Thalidomide modulates Mycobacterium leprae-induced NF-κB pathway and lower cytokine response

AbstractIt is widely accepted that tumor necrosis factor alpha (TNF-α) plays a critical role in the development of tissue and nerve damage in leprosy and during the reactional episodes of acute inflammation. Thalidomide (N-α-phthalimidoglutarimide), a drug used to treat leprosy reaction, modulates immune response, inhibits inflammation and NF-κB activity. Here we investigated whether thalidomide inhibits NF-κB activation induced by Mycobacterium leprae, p38 and ERK1/2 MAPK activation. EMSA and supershift assays were performed to investigate NF-κB activation in response to M. leprae and its modulation following in vitro treatment with thalidomide. Luciferase assay was assayed in transfected THP-1 cells to determine NF-κB transcriptional activity. Flow cytometry and immunofluorescence were used to investigate p65 accumulation in the nucleus. Immunoblotting was used to investigate p38 and ERK1/2 phosphorylation. Following activation of PBMC and monocytes with M. leprae, the formation and nuclear localization of NF-κB complexes composed mainly of p65/p50 and p50/p50 dimers was observed. Induction of NF-κB activation and DNA binding activity was inhibited by thalidomide. The drug also reduced M. leprae-induced TNF-α production and inhibited p38 and ERK1/2 activation. Definition of the activation mechanisms in cells stimulated with M. leprae can lead to the development of new therapy applications to modulate NF-κB activation and to control the inflammatory manifestations due to enhanced TNF-α response as observed in leprosy and in leprosy reactions

Detección y caracterización de enterobacterias resistentes a múltiples fármacos, portadoras del gen modificador de aminoglucósidos en un hospital universitario de Río de Janeiro, Brasil, durante tres décadas

Introduction: Multidrug-resistant Enterobacteriaceae, particularly those resistant to gentamicin, have become one of the most important causes of nosocomial infections.Objective: We sought to investigate the presence of genes conferring resistance to aminoglycosides, specially to gentamicin, in Klebsiella pneumoniae and Escherichia coli multidrug-resistant strains isolated from different clinical materials among patients hospitalized in a university hospital in Rio de Janeiro, Brazil.Materials and methods: Ten colonization strains and 20 infection strains were evaluated during three decades (1980 to 2010) using selective media containing 8 μg/ml of gentamicin. Thirty strains were tested for antimicrobial susceptibility. Twenty two strains were subjected to plasmid DNA extraction and 12 to hybridization assays using as probe a 1.9 kb plasmid DNA fragment from one of the K. pneumoniae strains isolated from faecal samples. This fragment was sequenced and assigned to the GQ422439 GenBank record. PCR was also performed using oligonucleotides designed for aminoglycoside-modifying enzymes.Results: An accC2 acetylase, besides transposons and insertion sequences, were evidenced. Twenty-four (80%) of the isolates were positive for the aacC2 gene in agreement with antibiotic susceptibility testing profiles, indicating the persistent presence of this gene throughout the three decades. We detected high molecular weight plasmids in 54,5% of the strains. Of the tested strains, 91% showed positive signal in the hybridization assays.Conclusion: A gene codifying for one specific aminoglycoside-modifying enzyme was detected all throughout the three decades. Our data back the adoption of preventive measures, such as a more conscious use of antimicrobial agents in hospital environments, which can contribute to control the dissemination of microorganisms harboring resistance gene plasmids.Introducción. Las enterobacterias resistentes a la gentamicina se asocian frecuentemente a infecciones hospitalarias.Objetivo. Verificar la presencia de los genes que confieren resistencia a los aminoglucósidos, específicamente a la gentamicina, en cepas de Klebsiella pneumoniae y Escherichia coli multirresistentes, obtenidas de pacientes internados en un hospital universitario de Río de Janeiro.Materiales y métodos. Se recolectaron y evaluaron 10 cepas de colonización y 20 de infección entre 1980 y 2010, utilizando medios selectivos enriquecidos con gentamicina (8 μg/ml). Se obtuvieron 30 cepas en las que se determinó la resistencia a los antibióticos por medios fenotípicos. Veintidós muestras se sometieron a extracción de ADN plasmídico y se hicieron ensayos de hibridización en 12 de ellas, usando como sonda un fragmento de ADN plasmídico de 1,9 kb obtenido de una cepa de K. pneumoniae aislada de muestra fecal. Este fragmento fue secuenciado y correspondió al registro GQ422439 del GenBank. Se verificó la presencia de genes de enzimas modificadoras de aminoglucósidos mediante reacción en cadena de la polimerasa.Resultados. En las cepas analizadas se evidenció la presencia de la acetilasa accC2, además de transposones y secuencias de inserción. Veinticuatro aislamientos (80 %) fueron positivos para el gen aacC2 en concordancia con los perfiles de sensibilidad a los antibióticos, lo que indicó su persistencia a lo largo de las tres décadas. Se detectaron plásmidos de alto peso molecular en 54,5 % de las cepas. El 91 % de las cepas analizadas mostró signos positivos en las pruebas de hibridación.Conclusión. Se detectó la persistencia de un gen codificador de una enzima modificadora de aminoglucósidos a lo largo de las tres décadas. Los resultados indican que las medidas de prevención, tales como un uso más responsable de los agentes antimicrobianos en el ambiente hospitalario, pueden contribuir al control de la diseminación de microorganismos que albergan plásmidos de genes de resistencia

Guidelines for the management of neuroendocrine tumours by the Brazilian gastrointestinal tumour group

Neuroendocrine tumours are a heterogeneous group of diseases with a significant variety of diagnostic tests and treatment modalities. Guidelines were developed by North American and European groups to recommend their best management. However, local particularities and relativisms found worldwide led us to create Brazilian guidelines. Our consensus considered the best feasible strategies in an environment involving more limited resources. We believe that our recommendations may be extended to other countries with similar economic standards.Univ Sao Paulo, Inst Canc Estado Sao Paulo, BR-01246000 Sao Paulo, BrazilUniv Sao Paulo, Fac Med, Dept Radiol & Oncol, BR-01246903 Sao Paulo, BrazilHosp Sirio Libanes, BR-01308050 Sao Paulo, BrazilHosp Moinhos de Vento Porto Alegre, BR-90035000 Porto Alegre, RS, BrazilOncoctr, BR-30360680 Belo Horizonte, MG, BrazilUniv Fed Rio Grande do Sul, Dept Cirurgia, BR-90040060 Porto Alegre, RS, BrazilHosp Clin Porto Alegre, BR-90035903 Porto Alegre, RS, BrazilUniv Fed Ceara, Fac Med, Dept Fisiol & Farmacol, BR-60020180 Fortaleza, Ceara, BrazilHosp Univ Walter Cantidio, BR-60430370 Fortaleza, Ceara, BrazilInst Nacl Canc, BR-20230240 Rio De Janeiro, BrazilUniv Sao Paulo, Fac Med, Disciplina Endocrinol & Metabol, BR-01246903 Sao Paulo, BrazilAC Camargo Canc Ctr, Dept Surg, BR-01509010 Sao Paulo, BrazilUniv Sao Paulo, Fac Med, Dept Gastroenterol, Sao Paulo, BrazilUniv Fed Ciencias Saude Porto Alegre, BR-90050170 Porto Alegre, RS, BrazilHosp Albert Einstein, BR-05652900 Sao Paulo, BrazilHosp Base, Fac Med Sao Jose do Rio Preto, BR-15090000 Sao Paulo, BrazilSanta Casa Sao Jose do Rio Preto, BR-15025500 Sao Jose Do Rio Preto, BrazilPontificia Univ Catolica Parana, Hosp Erasto Gaertner, BR-81520060 Curitiba, Parana, BrazilUniv Fed Rio Grande do Norte, BR-59300000 Natal, RN, BrazilUniv Sao Paulo, Inst Coracao, BR-05403900 Sao Paulo, BrazilAC Camargo Canc Ctr, Med Oncol, BR-01509010 Sao Paulo, BrazilUniv Fed Sao Paulo, Disciplina Gastroenterol, BR-04021001 Sao Paulo, BrazilHosp Sao Rafael, BR-41253190 Salvador, BA, BrazilHosp Canc Barretos, Dept Cirurgia Aparelho Digest Alto & Hepatobiliop, BR-14784400 Sao Paulo, BrazilUniv Sao Paulo, Fac Med, Dept Patol, BR-01246903 Sao Paulo, BrazilClin AMO, BR-1950640 Salvador, BA, BrazilHosp Sao Jose, BR-01323001 Sao Paulo, BrazilUniv Nove de Julho, BR-02111030 Sao Paulo, BrazilUniv Fed Sao Paulo, Disciplina Gastroenterol, BR-04021001 Sao Paulo, BrazilWeb of Scienc

ExoU activates NF-κB and increases IL-8/KC secretion during Pseudomonas aeruginosa infection.

ExoU, a Pseudomonas aeruginosa cytotoxin injected into host cytosol by type III secretion system, exhibits a potent proinflammatory activity that leads to a marked recruitment of neutrophils to infected tissues. To evaluate the mechanisms that account for neutrophil infiltration, we investigated the effect of ExoU on IL-8 secretion and NF-κB activation. We demonstrate that ExoU increases IL-8 mRNA and protein levels in P. aeruginosa-infected epithelial and endothelial cell lines. Also, ExoU induces the nuclear translocation of p65/p50 NF-κB transactivator heterodimer as well as NF-κB-dependent transcriptional activity. ChIP assays clearly revealed that ExoU promotes p65 binding to NF-κB site in IL-8 promoter and the treatment of cultures with the NF-κB inhibitor Bay 11-7082 led to a significant reduction in IL-8 mRNA levels and protein secretion induced by ExoU. These results were corroborated in a murine model of pneumonia that revealed a significant reduction in KC secretion and neutrophil infiltration in bronchoalveolar lavage when mice were treated with Bay 11-7082 before infection with an ExoU-producing strain. In conclusion, our data demonstrate that ExoU activates NF-κB, stimulating IL-8 expression and secretion during P. aeruginosa infection, and unveils a new mechanism triggered by this important virulence factor to interfere in host signaling pathways

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR

Submitted by sandra infurna ([email protected]) on 2016-04-05T13:40:01Z No. of bitstreams: 1 jairo_temerozo_etal_IOC_2015.pdf: 1066333 bytes, checksum: 1894875d5be6eb5dea98e3f11ac0ce98 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-05T13:55:25Z (GMT) No. of bitstreams: 1 jairo_temerozo_etal_IOC_2015.pdf: 1066333 bytes, checksum: 1894875d5be6eb5dea98e3f11ac0ce98 (MD5)Made available in DSpace on 2016-04-05T13:55:25Z (GMT). No. of bitstreams: 1 jairo_temerozo_etal_IOC_2015.pdf: 1066333 bytes, checksum: 1894875d5be6eb5dea98e3f11ac0ce98 (MD5) Previous issue date: 2015Universidade Federal do Rio Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Instituto de Microbiologia Paulo Góes. Laboratório de Imunobiologia de Leishmanioses. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Instituto de Microbiologia Paulo Góes. Laboratório de Imunobiologia de Leishmanioses. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Faculdade de Ciências Médicas. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling

ExoU activates p65/p50 NF-κB and increases IL-8 expression and secretion in HMEC-1 capillary endothelial cells.

<p>In (A), representative agarose gels of three different semi-quantitative RT-PCR assays carried out in duplicate. In (B), graph shows the means ± SEM of values obtained in three Real Time qRT-PCR assays. **p<0.01 and ***p<0.001 when the values obtained from PA103Δ<i>exoU</i>-infected cultures and non-infected cultures, respectively, were compared with those obtained from cultures infected with the ExoU-producing strain. In (C), representative EMSAs showing NF-κB nuclear translocation after 2 and 12 hours of <i>P. aeruginosa</i> infection or treatment with culture medium. In (D), extracts obtained from PA103-infected cells were incubated with specific antibodies for NF-κB subunits, p50, p52, p65 or cRel, but only supershifted with p50 and p65. In non-infected cells, antibodies were not added. EMSA and supershift assays were performed in triplicate. In (E), IL-8 concentrations, detected by ELISA, in supernatants of cultures pretreated or not with 5 µM Bay 11-7082, for 1 hour, and exposed to PA103, PA103Δ<i>exoU</i> or culture medium, for 21 hours. The graph represents the means ± SEM of two assays performed in quadruplicate and shows ***p<0.001 when the values obtained from untreated PA103-infected cultures were compared with those from the other untreated cultures or with those from Bay 11-7082-treated cultures infected with the ExoU-producing strain.</p

ExoU promotes p65/p50 nuclear translocation and NF-κB-dependent transcriptional activity in <i>P. aeruginosa</i>-infected A549 cells.

<p>In (A), representative EMSAs showing NF-κB nuclear translocation after 2 and 12 hours of <i>P. aeruginosa</i> infection or treatment with culture medium. To evaluate NF-κB inhibition by Bay 11-7082, some cultures were treated with 10 µM Bay 11-7082, 1 hour prior PA103 infection. As a control for non-specific interactions, nuclear extracts from PA103-infected cells were also incubated with a probe mutated in a single nucleotide (Mut). In (B), extracts obtained from PA103-infected cells were supershifted with specific antibodies against NF-κB subunits, p50 and p65. In “Non-infected cells” and “PA103-infected cells” lanes, antibodies were not added. EMSA and supershift assays were performed in triplicate. In (C), the graph shows the luciferase activity (in arbitrary values) of whole-cell lysates obtained from A549 cultures transfected with p6κB-LUC and pRL-CMV plasmids and then infected with <i>P. aeruginosa</i> strains or treated with cultured medium for 24 hours. Data represent means ± SEM of three different assays carried out in quadruplicate. **p<0.01 and ***p<0.001 when the values obtained from PA103Δ<i>exoU</i>-infected cultures and non-infected cultures, respectively, were compared with those obtained from PA103-infected cultures.</p

ExoU induces NF-κB-dependent KC secretion and neutrophil infiltration in mice airways at 24 hours post-infection.

<p>In (A), concentrations of KC assessed by ELISA, in (B) total leukocytes and in (C) neutrophils (PMN), both assessed by light microscopy, in BALF of mice pretreated or not with Bay 11-7082 at 20 mg/Kg, for 1 hour, and inoculated intratracheally with PA103, PA103Δ<i>exoU</i> or saline. The graphs show the mean values ± SEM of three independent assays (n = 16 for each group). *p<0,05 or **p<0.01 when the values obtained in untreated mice infected with PA103 strain were compared with the values obtained in the other groups.</p

ExoU increases IL-8 mRNA in A549 cells.

<p>In (A), representative agarose gels obtained from three different semi-quantitative RT-PCR assays carried out in duplicate. In (B) and (C), graph represents the means ± SEM of values obtained by three different Real Time qRT-PCR assays performed in triplicate. **p<0.01 or ***p<0.001 when the values obtained from cultures infected with the ExoU-producing PA103 strain were compared with those obtained from the other cultures.</p

Inhibition of NF-κB reduces IL-8 expression and secretion induced by ExoU in <i>P. aeruginosa</i>-infected A549 cultures.

<p>In (A), representative agarose gels of RT-PCR assays in which A549 cells were treated or not with 10 µM Bay 11-7082, 1 hour prior infection with PA103 or PA103Δ<i>exoU</i> strains or treatment with culture medium (non-infected cells) for 18 hours. In (B), graph shows the means ± SEM of values obtained by three Real Time qRT-PCR assays. In (C), IL-8 concentrations detected in supernatants of cultures pretreated or not with 10 µM Bay 11-7082 for 1 hour and then exposed for 21 hours to PA103, PA103Δ<i>exoU</i> or culture medium, as assessed by ELISA. In (D), IL-8 concentrations, as detected by ELISA, in supernatants of cultures pretreated or not with 10 µM Bay 11-7082 and/or 10 µM SP600125 for 1 hour and then exposed for 21 hours to bacterial strains or culture medium. In (C) and (D), data represent means ± SEM of three assays performed in quadruplicate. ***p<0.001 when the values obtained from untreated PA103-infected cultures were compared with those from the other cultures.</p