Sub-telomere directed gene expression during initiation of invasive aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*001 was the most common allele found, followed by ABO*022 and by ABO*A103. We identified 22 new ABO*(variants) in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

Lack of Galectin-3 Disturbs Mesenteric Lymph Node Homeostasis and B Cell Niches in the Course of Schistosoma mansoni Infection

Galectin-3 is a beta-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAc beta 1-4(Fuc alpha 1-3) GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine beta 1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in nonlymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S. mansoni.

Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis

Background: Chagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi. Methodology/Principal Findings: First, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4(+), CD8(+) and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-gamma and TNF-alpha and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, ROR gamma t and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice. Conclusion/Significance: These results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88(-/-)) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88(-/-) mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88(-/-) mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-gamma responses by NKT, CD4(+) and CD8(+) T lymphocytes, and production of high levels of IL-10. MyD88(-/-) mice replenished with IL-12 and IFN-gamma abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-gamma-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

The role of major histocompatibility complex Alleles in the susceptibility of Brazilian patients to develop the myogenic type of Graves' orbitopathy

Objective: To assess the frequency of the genetic markers HLA-DRB1 and DQB1 in patients with Graves' orbitopathy ( GO) with and without extraocular muscle involvement. Design: The frequencies of class II HLA-DRB1 and DQB1 allele groups were determined for 81 Brazilian patients with GO and 161 normal subjects. The patients were divided into myogenic and nonmyogenic groups based on the clinical characteristics of the orbitopathy and quantitative computed tomography analysis of the extraocular muscle ( EOM) dimensions. Main outcome: Compared to the frequency obtained for samples of normal subjects of the Brazilian population, HLA-DRB1*16 (p(c)= 0.008) was overrepresented in myogenic and HLA-DRB1*03 (p(c)= 0.02) in nonmyogenic patients. Conclusions: The association between the HLA-DRB1* 16 and the myogenic subtype of GO suggests that EOM involvement in GO may be genetically predisposed.

Primary Hyperparathyroidism as the First Clinical Manifestation of Multiple Endocrine Neoplasia Type 2A in a 5-Year-Old Child

Background: Primary hyperparathyroidism occurs in only 10%-30% of patients with multiple endocrine neoplasia type 2A (MEN2A), rarely as the sole clinical manifestation, and is usually diagnosed after the third decade of life. Summary: A5-year-old girl was referred for prophylactic thyroidectomy as she carried the p.C634R RET mutation. She was clinically asymptomatic, with a normally palpable thyroid and with the cervical region free of lymphadenopathy or other nodules. Preoperative tests revealed hypercalcemia associated with elevation of parathyroid hormone (PTH) (calcium = 11.2mg/dL, calcium ion = 1.48mmol/L, phosphorus = 4.0 mg/dL, alkaline phosphatase = 625U/L, parathyroid hormone (PTH) PTH = 998 pg/mL). A thyroid ultrasound was normal and parathyroid scintigraphy with (99m)Tc-Sestamibi revealed an area of radioconcentration in the upper half of the left thyroid lobe suggesting hyperfunctioning parathyroid tissue. She underwent total thyroidectomy and parathyroidectomy and developed hypocalcemia. The anatomopathological examination showed no histopathological changes in the thyroid tissue and an adenoma of the parathyroid gland, confirming the diagnosis of hyperparathyroidism. Conclusions: Primary hyperparathyroidism can be a precocious manifestation of MEN2A. This case report highlights that asymptomatic hypercalcemia should be scrutinized in children related to patients with MEN2A who carry a mutation in the RET proto-oncogene, especially mutations in the codon 634, before the currently recommended age of 8 years.

Serologic Survey for Rickettsiosis in Bats from Sao Paulo City, Brazil

Blood serum samples were collected from 451 bats captured within the Sao Paulo city from April 2007 to November 2008, and individually tested by indirect immunofluorescence assay against antigens derived from five Rickettsia species reported to occur in Brazil: the spotted fever group (SFG) species R. rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, and the ancestral group species R. bellii. For this purpose, an anti-bat immunoglobulin G was produced and used in the present study. Overall, 8.6% (39/451), 9.5% (34/358), 7.8% (28/358), 1.1% (4/358), and 0% (0/358) serum samples were reactive to R. rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, and R. bellii, respectively. Endpoint titers of reactive sera ranged from 64 to 256. From 20 bat species of 3 different families (Molossidae, Vespertilionidae, and Phyllostomidae), 46 animals were shown to be reactive to at least one rickettsial antigen. Seropositivity per bat species ranged from 0% to 33.3%. Most of the serologically positive sera reacted with two or more rickettsial antigens. Seropositivity for SFG rickettsial antigens in the absence of reactivity against R. bellii (ancestral group species) suggests that bats from Sao Paulo city can be infected by SFG rickettsiae. The possible role of soft ticks in serving as vectors of SFG rickettsiae to bats within the Sao Paulo city, associated to its public health risks, is discussed.

Tick-Borne Bacteria in Free-Living Jaguars (Panthera onca) in Pantanal, Brazil

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer >= 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8 +/- 25.2 nM and 167.39 +/- 60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4 +/- 15.9 nM to laminin and of 290.8 +/- 11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake) venom

Background: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization. Results: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens. Conclusion: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.