Challenges and perspectives in research and teaching of host pathogen interaction topics: new post-pandemic times to Brazil and other South American countries.

Here is our proposal to improve learning in biomedical sciences for graduate and undergraduate courses with a broad vision integrating disciplines such as molecular cell biology, biochemistry, and biophysics around concepts of pathogen interaction within vertebrate and invertebrate hosts. Our paradigm is based on the possibility offered by the pandemic to have remote activities that give access to students and researchers from different places in Brazil and Latin American countries to discuss science. A multidisciplinary view of host-pathogen interaction allows us to understand better the mechanisms involved in the pathology of diseases, as well as to formulate broad strategies for the diagnosis, treatment, and control of thereof. The approach to integrating heterogeneous groups in science involves the critical analysis of national scientific resource distribution, where only some have the possibilities to conduct competitive scientific research. Solid theoretical training, contact, collaboration with groups of excellence, and training within a multidisciplinary network are our proposals for a permanent platform of scientific strengthening and dissemination for Latin America. Here we will review the concept of host-pathogen interaction, the type of institutions where it is taught and researched, new trends in active teaching methodologies, and the current political context in science.

Challenges and perspectives in research and teaching of host pathogen interaction topics: new post-pandemic times to Brazil and other South American countries

Here is our proposal to improve learning in biomedical sciences for graduate and undergraduate courses with a broad vision integrating disciplines such as molecular cell biology, biochemistry, and biophysics around concepts of pathogen interaction within vertebrate and invertebrate hosts. Our paradigm is based on the possibility offered by the pandemic to have remote activities that give access to students and researchers from different places in Brazil and Latin American countries to discuss science. A multidisciplinary view of host-pathogen interaction allows us to understand better the mechanisms involved in the pathology of diseases, as well as to formulate broad strategies for the diagnosis, treatment, and control of thereof. The approach to integrating heterogeneous groups in science involves the critical analysis of national scientific resource distribution, where only some have the possibilities to conduct competitive scientific research. Solid theoretical training, contact, collaboration with groups of excellence, and training within a multidisciplinary network are our proposals for a permanent platform of scientific strengthening and dissemination for Latin America. Here we will review the concept of host-pathogen interaction, the type of institutions where it is taught and researched, new trends in active teaching methodologies, and the current political context in science.

Challenges and perspectives in research and teaching of host pathogen interaction topics: new post-pandemic times to Brazil and other South American countries

Here is our proposal to improve learning in biomedical sciences for graduate and undergraduate courses with a broad vision integrating disciplines such as molecular cell biology, biochemistry, and biophysics around concepts of pathogen interaction within vertebrate and invertebrate hosts. Our paradigm is based on the possibility offered by the pandemic to have remote activities that give access to students and researchers from different places in Brazil and Latin American countries to discuss science. A multidisciplinary view of host-pathogen interaction allows us to understand better the mechanisms involved in the pathology of diseases, as well as to formulate broad strategies for the diagnosis, treatment, and control of thereof. The approach to integrating heterogeneous groups in science involves the critical analysis of national scientific resource distribution, where only some have the possibilities to conduct competitive scientific research. Solid theoretical training, contact, collaboration with groups of excellence, and training within a multidisciplinary network are our proposals for a permanent platform of scientific strengthening and dissemination for Latin America. Here we will review the concept of host-pathogen interaction, the type of institutions where it is taught and researched, new trends in active teaching methodologies, and the current political context in science.

In Silico Prediction and Design of Uropathogenic Escherichia coli Alpha-Hemolysin Generate a Soluble and Hemolytic Recombinant Toxin.

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542-723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555-565, aa600-610, and aa674-717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Secreted autotransporter toxin (Sat) mediates innate immune system evasion

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

In silico prediction and design of uropathogenic Escherichia coli alpha-hemolysin generate a soluble and hemolytic recombinant toxin

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542–723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555–565, aa600–610, and aa674–717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Anti-Biofilm action of biological silver nanoparticles produced by aspergillus tubingensis and antimicrobial activity of fabrics carrying it

Biological silver nanoparticles (AgNPs) were synthesized using the marine endophytic fungus Aspergillus tubingensis and inhibited Bacillus subtilis biofilm formation at low concentrations. Cotton and polyester fabrics impregnated with AgNPs were analyzed by transmission electron microscopy (TEM), and the concentration of AgNPs in both fabrics was determined using inductivelycoupled plasma atomic emission spectrometry (ICP-AES). The fabrics carrying the AgNPs inhibited the Staphylococcus aureus and Escherichia coli growth by 100%. Both fabrics impregnated one time with AgNPs inhibited yeasts' clinical species' growth, Candida albicans, Candida glabrata, and Candida parapsilosis, from 80.1% to approximately 98.0%. Besides the anti-biofilm effect, the AgNPs impregnation process on cotton and polyester fabrics was highly efficient, and both fabrics presented antimicrobial effects against clinically relevant bacteria and yeast species. The results evidence that functionalized textiles containing these biological AgNPs can play an essential role in combating pathogenic microorganisms. Thereby offering an alternative to design effective solutions, mainly for hospital garments and biomedical devices, to avoid microorganisms transmissions and hospitalacquired nosocomial infections.

Secreted autotransporter toxin (Sat) induces cell damage during enteroaggregative Escherichia coli infection

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77,O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.

Secreted autotransporter toxin (Sat) induces cell damage during enteroaggregative Escherichia coli infection

Secreted autotransporter toxin (Sat) is a 107-kDa serine protease autotransporter of Enterobacteriaceae (SPATE) presenting cytotoxic activity in renal and bladder cells. Further studies have detected the Sat-encoding gene (sat) in enteroaggregative Escherichia coli (EAEC) and in E. coli strains isolated from neonatal septicemia and meningitis. Here, we investigated the role of Sat as a cytotoxin of EAEC. Sat was purified from a strain of E. coli harboring sat (DEC/Sat+, O126:H2) and used to raise antibodies in rabbit. The presence of Sat was detected by ELISA in the supernatant of 93.7% of EAEC strains harboring sat and in none lacking the gene. The effect of Sat during infection was investigated in polarized Caco-2 cells infected with Sat-producing EAEC (CV323/77,O125ab:H21). This strain induced intense cell detachment, which was inhibited by PMSF or Sat antiserum. Also, sat transcription and Sat production were detected during infection. Here we demonstrate that Sat is internalized in polarized cells leading to F-actin disruption which preceded cell detachment. A comparative study of the toxin action in cell lines corresponding to the infection sites in which bacteria carrying the sat gene have been isolated was performed. Cells originating from the gastrointestinal tract (Caco-2), urinary (LLC-PK1) and endothelium (HUVEC) were incubated with purified Sat. The time required for observation of cell damage differed according to the cell line. HUVEC cells were more sensitive to Sat than cells derived from urinary and intestinal tracts. The intense activity of Sat on the endothelial cells suggests that Sat could also be a virulence factor for the bacteria in the bloodstream. In addition, this is the first work demonstrating that Sat induces cytotoxic effect during EAEC infection in vitro. The cell damage observed during infection indicates that Sat may be another toxin with cytotoxic role in the EAEC pathogenesis.

Functional textiles impregnated with biogenic silver nanoparticles from Bionectria ochroleuca and its antimicrobial activity

Biogenic silver nanoparticles (AgNPs) were obtained throughout the fungal biosynthesis using extracellular filtrate of the epiphytic fungus B. ochroleuca and were incorporated in cotton and polyester fabrics by common impregnation procedure that was repeated once, twice or four times. Both fabrics were analyzed by scanning electron microscopy (SEM), and the effectiveness of impregnation was determined using inductively coupled plasma optical emission spectrometry (ICP OES). The AgNPs loaded fabrics showed potent antimicrobial activity on Staphylococcus aureus and Escherichia coli as well as on clinically relevant Candida albicans, Candida glabrata, and Candida parapsilosis, indicating that the AgNPs impregnation of cotton and polyester fabrics was efficient. AgNPs effectively inhibited the biofilm formation by Pseudomonas aeruginosa and was not toxic to Galleria mellonella larvae indicating a promising probability of biotechnological application.

Functional textiles impregnated with biogenic silver nanoparticles from Bionectria ochroleuca and its antimicrobial activity

Biogenic silver nanoparticles (AgNPs) were obtained throughout the fungal biosynthesis using extracellular filtrate of the epiphytic fungus B. ochroleuca and were incorporated in cotton and polyester fabrics by common impregnation procedure that was repeated once, twice or four times. Both fabrics were analyzed by scanning electron microscopy (SEM), and the effectiveness of impregnation was determined using inductively coupled plasma optical emission spectrometry (ICP OES). The AgNPs loaded fabrics showed potent antimicrobial activity on Staphylococcus aureus and Escherichia coli as well as on clinically relevant Candida albicans, Candida glabrata, and Candida parapsilosis, indicating that the AgNPs impregnation of cotton and polyester fabrics was efficient. AgNPs effectively inhibited the biofilm formation by Pseudomonas aeruginosa and was not toxic to Galleria mellonella larvae indicating a promising probability of biotechnological application.

Ferromagnetic resonance for the quantification of superparamagnetic iron oxide nanoparticles in biological materials

The aim of the present work is the presentation of a quantification methodology for the control of the amount of superparamagnetic iron oxide nanoparticles (SPIONs) administeredin biological materials by means of the ferromagnetic resonance technique (FMR) applied to studies both in vivo and in vitro. The in vivo study consisted in the analysis of the eliminationand biodistribution kinetics of SPIONs after intravenous administration in Wistar rats. The results were corroborated by X-ray fluorescence. For the in vitro study, a quantitative analysisof the concentration of SPIONs bound to the specific AC133 monoclonal antibodies was carriedout in order to detect the expression of the antigenic epitopes (CD133) in stem cells from human umbilical cord blood. In both studies FMR has proven to be an efficient technique forthe SPIONs quantification per volume unit (in vivo) or per labeled cell (in vitro).

Trypanosoma cruzi: associação entre infectividade e expressão de glicoproteínas de superfície com atividade sinalizadora de cálcio / Trypanosoma cruzi: association between infectivity and expression of surface glycoproteins with calcium signaling activity

Neste trabalho investigamos alguns aspectos do processo de invasão celular do T cruzi associados à mobilização de cálcio no parasita e na célula hospedeira. Traçamos como principais objetivos: a) determinar se existe correlação entre a capacidade de formas metacíclicas de diferentes cepas de T cruzi em invadir células de mamífero e a expressão de moléculas de superfície gp9O, gp82 e gp35/50. b) analisar comparativamente a capacidade da gp9O, gp82 e gp35/50 de induzir sinal de Ca 2+ na célula hospedeira e/ou no parasita. Os resultados de ensaio de invasão celular mostraram que as formas metacíclicas da cepa CL invadem células de mamífero (Hela, Vero) em maior número (6-8 vezes) em comparação à cepa G. A análise da expressão das glicoproteínas de superfície gp9O, gp82 e gp35/50 através da marcação das formas metacíclicas com 125I seguida de imunoprecipitação, "immunoblotting" e citometria de fluxo, revelou que a cepa G difere da cepa CL pela expressão de gp9O e pela presença de maiores níveis de gp35/50. Quanto à gp82, cuja expressão é comparável nas duas cepas. a análise da seqüência de amlnoácidos, deduzida a partir da seqüência de CDNA. revelou 97,9 por cento de identidade entre a cepa G e CL. Mostramos que o extrato sonicado de formas metacíclicas (G e CL) é capaz de induzir o aumento de [Ca2-]i em células Hela, e que grande parte dessa atividade é devida à gp82, a julgar pela inibição de 50-70 por cento que resulta da incubação do extrato parasitário com o anticorpo monoclonal 3F6 dirigido contra a gp82. Nos experimentos com a glicoproteína purificada de tripomasstígotas metacíclicos, observamos que: a) a gp9O, gp82 e gp35/50, ligam-se às células Hela de maneira dose dependente e saturável, o que caracteriza uma ligação do tipo receptor-dependente. b) a gp82 induz maior aumento de [Ca2+]i nas células Hela quando comparada à gp35/50, enquanto a gp9O não induz a mobilização de Ca2+ de maneira significativa, do mesmo modo que a gp63 de Leishmania (L.) amazonensis, utilizada como controle. c) as proteínas recombinantes contendo as sequencias da gp9O ou da gp82, geradas em bactérias, ligam-se às células Hela do mesmo modo que as moléculas nativas, indicando que os carboidratos das cadeias oligossacarídicas ou a âncora GPI não estão envolvidas na adesão à célula hospedeira. Observamos nos ensaios de mobilização de Ca2+ intracelular nas formas metacíclicas observamos que a mesma é induzida pelo extrato ...(au)

Trypanosoma cruzi: course of infection in platelets-depleted mice

The effect of platelet depletion on the course of Trypanosoma cruzi infection in BALB/c mice was investigated. Thrombocytopenia was achieved by inoculation of rabbit anti-platelet IgG during the parasitemic phase of the infection. The number of parasites in the blood of anti-platelet IgG treated was significantly higher than that of non-treated control mice, during the phase of high parasitemia. Cumulative mortality of platelet-depleted mice was consistently but not significantly higher than that of control mice up to the 32nd day of infection; from the 33rd day on they were equivalent, no mortalities occurring from then on, until observations were discontinued on the 60th day. These results suggest that platelets participate of the mechanisms of parasites removal from the bloodstream, but do not have an effective role in the mechanisms of defence against T. cruzi, during the acute phase of infection.

Contagem de linfocitos timo dependentes e bursas equivalentes em criancas normais / Count of lymphocytes thymos dependents and bursa equivalentes in normals childreens

Foram realizadas contagens de linfocitos timo dependentes(T) e bursa equivalentes (B) no sangue periferico de oitenta criancas normais entre dois e doze anos de idade. As tecnicas utilizads foram as de formacao de rosaceas com eritrocitos de carneiro para determinacao de linfocitos T e de rosaceas com zimosan para linfocitos B. Os resultados obtidos foram: 34,1 a 85,7% de linfocitos T e 2,78 a 19,6 de linfocitos B para o grupo etario de dois anos a quatroanos e onze meses: 40,5% a 81,7% de T e 1,7 a 19,3% de B para criancas de cinco anos a oito e onze meses; 43,1 a 85,9% de T e 3,8 a 19,0% de B entre criancasde nove a doze anos de idade. Esses valores podem ser utilizados como parametrospara comparacao com resultados de exames que pesquisem estados de imunodeficiencias em criancas .