Resistência contra ectoparasitas em bovinos da raça Crioula Lageana e meio-sangue Angus avaliada em condições naturais

This study was carried out to evaluate the breed resistance against ectoparasites infestations in Crioula Lageana and crossbred Angus male calves under natural condition. Ten weaned and neutered male calves (6-8 months) of each group were kept together in winter and summer pastures in Monte Castelo, Santa Catarina State. Every 28 days, female ticks (Rhipicephalus (Boophilus) microplus) more than 4 mm long were counted on the right and left side of the body of each animal, as well as the D. hominis nodules. Coat thickness also was measured at each sampling and all animals were classified according coat color. Crioula Lageana group was less infested by D. hominis than crossbred Angus in five times and by ticks in the infestation peak (P<0.05). There were no differences in the distribution of the ticks and grubs between animal body sides. Dark-haired animal in both groups were the most susceptible.

Maturation of mononuclear phagocytes in the lungs of young calves-In vitro study

The influences of age in calves' immune system are described in their first phase of life. We hypothesized that variations that occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. This study aimed to evaluate the innate immune system. Nine healthy calves were monitored for 3 mo and 8 immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. The alveolar macrophages in samples were identified by protein expression of cluster of differentiation 14 (CD14) and underwent functional evaluation of phagocytosis (Staphylococcus aureus stained with propidium iodide and Escherichia coli). Data was assessed by one-way ANOVA (unstacked and parametric) and the Mann-Whitney test (nonparametric). Functional alterations in CD14-positive phagocytes were observed, with punctual higher intensity of phagocytosis in the third week and its decrease starting at 45 d of life. A gradual increase in phagocytosis rate was observed starting at this date. It is concluded that from 45 d of life on, alveolar macrophages have less phagocytic capacity but more cells perform this function. We suggest that this occurs because lung macrophages of calves start to maintain their immune response without passive immunity influence. Until 90 d of life, calves did not achieve the stability to conclude the maturation of local innate immune response.

Mecanismo oncológico de escape à vigilância do sistema imunitário

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

Primary dengue haemorrhagic fever in patients from northeast of Brazil is associated with high levels of interferon-b during acute phase

Dengue is an acute febrile disease caused by the mosquito-borne dengue virus (DENV) that according to clinical manifestations can be classified as asymptomatic, mild or severe dengue. Severe dengue cases have been associated with an unbalanced immune response characterised by an over secretion of inflammatory cytokines. In the present study we measured type I interferon (IFN-I) transcript and circulating levels in primary and secondary DENV infected patients. We observed that dengue fever (DF) and dengue haemorrhagic fever (DHF) patients express IFN-I differently. While DF and DHF patients express interferon-a similarly (52,71 ± 7,40 and 49,05 ± 7,70, respectively), high levels of circulating IFN-b were associated with primary DHF patients. On the other hand, secondary DHF patients were not able to secrete large amounts of IFN-b which in turn may have influenced the high-level of viraemia. Our results suggest that, in patients from our cohort, infection by DENV serotype 3 elicits an innate response characterised by higher levels of IFN-b in the DHF patients with primary infection, which could contribute to control infection evidenced by the low-level of viraemia in these patients. The present findings may contribute to shed light in the role of innate immune response in dengue pathogenesis.

The macrophage-inducible C-type lectin, Mincle, is an essential component of the innate immune response to Candida albicans

The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-_ by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.

Peripheral compartment innate immune response to Haemophilus influenzae and Streptococcus pneumoniae in chronic obstructive pulmonary disease patients.

Alterations in innate immunity that predispose to chronic obstructive pulmonary disease (COPD) exacerbations are poorly understood. We examined innate immunity gene expression in peripheral blood polymorphonuclear leukocytes (PMN) and monocytes stimulated by Haemophilus influenzae and Streptococcus pneumoniae. Thirty COPD patients (15 rapid and 15 non-rapid lung function decliners) and 15 smokers without COPD were studied. Protein expression of IL-8, IL-6, TNF-α and IFN-γ (especially monocytes) increased with bacterial challenge. In monocytes stimulated with S. pneumoniae, TNF-α protein expression was higher in COPD (non-rapid decliners) than in smokers. In co-cultures of monocytes and PMN, mRNA expression of TGF-β1 and MYD88 was up-regulated, and CD14, TLR2 and IFN-γ down-regulated with H. influenzae challenge. TNF-α mRNA expression was increased with H. influenzae challenge in COPD. Cytokine responses were similar between rapid and non-rapid decliners. TNF-α expression was up-regulated in non-rapid decliners in response to H. influenzae (monocytes) and S. pneumoniae (co-culture of monocytes and PMN). Exposure to bacterial pathogens causes characteristic innate immune responses in peripheral blood monocytes and PMN in COPD. Bacterial exposure significantly alters the expression of TNF-α in COPD patients, although not consistently. There did not appear to be major differences in innate immune responses between rapid and non-rapid decliners.

Hyperlipidemia impaired innate immune response to periodontal pathogen Porphyromonas gingivalis in apolipoprotein E knockout mice

A finely-tuned innate immune response plays a pivotal role in protecting host against bacterial invasion during periodontal disease progression. Hyperlipidemia has been suggested to exacerbate periodontal health condition. However, the underlying mechanism has not been addressed. In the present study, we investigated the effect of hyperlipidemia on innate immune responses to periodontal pathogen Porphyromonas gingivalis infection. Apolipoprotein E-deficient and wild-type mice at the age of 20 weeks were used for the study. Peritoneal macrophages were isolated and subsequently used for the study of viable P. gingivalis infection. ApoE−/− mice demonstrated inhibited iNOS production and impaired clearance of P. gingivalis in vitro and in vivo; furthermore, ApoE−/− mice displayed disrupted cytokine production pattern in response to P. gingivalis, with a decreased production of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β and monocyte chemotactic protein-1. Microarray data demonstrated that Toll-like receptor (TLR) and NOD-like receptor (NLR) pathway were altered in ApoE−/− mice macrophages; further analysis of pattern recognition receptors (PRRs) demonstrated that expression of triggering receptors on myeloid cells-1 (TREM-1), an amplifier of the TLR and NLR pathway, was decreased in ApoE−/− mice macrophages, leading to decreased recruitment of NF-κB onto the promoters of the TNF-α and IL-6. Our data suggest that in ApoE−/− mice hyperlipidemia disrupts the expression of PRRs, and cripples the host’s capability to generate sufficient innate immune response to P. gingivalis, which may facilitate immune evasion, subgingival colonization and establishment of P. gingivalis in the periodontal niche.

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity.

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.

Lack of effect of glutamine administration to boost the innate immune system response in trauma patients in the intensive care unit

The use of glutamine as a dietary supplement is associated with a reduced risk of infection. We hypothesized that the underlying mechanism could be an increase in the expression and/or functionality of Toll-like receptors (TLR), key receptors sensing infections. The objective of this study was to evaluate whether glutamine supplementation alters the expression and functionality of TLR2 and TLR4 in circulating monocytes of trauma patients admitted to the intensive care unit (ICU).

Innate immune response in the sea urchin Echinometra lucunter (Echinodermata)

Echinometra lucunter, (Pinda) is a sea urchin encountered in the Brazilian coast and exposed to high and low temperatures related to low and high tides. Despite their great distribution and importance, few studies have been done on the biological function of their coelomocytes. Thus, Echinometra lucunter perivisceral coelomocytes were characterized under optical and transmission electron microscopy. Phagocytic amoebocytes in the perivisceral coelom were labelled by injecting ferritin, and ferritin labelled phagocytic amoebocytes were found in the peristomial connective tissue after injecting India ink into the tissue, indicating the amoebocytes ability to respond to an inflammatory stimulus. Results showed that the phagocytic amoebocytes were the main inflammatory cells found in the innate immune response of E lucunter. While other works have recorded these phenomena in sea urchins found in moderate and constant temperature, this study reports on these same phenomena in a tropical sea urchin under great variation of temperature, thus providing new data to inflammatory studies in invertebrate pathology. (C) 2007 Elsevier Inc. All rights reserved.