RESUMO
Kaposi sarcoma (KS) is the most common cancer in people living with HIV (PLWH) in many countries where KS-associated herpesvirus is endemic. Treatment has changed little in 20 years, but the disease presentation has. This prospective cohort study enrolled 122 human immunodeficiency virus (HIV) positive KS patients between 2017 and 2019 in Malawi. Participants were treated with bleomycin, vincristine and combination antiretroviral therapy, the local standard of care. One-year overall survival was 61%, and progression-free survival was 58%. The 48-week complete response rate was 35%. RNAseq (n = 78) differentiated two types of KS lesions, those with marked endothelial characteristics and those enriched in inflammatory transcripts. This suggests that different KS lesions are in different disease states consistent with the known heterogeneous clinical response to treatment. In contrast to earlier cohorts, the plasma HIV viral load of KS patients in our study was highly variable. A total of 25% of participants had no detectable HIV; all had detectable KSHV viral load. Our study affirms that many KS cases today develop in PLWH with well-controlled HIV infection and that different KS lesions have differing molecular compositions. Further studies are needed to develop predictive biomarkers for this disease.
Assuntos
Infecções por HIV , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Sarcoma de Kaposi/complicações , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/epidemiologia , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , HIV , Estudos Prospectivos , Herpesvirus Humano 8/fisiologiaRESUMO
Female Fischer 344 (F344) rats were exposed to N-nitrosodiphenylamine (NDPA) by dietary feed at concentrations of 0, 250, 1000, 2000, 3000 or 4000 ppm for 5 days, 2, 4 and 13 weeks duration. Endpoints evaluated included clinical observations, body weights, urinary bladder weights, blood NDPA, gross pathology and urinary bladder histopathology. There were no NDPA exposure-related clinical signs of toxicity. The mean body weight decreased 3% to 5% compared with the control in the 4000 ppm group during study weeks 2 through to 13. Statistically significant increases in urinary bladder weight were observed as early as after 5 days exposure and were concentration dependent at ≥ 3000 ppm. NDPA-related urinary bladder microscopic alterations consisted of mixed cell infiltrates, increased mitosis, increased necrosis of epithelial cells, diffuse and/or nodular transitional epithelial hyperplasia and squamous metaplasia of transitional epithelium. These changes affected only rats exposed to NDPA concentrations ≥ 2000 ppm. Blood NDPA concentrations were negligible in animals exposed to ≤ 1000 ppm and ranged from 0.12 to 0.19 µg ml(-1) in rats of the ≥ 2000 ppm groups at the 5 days and 2 weeks time points. A no observable adverse effect level (NOAEL) of 1000 ppm NDPA (60 mg kg(-1) day(-1) ) was selected based on the absence of urinary bladder histopathology.
Assuntos
Nitrosaminas/toxicidade , Testes de Toxicidade Subcrônica , Bexiga Urinária/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Feminino , Masculino , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Bexiga Urinária/metabolismo , Bexiga Urinária/patologiaRESUMO
Male Fischer 344 (F344) rats were exposed to bromobenzene (BB) for 5 days and 2, 4 and 13 weeks. BB was administered by gavage (corn oil vehicle) at doses of 0, 25, 100, 200, 300 and 400 mg kg(-1) per day. Endpoints evaluated included clinical observations, body weights, liver weights, serum chemistry, blood BB, gross pathology and liver histopathology. There were no BB exposure-related clinical signs of toxicity. Mean body weight decreased by 5-10% compared with control in the 400 mg kg(-1) per day group. Liver weight increases were dose- and exposure time-related and statistically significant at ≥25 mg kg(-1) per day. Incidence and severity of centrilobular cytoplasmic alteration and hepatocyte hypertrophy were related to dose and exposure time. At early time points (5 days and 2 weeks), centrilobular inflammation, including granulomatous areas, and necrotic and anisokaryocytic hepatocytes were observed in rats of the two highest BB dose groups. Blood BB concentrations increased linearly with dose and at 13 weeks ranged from 8 to 136 µg ml(-1) (25-400 mg kg(-1) per day). In conclusion, rats administered BB doses up to 400 mg kg(-1) per day for up to 13 weeks had mild liver effects. A NOAEL of 200 mg kg(-1) per day was selected based on the statistically significant incidence of hepatocyte hypertrophy at doses ≥ 400 mg kg(-1) per day.
Assuntos
Bromobenzenos/toxicidade , Fígado/efeitos dos fármacos , Testes de Toxicidade Subcrônica , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Óleo de Milho/química , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344RESUMO
Variants of concern (VOC) in SARS-CoV-2 refer to viruses whose viral genomes differ from the ancestor virus by ≥3 single-nucleotide variants (SNVs) and that show the potential for higher transmissibility and/or worse clinical progression. VOC have the potential to disrupt ongoing public health measures and vaccine efforts. Still, too little is known regarding how frequently new viral variants emerge and under what circumstances. We report a study to determine the degree of SARS-CoV-2 sequence evolution in 94 patients and to estimate the frequency at which highly diverse variants emerge. Two cases accumulated ≥9 SNVs over a 2-week period and one case accumulated 23 SNVs over 3 weeks, including three nonsynonymous mutations in the spike protein (D138H, E554D, D614G). The remainder of the infected patients did not show signs of intra-host evolution. We estimate that in as much as 2% of hospitalized COVID-19 cases, variants with multiple mutations in the spike glycoprotein emerge in as little as 1 month of persistent intra-host virus replication. This suggests the continued local emergence of variants with multiple nonsynonymous SNVs, even in patients without overt immune deficiency. Surveillance by sequencing for (i) viremic COVID-19 patients, (ii) patients suspected of reinfection, and (iii) patients with diminished immune function may offer broad public health benefits. IMPORTANCE New SARS-CoV-2 variants can potentially disrupt ongoing public health measures and vaccine efforts. Still, little is known regarding how frequently new viral variants emerge and under what circumstances. Based on this study, we estimate that in hospitalized COVID-19 cases, variants with multiple mutations may emerge locally in as little as 1 month, even in patients without overt immune deficiency. Surveillance by sequencing for continuously shedding patients, patients suspected of reinfection, and patients with diminished immune function may offer broad public health benefits.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Reinfecção , Família , Mutação , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Female F344 rats were exposed to 4,4'-methylenebis(N,N'-dimethyl)aniline (MDA) by dietary feed at concentrations of 0, 50, 200, 375, 500, or 750 ppm for 5 d, 2 wk, 4 wk, and 13 wk duration. Endpoints evaluated included clinical observations, body weights, thyroid weights, serum thyroid hormones, blood MDA, gross pathology, and thyroid histopathology. There were no MDA exposure-related clinical signs of toxicity. Mean body weight decreased 5% compared to control in the 750 ppm group during study wk 6 through 13. Serum TSH increased and serum T4 and T3 levels decreased with increasing feed concentrations of MDA and time of exposure. Thyroid weight increases were both concentration- and exposure time-dependent and statistically significant at ≥375 ppm. Incidence and severity of decreased colloid, follicular cell hypertrophy and follicular cell hyperplasia were also related to MDA concentration and exposure time. A no-observed-adverse-effect level (NOAEL) of 200 ppm was selected based on the statistically significant increase in incidence of follicular cell hyperplasia at concentrations ≥375 ppm.
Assuntos
Compostos de Anilina/toxicidade , Indicadores e Reagentes/toxicidade , Glândula Tireoide/efeitos dos fármacos , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/sangue , Compostos de Anilina/farmacocinética , Animais , Carcinógenos/administração & dosagem , Carcinógenos/análise , Carcinógenos/farmacocinética , Carcinógenos/toxicidade , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/sangue , Poluentes Ambientais/farmacocinética , Poluentes Ambientais/toxicidade , Feminino , Hiperplasia , Hipertrofia , Indicadores e Reagentes/administração & dosagem , Indicadores e Reagentes/análise , Indicadores e Reagentes/farmacocinética , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/crescimento & desenvolvimento , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/induzido quimicamente , Tireotropina/sangue , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
Male Sprague-Dawley rats were exposed to 1,2,4-tribromobenzene (TBB) by gavage for 5 days, 2, 4, and 13 weeks at 0, 2.5, 5, 10, 25, or 75 mg/kg per d. There were no TBB exposure-related clinical signs of toxicity or changes in body weight. Liver weight increases were dose and exposure time related and statistically significant at ≥10 mg/kg per d. Incidence and severity of centrilobular cytoplasmic alteration and hepatocyte hypertrophy were dose and time related. The 75 mg/kg per d group had minimally increased mitoses within hepatocytes (5 days only). Hepatocyte vacuolation was observed (13 weeks) and was considered TBB exposure related at ≥25 mg/kg per d. Concentrations of blood TBB increased linearly with dose and at 13 weeks, ranged from 0.5 to 17 µg/mL (2.5-75 mg/kg per d). In conclusion, rats administered TBB doses of 10-75 mg/kg per d for 13 weeks had mild liver effects. A no observed adverse effect level of 5 mg/kg per d was selected based on the statistically significant incidence of hepatocyte hypertrophy at doses ≥10 mg/kg per d.
Assuntos
Bromobenzenos/toxicidade , Fígado/efeitos dos fármacos , Animais , Bromobenzenos/sangue , Bromobenzenos/farmacocinética , Fígado/patologia , Masculino , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade SubcrônicaRESUMO
Male F344 rats were exposed to hydrazobenzene (HZB) by dietary feed at concentrations of 0, 5, 20, 80, 200, or 300 ppm for 5 days, 2 weeks, 4 weeks, or 13 weeks duration. End points evaluated included clinical observations, body weights, liver weights, serum chemistry, blood HZB, gross pathology, and liver histopathology. There were no HZB exposure-related clinical signs of toxicity. During study weeks 8 through 13, body weight means in rats of the 300 ppm group were 6% lower compared to control rat means. Serum alkaline phosphatase concentrations were decreased in rats of the 300 ppm group at all time points. Relative (to body weight) liver weight increases were observed in rats of the 200 and 300 ppm groups following 5 days (300 ppm only), 2 weeks, 4 weeks, and 13 weeks of exposure. Following 13 weeks of exposure, microscopic findings in the liver were observed only in rats of the 200 and 300 ppm groups and consisted of hypertrophy, macrovesiculation, eosinophilic granular cytoplasm, and bile duct duplication. Blood HZB concentrations ranged from 0.002 to 0.006 µg/mL in rats of the 200 or 300 ppm groups. A no observed effect level of 80 ppm (4.80 mg/kg per d) was selected based on the observation of microscopic hepatocyte alterations at ≥200 ppm HZB.
Assuntos
Carcinógenos Ambientais/toxicidade , Fenil-Hidrazinas/toxicidade , Testes de Toxicidade/métodos , Administração Oral , Fosfatase Alcalina/sangue , Animais , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/patologia , Peso Corporal/efeitos dos fármacos , Carcinógenos Ambientais/farmacocinética , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fenil-Hidrazinas/farmacocinética , Ratos , Ratos Endogâmicos F344 , Fatores de TempoRESUMO
Kaposi sarcoma (KS)-associated herpesvirus (KSHV/HHV-8) was first sequenced from the body cavity (BC) lymphoma cell line, BC-1, in 1996. Few other KSHV genomes have been reported. Our knowledge of sequence variation for this virus remains spotty. This study reports additional genomes from historical US patient samples and from African KS biopsies. It describes an assay that spans regions of the virus that cannot be covered by short read sequencing. These include the terminal repeats, the LANA repeats, and the origins of replication. A phylogenetic analysis, based on 107 genomes, identified three distinct clades; one containing isolates from USA/Europe/Japan collected in the 1990s and two of Sub-Saharan Africa isolates collected since 2010. This analysis indicates that the KSHV strains circulating today differ from the isolates collected at the height of the AIDS epidemic. This analysis helps experimental designs and potential vaccine studies.
Assuntos
Genoma Viral , Genômica , Genótipo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/classificação , Herpesvirus Humano 8/genética , Sarcoma de Kaposi/virologia , Adulto , Linhagem Celular , Feminino , Regulação Viral da Expressão Gênica , Genômica/métodos , Infecções por Herpesviridae/diagnóstico , Herpesvirus Humano 8/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Filogenia , Recombinação GenéticaRESUMO
BACKGROUND: Early in the pandemic, transmission risk from asymptomatic infection was unclear, making it imperative to monitor infection in workplace settings. Further, data on SARS-CoV-2 seroprevalence within university populations has been limited. METHODS: We performed a longitudinal study of University research employees on campus July-December 2020. We conducted questionnaires on COVID-19 risk factors, RT-PCR testing, and SARS-CoV-2 serology using an in-house spike RBD assay, laboratory-based Spike NTD assay, and standard nucleocapsid platform assay. We estimated prevalence and cumulative incidence of seroconversion with 95% confidence intervals using the inverse of the Kaplan-Meier estimator. RESULTS: 910 individuals were included in this analysis. At baseline, 6.2% (95% CI 4.29-8.19) were seropositive using the spike RBD assay; four (0.4%) were seropositive using the nucleocapsid assay, and 44 (4.8%) using the Spike NTD assay. Cumulative incidence was 3.61% (95% CI: 2.04-5.16). Six asymptomatic individuals had positive RT-PCR results. CONCLUSIONS: Prevalence and incidence of SARS-CoV-2 infections were low; however, differences in target antigens of serological tests provided different estimates. Future research on appropriate methods of serological testing in unvaccinated and vaccinated populations is needed. Frequent RT-PCR testing of asymptomatic individuals is required to detect acute infections, and repeated serosurveys are beneficial for monitoring subclinical infection.
Assuntos
COVID-19 , COVID-19/diagnóstico , COVID-19/epidemiologia , Humanos , Estudos Longitudinais , Pandemias , Estudos Prospectivos , SARS-CoV-2/genética , Estudos SoroepidemiológicosRESUMO
Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 µM for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKKα). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-κB), leading to inhibition of P53 function.
Assuntos
Arsenitos/toxicidade , Células Epiteliais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/genética , Urotélio/efeitos dos fármacos , Adulto , Arsenitos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/efeitos dos fármacos , Ureter/citologia , Ureter/efeitos dos fármacos , Urotélio/citologia , Adulto JovemRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constantly evolving. Prior studies focused on high-case-density locations, such as the northern and western metropolitan areas of the United States. This study demonstrates continued SARS-CoV-2 evolution in a suburban southern region of the United States by high-density amplicon sequencing of symptomatic cases. 57% of strains carry the spike D614G variant, which is associated with higher genome copy numbers, and its prevalence expands with time. Four strains carry a deletion in a predicted stem loop of the 3' UTR. The data are consistent with community spread within local populations and the larger continental United States. The data instill confidence in current testing sensitivity and validate "testing by sequencing" as an option to uncover cases, particularly nonstandard coronavirus disease 2019 (COVID-19) clinical presentations. This study contributes to the understanding of COVID-19 through an extensive set of genomes from a non-urban setting and informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the United States.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Glicoproteína da Espícula de Coronavírus/genética , COVID-19 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pandemias , Filogenia , SARS-CoV-2 , Estados UnidosRESUMO
Chloroquine (CQ), a drug that has been used extensively for the prevention and treatment of malaria, is currently considered safe for use during pregnancy. However, CQ has been shown to disrupt steroid homeostasis in adult rats and similar compounds, such as quinacrine, inhibit steroid production in the Leydig cell in vitro. To explore the effect of in utero CQ exposure on fetal male sexual development, pregnant Sprague-Dawley rats were given a daily dose of either water or chloroquine diphosphate from GD 16-18 by oral gavage. Chloroquine was administered as 200 mg/kg CQ base on GD 16, followed by two maintenance doses of 100 mg/kg CQ base on GD 16 and 18. Three days of CQ treatment resulted in reduced maternal and fetal weight on GD 19 and increased necrosis and steatosis in the maternal liver. Fetal livers also displayed mild lipid accumulation. Maternal serum progesterone was increased after CQ administration. Fetal testes testosterone, however, was significantly decreased. Examination of the fetal testes revealed significant alterations in vascularization and seminiferous tubule development after short-term CQ treatment. Anogenital distance was not altered. Microarray and RT-PCR showed down-regulation of several genes associated with cholesterol transport and steroid synthesis in the fetal testes. This study indicates that CQ inhibits testosterone synthesis and normal testis development in the rat fetus at human relevant doses.
Assuntos
Cloroquina/análogos & derivados , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Desenvolvimento Sexual/efeitos dos fármacos , Animais , Cloroquina/toxicidade , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Desenvolvimento Fetal/fisiologia , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos Sprague-Dawley , Desenvolvimento Sexual/fisiologia , Testículo/efeitos dos fármacos , Testículo/embriologiaRESUMO
Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (<0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1beta, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways.
Assuntos
Ácido Hipocloroso/toxicidade , Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Ácido Hipocloroso/administração & dosagem , Imunidade Inata/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Análise em Microsséries , Fator 2 Relacionado a NF-E2/metabolismo , Oxidantes/administração & dosagem , Ubiquitinação/efeitos dos fármacosRESUMO
The primary function of toll-like receptor 8 (TLR-8) is the detection of viruses and other microbial pathogens. Recent evidence suggests that TLR-8 also senses host microRNAs (miRNAs) and implicate TLR-8 in autoimmune disorders. This study examined the interaction between miR-1294 and TLR-8. We first performed a BLAST search to identify miRNAs with the same sequences as two core motifs of miR-1294. Next, we examined NFkB activation induced by the binding of miR-1294 mimic to endosomal TLR-8. HEK-Blue™ hTLR-8 cells (Invivogen), a HEK293 cell line co-transfected with human TLR-8 gene, were incubated with miR-1294 mimic. A TLR-8 agonist ssRNA40, was used as a positive control. Using the same experimental set up, we also examined the effects of miR-1294 and its two core motifs (Integrated DNA Technologies) on IL-8, IL-1ß, and TNFα. Data were analyzed using t-test or one-way ANOVA and Dunnets post-hoc test. Using miRCarta we identified 29 other mature human miRNAs or their precursors which contain the same core motifs as miR-1294. Our data show that miR-1294 activates NFkB in cells expressing TLR-8 (p < 0.05). miR-1294, and its core motifs induce expression of IL-8, IL-1ß, and TNFα via TLR8 activation (p < 0.05). This constitutes a novel mechanism by which endosomal TLR-8 senses host miRNAs resulting in the release of pro-inflammatory cytokines and thus potentially contributing to autoimmune disorders.
Assuntos
Citocinas/imunologia , MicroRNAs/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Receptor 8 Toll-Like/imunologia , Citocinas/genética , Células HEK293 , Humanos , MicroRNAs/genética , NF-kappa B/genética , Transdução de Sinais/genética , Receptor 8 Toll-Like/genéticaRESUMO
BACKGROUND: Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V2O5-induced bronchitis. METHODS: Normal human lung fibroblasts were exposed to V2O5 in a time course experiment. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Selected genes that were significantly changed in the microarray experiment were validated by RT-PCR. RESULTS: V2O5 altered more than 1,400 genes, of which ~300 were induced while >1,100 genes were suppressed. Gene ontology categories (GO) categories unique to induced genes included inflammatory response and immune response, while GO categories unique to suppressed genes included ubiquitin cycle and cell cycle. A dozen genes were validated by RT-PCR, including growth factors (HBEGF, VEGF, CTGF), chemokines (IL8, CXCL9, CXCL10), oxidative stress response genes (SOD2, PIPOX, OXR1), and DNA-binding proteins (GAS1, STAT1). CONCLUSION: Our study identified a variety of genes that could play pivotal roles in inflammation, fibrosis and repair during V2O5-induced bronchitis. The induction of genes that mediate inflammation and immune responses, as well as suppression of genes involved in growth arrest appear to be important to the lung fibrotic reaction to V2O5.
Assuntos
Bronquite/induzido quimicamente , Bronquite/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Doenças Profissionais/metabolismo , Proteoma/metabolismo , Compostos de Vanádio/intoxicação , Bronquite/genética , Células Cultivadas , Mapeamento Cromossômico/métodos , Humanos , Doenças Profissionais/genética , Exposição Ocupacional/efeitos adversos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteoma/genéticaRESUMO
Rodent cancer bioassays are part of a legacy of safety testing that has not changed significantly over the past 30 years. The bioassays are expensive, time consuming, and use hundreds of animals. Fewer than 1500 chemicals have been tested in a rodent cancer bioassay compared to the thousands of environmental and industrial chemicals that remain untested for carcinogenic activity. In this study, we used existing data generated by the National Toxicology Program (NTP) to identify gene expression biomarkers that can predict results from a rodent cancer bioassay. A set of 13 diverse chemicals was selected from those tested by the NTP. Seven chemicals were positive for increased lung tumor incidence in female B6C3F1 mice and six were negative. Female mice were exposed subchronically to each of the 13 chemicals, and microarray analysis was performed on the lung. Statistical classification analysis using the gene expression profiles identified a set of eight probe sets corresponding to six genes whose expression correctly predicted the increase in lung tumor incidence with 93.9% accuracy. The sensitivity and specificity were 95.2 and 91.8%, respectively. Among the six genes in the predictive signature, most were enzymes involved in endogenous and xenobiotic metabolism, and one gene was a growth factor receptor involved in lung development. The results demonstrate that increases in chemically induced lung tumor incidence in female mice can be predicted using gene biomarkers from a subchronic exposure and may form the basis of a more efficient and economical approach for evaluating the carcinogenic activity of chemicals.
Assuntos
Biomarcadores Tumorais/genética , Carcinógenos/toxicidade , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Neoplasias Pulmonares/genética , Algoritmos , Animais , Biomarcadores Tumorais/metabolismo , Testes de Carcinogenicidade/métodos , Carcinógenos/química , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Feminino , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Modelos Estatísticos , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Two-year rodent bioassays play a central role in evaluating the carcinogenic potential of both commercial products and environmental contaminants. The bioassays are expensive and time consuming, requiring years to complete and costing $2-4 million. In this study, we compare transcriptomic and metabonomic technologies for discovering biomarkers that can efficiently and economically identify chemical carcinogens without performing a standard two-year rodent bioassay. Animals were exposed subchronically to two chemicals (one genotoxic and one nongenotoxic) that were positive for lung and liver tumors in a standard two-year bioassay, two chemicals that were negative, and two control groups. Microarray analysis performed on liver and lung tissues identified multiple biomarkers in each tissue that could discriminate between carcinogenic and noncarcinogenic treatments. The discriminating biomarkers shared a common expression profile among carcinogenic treatments despite different genotoxicity categories and potential modes of action, suggesting that they reflect underlying cellular changes in the transition toward neoplasia. Statistical classification analysis exhibited 100% accuracy in both tissues when the number of genes was less than 5000. Additional genes reduced the predictive accuracy of the model. Serum samples were analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy, and chemical-specific metabolites were removed from the spectra. The statistical classification analysis of the endogenous serum metabolites showed relatively low predictive accuracy with few metabolites in the model, but the accuracy increased to a maximum of 94% when all metabolites were added. These results suggest that individual endogenous metabolites are relatively poor biomarkers, but the metabolite profile as a whole is altered following carcinogen treatment.
Assuntos
Carcinógenos/toxicidade , Genômica/métodos , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Bioensaio , Biomarcadores/metabolismo , Testes de Carcinogenicidade/métodos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Análise Serial de Proteínas , Distribuição Aleatória , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Although HSF1 plays an important role in the cellular response to proteotoxic stressors, little is known about the structure and function of the human HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 to comprehensively identify genes regulated directly and indirectly by HSF1. The correlation between promoter binding and gene expression was not significant for all genes bound by HSF1, suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown. Among promoters bound by HSF1 following heat shock, a gene ontology analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network following siRNA knockdown showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitinylation and others, highlighting a complex transcriptional program regulated directly and indirectly by HSF1.
Assuntos
Adaptação Fisiológica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sobrevivência Celular , Imunoprecipitação da Cromatina , Genoma Humano , Células HeLa , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Fatores de Tempo , TransfecçãoRESUMO
Enzymes that activate and detoxify benzene are likely genetic determinants of benzene-induced toxicity.NAD(P)H: quinone oxidoreductase-1 (NQO1) detoxifies benzoquinones, proposed toxic metabolites of benzene. NQO1 deficiency in humans is associated with an increased risk of leukemia, specifically acute myelogenous leukemia, and benzene poisoning. We examined the importance of NQO1 in benzene-induced toxicity by hypothesizing that NQO1-deficient (NQO1-/-) mice are more sensitive to benzene than mice with wild-type NQO1 (NQO1+/+; 129/Sv background strain). Male and female NQO1-/- and NQO1+/+ mice were exposed to inhaled benzene (0, 10, 50, or 100 ppm) for 2 weeks, 6 h/day, 5 days/week. Micronucleated peripheral blood cells were counted to assess genotoxicity. Peripheral blood counts and bone marrow histology were used to assess hematotoxicity and myelotoxicity. p21 mRNA levels in bone marrow cells were used as determinants of DNA damage response. Female NQO1-/- mice were more sensitive (6-fold) to benzene-induced genotoxicity than the female NQO1+/+ mice. Female NQO1-/- mice had a 9-fold increase (100 versus 0 ppm) in micronucleated reticulocytes compared with a 3-fold increase in the female NQO1+/+ mice. However, the induced genotoxic response in male mice was similar between the two genotypes (> or = 10-fold increase at 100 ppm versus 0 ppm). Male and female NQO1-/- mice exhibited greater hematotoxicity than NQO1+/+ mice. p21 mRNA levels were induced significantly in male mice (>10-fold) from both strains and female NQO1-/- mice (> 8-fold), which indicates an activated DNA damage response. These results indicate that NQO1 deficiency results in substantially greater benzene-induced toxicity. However, the specific patterns of toxicity differed between the male and female mice.
Assuntos
Benzeno/toxicidade , NAD(P)H Desidrogenase (Quinona)/metabolismo , Animais , Benzeno/farmacocinética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Citocromo P-450 CYP2E1/metabolismo , DNA/efeitos dos fármacos , Dano ao DNA , Feminino , Predisposição Genética para Doença , Doenças Hematológicas/induzido quimicamente , Doenças Hematológicas/patologia , Inativação Metabólica/genética , Masculino , Camundongos , Microssomos Hepáticos/enzimologia , NAD(P)H Desidrogenase (Quinona)/deficiência , NAD(P)H Desidrogenase (Quinona)/genéticaRESUMO
Benzene, a carcinogen that induces chromosomal breaks, is strongly associated with leukemias in humans. Possible genetic determinants of benzene susceptibility include proteins involved in repair of benzene-induced DNA damage. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), encoded by Prkdc, is one such protein. DNA-PKcs is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here we compared the toxic effects of benzene on mice (C57BL/6 and 129/Sv) homozygous for the wild-type Prkdc allele and mice (129/SvJ) homozygous for a Prkdc functional polymorphism that leads to diminished DNA-PK activity and enhanced apoptosis in response to radiation-induced damage. Male and female mice were exposed to 0, 10, 50, or 100 ppm benzene for 6 h/d, 5 d/week for 2 weeks. Male mice were more susceptible to benzene toxicity compared with females. Hematotoxicity was evident in all male mice but was not seen in female mice. We observed similar, large increases in both micronucleated erythrocyte populations in all male mice. Female mice had smaller but significant increases in micronucleated cells. The p53-dependent response was induced in all strains and genders of mice following benzene exposure, as indicated by an increase in p21 mRNA levels in bone marrow that frequently corresponded with cell cycle arrest in G2/M. Prkdc does not appear to be a significant genetic susceptibility factor for acute benzene toxicity. Moreover, the role of NHEJ, mediated by DNA-PK, in restoring genomic integrity following benzene-induced DSB remains equivocal.