Type IV secretion systems have been identified in several human pathogens including Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila. These systems are responsible for the translocation of virulence proteins and/or DNA, thereby playing an important role in the pathogenesis of infection and plasticity of genomes. Burkholderia cenocepacia is an important opportunistic human pathogen, particularly in persons with cystic fibrosis (CF). Respiratory tract infection by B. cenocepacia in CF patients is often associated with a decline in respiratory function, and can result in acute systemic infection. Burkholderia cenocepacia strain K56-2 is part of the epidemic and clinically problematic ET12 lineage. Two type IV secretion systems have been identified in this strain; one system is plasmid encoded (designated the Ptw type IV secretion system) whereas the other is chromosomally encoded (designated the VirB/D type IV secretion system) and shows homology to the Agrobacterium tumefaciens VirB/D4 type IV secretion system. It was determined that the plasmid encoded Ptw system is a chimeric type IV secretion system composed of VirB/D4-like elements and F-specific subunits. More recently, it was found that this system translocates a protein effector (PtwE1) that is cytotoxic to plant cells. It was also determined that the positively charged C-terminal region of PtwE1 is important for translocation via the Ptw type IV secretion system. Strains of the epidemic B. cenocepacia PHDC lineage contain only a chromosomal VirB/D4-like type IV secretion system (designated BcVirB/D); and a putative effector protein associated with this system has been identified that has C-terminal transport signal and sequences different from the effectors of the Ptw type IV secretion system. It has also been shown that a competing plasmid substrate and a plasmid fertility inhibition factor act to render B. cenocepacia of the PHDC lineage incapable of expressing a plant phenotype. Thus, three type IV secretion systems have been identified in epidemic B. cenocepacia lineages. From two of these, an effector has been identified that has cytotoxic effects on eukaryotic cells, and at least one of these type IV secretion systems is able to translocate DNA substrates.

Type IV secretion systems have been identified in several human pathogens including Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila. These systems are responsible for the translocation of virulence proteins and/or DNA, thereby playing an important role in the pathogenesis of infection and plasticity of genomes. Burkholderia cenocepacia is an important opportunistic human pathogen, particularly in persons with cystic fibrosis (CF). Respiratory tract infection by B. cenocepacia in CF patients is often associated with a decline in respiratory function, and can result in acute systemic infection. Burkholderia cenocepacia strain K56-2 is part of the epidemic and clinically problematic ET12 lineage. Two type IV secretion systems have been identified in this strain; one system is plasmid encoded (designated the Ptw type IV secretion system) whereas the other is chromosomally encoded (designated the VirB/D type IV secretion system) and shows homology to the Agrobacterium tumefaciens VirB/D4 type IV secretion system. It was determined that the plasmid encoded Ptw system is a chimeric type IV secretion system composed of VirB/D4-like elements and F-specific subunits. More recently, it was found that this system translocates a protein effector (PtwE1) that is cytotoxic to plant cells. It was also determined that the positively charged C-terminal region of PtwE1 is important for translocation via the Ptw type IV secretion system. Strains of the epidemic B. cenocepacia PHDC lineage contain only a chromosomal VirB/D4-like type IV secretion system (designated BcVirB/D); and a putative effector protein associated with this system has been identified that has C-terminal transport signal and sequences different from the effectors of the Ptw type IV secretion system. It has also been shown that a competing plasmid substrate and a plasmid fertility inhibition factor act to render B. cenocepacia of the PHDC lineage incapable of expressing a plant phenotype. Thus, three type IV secretion systems have been identified in epidemic B. cenocepacia lineages. From two of these, an effector has been identified that has cytotoxic effects on eukaryotic cells, and at least one of these type IV secretion systems is able to translocate DNA substrates.

Lėtinė obstrukcinė plaučių liga (LOPL) – šiuo metu pasaulyje viena svarbiausių didelį sergamumą ir mirtingumą sąlygojančių ligų. Pacientams, kuriems pasunkėja kasdieniai LOPL simptomai pasireiškia LOPL paūmėjimas. Šio tyrimo tikslas – įvertinti nespecifinį uždegimą paūmėjus lėtinei obstruk¬cinei plaučių ligai. Nustatyti periferinio kraujo ir indukuotų skreplių ląstelių sudėties skirtumai ir įvertintos sąsajos su plaučių funkcijos rodikliais bakterinio ir nebakterinio lėtinės obstrukcinės plaučių ligos paūmėji¬mo metu. Tirtos sergančiųjų bakterinės kilmės lėtinės obstrukcinės plaučių li¬gos paūmėjimo indukuotų skreplių neutrofilų ir makrofagų funkcijos (apoptozė, fagocitozė ir reaktyvių deguonies formų susidarymas) bei palygintos su nebakteriniu paūmėjimu. Įvertinti sergančiųjų bakterinės ir nebakterinės kilmės lėtinės obs¬trukcinės plaučių ligos paūmėjimo periferinio kraujo neutrofilų ir monocitų apoptozė ir chemotaksis, bei periferinio kraujo neutrofilų fagocitozė ir reaktyvių deguonies formų susidarymas. Ištirta sergančiųjų bakterinės ir nebakterinės kilmės lėtinės obstruk¬cinės plaučių ligos paūmėjimu interleukino-8 koncentracija ir sąsajos su periferinio kraujo neutrofilų chemotaksiu bei C reaktyviojo baltymo koncentracijos periferinio kraujo serume sąsajos su plaučių funkcija ir rūkymo intensyvumu. / Chronic obstructive pulmonary disease (COPD) – a common preventable and treatable disease, is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. An acute exacerbation of COPD (AECOPD) is an acute event characterized by a worsening of the patient’s respiratory symptoms that is beyond normal day-to-day variations and leads to a change in medication. The aim of this study was to evaluate the non-specific inflammation during acute exacerbation of COPD to investigate change of cellular activity (apoptosis, chemotaxis, phagocytosis and production of reactive oxygen species) during AECOPD depending on infection agent. Subjects with AECOPD and the same in remission were investigated. Increased count of induced sputum neutrophils and macrophages as well peripheral blood neutrophils and monocytes during bacterial and non-bacterial AECOPD was related with impaired pulmonary function and smoking history. Induced sputum neutrophils and macrophage apoptosis and phagocytosis were weaker, but production of reactive oxygen species was strongly activated during bacterial acute exacerbation of chronic obstructive pulmonary disease than non-bacterial AECOPD. This study showed differences of peripheral blood neutrophil and monocyte apoptosis, chemotaxis, as well as peripheral blood neutrophil phagocytosis and the production of reactive oxygen species... [to full text]

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.

Data compiled by the Centers for Disease Control demonstrates that African American women, in particular young people between the ages of 14-25, have an increased incidence of infection with the sexually transmitted pathogen Neisseria gonorrhoeae. Estradiol is a key regulatory hormone in female reproductive function. It has been studied extensively in the cardiovascular field, and has been linked to breast and endometrial cancers in women. However, its impact on infectious diseases is largely unknown. Given what is known about the effect of estradiol on immunologic and inflammatory disorders in women, I hypothesize that estradiol alters the infectivity of Neisseria gonorrhoeae in the female reproductive tract by altering the host inflammatory immune response. This may explain a risk factor for increased rates of infection in some populations. I sought to develop a relevant in vitro model. After screening a number of candidate cell lines, I selected the human endometrial adenocarcinoma cell line, Ishikawa. These cells express specific estrogen receptors and respond to exogenous estrogen stimulation. Estrogen treatment of Ishikawa cells did not have an impact on the invasion of N. gonorrhoeae, nor did it impact bacterial growth. However, gonococcal induced chemokine secretion was reduced by estrogen, as measured by interleukin-8 secretion. I conclude that estrogen blunts the inflammatory response to Neisseria gonorrhoeae without altering bacterial infectivity.

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Orientador: Claudia Aparecida Rainho / Resumo: O carcinoma mamário é o tipo de câncer mais frequente entre as mulheres, com estimativas de aumento de sua incidência mundial nas próximas décadas. O câncer de mama triplo negativo (CMTN) é um subtipo definido pela falta de receptores para os hormônios estrógeno e progesterona e sem superexpressão de HER-2. Ocorre geralmente em mulheres com menos de 50 anos e está relacionado a uma alta taxa de recorrência e mortalidade. As terapias convencionais são hormônio-dependentes ou possuem a proteína HER2 como alvo, logo o CMTN não é responsivo a essas modalidades terapêuticas. Devido a biologia deste tumor, novas abordagens são necessárias para o seu tratamento. Plantas são importantes fontes de compostos antitumorais. Os sesquiterpenos α-humuleno (HUM) e β-cariofileno (CAR) geralmente estão presentes em conjunto no óleo essencial de diversas plantas. Um número limitado de estudos foi realizado sobre os seus efeitos em células derivadas de carcinomas mamários, porém um efeito imunomodulador foi descrito para diferentes sesquiterpenos em células derivadas de cânceres humano. A hipótese desse estudo é que os efeitos in vitro dos sesquiterpenos HUM e CAR nas linhas celulares derivadas de CMTP são mediados por alterações nos níveis de expressão de citocinas produzidas pelas células tumorais. Foram selecionadas quatro linhagens celulares triplo-negativas (BT-20, BT-549, MDA-MB-231 e MDA-MB-436). Inicialmente, os efeitos isolados ou combinados do HUM e CAR foram avaliados pelo ensaio de v... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Breast cancer is the most common type of cancer amongst women. Worldwide, its incidence is estimated to increase in the coming decades. Triple negative breast cancer (TNBC) is a subtype defined by the absence of receptors for estrogen and progesterone hormones and without HER-2 overexpression. It usually occurs in women under 50 years old and it is related to a high recurrence and mortality rates. As conventional therapies are hormone-dependent or target HER2, TNBC are not responsive to these therapeutic approaches. Due to the biology of this tumor, new targets are needed for its treatment. Plants are important sources of antitumor compounds, the sesquiterpenes α-humulene (HUM) and β-caryophyllene (CAR) are usually present together in the essential oil of several plants. Few studies have been conducted on their effect in breast cancer cells, but an immunomodulatory effect of different sesquiterpenes was described on human cancer cells. The hypothesis of this study is that the in vitro effects of sesquiterpenes HUM and CAR on cell lines derived from TNBC are mediated by changes in cytokine expression levels produced by tumor cells. For this, four triple-negative cell lines were selected (BT-20, BT-549, MDA-MB-231, and MDA-MB-436). Initially, the isolated or combined effects of HUM and CAR were evaluated by the MTT cell viability assay upon different concentrations (6.25, 12.5, 25, 50, and 100µM), after 24, 48, 72, and 96 hours of exposure. Based on the effect of the sesquiterp... (Complete abstract click electronic access below) / Mestre

The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IκB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis.

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