Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-alpha, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this beta-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

Fatal septic shock due to a disseminated chronic form of paracoccidioidomycosis in an aged woman

Once rare, septic shock (SS) due to disseminated fungal infections has been increasingly reported due to a growing number of immunocompromised patients, but remains rare in non-immune-compromised individuals. In paracoccidioidomycosis, it has been described in only three patients with the severe, acute form of the disease. We describe the development of a refractory, fatal septic shock due to a severe disseminated chronic form of paracoccidioidomycosis in an older woman without any other microbial insults. A striking event in the evolution of her case was the severe depletion of lymphocytes from the peripheral blood and lymphoid organs. Lymphocyte depletion due to apoptosis is described in the late phase of sepsis and can contribute both to immunosuppression and the progression of SS. The possible mechanisms involved in the induction of SS in the chronic form of paracoccidioidomycosis are discussed.

Candida spp. isolated from inpatients, the environment, and health practitioners in the Pediatric Unit at the Universitary Hospital of the Jundiai Medical College, State of Sao Paulo, Brazil

Introduction: This study aimed to isolate and identify Candida spp. from the environment, health practitioners, and patients with the presumptive diagnosis of candidiasis in the Pediatric Unit at the Universitary Hospital of the Jundiai Medical College, to verify the production of enzymes regarded as virulence factors, and to determine how susceptible the isolated samples from patients with candidiasis are to antifungal agents. Methods: Between March and November of 2008 a total of 283 samples were taken randomly from the environment and from the hands of health staff, and samples of all the suspected cases of Candida spp. hospital-acquired infection were collected and selected by the Infection Control Committee. The material was processed and the yeast genus Candida was isolated and identified by physiological, microscopic, and macroscopic attributes. Results: The incidence of Candida spp. in the environment and employees was 19.2%. The most frequent species were C. parapsilosis and C. tropicalis among the workers, C. guilliermondii and C. tropicalis in the air, C. lusitanae on the contact surfaces, and C. tropicalis and C. guilliermondii in the climate control equipment. The college hospital had 320 admissions, of which 13 (4%) presented Candida spp. infections; three of them died, two being victims of a C. tropicalis infection and the remaining one of C. albicans. All the Candida spp. in the isolates evidenced sensitivity to amphotericin B, nystatin, and fluconazole. Conclusions: The increase in the rate of hospital-acquired infections caused by Candida spp. indicates the need to take larger measures regarding recurrent control of the environment.

Posaconazole salvage treatment in paediatric patients: a multicentre survey

While a paediatric dosage has not been defined, posaconazole is occasionally being used in children. We conducted a multicentre retrospective survey and identified 15 patients (median age 10 years [range 3.6-17.5]) who received posaconazole salvage therapy for proven (9 patients) or probable (6 patients) invasive fungal infections. Posaconazole was administered for a median of 32 days (range 4-262) at a median dosage of 21 mg/kg (range 4.8-33.3). None of the patients discontinued therapy due to adverse events, which were mostly mild and observed in 11 patients. Complete or partial responses were observed in 4/7 patients with zygomycosis, 3/4 patients with invasive mould infection, 1/2 patients with invasive aspergillosis and 1/2 patients with chronic disseminated candidiasis. We conclude from the data that posaconazole displays favourable safety and tolerance and may be useful for management of individual paediatric patients with invasive infections.

Experimental Infection of Bats with Geomyces destructans Causes White-nose Syndrome

White-nose syndrome (WNS) has caused recent catastrophic declines among multiple species of bats in eastern North America1, 2. The disease’s name derives from a visually apparent white growth of the newly discovered fungus Geomyces destructans on the skin (including the muzzle) of hibernating bats1, 3. Colonization of skin by this fungus is associated with characteristic cutaneous lesions that are the only consistent pathological finding related to WNS4. However, the role of G. destructans in WNS remains controversial because evidence to implicate the fungus as the primary cause of this disease is lacking. The debate is fuelled, in part, by the assumption that fungal infections in mammals are most commonly associated with immune system dysfunction5, 6, 7. Additionally, the recent discovery that G. destructans commonly colonizes the skin of bats of Europe, where no unusual bat mortality events have been reported8, 9, 10, has generated further speculation that the fungus is an opportunistic pathogen and that other unidentified factors are the primary cause of WNS11, 12. Here we demonstrate that exposure of healthy little brown bats (Myotis lucifugus) to pure cultures of G. destructans causes WNS. Live G. destructans was subsequently cultured from diseased bats, successfully fulfilling established criteria for the determination ofG. destructans as a primary pathogen13. We also confirmed that WNS can be transmitted from infected bats to healthy bats through direct contact. Our results provide the first direct evidence that G. destructans is the causal agent of WNS and that the recent emergence of WNS in North America may represent translocation of the fungus to a region with a naive population of animals8. Demonstration of causality is an instrumental step in elucidating the pathogenesis14 and epidemiology15 of WNS and in guiding management actions to preserve bat populations against the novel threat posed by this devastating infectious disease.

STAT3 CONTROLS THE NEUTROPHIL MIGRATORY RESPONSE TO CXCR2 AND ITS LIGAND MIP-2 (CXCL2)

Among the first white blood cells to respond to bacterial and fungal infections, neutrophils are produced in the bone marrow, released into circulating blood, and recruited to inflamed tissue. The cytokine granulocyte colony-stimulating factor (G

CARD9 functions as a key regulator in monocyte homeostasis and pathogen clearance

Mononuclear phagocytes are designed to neutralize systemic bacterial and fungal infections. However, the exact regulation of these functions are largely unknown. CARD9 was first identified as an immune-specific adaptor protein of unclear function. Here, we have found that Card9 is specifically expressed in monocyte-origin cell populations. To better understand the biological function of Card9, we have generated Card9-deficient (Card9-/-) mice. Hematologic profiling and histological analysis of Card9-/- mice revealed a decreased leukocyte/myeloid cell count, delayed monocyte maturation in bone marrow as well as monocyte counts in the peripheral blood. Upon M-CSF stimulation, Card9-/- macrophages further exhibit a partial loss in IKK phosphorylation. As a consequence, in vivo challenge with Listeria monocytogenes in Card9-/- mice results in a higher susceptibility to infection-associated inflammation and fatality. Collectively, these data suggest that CARD9 is required for monocyte development and function. ^ At the cellular level, Card9-/- macrophages are defective in killing Listeria and the production of pro-inflammatory cytokines. Molecular characterizations have further demonstrated that CARD9 inducibly interacts with NOD2, controls p38 MAPK activation, and regulates ROS production during Listeria infections. Cytotrap screening showed that CARD9 could physically associate with various g&barbelow;uanine e&barbelow;xchange f&barbelow;actor (GEF) proteins that are essential for regulating ROS production. In summary, we have first identified and provided genetic evidence that CARD9 functions as a novel regulator during monocyte development and serves as an essential protein adaptor for p38 MAPK activation during bacterial clearance processes in macrophages. ^

Deciphering the C-Type Lectin Receptor Signaling Pathway in Macrophages in Response to Candida albicans

Candida albicans causes opportunistic fungal infections in humans and is a significant cause of mortality and morbidity in immune-compromised individuals. Dectin-2, a C-type lectin receptor, is required for recognition of C. albicans by innate immune cells and is required for initiation of the anti-fungal immune response. We set out to identify components of the intracellular signaling cascade downstream of Dectin-2 activation in macrophages and to understand their importance in mediating the immune response to C. albicans in vivo. Using macrophages derived from Phospholipase-C-gamma 1 and 2 (PLCγ1and PLCγ2) knockout mice, we demonstrate that PLCγ2, but not PLCγ1, is required for activation of NF-κB and MAPK signaling pathways after C. albicans stimulation, resulting in impaired production of pro-inflammatory cytokines and reactive oxygen species. PLCγ2-deficient mice are highly susceptible to infections with C. albicans, indicating the importance of this pathway to the anti-fungal immune response. TAK1 and TRAF6 are critical nodes in NF-κB and MAPK activation downstream of immune surveillance and may be critical to the signaling cascade initiated by C-type lectin receptors in response to C. albicans. Macrophages derived from both TAK1 and TRAF6-deficient mice were unable to activate NF-κB and MAPK and consequently failed to produce inflammatory cytokines characteristic of the response to C. albicans. In this work we have identified PLCγ2, TAK1 and TRAF6 as components of a signaling cascade downstream of C. albicans recognition by C-type lectin receptors and as critical mediators of the anti-fungal immune response. A mechanistic understanding of the host immune response to C. albicans is important for the development of anti-fungal therapeutics and in understanding risk-factors determining susceptibility to C. albicans infection.

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.

Candidemia : revisão e atualização

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

Population pharmacokinetics of itraconazole and its active metabolite hydroxy-itraconazole in paediatric cystic fibrosis and bone marrow transplant patients

Objective: The objective of the study was to characterise the population pharmacokinetic properties of itraconazole and its active metabolite hydroxyitraconazole in a representative paediatric population of cystic fibrosis and bone marrow transplant (BMT) patients and to identify patient characteristics influencing the pharmacokinetics of itraconazole. The ultimate goals were to determine the relative bioavailability between the two oral formulations (capsules vs oral solution) and to optimise dosing regimens in these patients. Methods: All paediatric patients with cystic fibrosis or patients undergoing BMT at The Royal Children's Hospital, Brisbane, QLD, Australia, who were prescribed oral itraconazole for the treatment of allergic bronchopulmonary aspergillosis (cystic fibrosis patients) or for prophylaxis of any fungal infection (BMT patients) were eligible for the study. Blood samples were taken from the recruited patients as per an empirical sampling design either during hospitalisation or during outpatient clinic visits. ltraconazole and hydroxy-itraconazole plasma concentrations were determined by a validated high-performance liquid chromatography assay with fluorometric detection. A nonlinear mixed-effect modelling approach using the NONMEM software to simultaneously describe the pharmacokinetics of itraconazole and its metabolite. Results: A one-compartment model with first-order absorption described the itraconazole data, and the metabolism of the parent drug to hydroxy-itraconazole was described by a first-order rate constant. The metabolite data also showed one-compartment characteristics with linear elimination. For itraconazole the apparent clearance (CLitraconazole) was 35.5 L/hour, the apparent volume of distribution (V-d(itraconazole)) was 672L, the absorption rate constant for the capsule formulation was 0.0901 h(-1) and for the oral solution formulation was 0.96 h-1. The lag time was estimated to be 19.1 minutes and the relative bioavailability between capsules and oral solution (F-rel) was 0.55. For the metabolite, volume of distribution, V-m/(F (.) f(m)), and clearance, CL/(F (.) fm), were 10.6L and 5.28 L/h, respectively. The influence of total bodyweight was significant, added as a covariate on CLitraconazoie/F and V-d(itraconazole)/F (standardised to a 70kg person) using allometric three-quarter power scaling on CLitraconazole/F, which therefore reflected adult values. The unexplained between-subject variability (coefficient of variation %) was 68.7%, 75.8%, 73.4% and 61.1% for CLitraconazoie/F, Vd(itraconazole)/F, CLm/(F (.) fm) and F-rel, respectively. The correlation between random effects of CLitraconazole and Vd((itraconazole)) was 0.69. Conclusion: The developed population pharmacokinetic model adequately described the pharmacokinetics of itraconazole and its active metabolite, hydroxy-itraconazole, in paediatric patients with either cystic fibrosis or undergoing BMT. More appropriate dosing schedules have been developed for the oral solution and the capsules to secure a minimum therapeutic trough plasma concentration of 0.5 mg/L for these patients.

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Síndrome de Sjögren Primário

A Síndrome de Sjögren (SS) é uma das três patologias autoimunes mais frequentes na população mundial, ainda assim frequentemente infradiagnosticada. Esta síndrome é uma doença autoimune crónica que afeta as glândulas exócrinas, particularmente as glândulas salivares e lacrimais. A SS primária (SSp) afeta especificamente as glândulas exócrinas, enquanto que a SS secundária (SSs) aparece associada a outras patologias autoimunes sistémicas. A SS é caracterizada, histopatologicamente, por um infiltrado inflamatório linfocitário que interfere com a função glandular normal. Afeta 0,5% da população mundial, sendo claramente mais predominante em mulheres (9:1 versus homens), principalmente por volta dos 50 anos (após a menopausa), ainda que também possa aparecer depois da menarca (entre os 20 e os 30 anos). Considera se que a etiologia da SS é multifatorial. Fatores genéticos, ambientais, hormonais e virais estão implicados na sua etiopatogénese. É muito importante o papel dos Médicos Dentistas no diagnóstico da SSp, uma vez que na maior parte dos casos são eles quem detetam os primeiros sintomas, mais propriamente a boca seca. Diversos testes auxiliares são utilizados para o diagnóstico desta doença, tais como: o teste de Schimer, o teste de Rosa Bengala, a Sialometria, a Sialografia, a Biópsia das glândulas salivares, entre outros. A SSp, regra geral, tem um curso não doloroso, sendo a boca seca e a secura ocular as suas duas características clínicas mais salientes. A maior parte das manifestações orais que se apresentam nestes pacientes são resultado de hipofunção das glândulas salivares (da boca seca), tais como: a cárie dentária, a doença periodontal, as infeções fúngicas, entre outras. Esta doença também se pode associar a problemas a nível sistémico, que podem ser subdivididos em não viscerais (pele, artralgia, mialgia) e viscerais (pulmão, coração, rim, sistema gastrointestinal, sistema endócrino e sistema nervoso central e periférico). O tratamento é empírico, sintomático e direcionado a tratar as complicações da doença mais inicial, que consiste em limitar os danos da xerostomia e da queratoconjuntivite. A prevenção dos sintomas a nível oral e ocular é fundamental em pacientes com SSp, para assim terem maior qualidade de vida.

Annotation of Gyrodactylus salaris transcriptome

Relationship between organisms within an ecosystem is one of the main focuses in the study of ecology and evolution. For instance, host-parasite interactions have long been under close interest of ecology, evolutionary biology and conservation science, due to great variety of strategies and interaction outcomes. The monogenean ecto-parasites consist of a significant portion of flatworms. Gyrodactylus salaris is a monogenean freshwater ecto-parasite of Atlantic salmon (Salmo salar) whose damage can make fish to be prone to further bacterial and fungal infections. G. salaris is the only one parasite whose genome has been studied so far. The RNA-seq data analyzed in this thesis has already been annotated by using LAST. The RNA-seq data was obtained from Illumina sequencing i.e. yielded reads were assembled into 15777 transcripts. Last resulted in annotation of 46% transcripts and remaining were left unknown. This thesis work was started with whole data and annotation process was continued by the use of PANNZER, CDD and InterProScan. This annotation resulted in 56% successfully annotated sequences having parasite specific proteins identified. This thesis represents the first of Monogenean transcriptomic information which gives an important source for further research on this specie. Additionally, comparison of annotation methods interestingly revealed that description and domain based methods perform better than simple similarity search methods. Therefore it is more likely to suggest the use of these tools and databases for functional annotation. These results also emphasize the need for use of multiple methods and databases. It also highlights the need of more genomic information related to G. salaris.