Molecular Verification of Trichophyton in the Brazilian URM Culture Collection.

Trichophyton species cause dermatophytosis in humans, with a high, worldwide frequency of reports and important public health relevance. We evaluated 61 Trichophyton strains from different sources deposited in the University Recife Mycology (URM) culture collection of the Universidade Federal de Pernambuco, Brazil. Strains were phenotypically identified and confirmed by sequencing Internal Transcribed Spacers rDNA and partial beta-tubulin 2-exon. Additionally, we evaluated their susceptibility to terbinafine and itraconazole. Physiological analyses included urease activity and growth in casein medium. Phenotypic methods allowed the reliable identification of T. rubrum only, whereas, for other species, molecular methods were mandatory. All Trichophyton species exhibited susceptibility profiles to itraconazole (0.04-5.33 µg/mL) and terbinafine (0.17-3.33 µg/mL). Our analyses revealed a heterogeneous distribution of T. mentagrophytes, which does not support the current distribution within the species complex of T. mentagrophytes and its genotypes.

Impact of repair protocols on the bond strength to composite resin.

This study evaluated the impact of different repair protocols on a composite resin substrate using distinct bonding agents submitted or not to artificial aging. Unopened sets of a single-step universal adhesive system (UA) and silane-coupling agents, a single-step pre-hydrolyzed (PH) or a two-step immediately hydrolyzed (IH), were used. Half of the sets were subjected to artificial aging being stored at 48 °C for 30 days, while the other half remained unaged. The composite resin substrates were prepared and aged in distilled water, sandblasted (Al2O3), and cleaned. Then the different repair protocols were applied according to the groups. UA was used without a previous silane layer, while PH and IH were applied followed by a single-step etch-and-rinse adhesive system. Adhesive systems were light-activated, and four composite resin cylinders were formed over the substrate. After 24 h, the specimens were subjected to microshear bond strength (µSBS) test and failure mode analysis. The µSBS data were subjected to two-way ANOVA followed by Tukey HSD; Kruskal-Wallis analysis was used for failure mode distribution (α = 0.05). After aging the products, UA showed higher bond strength, while PH had significantly lower results, and IH showed no significant differences (p = 0.157). No significant differences were found for bond strength among the repair protocols when using non-aged products (p > 0.05). The protocols using UA and IH showed no significant differences between aged and non-aged bottles, whereas PH exhibited lower bond strength when comparing aged and non-aged products. More cohesive failures were observed in the resin substrate for the IH group without aging.

Organotypic hippocampal culture model reveals differential responses to highly similar Zika virus isolates.

INTRODUCTION: Zika virus (ZIKV) caused an outbreak in Brazil, in 2015, being associated to microcephaly. ZIKV has a strong neurotropism leading to death of infected cells in different brain regions, including the hippocampus, a major site for neurogenesis. The neuronal populations of the brain are affected differently by ZIKV from Asian and African ancestral lineages. However, it remains to be investigated whether subtle variations in the ZIKV genome can impact hippocampus infection dynamics and host response. OBJECTIVE: This study evaluated how two Brazilian ZIKV isolates, PE243 and SPH2015, that differ in two specific missense amino acid substitutions, one in the NS1 protein and the other in the NS4A protein, affect the hippocampal phenotype and transcriptome. METHODS: Organotypic hippocampal cultures (OHC) from infant Wistar rats were infected with PE243 or SPH2015 and analyzed in time series using immunofluorescence, confocal microscopy, RNA-Seq and RT-qPCR. RESULTS: Unique patterns of infection and changes in neuronal density in the OHC were observed for PE243 and SPH2015 between 8 and 48 h post infection (p.i.). Phenotypic analysis of microglia indicated that SPH2015 has a greater capacity for immune evasion. Transcriptome analysis of OHC at 16 h p.i. disclosed 32 and 113 differentially expressed genes (DEGs) in response to infection with PE243 and SPH2015, respectively. Functional enrichment analysis suggested that infection with SPH2015 activates mostly astrocytes rather than microglia. PE243 downregulated biological process of proliferation of brain cells and upregulated those associated with neuron death, while SPH2015 downregulated processes related to neuronal development. Both isolates downregulated cognitive and behavioral development processes. Ten genes were similarly regulated by both isolates. They are putative biomarkers of early hippocampus response to ZIKV infection. At 5, 7, and 10 days p.i., neuronal density of infected OHC remained below controls, and mature neurons of infected OHC showed an increase in the epigenetic mark H3K4me3, which is associated to a transcriptionally active state. This feature is more prominent in response to SPH2015. CONCLUSION: Subtle genetic diversity of the ZIKV affects the dynamics of viral dissemination in the hippocampus and host response in the early stages of infection, which may lead to different long-term effects in neuronal population.

First report of Colletotrichum plurivorum causing anthracnose on Cucumis sativus in Brazil.

Cucumbers have great economic and social importance. Annual worldwide production is approximately 80 million tons (FAOSTAT, 2019), 184 thousand tons of which are produced in Brazil (IBGE, 2020). Leaves with symptoms of anthracnose (necrotic brown or angular spots) were observed on cucumber plants grown in organic systems in September 2021, Pernambuco, Brazil (8°7'45''S, 35°16'167''W). About 40% of the plants fields were infected. Samples were collected and fragments were cut from the margins of the symptomatic tissue. The fragments were superficially disinfected with 70% ethanol (30 s) and 2% sodium hypochlorite (2 min), then washed three times with sterile distilled H2O and dried on sterile filter paper. The fragments were placed on potato dextrose agar (PDA) containing chloramphenicol (50 mg/L) and incubated at 28 ± 2 °C for 3 days. From the fungal isolates obtained, a representative specimen of Colletotrichum spp. was isolated, purified by subculturing from emergent hyphae tips and used for morphological characterization, phylogenetic analysis, and pathogenicity testing. The fungus isolated on PDA formed gray to grayish-black colonies with white aerial mycelia after 7 days. Ascomata were globose to subglobose, 120-200 × 100-150 µm in size (n = 10). Setae formed directly on the hyphae. Asci were 50-70 × 10-12 µm in size, 8-spored, unitunicate, thin-walled, and clavate. Ascospores were 14-22 × 4-5 µm in size (n = 30), hyaline, slightly curved to curved with obtuse to slightly rounded ends. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, the apex and base rounded, and 12-15 × 5 µm in size, (n = 30). For molecular identification, the nuclear ribosomal internal transcribed spacers (nrITS), actin (ACT), beta-tubulin (TUB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were sequenced (Damm et al. 2019). The sequences obtained were deposited in GenBank (nrITS: OP720945, ACT: OP723523, TUB: OP723525, and GAPDH: OP723524). The sequences from the nrITS region, ACT, TUB2, and GAPDH were highly similar to those from C. plurivorum: nrITS - CBS 125474 (539/539 - 100%; NR_160828); ACT - CBS 125474 (270/271 - 99%; MG600925), TUB2 - CBS 125474 (517/518 - 99%; MG600985); and GAPDH - CBS 125474 (197/197 - 100%; MG600781), respectively. Multilocus phylogenetic analysis was performed using Bayesian inference, which showed that the isolate C. plurivorum FPO04 clustered in the same clade as the ex-type of C. plurivorum (CBS 125474). In the pathogenicity test, leaves of five healthy cucumber plants, previously injured in the middle region with sterile needles, were inoculated with 50 µl of a conidial suspension (1 × 106 spores mL -1) prepared from 7-day-old of colonies of C. plurivorum. Sterile distilled water was used as negative controls. The inoculated plants were maintained in a humid greenhouse chamber for 24 hours. After 7 days, the same anthracnose symptoms seen in the field were observed on the inoculated plants. Control plants remained healthy. Colletotrichum plurivorum was reisolated from symptomatic leaves, fulfilling Koch's postulates. This species has been reported from several crops, including Abelmoschus esculentus (okra) (Damm et al. 2019) and Glycine max (soybeans) (Zaw et al. 2019). To our knowledge, this is the first report of C. plurivorum causing anthracnose on cucumber leaves in Brazil. This report lays the groundwork for future studies to determine management practices for control of this disease in C. sativus.

Mitochondrial DNA as a Possible Ligand for TLR9 in Irinotecan-induced Small Intestinal Mucositis.

Cancer chemotherapy and radiotherapy may result in mucositis characterized by stem cell damage and inflammation in the gastrointestinal tract. The molecular mechanisms underlying this pathology remain unknown. Based on the assumption that mitochondrial CPG-DNA (mtDNA) released and sensed by TLR9 could underlie mucositis pathology, we analyzed the mtDNA levels in sera as well as inflammatory and disease parameters in the small intestine from wild-type (WT) and TLR9-deficient mice (TLR9-/-) in an experimental model of intestinal mucositis induced by irinotecan. Additionally, we verified the ability of WT and TLR9-/- macrophages to respond to CpG-DNA in vitro. WT mice injected with irinotecan presented a progressive increase in mtDNA in the serum along with increased hematocrit, shortening of small intestine length, reduction of intestinal villus:crypt ratio and increased influx of neutrophils, which were followed by higher expression of Nlrp3 and Casp1 mRNA and increased IL-1ß levels in the ileum when compared to vehicle-injected mice. TLR9-deficient mice were protected in all these parameters when compared to WT mice. Furthermore, TLR9 was required for the production of IL-1ß and NO after macrophage stimulation with CpG-DNA. Overall, our findings show that the amount of circulating free CpG-DNA is increased upon chemotherapy and that TLR9 activation is important for NLRP3 inflammasome transcription and further IL-1ß release, playing a central role in the development of irinotecan-induced intestinal mucositis. We suggest that TLR9 antagonism may be a new therapeutic strategy for limiting irinotecan-induced intestinal inflammation.

Relevance of angiotensin-(1-7) and its receptor Mas in pneumonia caused by influenza virus and post-influenza pneumococcal infection.

Resolution failure of exacerbated inflammation triggered by Influenza A virus (IAV) prevents return of pulmonary homeostasis and survival, especially when associated with secondary pneumococcal infection. Therapeutic strategies based on pro-resolving molecules have great potential against acute inflammatory diseases. Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator that acts on its Mas receptor (MasR) to promote resolution of inflammation. We investigated the effects of Ang-(1-7) and the role of MasR in the context of primary IAV infection and secondary pneumococcal infection and evaluated pulmonary inflammation, virus titers and bacteria counts, and pulmonary damage. Therapeutic treatment with Ang-(1-7) decreased neutrophil recruitment, lung injury, viral load and morbidity after a primary IAV infection. Ang-(1-7) induced apoptosis of neutrophils and efferocytosis of these cells by alveolar macrophages, but had no direct effect on IAV replication in vitro. MasR-deficient (MasR-/-) mice were highly susceptible to IAV infection, displaying uncontrolled inflammation, increased viral load and greater lethality rate, as compared to WT animals. Ang-(1-7) was not protective in MasR-/- mice. Interestingly, Ang-(1-7) given during a sublethal dose of IAV infection greatly reduced morbidity associated with a subsequent S. pneumoniae infection, as seen by decrease in the magnitude of neutrophil influx, number of bacteria in the blood leading to a lower lethality. Altogether, these results show that Ang-(1-7) is highly protective against severe primary IAV infection and protects against secondary bacterial infection of the lung. These effects are MasR-dependent. Mediators of resolution of inflammation, such as Ang-(1-7), should be considered for the treatment of pulmonary viral infections.

Influence of material and loading location on stress distribution of inlays.

PURPOSE: To evaluate the stress distribution of inlays fabricated with restorative materials of different elastic moduli under two functional loading conditions by using three-dimensional (3D) finite element analysis (FEA) models of a second maxillary premolar. METHODS: A 3D model of a sound maxillary left second premolar and its supporting bone was generated in a previous study and reutilized under permission of the authors for the present study. Inlay models obtained from the sound tooth were then assigned according to the type and inherent elastic modulus of the restorative material used, as follows: microhybrid composite (Filtek Z250); indirect resin composite (Paradigm); lithium disilicate reinforced glass ceramic (IPS e.max Press); and type III gold alloy. The geometric models were then exported for 3D FEA. All materials were considered isotropic, homogeneous, and linear-elastic. A static load of 100 N was applied in two conditions: axially at both cusps (axial loading) and at the mesial marginal ridge (proximal loading). Maximum principal and von Mises stresses were used to analyze the stress distribution pattern in inlays and sound premolar models. RESULTS: Under axial loading condition, either resin composite, ceramic or type III gold alloy inlays generated a similar biomechanical behavior, especially in terms of stress distribution in the remaining tooth structure and cusp deflection. However, higher tensile stresses were observed along the proximal gingival margin of the preparation under proximal loading in the Z250 and Paradigm models, as well as a greater cusp deflection. In contrast, a deflection like the sound model was observed in the ceramic and gold inlay models. CLINICAL SIGNIFICANCE: Restorative materials with higher elastic modulus, such as dental ceramics and type III gold alloys, seem to be the best choice for maxillary premolars restored with inlays in the presence of occlusal contact on the marginal ridge.

ACKR2 contributes to pulmonary dysfunction by shaping CCL5:CCR5-dependent recruitment of lymphocytes during influenza A infection in mice.

Inflammation triggered by influenza A virus (IAV) infection is important for viral clearance, induction of adaptive responses, and return to lung homeostasis. However, an exaggerated immune response, characterized by the overproduction of chemokines, can lead to intense lung injury, contributing to mortality. Chemokine scavenger receptors, such as ACKR2, control the levels of CC chemokines influencing the immune responses. Among the chemokine targets of ACKR2, CCL5 is important to recruit and activate lymphocytes. We investigated the role of ACKR2 during IAV infection in mice. Pulmonary ACKR2 expression was increased acutely after IAV infection preceding the virus-induced lung dysfunction. ACKR2-knockout (ACKR2-/-) mice were protected from IAV, presenting decreased viral burden and lung dysfunction. Mechanistically, the absence of ACKR2 resulted in augmented airway CCL5 levels, secreted by mononuclear and plasma cells in the lung parenchyma. The higher chemokine gradient led to an augmented recruitment of T and B lymphocytes, formation of inducible bronchus-associated lymphoid tissue and production of IgA in the airways of ACKR2-/- mice post-IAV. CCL5 neutralization in ACKR2-/- mice prevented lymphocyte recruitment and increased bronchoalveolar lavage fluid protein levels and pulmonary dysfunction. Finally, CCR5-/- mice presented increased disease severity during IAV infection, displaying increased neutrophils, pulmonary injury and dysfunction, and accentuated lethality. Collectively, our data showed that ACKR2 dampens CCL5 levels and the consequent recruitment of CCR5+ T helper 1 (Th1), T regulatory cells (Tregs), and B lymphocytes during IAV infection, decreasing pathogen control and promoting lung dysfunction in wild type mice. Therefore, ACKR2 is detrimental and CCR5 is protective during IAV infection coordinating innate and adaptive immune responses in mice.

Development of a fiber-reinforced material for fiber posts: Evaluation of stress distribution, fracture load, and failure mode of restored roots.

STATEMENT OF PROBLEM: The biomechanical behavior of post-restored roots with an experimental fiber-reinforced composite resin is unknown. PURPOSE: The purpose of this in vitro study was to investigate the biomechanical behavior of an experimental composite resin (3-mm short glass fiber incorporated in methacrylate matrix with filler particles) used to produce the custom post itself or to reline fiber posts. MATERIAL AND METHODS: Four testing groups (n=10) were created according to the root restoration method: FG, commercially available fiber post; FG+RC, fiber post relined with conventional composite resin; FG+EXP, fiber post relined with the experimental composite resin; and EXP, a custom post made of experimental composite resin. A three-dimensional finite element linear elastic analysis was performed by using geometric representations of groups, and the results were analyzed by von Mises (σvM) and maximum principal stress criteria. In sequence, 40 bovine incisors were assigned to these groups and subjected to a fracture load test (Instron 5965; 0.5 mm/min), and the failure mode was determined. RESULTS: The EXP group showed more homogeneous stress distribution for σvM. ANOVA and the Tukey honestly significant difference (HSD) tests showed significant differences (P<.001) in fracture load (mean ±standard deviation; different superscript letters indicate statistical difference): FG+EXP (669.5 ±107.7)A; FG (620.7 ±59.2)A; EXP (506.5 ±27.0)B; FG+RC (452.7 ±81.6)B. No differences were found for failure mode (P=.595). CONCLUSIONS: The experimental composite resin significantly increases fracture load when used to reline commercially available fiber posts and, irrespective of its use, presented lower stress concentration.

Therapeutical effects of vaccine from Trypanosoma cruzi amastigote surface protein 2 by simultaneous inoculation with live parasites.

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 - Control Group (CTRL); Group 2 - Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 - Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 8 plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 8 pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection.

The Combined Deficiency of Immunoproteasome Subunits Affects Both the Magnitude and Quality of Pathogen- and Genetic Vaccination-Induced CD8+ T Cell Responses to the Human Protozoan Parasite Trypanosoma cruzi.

The ß1i, ß2i and ß5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected ß1i, ß2i and ß5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.

Endovascular management of tracheo-innominate artery fistula: a case report and literature review.

Tracheo-innominate artery fistula (TIF) is a rare complication of tracheostomy, with incidence ranging from 0.1 to 1%, but mortality is high in untreated cases. Early signs range from self-limited bleeding to massive hemorrhage with hypovolemic shock. The caliber of the tracheostomy cannula, its position in contact with the tracheal wall, and tracheal cuff pressure can traumatize the mucosa and trigger development of a TIF. We describe the case of a 14-year-old female patient who had been tracheostomized at the age of eight because of head trauma. She later developed subglottic stenosis requiring dilation sessions for six years. During the fifth year of these sessions, she presented repetitive hemoptysis, initially treated by surgery to implant an expanded polytetrafluoroethylene graft. One year later, she had an intense hemorrhage, which was controlled using endovascular techniques followed by definitive surgery, performed electively. The patient was followed up for six months, without complications.

A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas' heart disease.

Dendritic cells, macrophages, NK and CD8+ T lymphocytes play pivotal roles in controlling HSV-1 in the trigeminal ganglia by producing IL1-beta, iNOS and granzyme B.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) cause not only mild symptoms but also blindness and encephalitis. It was previously shown that the immune response against HSV-1 occurs mainly in the trigeminal ganglia (TG) and that Toll-like receptors 2 and 9 (TLR2/9) are important in mediating this response. It was also demonstrated that iNOS (nitric oxide synthase) and interleukin 1 beta (IL-1ß) play an essential role in the defense against HSV-1 infection. Importantly, the present work aimed to identify the primary cells responsible for iNOS and IL-1ß production and search for other important molecules and cells that might or might not depend on TLR2/9 receptors to mediate the immune response against HSV-1. METHODS: C57BL/6 (wild type, WT) and TLR2/9-/- mice were infected by the intranasal route with HSV-1 (1 × 106 p.f.u.). Cells were obtained from the TG and spleen tissues and the profile of immune cells was determined by flow cytometry in infected and mock infected WT and knockout mice. The percentage of cells producing iNOS, IL-1ß, granzyme B and perforin was also determined by flow cytometry. Chemokine monocyte chemoattractant protein-1 (MCP1) was measured by Cytometric Bead Array (CBA) in the TG, spleen and lung. Expression of type I interferons (IFNs), interleukins (IL) 5 and 10, IL-1ß and granzyme B were quantified by real time PCR. RESULTS: The results indicate that dendritic cells (DCs) and monocytes/macrophages (Mo/Mϕ) were the main sources of IL-1ß and iNOS, respectively, which, together with type I IFNs, were essential for the immune response against HSV-1. Additionally, we showed that granzyme B produced by CD8+ T and NK lymphocytes and MCP-1 were also important for this immune response. Moreover, our data indicate that the robust production of MCP-1 and granzyme B is either TLR-independent or down regulated by TLRs and occurs in the TG of TLR2/9-/- infected mice. CONCLUSION: Taken together, our data provide strong evidence that the responses mediated by DCs, Mo/Mϕ, NK and CD8+ T lymphocytes through IL-1ß, iNOS and granzyme B production, respectively, together with the production of type I IFN early in the infection, are crucial to host defense against HSV-1.

Phylogeny, Identification, and Pathogenicity of Lasiodiplodia Associated with Postharvest Stem-End Rot of Coconut in Brazil.

Coconut palm (Cocos nucifera L.) is one of the most important perennial tropical crops. Stem-end rot is the major postharvest disease of coconut in Brazil. The fungus Lasiodiplodia theobromae is the only species that has been reported to be associated with this disease. However, a comprehensive study elucidating the true identity of this pathogen with molecular tools has never been conducted. In recent years, new species of Lasiodiplodia have been proposed after molecular studies were performed, indicating the existence of a species complex. The aims of this research were to study the etiology of the postharvest stem-end rot of immature coconut based on a combination of morphological and phylogenetic analyses, to establish the phylogenetic position of such taxa, and to assess the pathogenicity of each taxon. Four species were identified: L. brasiliense, L. egyptiacae, L. pseudotheobromae, and L. theobromae. All of the species were distinguished morphologically and phylogenetically and were proven to be pathogenic to coconut following artificial inoculation. L. theobromae was the most common and the most aggressive species. This study represents the first report of three additional species of Lasiodiplodia as causal agents of postharvest stem-end rot of immature coconut in Brazil.

Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.

MHC class Ia-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Genetic vaccination against experimental infection with myotropic parasite strains of Trypanosoma cruzi.

In earlier studies, we reported that a heterologous prime-boost regimen using recombinant plasmid DNA followed by replication-defective adenovirus vector, both containing Trypanosoma cruzi genes encoding trans-sialidase (TS) and amastigote surface protein (ASP) 2, provided protective immunity against experimental infection with a reticulotropic strain of this human protozoan parasite. Herein, we tested the outcome of genetic vaccination of F1 (CB10XBALB/c) mice challenged with myotropic parasite strains (Brazil and Colombian). Initially, we determined that the coadministration during priming of a DNA plasmid containing the murine IL-12 gene improved the immune response and was essential for protective immunity elicited by the heterologous prime-boost regimen in susceptible male mice against acute lethal infections with these parasites. The prophylactic or therapeutic vaccination of resistant female mice led to a drastic reduction in the number of inflammatory infiltrates in cardiac and skeletal muscles during the chronic phase of infection with either strain. Analysis of the electrocardiographic parameters showed that prophylactic vaccination reduced the frequencies of sinus arrhythmia and atrioventricular block. Our results confirmed that prophylactic vaccination using the TS and ASP-2 genes benefits the host against acute and chronic pathologies caused by T. cruzi and should be further evaluated for the development of a veterinary or human vaccine against Chagas disease.

Influence of different finishing, aging with coffee, and repolishing protocols on the properties of nanoparticle composite resins.

Background: Considering the variability of finishing protocols for composite resins, the literature does not offer a consensus about the influence of these approaches to obtain a final polishing and whether the physical properties of these composite resins change at different analysis times. Therefore, the study analyzed the microhardness, roughness, color stability, and gloss of a nanocomposite resin with different finishing, aging with coffee, and repolishing protocols. Material and Methods: Nanocomposite resin samples were divided into three finishing protocol groups: Diamond burs (F and FF), multi-fluted tungsten carbide burs (18 and 30 flutes), and coarse and medium abrasive discs (Soflex-3M). All protocols used spiral rubber tips (F and FF) for polishing. Knoop microhardness (KHN), roughness (Ra), color changes (ΔE00 and YI), and gloss (GU) were analyzed. Scanning electron microscopy provided images of resins and finishing and polishing instruments. Results: Resin KHN (p<0.001) decreased, and Ra (p<0.001), ΔE00 (p<0.001), and YI (p<0.001) increased after aging with coffee, regardless of finishing protocol. Abrasive discs showed lower color changes, YI, and Ra and higher GU. Repolishing restored KHN and Ra but not ΔE00 (p>0.05) and YI (p>0.05). Conclusions: Abrasive disc finishing reduced roughness and yellowness and increased nanocomposite resin gloss after aging with coffee. Key words:Color, Composite resins, Dental materials, Staining, Surface properties.

The impact of an effective 3-step hand hygiene technique in reducing potentially pathogenic microorganisms found on nursing professionals' hands.

INTRODUCTION: Healthcare-associated infections are concerning adverse events and hand hygiene is considered an essential preventive measure. The objective of the present study was to assess the effect of a correct 3-step hand hygiene technique on reducing of potentially pathogenic microorganisms on hands related to the WHO five moments for hand hygiene. METHODOLOGY: A cross-sectional study was performed by means of direct observation involving 60 Intensive Care Units (ICU) and clinical nursing professionals in a Brazilian hospital. Observations were performed in order to ascertain the adherence rate and the correct technique during health assistance. Additionally, microbiological analysis of material collected from the nursing professional's hands was carried out. Exploratory and inferential analyses were performed on R software and binomial analysis was carried out by using the Z-test. The study was approved by the research ethics committee and covered all the legal principles for the protection of human subjects. RESULTS: Hand hygiene adherence rate was 63.3%. However, only 13.3% of the professionals performed the correct 3-step hand hygiene technique regarding steps and time. Sixty-five microorganisms were isolated, among which 56.9% were coagulase-negative Staphylococcus, 26.2% were Gram-negative bacilli, 7.7% were Enterococcus faecalis, and 6.2% were Candida parapsilosis. There was no presence of potentially pathogenic microorganisms on the nursing professional's hands who performed the correct three-step technique. CONCLUSIONS: Overall correct hand hygiene technique was poor. The results indicated the presence of potentially pathogenic microorganisms at moments in which hand hygiene was mandatory but was not executed or was executed incorrectly. The 3-step hand hygiene technique proved to be effective when correctly performed since there were no microorganism growth. Larger studies are needed to test if these results can be replicated at a larger scale, since streamlining hand hygiene technique yielded encouraging results.

Exercise-Induced Muscle Damage after a High-Intensity Interval Exercise Session: Systematic Review.

High-intensity interval training (HIIT) is considered an effective method to improve fitness and health indicators, but its high-intensity exercises and the mechanical and metabolic stress generated during the session can lead to the occurrence of exercise-induced muscle damage. Therefore, this study aimed to describe, by means of a systematic review, the effects of a single HIIT session on exercise-induced muscle damage. A total of 43 studies were found in the Medline/PubMed Science Direct/Embase/Scielo/CINAHL/LILACS databases; however, after applying the exclusion criteria, only 15 articles were considered eligible for this review. The total sample was 315 participants. Among them, 77.2% were men, 13.3% were women and 9.5 uninformed. Their age ranged from 20.1 ± 2 to 47.8 ± 7.5 years. HIIT protocols included running with ergometers (n = 6), CrossFit-specific exercises (n = 2), running without ergometers (n = 3), swimming (n = 1), the Wingate test on stationary bicycles (n = 2), and cycling (n = 1). The most applied intensity controls were %vVO2max, "all out", MV, MAV, Vmax, and HRreserve%. The most used markers to evaluate muscle damage were creatine kinase, myoglobin, and lactate dehydrogenase. The time for muscle damage assessment ranged from immediately post exercise to seven days. HIIT protocols were able to promote changes in markers of exercise-induced muscle damage, evidenced by increases in CK, Mb, LDH, AST, ALT, pain, and muscle circumference observed mainly immediately and 24 h after the HIIT session.