Lung infection rates in two sympatric Tropiduridae lizard species by pentastomids and nematodes in northeastern Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: Representational difference analysis identifies candidate genes associated with fungal pathogenesis

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans. (c) 2006 Elsevier Masson SAS. All rights reserved.

The therapeutic potential of the humoral pattern recognition molecule PTX3 in chronic lung infection caused by Pseudomonas aeruginosa

Chronic lung infections by Pseudomonas aeruginosa strains are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Although there is no clear evidence for a primary defect in the immune system of CF patients, the host is generally unable to clear P. aeruginosa from the airways. PTX3 is a soluble pattern recognition receptor that plays nonredundant roles in the innate immune response to fungi, bacteria, and viruses. In particular, PTX3 deficiency is associated with increased susceptibility to P. aeruginosa lung infection. To address the potential therapeutic effect of PTX3 in P. aeruginosa lung infection, we established persistent and progressive infections in mice with the RP73 clinical strain RP73 isolated from a CF patient and treated them with recombinant human PTX3. The results indicated that PTX3 has a potential therapeutic effect in P. aeruginosa chronic lung infection by reducing lung colonization, proinflammatory cytokine levels (CXCL1, CXCL2, CCL2, and IL-1β), and leukocyte recruitment in the airways. In models of acute infections and in in vitro assays, the prophagocytic effect of PTX3 was maintained in C1q-deficient mice and was lost in C3- and Fc common γ-chain-deficient mice, suggesting that facilitated recognition and phagocytosis of pathogens through the interplay between complement and FcγRs are involved in the therapeutic effect mediated by PTX3. These data suggested that PTX3 is a potential therapeutic tool in chronic P. aeruginosa lung infections, such as those seen in CF patients.

Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.

Misleading high tobramycin plasma concentrations can be caused by skin contamination of fingerprick blood following inhalation of nebulized tobramycin (TOBI (R)) - A short report

We observed unexpected high plasma concentrations of tobrarriycin (48.5 and 28.1 mg/L) in fingerprick blood samples after the nebulization of tobramycin solution for inhalation (tobramycin 300 mg/5 mL, TOBI(R)) by 2 young children aged 3 years. To investigate whether dermal contamination could be the source of error, 3 adult volunteers were present during another nebulization by a third child (age 2 years). The volunteers had exposure to tobramycin by handling the nebulizer or the nebule and also by inhalation from holding the child and being in close proximity while TOBI(R) was being administered. Five blood samples by fingerprick and 2 by venipuncture were collected and assayed for tobramycin concentration. On each occasion the site was swabbed with alcohol wipes to mimic standard patient sampling methods. One site was resampled after cleaning of hands with 2% chlorhexidine gluconate and water. Tobramycin concentrations from venipuncture 1-2 hours after nebulization were all < 0.2 mg/L except for 1 result of 1.2 mg/L. The tobramycin concentrations from fingerpricks before hand washing varied between 6.8 and 172 mg/L, and after hand washing between 0.3 and 17.6 mg/L. Contamination of fingers with tobramycin is likely to have caused the error in the 2 initial cases and did cause misleadingly elevated levels in the adult volunteers. We caution that therapeutic drug monitoring of nebulized tobramycin should not be done by fingerprick sampling, and care should be taken to avoid contamination of the venipuncture site.

A mathematical model of chlamydial infection incorporating movement of chlamydial particles

We present a spatiotemporal mathematical model of chlamydial infection, host immune response and spatial movement of infectious particles. The re- sulting partial differential equations model both the dynamics of the infection and changes in infection profile observed spatially along the length of the host genital tract. This model advances previous chlamydia modelling by incorporating spatial change, which we also demonstrate to be essential when the timescale for movement of infectious particles is equal to, or shorter than, the developmental cycle timescale. Numerical solutions and model analysis are carried out, and we present a hypothesis regarding the potential for treatment and prevention of infection by increasing chlamydial particle motility.

Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection

Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of the C. trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.

Chlamydia pneumoniae infection of Dentritic cells and its role in asthma exacerbations

Chlamydia infections are associated with exacerbations of asthma however the mechanisms are poorly understood. In this thesis we infected dendritic cells from healthy controls and asthmatic patients to determine if the immune response to chlamydial infection by these key immune cells could explain this association of chlamydial infection with asthma attacks. Infected dendritic cells from asthmatic patients showed increased expression of multiple inflammatory cytokine genes and genes for several tissue remodelling proteins, suggesting that infected dendritic cells play a central role in driving the airways damage associated with asthma. The findings provide a greater understanding of the role of infections in asthma and may provide a basis for new therapies to treat this important disease.

Rejection by pigs of mouldy grain containing deoxynivalenol

Weaner pigs on a farm near Beaudesert in south eastern Queensland refused to eat feed comprised largely of wheat and barley. Older pigs consumed small amounts and some prepubertal gilts subsequently displayed enlarged and reddened vulvas. Wheat, barley and triticale were grown on the farm during 1983, which was unusually and persistently wet. The wheat and triticale were harvested and stored for about 3 weeks with moisture contents above 14% before being fed. Samples of the wheat and triticale contained pale pink grains, which can indicate infection by the fungus Fusariurn grarninearurn Schw. On analysis 2 mycotoxins known to be produced by F. graminearurn were detected, deoxynivalenol (vomitoxin) which causes feed refusal and vomiting, and zearalenone which causes oestrogenic effects. Concentrations of deoxynivalenol in the wheat, triticale and barley were 34, 10, and <0.1 mg/kg respectively. Concentrations of zearalenone were 6.2, 2.8 and 0.1 mg/kg respectively. Subsequently, F. grarninearurn was isolated from grains and crop residues. Although the wet weather contributed to F. grarninearurn infection of the crops before harvest, most of the toxins probably developed during storage.

Tan spot: A new disease of pyrethrum caused by Microsphaeropsis tanaceti sp. nov

The isolation frequency of Microsphaeropsis sp. in spring in association with necrotic lesions on leaves in Tasmanian pyrethrum (Tanacetum cinerariifolium) fields has increased substantially since first identification in 2001. Examination of morphological features and sequencing of the internal transcribed spacer region (ITS) resulted in the identification of a new species, herein described as Microsphaeropsis tanaceti sp. nov. The pathogenicity of three M. tanaceti isolates to two pyrethrum cultivars was confirmed by inoculating glasshouse-grown plants in three experiments. No significant differences in the susceptibility of the two cultivars to infection by M. tanaceti were found. Symptoms were tan-coloured spots which coalesced around the margins of the leaves. Therefore, the name 'tan spot' is proposed for this new disease of pyrethrum. The sensitivity of seven M. tanaceti isolates to difenoconazole and azoxystrobin, commonly used fungicides for the management of foliar diseases in spring, was assessed under in vitro conditions. Sensitivity testing for difenoconazole was conducted using a mycelial growth assay on potato dextrose agar, whilst testing for sensitivity to azoxystrobin used a conidial germination assay on water agar. Microsphaeropsis tanaceti was found to be more sensitive to azoxystrobin than difenoconazole, with complete inhibition of conidial germination at concentrations above 0.625 µg a.i. mL-1. By comparison, concentrations of 50 µg a.i. difenoconazole mL-1 or greater were required for significant inhibition of mycelial growth. It therefore appears likely that there is currently some control of tan spot as a result of the use of azoxystrobin and to a lesser extent, difenoconazole, for the control of other diseases.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Interferon-gamma- and glucocorticoid-mediated pathways synergize to enhance death of CD4(+) CD8(+) thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S.typhimurium). Significant death of CD4+CD8+ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4+CD8+ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon- (IFN-), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifn/ mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN- and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifn/ mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifn/ mice. Importantly, the number of CD4+CD8+ thymocytes is significantly higher in Ifn/ mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN--mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S.typhimurium infection. The implications of this study for host responses during infection are discussed.

Distribución de "coxiella burnetii" en los rumiantes domésticos y en la fauna silvestre de la Comunidad Atutónoma Vasca. Evaluacióndel efecto del tratamiento antibiótico y de la vacunación en el control de la fiebre Q en rebaños ovinos naturalmente infectados

243 p. : il.

Polymorphisms in the C-type lectin genes cluster in chromosome 19 and predisposition to severe acute respiratory syndrome coronavirus (SARS-CoV) infection

Background: Polymorphisms of CLEC4M have been associated with predisposition for infection by the severe acute respiratory syndrome coronavirus (SARS-CoV). DC-SIGNR, a C-type lectin encoded by CLEC4M, is a receptor for the virus. A variable number tandem