<i>Mycobacterium bovis</i>–Infected White-Tailed Deer (<i>Odocoileus Virginianus</i>): Detection of Immunoglobulin Specific to Crude Mycobacterial Antigens by ELISA

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis–infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis–infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K–digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., ~11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K–digested M. bovis antigens in the antibody-based assays of tuberculosis.

Recombinant Factors for Hemostasis

Trauma deaths are a result of hemorrhage in 37% of civilians and 47% military personnel and are the primary cause of death for individuals under 44 years of age. Current techniques used to treat hemorrhage are inadequate for severe bleeding. Preliminary research indicates that fibrin sealants (FS) alone or in combination with a dressing may be more effective; however, it has not been economically feasible for widespread use because of prohibitive costs related to procuring the proteins. To meet future demands for hemostatic therapies, FS will likely include recombinant human fibrinogen (rFI) and recombinant human Factor XIII (rFXIII). The underlying hypothesis of the research presented in this dissertation is that a liquid fibrin sealant (LFS) composed of recombinant FI, FXIII and FIIa in optimized proportions can assist hemostasis in the presence and absence of a bioresorbable bandage while using considerably fewer biologics than commercial products currently available. This dissertation characterized rFI produced in the milk of transgenic cows, plasma-derived thrombin (pdFIIa) activated by sodium citrate and rFXIIIa expressed in genetically engineered Pichia pastoris with respect to their capacity to serve as components in a LFS. The ratios of these factors were optimized to yield a LFS with a rapid clot formation rate and high viscoelastic strength. This optimized LFS was preliminarily tested ex vivo and in vivo. The clotting kinetics and viscoelastic strength of our optimized LFS was equivalent to those of a commercially available LFS; however, it uses approximately 75% less fibrinogen and thrombin. Our optimal LFS successfully achieved hemostasis in a significant number of the wounds that included extensive tissue and vascular damage. LFS applied without the assistance of a dressing was able to stop bleeding of oozing wounds or those with small vessels; however, a scaffold was needed when wounds contained large vasculature.

Avaliação visceral da técnica sorológica para Leishmaniose visceral e doença de Chagas em animais silvestres e identificação molecular

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds of Dimorphandra gardneriana and Platymiscium floribundum, native plants from Caatinga biome

Nos últimos anos, o Ministério da Saúde do Brasil e a Organização Mundial da Saúde tem apoiado a investigação de novas tecnologias que possam contribuir para a vigilância, novos tratamentos e controle da leishmaniose visceral no país. Assim, o objetivo deste trabalho foi isolar compostos de plantas do bioma Caatinga, e investigar a toxicidade destes compostos contra as formas promastigotas e amastigotas de Leishmania infantum chagasi, principal parasita responsável pela leishmaniose visceral na América do Sul, e avaliar a sua capacidade para inibir a enzima acetil-colinesterase (AChE). Após a exposição aos compostos em estudo, foram realizados testes utilizando a forma promastigota que expressa luciferase e ELISA in situ para medir a viabilidade das formas promastigotas e amastigota, respectivamente. O ensaio colorimétrico MTT foi realizado para determinar a toxicidade destas substâncias utilizando células monocíticas murina RAW 264.7. Todos os compostos foram testados in vitro para as sua propriedade anti-colinesterásica. Um cumarina, escoparona, foi isolada a partir de hastes de Platymiscium floribundum, e os flavonóides, rutina e quercetina, foram isolados a partir de grãos de Dimorphandra gardneriana. Estes compostos foram purificados, utilizando cromatografia em coluna gel eluída com solventes orgânicos em misturas de polaridade crescente, e identificados por análise espectral. Nos ensaios leishmanicidas, os compostos fenólicos mostraram eficácia contra as formas extracelulares promastigotas, com EC50 para escoporona de 21.4µg/mL e para quercetina e rutina 26 e 30.3µg/mL, respectivamente. Os flavonóides apresentaram resultados comparáveis à droga controle, a anfotericina B, contra as formas amastigotas com EC50 para quercetina e rutina de 10.6 e 43.3µg/mL, respectivamente. Os compostos inibiram a enzima AChE com halos de inibição variando de 0,8 a 0,6cm, indicando um possível mecanismo de ação para a atividade leishmanicida.

In silico identification of conserved intercoding sequences in Leishmania genomes: Unraveling putative cis-regulatory elements

In silico analyses of Leishmania spp. genome data are a powerful resource to improve the understanding of these pathogens' biology. Trypanosomatids such as Leishmania spp. have their protein-coding genes grouped in long polycistronic units of functionally unrelated genes. The control of gene expression happens by a variety of posttranscriptional mechanisms. The high degree of synteny among Leishmania species is accompanied by highly conserved coding sequences (CDS) and poorly conserved intercoding untranslated sequences. To identify the elements involved in the control of gene expression, we conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L major, L infantum, and L braziliensis. We used a combination of computational tools, such as Linux-Shell, PERL and R languages, BLAST, MSPcrunch, SSAKE, and Pred-A-Term algorithms to construct a pipeline which was able to: (i) search for conservation in target-regions, (ii) eliminate CICS redundancy and mask repeat elements, (iii) predict the mRNA's extremities, (iv) analyze the distribution of orthologous genes within the generated LeishCICS-clusters, (v) assign GO terms to the LeishCICS-clusters. and (vi) provide statistical support for the gene-enrichment annotation. We associated the LeishCICS-cluster data, generated at the end of the pipeline, with the expression profile oft. donovani genes during promastigote-amastigote differentiation, as previously evaluated by others (GEO accession: GSE21936). A Pearson's correlation coefficient greater than 0.5 was observed for 730 LeishCICS-clusters containing from 2 to 17 genes. The designed computational pipeline is a useful tool and its application identified potential regulatory cis elements and putative regulons in Leishmania. (C) 2012 Elsevier B.V. All rights reserved.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Cysticercosis in experimentally and naturally infected pigs: Parasitological and immunological diagnosis

Silva M. R. M., Uyhara C.N.S., Silva F. H., Espindola N.M., Poleti M. D., Vaz A.J., Meirelles F. V. & Maia A. A. M. 2012. Cysticercosis in experimentally and naturally infected pigs: Parasitological and immunological diagnosis. Pesquisa Veterinaria Brasileira 32(4): 297-302. Departamento de Ciencias Basicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de Sao Paulo, Av. Duque de Caxias Norte 225, Pirassununga, SP 13635-900, Brazil. E-mail: antomaia@usp.br. Our objective was to evaluate the diagnosis of swine cysticercosis by examining "ante mortem" (inspection of the tongue), "post mortem" (inspection and detailed necropsy) and ELISA for research in serum of antibodies (Ab-ELISA) and antigens (Ag-ELISA). Seven (7) pigs were experimentally infected orally with eggs of Taenia solium and another 10 were naturally infected. In the pigs experimentally infected, inspection of the tongue was negative in all animals, in the routine inspection detailed necropsy and cysticercis were identified in all of them. In pigs with heavy natural infection, inspection of the tongue identified cysticerci in two (20%), while at inspection with necropsy the parasites were identified in large quantities in all animals. In ELISA for antibody search (Ab-ELISA) TS-14 recombinant protein was used, and in search for antigen (Ag-ELISA) a monoclonal antibody against this protein. In animals experimentally infected, blood was collected weekly for 140 days. The Ab-ELISA identified an increase in titers of antibody to cysticerci 21 days after infection, and at the end of the experimental period six animals (86%) were positive to the test. The search for circulating antigens (Ag-ELISA) was positive in two pigs 28 to 91 days after infection. All naturally infected pigs were positive for Ag-ELISA and Ab-ELISA. The search for antibodies and antigens by ELISA in serum from 30 pigs of a local farm and without history of cysticercosis was negative. Thus, the use of TS-14 antigen in ELISA test (Ab-ELISA) can be useful for the diagnosis of cysticercosis in pigs with low infection.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

CE-ESI-MS metabolic fingerprinting of Leishmania resistance to antimony treatment

Metabolomics has become an invaluable tool to unveil biology of pathogens, with immediate application to chemotherapy. It is currently accepted that there is not one single technique capable of obtaining the whole metabolic fingerprint of a biological system either due to their different physical-chemical properties or concentrations. In this work, we have explored the capability of capillary electrophoresis mass spectrometry with a sheathless interface with electrospray ionization (CE-ESI-TOF-MS) to separate metabolites in order to be used as a complementary technique to LC. As proof of concept, we have compared the metabolome of Leishmania infantum promastigotes BCN 150 (Sb (III) IC50 = 20.9 mu M) and its variation when treated with 120 mu M of Sb(III) potassium tartrate for 12 h, as well as with its Sb(III) resistant counterpart obtained by growth of the parasites under increasing Sb(III) in a step-wise manner up to 180 mu M. The number of metabolites compared were of 264 for BCN150 Sb(III) treated versus nontreated and of 195 for Sb(III) resistant versus susceptible parasites. After successive data filtering, differences in seven metabolites identified in databases for Leishmania pathways, showed the highest significant differences, corresponding mainly to amino acids or their metabolite surrogates. Most of them were assigned to sulfur containing amino acids and polyamine biosynthetic pathways, of special relevance considering the deterioration of the thiol-dependent redox metabolism in Leishmania by Sb(III). Given the low concentrations typical for most of these metabolites, the assay can be considered a success that should be explored for new biological questions.

Leishmania (Viannia) braziliensis is the main species causing cutaneous leishmaniasis in the Federal District of Brazil

The first autochthonous case of American cutaneous leishmaniasis was reported in the Federal District in 1980, and the species involved in this type of leishmaniasis was unknown. This study aimed to identify the species that causes the disease in the Federal District and to investigate its clinical and epidemiological aspects. Between 2000 and 2007, 71 autochthonous cases of leishmaniasis were reported in the Federal District. Leishmania species were identified by means of direct immunofluorescence reactions using monoclonal antibodies and restriction fragment length polymorphism. The species of 40 (56.33%) out of 71 samples were identified. Thirty-six (90%) were identified as Leishmania (Viannia) braziliensis and four (10%) were identified as Leishmania (Leishmania) amazonensis. In this area, the disease had clinical and epidemiological characteristics similar to those found in other Brazilian regions.

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4+ and CD8+ immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207+, CD11c+, CD4+, CD8+, iNOS+ cellular densities. Cytokine (IFN-?, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207+ and CD11c+ were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4+ and CD8+ were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS+ was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-? was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

Bladder expression of CD cell surface antigens and cell-type-specific transcriptomes

Many cell types have no known functional attributes. In the bladder and prostate, basal epithelial and stromal cells appear similar in cytomorphology and share several cell surface markers. Their total gene expression (transcriptome) should provide a clear measure of the extent to which they are alike functionally. Since urologic stromal cells are known to mediate organ-specific tissue formation, these cells in cancers might exhibit aberrant gene expression affecting their function. For transcriptomes, cluster designation (CD) antigens have been identified for cell sorting. The sorted cell populations can be analyzed by DNA microarrays. Various bladder cell types have unique complements of CD molecules. CD9(+) urothelial, CD104(+) basal and CD13(+) stromal cells of the lamina propria were therefore analyzed, as were CD9(+) cancer and CD13(+) cancer-associated stromal cells. The transcriptome datasets were compared by principal components analysis for relatedness between cell types; those with similarity in gene expression indicated similar function. Although bladder and prostate basal cells shared CD markers such as CD104, CD44 and CD49f, they differed in overall gene expression. Basal cells also lacked stem cell gene expression. The bladder luminal and stromal transcriptomes were distinct from their prostate counterparts. In bladder cancer, not only the urothelial but also the stromal cells showed gene expression alteration. The cancer process in both might thus involve defective stromal signaling. These cell-type transcriptomes provide a means to monitor in vitro models in which various CD-isolated cell types can be combined to study bladder differentiation and bladder tumor development based on cell-cell interaction.

Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.