Estudo da Influência de polimorfismos nos genes IL-10, IL-12, MIF e TNF na Imunopatogênese da Leishmaniose tegumentar americana

Submitted by Anderson Silva ([email protected]) on 2012-11-06T15:56:00Z No. of bitstreams: 1 claudia_j_f_covas_ioc_bcm_0023_2010.pdf: 6745296 bytes, checksum: cb60c41970a2659edc6c7fd4600969bf (MD5)Made available in DSpace on 2012-11-06T15:56:00Z (GMT). No. of bitstreams: 1 claudia_j_f_covas_ioc_bcm_0023_2010.pdf: 6745296 bytes, checksum: cb60c41970a2659edc6c7fd4600969bf (MD5) Previous issue date: 2010Fundação Oswaldo Cruz Instituto Oswaldo Cruz. Laboratório Interdisciplinar em Pesquisas Médicas. Rio de Janeiro, RJ, BrasilA leishmaniose tegumentar Americana (LTA) representa um conjunto de doenças com características clínicas e imunopatológicas distintas, cuja evolução depende, pelo menos em parte, de fatores relacionados à resposta imune do hospedeiro. A imunopatogênese da leishmaniose mucosa (LM), causadas principalmente pela L. braziliensis, é dependente de uma intensa resposta celular do tipo 1, dirigida peincipalmente pela elevada secreção das citocinas IFN-γ e TNF, que apresentam expressão aumentada em presença de IL-12. Por outro lado, várias evidências apontam que a IL-10 é a principal citocina envolvida na regulação da resposta imune em pacientes com leishmaniose, geralmente suprimindo a imunidade mediada por células. Estudos recentes vêm demonstrando que a susceptibilidade ou resistência a dor do hospedeiro a patógenos intracelulares, bem como a gravidade a várias doenças infecciosas podem ser associados com polimorfismos em genes de citocinas. Assim, com o propósito de estabelecer marcadores que possuam ter influência genética na evolução clínica da LTA, o presente trabalho teve como objetivo avaliar as freqüências dos SNPs nos genes da IL-10 -819C>T, IL-12 -1188ª>C 3’UTR, MIF -173G>C e TNF -308G>A em pacientes com histórico de LTA e controles saudáveis. Além diss, buscou-se analisar a associação dos genótipos com a produção das citocinas IL-10, IL-12p70, MIF e TNF nas diferentes formas clínicas da LTA e nos controles saudáveis.. No que se refere aos SNPs IL-10 -819 e IL-12 +1188 não foi observada uma associação dos mesmos com a LTA per se, no entanto, os SNP MIF -17 e TNF -308 sugerem risco à doença: MIF -173GC (OR=0,04; p=0,04) e TNF – 308AA (OR=3,92; p=0,0001). Nenhum dos SNPs estudados apresentou relação com a progressão para a LM, considerada a forma mais grave da LTA. Em relação à dosagem das citocinas IL-10, IL-12, MIF e TNF os indivíduos controles apresentaram níveis menores do que os pacientes com LTA (pC e TNF – 308G>A, e sinaliza a relevância da continuidade de estudos dessa natureza para ampliar o nosso entendimento sobre a dinâmica da doença no Brasil no contexto genético do hospedeir

Potencial biotecnológico de Pedobacter lusitanus NL19 uma nova espécie isolada de lamas de uma mina de urânio em Portugal

Bioactive natural products (BNPs) play an important role in our daily lives, especially as therapeutic agents. One example is antibiotics, which have been widely used in human health. The inappropriate use of these antibiotics has led to an increase in bacterial resistance, which is a problem of worldwide concern, warning for the urgent need for new effective alternatives. Extreme environments have already proven to be a rich source of microorganisms producing new chemical entities with important biological properties, namely antimicrobials. In the present study one such environment was explored. We have isolated several bacteria from sludges contaminated with radionuclides and metals, collected from a uranium mine run-off in Quinta do Bispo, Viseu, Portugal. One strain was selected based on its broad activity against Grampositive and Gram-negative bacteria, and also yeasts, with relevance in the clinical, food, veterinary and aquaculture settings. The phylogenetic analysis of the 16S rRNA, gyrB and Internal Transcribed Spacer (ITS) gene sequences identified this strain as belonging to the genus Pedobacter, and having P. himalayensis, P. cryoconitis, P. westerhofensis and P. hartonius as closely related species. Further polyphasic analysis showed that the strain is a new bacterial species, and which was named Pedobacter lusitanus NL19. In silico analysis of NL19 genome sequence revealed the presence of 17 biosynthetic gene clusters (BGCs) for different classes of BNPs, including nonribosomal peptide (NRPs), polyketides (PKs), ribosomally synthesized and posttranslationally modified peptides (RiPPs), siderophores, terpenes and several hybrid NRPs-PKs. NL19 bioactivity was detected in solid medium but not in its equivalent broth. Further investigations showed that a high concentration of peptone from casein (PC) was the main factor associated with the lack of bioactivity by NL19 in broth. On the contrary, in broth with a low concentration of PC, the compounds produced by NL19 showed a broad spectrum of activity against bacteria and yeasts. Furthermore, the bioactivity detected was mainly due to the presence of pedopeptins, which are NRPs and whose genes are present in the NL19 genome. These results were supported by RNA-seq and validated by qPCR, which showed that the transcription of pedo_nrps genes was significantly downregulated in cells grown in a high concentration of PC. Also, NL19´ RNA-seq transcriptome data in high and low PC concentrations, allowed us to identify 261 differentially expressed genes (DEGs). In general, at high concentrations of PC, in addition to the genes involved in pedopeptins biosynthesis, genes involved in the de novo biosynthesis of biotin were also found to be significantly downregulated. In this condition genes that encode putative efflux pumps as well as ferrous iron capture system, were significantly upregulated, which suggests redirection of cellular metabolism. Genome mining approaches also revealed that NL19 and its closely related species have an important collection of new NRPS-BGCs. Finally, the pan-genome analyses highlight the genus Pedobacter as a prolific source of relevant new BNPs including NRPs, PKs, RiPPs, hidroxamate, carotenoids and squalene, mainly the NL19 and P. cryoconitis species. The work developed reinforces the relevance of bioprospecting extreme environments. The characterization of the strain P. lusitanus NL19 reveals the enormous, yet still untapped, biotechnological potential of this strain, and also of other members of the genus Pedobacter, for the discovery of new compounds with diverse biotechnological applications, including therapeutic ones.Os produtos naturais bioativos (BNPs) têm um grande impacto no nosso quotidiano, principalmente como agentes terapêuticos. Um exemplo são os antibióticos, que têm sido amplamente usados na melhoria da saúde humana. O uso inadequado destes antibióticos tem levado ao crescente aumento de resistência bacteriana, sendo esta uma preocupação mundial que obriga a explorar novas alternativas eficazes. Os ambientes extremos têm mostrado ser uma rica fonte de microrganismos produtores de novas entidades químicas, com importantes propriedades biológicas, nomeadamente, antimicrobianas. No presente estudo explorámos um desses ambientes. Isolámos várias bactérias a partir de lamas contaminadas com radionuclídeos e metais, que foram recolhidas na mina de urânio desativada na Quinta do Bispo, Viseu-Portugal. Um isolado bacteriano foi selecionado por apresentar amplo espetro de atividade contra bactérias de Gram-positivo, de Gram-negativo, e também leveduras, com relevância nas áreas clínica, alimentar, veterinária e aquacultura. Esse isolado foi identificado e caracterizado por análises filogenéticas das sequências dos genes 16S rRNA, gyrB e de espaçadores do transcrito interno (ITSs), como pertencendo ao género Pedobacter e tendo como espécies próximas P. himalayensis, P. cryoconitis, P. westerhofensis e P. hartonius. A análise polifásica revelou ser uma nova espécie bacteriana a que se deu o nome Pedobacter lusitanus NL19. A análise in silico do seu genoma revelou a presença de 17 agrupamentos de genes biossintéticos (BGCs) para diferentes classes de BNPs, incluindo péptidos não ribossomais (NRPs), policétidos (PKs), péptidos ribossomais pós-traducionalmente modificados (RiPPs), sideróforos, terpenos e vários NRPs-PKs híbridos. A bioatividade da NL19 foi detectada quando a bactéria foi cultivada em meio sólido, mas não no meio líquido equivalente. Verificámos ainda que elevadas concentrações de peptona de caseína (PC) afectam a bioatividade da NL19 quando cultivada em meio líquido e que os compostos produzidos a baixas concentrações de PC têm atividade contra bactérias e leveduras. A bioatividade detectada foi associada à presença das pedopeptinas, que são NRPs e cujos genes estão presentes no genoma da NL19. Estes dados foram suportados pelos resultados da análise por RNA-seq e validados por qPCR, que mostram que os genes pedo_nrps se encontram significativamente subexpressos em células cultivadas em elevadas concentrações de PC. Para além disso, os dados obtidos da análise do transcriptoma da NL19 cultivada em elevadas e baixas concentrações de PC, permitiram identificar 261 genes diferencialmente expressos (DEGs). De uma forma geral, em concentrações elevadas de PC, para além dos genes envolvidos na biossíntese das pedopeptinas, também os genes envolvidos na biossíntese de novo da biotina se encontravam significativamente subexpressos. Nesta condição, os genes codificantes para bombas de efluxo, bem como para um sistema de captura de ferro ferroso, estavam significativamente sobreexpressos, sugerindo um redirecionamento do metabolismo celular. A mineração do genoma revelou ainda que a NL19 e as espécies próximas atrás referidas, possuem uma importante coleção de novos NRPS-BGCs. Finalmente, a análise dos pangenomas revelaram que o gênero Pedobacter, nomeadamente, as espécies NL19 e P. cryoconitis, é fonte prolífica de novas classes de BNPs, incluindo NRPs, PKs, RiPPs, hidroxamato, carotenóides e esqualeno. Os resultados obtidos ao longo deste trabalho reforçam a relevância da bioprospecção dos ambientes extremos. Para além disso, a caracterização da estirpe P. lusitanus NL19, e de outras espécies próximas, revela o enorme, mas ainda inexplorado, potencial biotecnológico desta estirpe, bem como de membros do género Pedobacter, para a descoberta de novos compostos com diversas aplicações biotecnológicas, inclusive terapêuticas.Programa Doutoral em Biologi

Peptone from casein, an antagonist of nonribosomal peptide synthesis: a case study of pedopeptins produced by Pedobacter lusitanus NL19

Novel natural products are urgently needed to address the worldwide incidence of bacterial resistance to antibiotics. Extreme environments are a major source of novel compounds with unusual chemical structures. Pedobacter lusitanus NL19 is a new bacterial species that was isolated from one such environment and which produces compounds with potent activity against relevant microorganisms in the clinical, food, veterinary and aquaculture areas. The production of antimicrobials by P. lusitanus NL19 was identified in tryptic soy agar (TSA), but not in its equivalent broth (TSB). It was observed that in TSB medium a high concentration of casein peptone (PC) repressed the production of antibacterial compounds. HPLC, MS and MS/MS spectra with de novo sequencing revealed that the bioactivity of P. lusitanus NL19 was due to the production of pedopeptins. Hence, biosynthesis of pedopeptins is inhibited by high concentrations of PC in the broth medium. Furthermore, a nonribosomal peptide synthetase (NRPS) gene cluster was identified in the genome of NL19 encoding the biosynthesis of the peptides. qPCR analysis confirmed that the transcription of these genes is repressed in cells cultivated in high concentrations of PC. It is shown that pedopeptins are nonribosomal peptides with a broad-spectrum activity, including against Gram-positive and Gram-negative bacteria and yeasts.publishe

Primary nodal peripheral T-cell lymphomas: diagnosis and therapeutic considerations

Nodal peripheral T-cell lymphomas are a rare group of neoplasms derived from post-thymic and activated T lymphocytes. A review of scientific articles listed in PubMed, Lilacs, and the Cochrane Library databases was performed using the term "peripheral T-cell lymphomas". According to the World Health Organization classification of hematopoietic tissue tumors, this group of neoplasms consists of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma-anaplastic lymphoma kinase positive (ALCL-ALK+), and a provisional entity called anaplastic large cell lymphoma-anaplastic lymphoma kinase negative (ALCL-ALK-). Because the treatment and prognoses of these neoplasms involve different principles, it is essential to distinguish each one by its clinical, immunophenotypic, genetic, and molecular features. Except for anaplastic large cell lymphoma-anaplastic lymphoma kinase positive, which has no adverse international prognostic index, the prognosis of nodal peripheral T-cell lymphomas is worse than that of aggressive B-cell lymphomas. Chemotherapy based on anthracyclines provides poor outcomes because these neoplasms frequently have multidrug-resistant phenotypes. Based on this, the current tendency is to use intensified cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP) regimens with the addition of new drugs, and autologous hematopoietic stem cell transplantation. This paper describes the clinical features and diagnostic methods, and proposes a therapeutic algorithm for nodal peripheral T-cell lymphoma patients

Pedobacter lusitanus sp. nov., isolated from sludge of a deactivated uranium mine

Strain NL19(T) is a Gram-stain-negative, aerobic bacterium that was isolated from sludge of a deactivated uranium mine in Portugal. 16S rRNA gene sequence analysis revealed that strain NL19(T) is a member of the genus Pedobacter and closely related to the strains Pedobacter himalayensis MTCC 6384(T), Pedobacter cryoconitis DSM 14825(T), Pedobacter westerhofensis DSM 19036 (T) and Pedobacter hartonius DSM 19033(T). It had a DNA G+C content of 40.8 mol%, which agreed with the genus description. The main fatty acids included C-16 : 1 omega 7c, C-14 : 1 omega 5c, C-4 : 0, iso-C-17 : 0, iso-C-17 : 0 3-OH, C-16 : 0, anteiso-C-15 : 0 and isoC(15 : 0) 3-OH. The main lipids present were phospholipids (60 %) and sphingolipids (35 %). The most abundant phospholipids included phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Menaquinone-7 (MK-7) was the only isoprenoid quinone detected. DNA-DNA hybridization similarities between strain NL19(T) and Pedobacter himalayensis MTCC 6384(T), Pedobacter cryoconitis DSM 14825(T), Pedobacter westerhofensis DSM 19036(T) and Pedobacter hartonius DSM 19033(T) were 15.3, 16.2, 11.5 and 16.0 %, respectively. Strain NL19(T) can also be distinguished from these four species based on gyrB and intergenic transcribed spacers (ITS) sequences and by some phenotypic traits such as NaCl tolerance, pH, growth temperature and carbon source utilization. Strain NL19(T) represents a novel species of the genus Pedobacter, for which the name Pedobacter lusitanus sp. nov. is proposed. The type strain is NL19(T) (= LMG 29220(T) = CECT 9028(T)). An amended description of Pedobacter himalayensis is also included.info:eu-repo/semantics/publishedVersio

Pedobacter lusitanus sp. nov., isolated from sludge of a deactivated uranium mine

Strain NL19 is a Gram-stain-negative, aerobic bacterium that was isolated from sludge of a deactivated uranium mine in Portugal. 16S rRNA gene sequence analysis revealed that strain NL19 is a member of the genus Pedobacter and closely related to the strains Pedobacter himalayensis MTCC 6384, Pedobacter cryoconitis DSM 14825, Pedobacter westerhofensis DSM 19036 and Pedobacter hartonius DSM 19033. It had a DNA G+C content of 40.8 mol%, which agreed with the genus description. The main fatty acids included Cω7c, Cω5c, C, iso-C, iso-C 3-OH, C, anteiso-C and iso- C 3-OH. The main lipids present were phospholipids (60%) and sphingolipids (35%). The most abundant phospholipids included phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Menaquinone-7 (MK-7) was the only isoprenoid quinone detected. DNA–DNA hybridization similarities between strain NL19 and Pedobacter himalayensis MTCC 6384, Pedobacter cryoconitis DSM 14825, Pedobacter westerhofensis DSM 19036 and Pedobacter hartonius DSM 19033 were 15.3, 16.2, 11.5 and 16.0%, respectively. Strain NL19 can also be distinguished from these four species based on gyrB and intergenic transcribed spacers (ITS) sequences and by some phenotypic traits such as NaCl tolerance, pH, growth temperature and carbon source utilization. Strain NL19 represents a novel species of the genus Pedobacter, for which the name Pedobacter lusitanus sp. nov. is proposed. The type strain is NL19 (=LMG 29220=CECT 9028). An amended description of Pedobacter himalayensis is also included.This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within the project UID/AMB/50017/2013. T. C. and C. C. were supported by FCT, POPH, and European Union grants SFRH/BPD/77900/2011 and SFRH/BD/98446/2013, respectively. A. C. was supported by a postdoctoral grant (BPD/UI88/2886/2013) from the project ‘Sustainable Use of Marine Resources’—MARES (CENTRO-07-ST24-FEDER002033), funded by QREN, Mais Centro-Programa Operacional Regional do Centro e Uniao Europeia/Fundo Europeu de Desenvolvimento Regional.Peer Reviewe

Analysis of renewal capacity of memory compartments and activated T cell replenishment of healed cutaneous leishmaniasis subjects.

<p>Renewal capacity of CD4⁺ (<b>A</b>) and CD8⁺ (<b>B</b>) T cells phenotipically characterized by central memory (Tcm, CD45RO⁺CCR7⁺) or effector memory (Tem, CD45RO⁺CCR7^-). The white box and whiskers represent those cells evaluated immediately after PBMC (<i>ex vivo</i>) and the gray boxes represent those cells after <i>in </i><i>vitro</i> stimuli with Lb-Ag. The central line represents median values. hCL= healed cutaneous leishmaniasis; HS= healthy subjects; *p<0.05, **p<0.01, ***p<0.001 (Kruskal-Wallis with post-test Dunns). Correlation analysis between the percentage of activated CD25⁺ (<b>C</b>, n=9) or CD69⁺ (<b>D</b>, n=12) in CD8⁺ T cells after <i>in </i><i>vitro</i> Lb-Ag stimuli and the percentage of effector memory (CD45RO⁺CCR7^-) CD8⁺ T cells after <i>in </i><i>vitro</i> Lb-Ag stimuli. Each point represents one subject. The graphs show the best fitted lines with 95% confidence intervals. r= correlation coefficient; p= significance level, (Spearman test).</p

Correlation analysis of clinical and/or immunological parameters from healed cutaneous leishmaniasis subjects.

<p>Correlation between the percentage of recently activated CD4⁺ (A, n=13) or CD8⁺ (B, n=14) T lymphocytes from PBMC after <i>in </i><i>vitro</i> Lb-Ag stimulation and the duration of clinical cure; Correlation between the percentage of activated CD4⁺ (C, n=8) or CD8⁺ (D, n=9) T lymphocytes and the concentration of IL-10 from cell culture supernatant; Each point represents one subject. The graphs show the best fitted lines with 95% confidence intervals. r= correlation coefficient; p= significance level, (Spearman test). </p

Peptone from casein, an antagonist of nonribosomal peptide synthesis: a case study of pedopeptins produced by Pedobacter lusitanus NL19

Novel natural products are urgently needed to address the worldwide incidence of bacterial resistance to antibiotics. Extreme environments are a major source of novel compounds with unusual chemical structures. Pedobacter lusitanus NL19 is a new bacterial species that was isolated from one such environment and which produces compounds with potent activity against relevant microorganisms in the clinical, food, veterinary and aquaculture areas. The production of antimicrobials by P. lusitanus NL19 was identified in tryptic soy agar (TSA), but not in its equivalent broth (TSB). It was observed that in TSB medium a high concentration of casein peptone (PC) repressed the production of antibacterial compounds. HPLC, MS and MS/MS spectra with de novo sequencing revealed that the bioactivity of P. lusitanus NL19 was due to the production of pedopeptins. Hence, biosynthesis of pedopeptins is inhibited by high concentrations of PC in the broth medium. Furthermore, a nonribosomal peptide synthetase (NRPS) gene cluster was identified in the genome of NL19 encoding the biosynthesis of the peptides. qPCR analysis confirmed that the transcription of these genes is repressed in cells cultivated in high concentrations of PC. It is shown that pedopeptins are nonribosomal peptides with a broad-spectrum activity, including against Gram-positive and Gram-negative bacteria and yeasts.publishe