Keratinocyte Response to Infection with Sporothrix schenckii.

Sporotrichosis is a subacute, or chronic mycosis caused by traumatic inoculation of material contaminated with the fungus Sporothrix schenckii which is part of the Sporothrix spp. complex. The infection is limited to the skin, although its progression to more severe systemic or disseminated forms remains possible. Skin is the tissue that comes into contact with Sporothrix first, and the role of various cell lines has been described with regard to infection control. However, there is little information on the response of keratinocytes. In this study, we used the human keratinocyte cell line (HaCaT) and evaluated different aspects of infection from modifications in the cytoskeleton to the expression of molecules of the innate response during infection with conidia and yeast cells of Sporothrix schenckii. We found that during infection with both phases of the fungus, alterations of the actin cytoskeleton, formation of membrane protuberances, and loss of stress fibers were induced. We also observed an overexpression of the surface receptors MR, TLR6, CR3 and TLR2. Cytokine analysis showed that both phases of the fungus induced the production of elevated levels of the chemokines MCP-1 and IL-8, and proinflammatory cytokines IFN-α, IFN-γ and IL-6. In contrast, TNF-α production was significant only with conidial infection. In late post-infection, cytokine production was observed with immunoregulatory activity, IL-10, and growth factors, G-CSF and GM-CSF. In conclusion, infection of keratinocytes with conidia and yeast cells of Sporothrix schenckii induces an inflammatory response and rearrangements of the cytoskeleton.

Analysis of inflammatory cytokine expression in the urinary tract of BALB/c mice infected with Proteus (P.) mirabilis and enteroaggregative Escherichia (E.) coli (EAEC) strains.

This study aimed to analyze the proinflammatory cytokine mRNA expression in the urinary tract of BALB/c mice infected with bacterial strains with uropathogenic potential. Groups of four 6-week-old female BALB/c mice were intraurethrally inoculated with 5 × 107 colony-forming units (CFU) of P. mirabilis ATCC29906, EAEC O42, P. mirabilis RTX339, or sterile saline (control group) and then sacrificed at 0, 2, 4, 7, or 10 days post-infection (p.i.). Samples were cultured to determine the CFU/mL in urine or CFU/g in the bladders and kidneys. Cytokine expression (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, -6, and -8) was evaluated in the target organs using real-time PCR and immunohistochemistry; histology was examined with hematoxylin and eosin staining. The results are presented as the means and standard deviations and were compared using one-way ANOVA, with p < 0.05 indicating significant differences. Bacteriuria was not detected in the infected groups; bacterial colonization occurred in the target organs at all time points, but was higher in mice infected with EAEC O42 or P. mirabilis RTX339 at 7 days p.i. The expression of all cytokine mRNAs was seen, but only the levels of the IL-8 protein increased in situ at 7 days p.i. in the P. mirabilis RTX339 and EAEC O42 groups in both organs. Morphological alterations, observed in all of the infected groups, were more prominent in the EAEC O42 and P. mirabilis RTX339 groups. The findings provide insights into the uropathogenicity and inflammatory cytokine expression in the urinary tract of mice infected with three previously untested bacterial strains.

Impact of chronic immobilization stress on parameters of colonic homeostasis in BALB/c mice.

The intestinal epithelium is a monolayer of cells arranged side­by­side and connected by tight junction (TJ) proteins expressed at the apical extreme of the paracellular membrane. This layer prevents stress­induced inflammatory responses, thus helping to maintain gut barrier function and gut homeostasis. The aim of the present study was to evaluate the effects of chronic immobilization stress on the colonic expression of various parameters of homeostasis. A total of two groups of female BALB/c mice (n=6) were included: A stressed group (short­term immobilization for 2 h/day for 4 consecutive days) and an unstressed (control) group. Colon samples were obtained to detect neutrophils and goblet cells by optical microscopy, TJ protein expression (occludin, and claudin ­2, ­4, ­7, ­12 and ­15) by western blotting, mRNA levels of TJ genes and proinflammatory cytokines [tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, ­6 and ­8] by reverse transcription­quantitative PCR, fecal lactoferrin by ELISA and the number of colony­forming units of aerobic bacteria. Compared with goblet cells in control mice, goblet cells were enlarged and reduced in number in stressed mice, whereas neutrophil cellularity was unaltered. Stressed mice exhibited reduced mRNA expression for all evaluated TJ mRNAs, with the exception of claudin­7, which was upregulated. Protein levels of occludin and all claudins (with the exception of claudin­12) were decreased in stressed mice. Fecal lactoferrin, proinflammatory cytokine mRNA levels and aerobic bacterial counts were all increased in the stressed group. These results indicated that immobilization stress induced proinflammatory and potential remodeling effects in the colon by decreasing TJ protein expression. The present study may be a useful reference for therapies aiming to regulate the effects of stress on intestinal inflammatory dysfunction.

Keratinocyte infection by Actinomadura madurae triggers an inflammatory response.

BACKGROUND: Actinomycetoma is a syndrome of the skin characterized by chronic inflammation and lesions with nodular grain-like structures. The most common aetiological agents are Nocardia brasiliensis and Actinomadura madurae. In response to infection with these organisms the body produces an inflammatory immune response in the skin. The aim of the present study was to determine the production of chemokines, pro-inflammatory cytokines, antimicrobial peptides and the expression of Toll-like receptors (TLRs) in keratinocytes infected by A. madurae. METHODS: A cell line of HaCaT keratinocytes was infected with A. madurae at a multiplicity of infection of 20:1 for 2 h and the samples were collected from 2 to 72 h post-infection. Intracellular replication of the bacterium was evaluated by counting of colony-forming units, the TLR expression and antimicrobial peptide production were assayed by confocal microscopy and chemokine and pro-inflammatory cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: Early in the infection, A. madurae was able to achieve intracellular replication in keratinocytes, however, the cells eventually controlled the infection. In response to the infection, keratinocytes overexpressed TLR2 and TLR6, produced high concentrations of cytokines monocyte chemoattractant protein-1, interleukin 8, human ß-defensin-1, human ß-defensin-2 and LL37 and low levels of tumour necrosis factor α. CONCLUSIONS: The human keratinocytes contribute to the inflammatory process in response to A. madurae infection by overexpressing TLRs and producing chemokines, pro-inflammatory cytokines and antimicrobial peptides.

[Péptidos antimicrobianos, una alternativa prometedora para el tratamiento de enfermedades infecciosas].

Microorganisms that cause diseases in humans are constantly evolving, which represents a challenge in the search for effective treatments against them. Even when currently there are several pharmacological alternatives available, sometimes they are inefficient for the control of infectious diseases, especially because pathogens have generated multiple resistance mechanisms against them. Antimicrobial peptides have been described in many species of organisms, from fungi, plants and insects to humans; currently, there are molecules that appear as a solution that can be effective in modern therapeutics. The advantage of these natural peptides lies in that they have been evolving almost the same amount of time than the species that produce them and their effect on the control of microorganisms is highly significant; some of these molecules are isolated from living organisms, others are starting to be produced by synthetic methods, which allows having access to an endless number of peptides with diverse therapeutic activities.

Los microorganismos causantes de enfermedades en humanos evolucionan constantemente, lo que representa un reto en la búsqueda de tratamientos efectivos contra estos patógenos. Aun cuando en la actualidad se cuenta con diversas alternativas farmacológicas, estas en ocasiones resultan ineficientes para el control de las enfermedades infecciosas, sobre todo porque los patógenos han generado múltiples mecanismos de resistencia. Los péptidos antimicrobianos se han descrito en muchas especies de organismos: hongos, plantas, insectos y humanos; en la actualidad se presentan como una solución terapéutica que puede ser efectiva. La ventaja de estos péptidos naturales es que llevan evolucionando casi la misma cantidad de tiempo que las especies que producen y su efecto en el control de los microorganismos es muy notable; algunas de estas moléculas son aisladas de organismos vivos y otras se comienzan a producir por métodos sintéticos, lo que permite tener acceso a un sinfín de posibles péptidos con actividades terapéuticas diversas.

Activation and IL-1ß secretion of human peripheral phagocytes infected with Actinomadura madurae, Nocardia asteroides and Candida albicans.

OBJECTIVE: To evaluate the ability of Actinomadura madurae (A. madurae) and Nocardia asteroides (N. asteroides), using Candida albicans (C. albicans) as prototypic control, to elicit the activation and IL-1ß secretion of blood phagocytic cells from healthy donors. METHODS: Microscopic evaluation of phagocytosis/activation, cell viability and spectrophotometric quantitation of endocytosis/activation, were assessed by using formazan blue test in human blood phagocytes infected with C. albicans, A. madurae or N. asteroides treated with either normal human serum (NHS) or with decomplemented NHS. Interlukin-1ß from culture supernatants of infected polymorphonuclear was tested by ELISA kit assay. RESULTS: Microscopic assay showed that phagocytosis and activation of adherent mononuclear phagocytes were greater with C. albicans followed by A. madurae and then by N. asteroides. Spectrophotometric assay in polymorphonuclear phagocytes infected with NHS-treated pathogens indicated that activation was similarly higher by C. albicans and A. madurae and lower by N. asteroides. Kinetic assays in infected polymorphonuclear cells showed that viability was decreased by C. albicans and N. asteroides or unaffected with A. madurae. Levels of IL-1ß at 8 h of incubation were higher with C. albicans followed by A. madurae whereas lower levels were found with N. asteroides. CONCLUSIONS: The extent of cell-viability and activation as well IL-1ß secretion may be related with the virulence of C. albicans and N. asteroides and other parameters remain to be explored for assessing the virulence of A. madurae.

Antibiotic resistance and multidrug-resistant efflux pumps expression in lactic acid bacteria isolated from pozol, a nonalcoholic Mayan maize fermented beverage.

Pozol is a handcrafted nonalcoholic Mayan beverage produced by the spontaneous fermentation of maize dough by lactic acid bacteria. Lactic acid bacteria (LAB) are carriers of chromosomal encoded multidrug-resistant efflux pumps genes that can be transferred to pathogens and/or confer resistance to compounds released during the fermentation process causing food spoiling. The aim of this study was to evaluate the antibiotic sensibility and the transcriptional expression of ABC-type efflux pumps in LAB isolated from pozol that contributes to multidrug resistance. Analysis of LAB and Staphylococcus (S.) aureus ATCC 29213 and ATCC 6538 control strains to antibiotic susceptibility, minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) to ethidium bromide were based in "standard methods" whereas the ethidium bromide efflux assay was done by fluorometric assay. Transcriptional expression of efflux pumps was analyzed by RT-PCR. LAB showed antibiotic multiresistance profiles, moreover, Lactococcus (L.) lactis and Lactobacillus (L.) plantarum displayed higher ethidium bromide efflux phenotype than S. aureus control strains. Ethidium bromide resistance and ethidium bromide efflux phenotypes were unrelated with the overexpression of lmrD in L. lactics, or the underexpression of lmrA in L. plantarum and norA in S. aureus. These findings suggest that, moreover, the analyzed efflux pumps genes, other unknown redundant mechanisms may underlie the antibiotic resistance and the ethidium bromide efflux phenotype in L. lactis and L. plantarum. Phenotypic and molecular drug multiresistance assessment in LAB may improve a better selection of the fermentation starter cultures used in pozol, and to control the antibiotic resistance widespread and food spoiling for health safety.

Candida glabrata survives and replicates in human osteoblasts.

Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell.

Molecular characterization of microbial contaminants isolated from Umbilical Cord Blood Units for transplant

ABSTRACT Disposal of Umbilical Cord Blood Units due to microbial contamination is a major problem in Cord Blood Banks worldwide as it reduces the number of units available for transplantation. Additionally, economic losses are generated as result of resources and infrastructure used to obtain such units. Umbilical Cord Blood Units that showed initial microbial contamination were subject to strains isolation, identification, and characterization by sequencing the 16S rRNA gene and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Moreover, tests of antimicrobial resistance/sensitivity and phenotypic activities that may play an important role in microbial infection were performed. Microbial contamination was detected in 120 Umbilical Cord Blood Units (2.31%) in the period from 2003 to 2013. The most frequently isolated strains were Enterococcus faecium, followed by Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, Staphylococcus haemoliticus, Klebsiella pneumoniae, Enterococcus durans, Lactobacillus helveticus, Enterococcus hiriae and Roseomonas genomospecies 5. The ERIC-PCR assays revealed a wide genetic diversity in some strains although belonging to the same genus and specie, indicating different sources of contamination. Broad-spectrum penicillins, third generation cephalosporins, aminoglycosides, and fluoroquinolones showed lower inhibitory activity on the tested strains. All strains were proteolytic, 67.69% were amylase-positive, 27.6% hemolysis-positive, and 34.71% nuclease-positive. The most common sources of contamination were: vaginal flora, digestive tract, and skin flora, highlighting the need for staff training in good manufacturing practices in collection SCU since all contaminants identified are part of the microbial flora of the donors. Implications and consequences in the therapeutic use of Umbilical Cord Blood Units for transplantation contaminated by multiresistant bacteria in immunocompromised patients are discussed.

Molecular characterization of microbial contaminants isolated from Umbilical Cord Blood Units for transplant.

Disposal of Umbilical Cord Blood Units due to microbial contamination is a major problem in Cord Blood Banks worldwide as it reduces the number of units available for transplantation. Additionally, economic losses are generated as result of resources and infrastructure used to obtain such units. Umbilical Cord Blood Units that showed initial microbial contamination were subject to strains isolation, identification, and characterization by sequencing the 16S rRNA gene and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR). Moreover, tests of antimicrobial resistance/sensitivity and phenotypic activities that may play an important role in microbial infection were performed. Microbial contamination was detected in 120 Umbilical Cord Blood Units (2.31%) in the period from 2003 to 2013. The most frequently isolated strains were Enterococcus faecium, followed by Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, Staphylococcus haemoliticus, Klebsiella pneumoniae, Enterococcus durans, Lactobacillus helveticus, Enterococcus hiriae and Roseomonas genomospecies 5. The ERIC-PCR assays revealed a wide genetic diversity in some strains although belonging to the same genus and specie, indicating different sources of contamination. Broad-spectrum penicillins, third generation cephalosporins, aminoglycosides, and fluoroquinolones showed lower inhibitory activity on the tested strains. All strains were proteolytic, 67.69% were amylase-positive, 27.6% hemolysis-positive, and 34.71% nuclease-positive. The most common sources of contamination were: vaginal flora, digestive tract, and skin flora, highlighting the need for staff training in good manufacturing practices in collection SCU since all contaminants identified are part of the microbial flora of the donors. Implications and consequences in the therapeutic use of Umbilical Cord Blood Units for transplantation contaminated by multiresistant bacteria in immunocompromised patients are discussed.

Macrophage Activation by Ursolic and Oleanolic Acids during Mycobacterial Infection.

Oleanolic (OA) and ursolic acids (UA) are triterpenes that are abundant in vegetables, fruits and medicinal plants. They have been described as active moieties in medicinal plants used for the treatment of tuberculosis. In this study, we analyzed the effects of these triterpenes on macrophages infected in vitro with Mycobacterium tuberculosis (MTB). We evaluated production of nitric oxide (NO), reactive oxygen species (ROS), and cytokines (TNF-α and TGF-ß) as well as expression of cell membrane receptors (TGR5 and CD36) in MTB-infected macrophages following treatment with OA and UA. Triterpenes caused reduced MTB growth in macrophages, stimulated production of NO and ROS in the early phase, stimulated TNF-α, suppressed TGF-ß and caused over-expression of CD36 and TGR5 receptors. Thus, our data suggest immunomodulatory properties of OA and UA on MTB infected macrophages. In conclusion, antimycobacterial effects induced by these triterpenes may be attributable to the conversion of macrophages from stage M2 (alternatively activated) to M1 (classically activated).

Macropinocytosis is responsible for the uptake of pathogenic and non-pathogenic mycobacteria by B lymphocytes (Raji cells).

BACKGROUND: The classical roles of B cells include the production of antibodies and cytokines and the generation of immunological memory, these being key factors in the adaptive immune response. However, their role in innate immunity is currently being recognised. Traditionally, B cells have been considered non-phagocytic cells; therefore, the uptake of bacteria by B cells is not extensively documented. In this study, we analysed some of the features of non-specific bacterial uptake by B lymphocytes from the Raji cell line. In our model, B cells were infected with Mycobacterium tuberculosis (MTB), Mycobacterium smegmatis (MSM), and Salmonella typhimurium (ST). RESULTS: Our observations revealed that the Raji B cells were readily infected by the three bacteria that were studied. All of the infections induced changes in the cellular membrane during bacterial internalisation. M. smegmatis and S. typhimurium were able to induce important membrane changes that were characterised by abundant filopodia and lamellipodia formation. These membrane changes were driven by actin cytoskeletal rearrangements. The intracellular growth of these bacteria was also controlled by B cells. M. tuberculosis infection also induced actin rearrangement-driven membrane changes; however, the B cells were not able to control this infection. The phorbol 12-myristate 13-acetate (PMA) treatment of B cells induced filopodia and lamellipodia formation, the production of spacious vacuoles (macropinosomes), and the fluid-phase uptake that is characteristic of macropinocytosis. S. typhimurium infection induced the highest fluid-phase uptake, although both mycobacteria also induced fluid uptake. A macropinocytosis inhibitor such as amiloride was used and abolished the bacterial uptake and the fluid-phase uptake that is triggered during the bacterial infection. CONCLUSIONS: Raji B cells can internalise S. typhimurium and mycobacteria through an active process, such as macropinocytosis, although the resolution of the infection depends on factors that are inherent in the virulence of each pathogen.