimurium/crecimiento & desarrollo , Proteínas Bacterianas/genética , Citocinas/metabolismo , Células Epiteliales/microbiología , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Modelos Teóricos , Imagen Óptica , Vacuolas/microbiologíaRESUMEN
Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.
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Diferenciación Celular/genética , Endopeptidasas/genética , Hematopoyesis/genética , Linfocitos/metabolismo , Animales , Recuento de Células Sanguíneas , Western Blotting , Endopeptidasas/metabolismo , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Genotipo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/metabolismo , Linfocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores , Proteína p53 Supresora de Tumor/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.
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Proteínas de Transporte de Anión/fisiología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/patología , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Proteínas de Transporte de Anión/deficiencia , Proteínas de Transporte de Anión/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Cruzamientos Genéticos , Marcación de Gen , Inmunofenotipificación , Subgrupos Linfocitarios/metabolismo , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Lisofosfolípidos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Esfingosina/genética , Esfingosina/metabolismoRESUMEN
A transposon-based, genomewide mutagenesis screen exploiting the killing activity of a lytic ViII bacteriophage was used to identify Salmonella enterica serovar Typhi genes that contribute to Vi polysaccharide capsule expression. Genes enriched in the screen included those within the viaB locus (tviABCDE and vexABCDE) as well as oxyR, barA/sirA, and yrfF, which have not previously been associated with Vi expression. The role of these genes in Vi expression was confirmed by constructing defined null mutant derivatives of S. Typhi, and these were negative for Vi expression as determined by agglutination assays with Vi-specific sera or susceptibility to Vi-targeting bacteriophages. Transcriptome analysis confirmed a reduction in expression from the viaB locus in these S. Typhi mutant derivatives and defined regulatory networks associated with Vi expression.
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Genes Bacterianos , Polisacáridos Bacterianos/biosíntesis , Salmonella typhi/genética , Salmonella typhi/metabolismo , Elementos Transponibles de ADN/genética , Perfilación de la Expresión Génica , Genoma Bacteriano , Mutagénesis , Mutación , Fagos de Salmonella/fisiologíaRESUMEN
MicroRNAs (miRNAs) are small noncoding molecules that control gene expression posttranscriptionally, with microRNA-155 (miR-155) one of the first to be implicated in immune regulation. Here, we show that miR-155-deficient mice are less able to eradicate a mucosal Citrobacter rodentium infection than wild-type C57BL/6 mice. miR-155-deficient mice exhibited prolonged colonization associated with a higher C. rodentium burden in gastrointestinal tissue and spread into systemic tissues. Germinal center formation and humoral immune responses against C. rodentium were severely impaired in infected miR-155-deficient mice. A similarly susceptible phenotype was observed in µMT mice reconstituted with miR-155-deficient B cells, indicating that miR-155 is required intrinsically for mediating protection against this predominantly luminal bacterial pathogen.
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Citrobacter rodentium , Infecciones por Enterobacteriaceae/microbiología , Predisposición Genética a la Enfermedad , MicroARNs/metabolismo , Animales , Colitis/genética , Colitis/microbiología , Colitis/patología , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.
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Toxinas Bacterianas , Proteínas F-Box , Humanos , Neutrófilos/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Exotoxinas/toxicidad , Inflamación/genética , Inflamación/metabolismo , Macrófagos/metabolismo , Muerte Celular/genética , Leucocidinas/farmacología , Leucocidinas/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMEN
The discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell culture model, (ii) two 3D cell culture models, and (iii) iPSC derived macrophages in place of primary macrophages or cell lines, to determine whether they offer improved approaches to anti-leishmanial drug discovery and development. Using a Leishmania major amastigote-macrophage assay the activities of standard drugs were investigated to show the effect of changing parameters in these assays. We determined that drug activity was reduced by media perfusion (EC50 values for amphotericin B shifted from 54 (51-57) nM in the static system to 70 (61-75) nM under media perfusion; EC50 values for miltefosine shifted from 12 (11-15) µM in the static system to 30 (26-34) µM under media perfusion) (mean and 95% confidence intervals), with corresponding reduced drug accumulation by macrophages. In the 3D cell culture model there was a significant difference in the EC50 values of amphotericin B but not miltefosine (EC50 values for amphotericin B were 34.9 (31.4-38.6) nM in the 2D and 52.3 (46.6-58.7) nM in 3D; EC50 values for miltefosine were 5.0 (4.9-5.2) µM in 2D and 5.9 (5.5-6.2) µM in 3D (mean and 95% confidence intervals). Finally, in experiments using iPSC derived macrophages infected with Leishmania, reported here for the first time, we observed a higher level of intracellular infection in iPSC derived macrophages compared to the other macrophage types for four different species of Leishmania studied. For L. major with an initial infection ratio of 0.5 parasites per host cell the percentage infection level of the macrophages after 72 h was 11.3% ± 1.5%, 46.0% ± 1.4%, 66.4% ± 3.5% and 75.1% ± 2.4% (average ± SD) for the four cells types, THP1 a human monocytic cell line, mouse bone marrow macrophages (MBMMs), human bone marrow macrophages (HBMMs) and iPSC derived macrophages respectively. Despite the higher infection levels, drug activity in iPSC derived macrophages was similar to that in other macrophage types, for example, amphotericin B EC50 values were 35.9 (33.4-38.5), 33.5 (31.5-36.5), 33.6 (30.5-not calculated (NC)) and 46.4 (45.8-47.2) nM in iPSC, MBMMs, HBMMs and THP1 cells respectively (mean and 95% confidence intervals). We conclude that increasing the complexity of cellular assays does impact upon anti-leishmanial drug activities but not sufficiently to replace the current model used in HTS/HCS assays in drug discovery programmes. The impact of media perfusion on drug activities and the use of iPSC macrophages do, however, deserve further investigation.
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CONTEXT: The University of Pittsburgh School of Dental Medicine incorporates a voluntary student peer-tutoring program as 1 resource available to pre-doctoral students. It uses peer-tutoring in didactic and preclinical courses in order to provide additional help to struggling students. OBJECTIVE: The goal of this article is to describe an initial program assessment using data collected between 2015 and 2017. In addition to assessing the program, this report also investigates the benefits of the program to the tutors. DESIGN: Data were collected using surveys from tutors (N = 133) and tutees (N = 115), as well as reflective journals written by the tutors (response rate varies across instruments and questions). Responses to the surveys were analyzed using quantitative analysis, and content analysis was completed for coding the open-ended short responses and reflective journals. RESULTS: Results show tutors' increased preparedness at the end of the term to provide tutoring, an increase in communication and teaching skills due to participation in the program, and other cited benefits to the tutor. CONCLUSIONS: In addition to academic credit, tutors benefit from reviewing the course materials and practicing skills relevant to their future careers. Tutees provided overwhelmingly positive feedback on the tutors' strengths and effectiveness, the program in general, and the extent to which tutoring helped their performance in the class. Based on the initial program assessment, the program provides benefits to both tutors and tutees; the tutors gain a valuable experience impacting them both academically and personally.
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Estudiantes de Medicina , Comunicación , Humanos , Grupo Paritario , Evaluación de Programas y Proyectos de Salud , Encuestas y Cuestionarios , EnseñanzaRESUMEN
Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.
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Dinoprostona/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Subunidad beta del Receptor de Interleucina-10/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Salmonella typhimurium/metabolismo , Transducción de Señal/genética , Diferenciación Celular/genética , Células Cultivadas , Dinoprostona/antagonistas & inhibidores , Femenino , Técnicas de Inactivación de Genes , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/genética , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Macrófagos/efectos de los fármacos , Mutación , Fosforilación/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1ß secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1ß levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1ß secretion after PVL exposure and controls S. aureus lung burdens.
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Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Exotoxinas/antagonistas & inhibidores , Células Madre Pluripotentes Inducidas/citología , Leucocidinas/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/efectos de los fármacos , Staphylococcus aureus , Animales , Antígeno CD11b/inmunología , Sistemas CRISPR-Cas , Diferenciación Celular , Células Cultivadas , Exotoxinas/deficiencia , Técnicas de Sustitución del Gen , Humanos , Interleucina-1beta/metabolismo , Antígenos Comunes de Leucocito/fisiología , Pulmón/inmunología , Pulmón/microbiología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Fragmentos de Péptidos/inmunología , Neumonía Estafilocócica/inmunología , Subunidades de Proteína , Receptor de Anafilatoxina C5a/deficiencia , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/fisiología , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/fisiologíaRESUMEN
The Gulf of Mexico (GoM) region is prone to disasters, including recurrent oil spills, hurricanes, floods, industrial accidents, harmful algal blooms, and the current COVID-19 pandemic. The GoM and other regions of the U.S. lack sufficient baseline health information to identify, attribute, mitigate, and facilitate prevention of major health effects of disasters. Developing capacity to assess adverse human health consequences of future disasters requires establishment of a comprehensive, sustained community health observing system, similar to the extensive and well-established environmental observing systems. We propose a system that combines six levels of health data domains, beginning with three existing, national surveys and studies plus three new nested, longitudinal cohort studies. The latter are the unique and most important parts of the system and are focused on the coastal regions of the five GoM States. A statistically representative sample of participants is proposed for the new cohort studies, stratified to ensure proportional inclusion of urban and rural populations and with additional recruitment as necessary to enroll participants from particularly vulnerable or under-represented groups. Secondary data sources such as syndromic surveillance systems, electronic health records, national community surveys, environmental exposure databases, social media, and remote sensing will inform and augment the collection of primary data. Primary data sources will include participant-provided information via questionnaires, clinical measures of mental and physical health, acquisition of biological specimens, and wearable health monitoring devices. A suite of biomarkers may be derived from biological specimens for use in health assessments, including calculation of allostatic load, a measure of cumulative stress. The framework also addresses data management and sharing, participant retention, and system governance. The observing system is designed to continue indefinitely to ensure that essential pre-, during-, and post-disaster health data are collected and maintained. It could also provide a model/vehicle for effective health observation related to infectious disease pandemics such as COVID-19. To our knowledge, there is no comprehensive, disaster-focused health observing system such as the one proposed here currently in existence or planned elsewhere. Significant strengths of the GoM Community Health Observing System (CHOS) are its longitudinal cohorts and ability to adapt rapidly as needs arise and new technologies develop.
Asunto(s)
COVID-19 , Desastres , Golfo de México , Humanos , Estudios Longitudinales , Pandemias , Salud Pública , SARS-CoV-2RESUMEN
Embryonic stem (ES) cells are susceptible to genetic manipulation and retain the potential to differentiate into diverse cell types, which are factors that make them potentially attractive cells for studying host-pathogen interactions. Murine ES cells were found to be susceptible to invasion by Salmonella enterica serovar Typhimurium and Shigella flexneri and to the formation of attaching and effacing lesions by enteropathogenic Escherichia coli. S. enterica serovar Typhimurium and S. flexneri cell entry was dependent on the Salmonella pathogenicity island 1 and Shigella mxi/spa type III secretion systems, respectively. Microscopy studies indicated that both S. enterica serovar Typhimurium and S. flexneri were located in intracellular niches in ES cells that were similar to the niches occupied in differentiated cells. ES cells were eventually killed following bacterial invasion, but no evidence of activation of classical caspase-associated apoptotic or innate immune pathways was found. To demonstrate the potential of mutant ES cells, we employed an ES cell line defective in cholesterol synthesis and found that the mutant cells were less susceptible to infection by Salmonella and Shigella than the parental ES cells. Thus, we highlighted the practical use of genetically modified ES cells for studying microbe-host interactions.
Asunto(s)
Células Madre Embrionarias/microbiología , Escherichia coli Enteropatógena/fisiología , Salmonella typhimurium/fisiología , Shigella flexneri/fisiología , Animales , Muerte Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/ultraestructura , Regulación de la Expresión Génica/fisiología , Interacciones Huésped-Patógeno , RatonesRESUMEN
The rapid development of genomics and other "-omics" approaches has significantly impacted how we have investigated host-pathogen interactions since the turn of the millennium. Technologies such as next-generation sequencing, stem cell biology, and high-throughput proteomics have transformed the scale and sensitivity with which we interrogate biological samples. These approaches are impacting experimental design in the laboratory and transforming clinical management in health care systems. Here, we review this area from the perspective of research on bacterial pathogens.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Biología de Sistemas/métodos , Animales , Manejo de la Enfermedad , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Proteómica/métodos , Células MadreRESUMEN
A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.