Absence of Wolbachia endobacteria in the non-filariid nematodes Angiostrongylus cantonensis and A. costaricensis

The majority of filarial nematodes harbour Wolbachia endobacteria, including the major pathogenic species in humans, Onchocerca volvulus, Brugia malayi and Wuchereria bancrofti. These obligate endosymbionts have never been demonstrated unequivocally in any non-filariid nematode. However, a recent report described the detection by PCR of Wolbachia in the metastrongylid nematode, Angiostrongylus cantonensis (rat lungworm), a leading cause of eosinophilic meningitis in humans. To address the intriguing possibility of Wolbachia infection in nematode species distinct from the Family Onchocercidae, we used both PCR and immunohistochemistry to screen samples of A. cantonensis and A. costaricensis for the presence of this endosymbiont. We were unable to detect Wolbachia in either species using these methodologies. In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes. This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples

Macrophage-epithelial crosstalk during alveolar epithelial repair following pathogen-induced pulmonary inflammation

Bacterial invasion of the alveolar air space is followed by the fast, tightly regulated immune response facilitating a successful pathogen clearance. Upon pathogen recognition activated resident alveolar macrophages (AMf;) early release pro-inflammatory cytokines, stimulating neighbouring alveolar cells to produce chemokines which in turn mediate the infiltration of neutrophils, exudate macrophages and lymphocytes. The following inflammatory reaction and the pathogen itself leave a damaged alveolar barrier associated with pulmonary oedema and impaired gas exchange. Consequently, epithelial repair processes are initiated to restore the normal lung homeostasis. During the later phase of infection AMf; have been shown to acquire an anti-inflammatory phenotype thereby enhancing alveolar repair processes. However, the potential of early activated, pro-inflammatory AMf to influence epithelial repair remains largely elusive. Therefore, in the present thesis it was investigated whether activated AMf contribute to alveolar epithelial repair upon LPS challenge in vitro and in vivo, as well as in K. pneumoniae pneumonia, and the molecular interaction pathways involved were analysed. The cross-talk between resident alveolar macrophages and alveolar epithelial cells during alveolar repair was assessed in an in vitro co-culture system and an in vivo model of LPS-induced acute lung injury. Gene expression and protein analysis showed that LPS-activated alveolar macrophages stimulated alveolar epithelial cells (AEC) to express growth factors, particularly GM-CSF upon co-culture. Antibody neutralization experiments revealed epithelial GM-CSF expression to be macrophage TNF-alpha dependent. GM-CSF elicited proliferative signalling in alveolar epithelial cells via autocrine activation of the transcription factor STAT 5 and Cyclin D1 expression. Notably, macrophage TNF-alphab induced epithelial proliferation in wild-type but not in GM-CSF-deficient alveolar epithelial cells as shown by [3H]-thymidine incorporation and cell counting. Matrigel:collagen AEC culture preserving the type II phenotype in vitro supported the concept that the proliferative response to GM-CSF is related to the type II AEC phenotype. Moreover, intra-alveolar TNF-alpha neutralization impaired alveolar epithelial type II cell proliferation in LPS-injured mice in vivo, as investigated by flow cytometric Ki67 and immunofluorescence staining of lung sections. Additionally, GM-CSF-deficient mice displayed reduced AEC II proliferation and sustained alveolar barrier dysfunction upon LPS treatment compared to wild-type and SPC-GM mice (overexpressing GM-CSF in AEC II in a GM-CSF-deficient background). Similarly, K. pneumoniae lung infection confirmed the findings in the LPS-model and resulted in early release of macrophage TNF-alpha and epithelial GM-CSF, as well as subsequent TNF-alpha-dependent AEC II proliferation during alveolar repair events. Collectively, these findings indicate that TNF-alpha released from activated resident alveolar macrophages induces epithelial GM-CSF expression, which in turn initiates alveolar epithelial type II cell proliferation and thus contributes to restore alveolar barrier function.Die bakterielle Infektion des Alveolarraumes ist regelhaft von einer schnellen, streng koordinierten Immunantwort gefolgt, deren Ziel die rasche Elimination des Erregers ist. Nach der Erkennung des Erregers über spezielle Pathogen-Rezeptoren setzen Alveolarmakrophagen (AMf;) pro-inflammatorische Zytokine frei und stimulieren benachbarte Parenchymzellen zur Produktion von Chemokinen, welche letztendlich die Chemotaxis neutrophiler Granulozyten, von Exudatmakrophagen und Lymphozyten vermitteln. Diese Immunreaktion, aber auch die Infektion selbst, führen zu einer Destruktion der alveolären Barriere mit konsekutivem alveolärem Ödem und eingeschränktem Gasaustausch. In der Folge werden alveoläre Reparaturprozesse in Gang gesetzt, um die Organfunktion wieder herzustellen. Alveolarmakrophagen aquirieren in der Spätphase der Entzündung einen anti-inflammatorischen Phänotyp und können solche Reparaturprozesse in Gang setzen. Jedoch war das Reparaturpotenzial früh aktivierter, pro-inflammatorischer Alveolarmakrophagen bis dato ungeklärt. In der vorliegenden Arbeit wurde deshalb untersucht, ob früh-inflammatorisch aktivierte residente Alveolarmakrophagen zur alveolarepithelialen Reparatur nach LPS-Applikation in vitro und in vivo und im Klebsiella-Pneumonie-Modell beitragen und welche die zugrunde liegenden molekularen Mechanismen sind. Es wurden die Mediatoren des Cross-talk zwischen Alveolarmakrophagen und Alveolarepithel in der alveolarepithelialen Reparatur in einem in vitro Ko-Kulturmodell und im Mausmodell der LPS-induzierten Acute Lung Injury ermittelt. Genexpressions- und Proteinanalysen zeigten hierbei, dass LPS-aktivierte Alveolarmakrophagen in der Ko-Kultur Alveolarepithelzellen zur Freisetzung epithelialer Wachstumsfaktoren, insbesondere von GM-CSF, stimulieren. Neutralisationsexperimente zeigten, dass die epitheliale GM-CSF Expression abhängig war von Makrophagen-sezerniertem TNF-alpha. GM-CSF induzierte autokrin eine STAT5-Cyclin D1-vermittelte proliferative Signalkaskade in Alveolarepithelzellen. Interessanterweise konnte mittels [3H]-Thymidin-Einbau und Zellzählung gezeigt werden, dass TNF-alpha eine epitheliale Proliferation in Wildtyp-, nicht jedoch in GM-CSF-defizienten Alveolarepithelzellen induziert. Ähnliche Experimente mit Alveolarepithelzellen, die auf einer Matrigel:Collagen-Matrix kultiviert wurden und dabei einen Phänotyp II (AEC II) behielten, zeigten, dass diese GM-CSF-vermittelte Proliferationsantwort an den Phänotyp II gekoppelt war. Darüberhinaus konnte im LPS-Mausmodell gezeigt werden, dass die intraalveoläre Neutralisation von TNF-alpha die Proliferation von Typ II Alveolarepithelzellen in vivo, gemessen anhand der Ki-67 Expression im FACS und in der Immunfluoreszenz, deutlich reduzierte. Zusätzlich zeigten GM-CSF-defiziente Mäuse eine eingeschränkte Alveolarepithelzellproliferation und eine deutlich prolongierte Dysfunktion der alveolären Barriere nach intratrachealer LPS-Gabe verglichen mit Wildtyp- oder SPC-GM-Mäusen (mit Überexpression von GM-CSF im Alveolarepithel, generiert in GM-CSF-defizienten Mäusen). Im Klebsiella-Pneumoniemodell konnten diese Mechanismen bestätigt werden. Zusammenfassend konnte gezeigt werden, dass TNF-alpha, welches von LPS-aktivierten residenten Alveolarmakrophagen freigesetzt wird, eine alveolarepitheliale GM-CSF-Expression induziert. GM-CSF wiederum initiiert über eine autokrine Signalkaskade die Proliferation von Typ II Alveolarepithelzellen und trägt somit wesentlich zur Erneuerung und Funktionalität der alveolarepithelialen Barriere bei

Environmental factors affecting larval fish community in the salt marsh area of Guadiana estuary (Algarve, Portugal)

Salt marsh areas in the Guadiana estuary are important nursery sites for many fish species of commercial and recreational value. More effective protection measures should be adopted as the area is highly affected by anthropogenic and natural threats. Studying larval fish communities in these impacted nursery areas will be relevant to the management of local ecosystems and to larval fish ecology in general. Spatial and seasonal distribution and the effect of environmental factors on the larval fish community of this ecosystem were studied for one year (April 2010 to March 2011). Larvae were sampled monthly in parallel with phytoplankton and zooplankton. Hydrological data and physical parameters were monitored. A decision tree model was used to assess the influence of environmental factors on the larval fish community. A total of 130 larvae and 1171 eggs were caught. Diplodus sargus, Sardina pilchardus, and Pomatoschistus microps were the most abundant larval fish species. The peaks of fish larvae abundance occurred in March and April. The output of the model demonstrates that the abundance of larval fish is determined by the abundance of eggs, zooplanktonic food, and water flood and flow. This study shows the importance of the Guadiana salt marsh as an area for fish nursery and highlights the need for conservation of this area.FCT (Portuguese Foundation for Science and Technology) [SFRH/BD/47985/2008

Akkermansia muciniphila ameliorates the age-related decline in colonic mucus thickness and attenuates immune activation in accelerated aging Ercc1(-/7) mice

BackgroundThe use of Akkermansia muciniphila as potential therapeutic intervention is receiving increasing attention. Health benefits attributed to this bacterium include an improvement of metabolic disorders and exerting anti-inflammatory effects. The abundance of A. muciniphila is associated with a healthy gut in early mid- and later life. However, the effects of A. muciniphila on a decline in intestinal health during the aging process are not investigated yet. We supplemented accelerated aging Ercc1(-/7) mice with A. muciniphila for 10weeks and investigated histological, transcriptional and immunological aspects of intestinal health.ResultsThe thickness of the colonic mucus layer increased about 3-fold after long-term A. muciniphila supplementation and was even significantly thicker compared to mice supplemented with Lactobacillus plantarum WCFS1. Colonic gene expression profiles pointed towards a decreased expression of genes and pathways related to inflammation and immune function, and suggested a decreased presence of B cells in colon. Total B cell frequencies in spleen and mesenteric lymph nodes were not altered after A. muciniphila supplementation. Mature and immature B cell frequencies in bone marrow were increased, whereas B cell precursors were unaffected. These findings implicate that B cell migration rather than production was affected by A. muciniphila supplementation. Gene expression profiles in ileum pointed toward a decrease in metabolic- and immune-related processes and antimicrobial peptide production after A. muciniphila supplementation. Besides, A. muciniphila decreased the frequency of activated CD80(+)CD273(-) B cells in Peyer's patches. Additionally, the increased numbers of peritoneal resident macrophages and a decrease in Ly6C(int) monocyte frequencies in spleen and mesenteric lymph nodes add evidence for the potentially anti-inflammatory properties of A. muciniphila.ConclusionsAltogether, we show that supplementation with A. muciniphila prevented the age-related decline in thickness of the colonic mucus layer and attenuated inflammation and immune-related processes at old age. This study implies that A. muciniphila supplementation can contribute to a promotion of healthy aging.Peer reviewe

Impairment of the Organization of Locomotor and Exploratory Behaviors in Bile Duct-Ligated Rats

Hepatic encephalopathy (HE) arises from acute or chronic liver diseases and leads to several problems, including motor impairment. Animal models of chronic liver disease have extensively investigated the mechanisms of this disease. Impairment of locomotor activity has been described in different rat models. However, these studies are controversial and the majority has primarily analyzed activity parameters. Therefore, the aim of the present study was to evaluate locomotor and exploratory behavior in bile duct-ligated (BDL) rats to explore the spatial and temporal structure of behavior. Adult female Wistar rats underwent common bile duct ligation (BDL rats) or the manipulation of common bile duct without ligation (control rats). Six weeks after surgery, control and BDL rats underwent open-field, plus-maze and foot-fault behavioral tasks. The BDL rats developed chronic liver failure and exhibited a decrease in total distance traveled, increased total immobility time, smaller number of rearings, longer periods in the home base area and decreased percentage of time in the center zone of the arena, when compared to the control rats. Moreover, the performance of the BDL rats was not different from the control rats for the elevated plus-maze and foot-fault tasks. Therefore, the BDL rats demonstrated disturbed spontaneous locomotor and exploratory activities as a consequence of altered spatio-temporal organization of behavior

Third brazilian consensus for autoantIbodies screening in HEp-2 Cells : historical perspectve, quality control and clinical associatons

O III Consenso Brasileiro para Pesquisa de Autoanticorpos em Células HEp-2 (FAN) objetivou discutir estratégias para controlar a qualidade do ensaio, promover a atualização das associações clínicas dos diversos padrões e avaliar as difculdades de implantação do II Consenso ocorrido no ano de 2002. Métodos: Nos dias 13 e 14 de abril de 2007 participaram do encontro em Goiânia pesquisadores e especialistas de diversos centros universitários e laboratórios clínicos de diferentes regiões do Brasil, com o propósito de discutir e aprovar as recomendações que visam a melhores padronização, interpretação e utilização do ensaio pelos clínicos. Foram convidados como ouvintes representantes comerciais de diferentes empresas produtoras de insumos para realização do teste de FAN. Resultados e conclusão: Dada a heterogeneidade de microscópios e reagentes disponíveis no mercado, o III Consenso enfatizou a necessidade do controle de qualidade em ensaios de imunofuorescência indireta. Foram também feitas algumas adequações na terminologia utilizada para classifcar os diferentes padrões. Finalmente, foi realizada uma atualização das associações clínicas com fnalidade de facilitar cada vez mais o melhor uso do ensaio pelos clínicos. _________________________________________________________________________________ ABSTRACTThe Third Brazilian Consensus for Autoantibodies Screening in HEp-2 Cells (ANA) had as purpose the evaluation of diffculties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the discussion of an update of the clinical associations of the several immunofuorescent patterns. Methods: Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2007 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop. Results and conclusion: The 3rd ANA Consensus emphasized the need for quality control in indirect immunofuorescent assays since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and fnally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians

Third Brazilian consensus for autoantibodies screening in HEp-2 cells (ANA) : recommendations for standardization of autoantibodies screening trial in HEp-2 cells, quality control and clinical associations

Objetivo: O 3º Consenso Brasileiro para pesquisa de autoanticorpos em Células HEp-2 (FAN) teve como propósito avaliar as dificuldades de implantação do 2º Consenso ocorrido no ano de 2002, discutir estratégias para controlar a qualidade do ensaio e promover a atualização das associações clínicas dos diversos padrões. Métodos: Participaram do encontro em Goiânia nos dias 13 e 14 de abril de 2008 pesquisadores e especialistas de diversos centros universitários e laboratórios clínicos de diferentes regiões do Brasil, com o propósito de discutir e aprovar as recomendações que visam à melhor padronização, interpretação e utilização do ensaio pelos clínicos. Representantes comerciais de diferentes empresas produtoras de insumos para realização do teste de FAN foram convidados como ouvintes. Resultados e Conclusões: O 3º Consenso enfatizou a necessidade do controle de qualidade em imunofluorescência dada a heterogeneidade de microscópios e reagentes disponíveis no mercado, promoveu adequações na terminologia utilizada para classificar os diferentes padrões e, finalmente, atualizou as associações clínicas com finalidade de facilitar cada vez mais o melhor uso do ensaio pelos clínicos. ________________________________________________________________________________ ABSTRACTObjective: The Third Brazilian Consensus for autoantibodies Screening in HEp-2 cells had as purpose the evaluation of difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the promotion of an update of the clinical associations of the several immunofluorescent patterns. Methods: Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2008 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop. Results and Conclusions: The 3rd Consensus emphasized the need for quality control in indirect immunofluorescent since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and finally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians

Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer

<p>Abstract</p> <p>Background</p> <p>CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. <it>CXCL12 </it>promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor α (<it>ESR1</it>) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the <it>CXCL12 </it>promoter and <it>ESR1 </it>in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data.</p> <p>Methods</p> <p>First, we analysed <it>CXCL12 </it>expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of <it>CXCL12 </it>in breast tumour cell lines. We evaluated <it>CXCL12 </it>and <it>ESR1 </it>methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis.</p> <p>Results</p> <p><it>CXCL12 </it>promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The <it>ESR1 </it>promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ERα protein expression diminished with increased frequency of <it>ESR1 </it>methylation (p < 0.0001). This study also demonstrated that <it>CXCL12 </it>island 4 and <it>ESR1 </it>methylation occur simultaneously at a high frequency (p = 0.0220).</p> <p>Conclusions</p> <p>This is the first study showing a simultaneous involvement of epigenetic regulation for both <it>CXCL12 </it>and <it>ESR1 </it>genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.</p