Leishmania amazonensis infection impairs VLA-4 clustering and adhesion complex assembly at the adhesion site of J774 cells.

Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site), and focal adhesion complex assembly. Phagocytes treated with Methyl-ß-Cyclodextrin (MßCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MßCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.

Environmental Biofilms from an Urban Community in Salvador, Brazil, Shelter Previously Uncharacterized Saprophytic Leptospira.

Biofilms are complex microecosystems with valuable ecological roles that can shelter a variety of microorganisms. Spirochetes from the genus Leptospira have been observed to form biofilms in vitro, in rural environments, and in the kidneys of reservoir rats. The genus Leptospira is composed of pathogenic and non-pathogenic species, and the description of new species is ongoing due to the advent of whole genome sequencing. Leptospires have increasingly been isolated from water and soil samples. To investigate the presence of Leptospira in environmental biofilms, we collected three distinct samples of biofilms formed in an urban setting with poor sanitation: Pau da Lima, in Salvador, Bahia, Brazil. All biofilm samples were negative for the presence of pathogenic leptospires via conventional PCR, but cultures containing saprophytic Leptospira were identified. Whole genomes were generated and analyzed for twenty isolates obtained from these biofilms. For species identification, we used digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis. The obtained isolates were classified into seven presumptive species from the saprophytic S1 clade. ANI and dDDH analysis suggest that three of those seven species were new. Classical phenotypic tests confirmed the novel isolated bacteria as saprophytic Leptospira. The isolates presented typical morphology and ultrastructure according to scanning electron microscopy and formed biofilms under in vitro conditions. Our data indicate that a diversity of saprophytic Leptospira species survive in the Brazilian poorly sanitized urban environment, in a biofilm lifestyle. We believe our results contribute to a better understanding of Leptospira biology and ecology, considering biofilms as natural environmental reservoirs for leptospires.

A comparative study of human glomerular basement membrane thickness using direct measurement and orthogonal intercept methods.

INTRODUCTION: Here we report estimates of glomerular basement membrane (GBM) thickness in the Brazilian population performed using direct (DM) and orthogonal interception methods (OIM), and comment on potential sources of variation among estimates made by different laboratories. METHODOLOGY: A total of 38 patients, ranging from 3 to 78 years of age, 26 (68%) males and 12 (32%) females, were submitted to kidney biopsy procedures for renal disease diagnosis. Glomeruli were diagnosed with minor histological changes by conventional, immunofluorescence and electron microscopy. GBM thickness was estimated using both DM and OIM methods. RESULTS: Estimates of GBM thickness obtained using DM were higher than those obtained by OIM. However, the application of a correction for non-perpendicular membrane sectioning to DM estimates yielded similar results to those obtained under OIM. The estimated GMB thickness using DM after correction was 289 + 44 nm, versus 287 + 48 nm by OIM. No statistically significant differences were detected in GMB thickness, nor with respect to patient age or sex. CONCLUSIONS: GBM thickness in the studied Brazilian population measured approximately 290 nm. The application of criteria for estimating the shortest distance between the endothelial and podocyte cell membranes with correction for non-perpendicular membrane sectioning can increase the accuracy of GBM thickness estimates using DM and OIM.

Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs.

Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05-1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20-9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs.

IGF-1 overexpression improves mesenchymal stem cell survival and promotes neurological recovery after spinal cord injury.

BACKGROUND: Survival and therapeutic actions of bone marrow-derived mesenchymal stem cells (BMMSCs) can be limited by the hostile microenvironment present during acute spinal cord injury (SCI). Here, we investigated whether BMMSCs overexpressing insulin-like growth factor 1 (IGF-1), a cytokine involved in neural development and injury repair, improved the therapeutic effects of BMMSCs in SCI. METHODS: Using a SCI contusion model in C57Bl/6 mice, we transplanted IGF-1 overexpressing or wild-type BMMSCs into the lesion site following SCI and evaluated cell survival, proliferation, immunomodulation, oxidative stress, myelination, and functional outcomes. RESULTS: BMMSC-IGF1 transplantation was associated with increased cell survival and recruitment of endogenous neural progenitor cells compared to BMMSC- or saline-treated controls. Modulation of gene expression of pro- and anti-inflammatory mediators was observed after BMMSC-IGF1 and compared to saline- and BMMSC-treated mice. Treatment with BMMSC-IGF1 restored spinal cord redox homeostasis by upregulating antioxidant defense genes. BMMSC-IGF1 protected against SCI-induced myelin loss, showing more compact myelin 28 days after SCI. Functional analyses demonstrated significant gains in BMS score and gait analysis in BMMSC-IGF1, compared to BMMSC or saline treatment. CONCLUSIONS: Overexpression of IGF-1 in BMMSC resulted in increased cell survival, immunomodulation, myelination, and functional improvements, suggesting that IGF-1 facilitates the regenerative actions of BMMSC in acute SCI.

Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums.

We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.

Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums

Abstract We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] µm(2) for uninfected and 41 [34-51] µm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 µm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 µm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

MORPHOLOGICAL AND ULTRASTRUCTURAL FEATURES OF A STRAIN OF BOTRYOCOCCUS TERRIBILIS (TREBOUXIOPHYCEAE) FROM BRAZIL(1).

The genus Botryococcus comprises a group of cosmopolitan species of freshwater colonial green algae, some of which synthesize and accumulate an unusually high level (15-76%) of liquid hydrocarbons. This characteristic suggests the possibility of exploiting species from this group as renewable sources for jet fuel. An oil-rich strain of Botryococcus (Trebouxiophyceae) was isolated from a freshwater pond in the state of Bahia, Brazil, and is presently maintained under standard conditions at the Culture Collection of the Institute of Biology, Federal University of Bahia. The taxonomic classification of the species was based on light microscopy (LM); and TEM and SEM were used to better characterize its features, which have never before been described at this level. The LM characterization included the size of the colonies (35.7-157 µm) and cells (8-10 × 5-9 µm) and their connection in sub-colonies by mucilaginous strands, as well as the presence of mucilaginous processes on the periphery of some of the colonies, with most of the cells included inside the colony. Reproduction occurred through divisions into two to four autospores. These features characterized the species as Botryococcus terribilis Komárek and Marvan. The TEM study showed, in addition to the presence of starch grains, pyrenoids that are penetrated by thick thylakoids. The pyrenoid bodies appear as electron-dense protein inclusions located in the chloroplast and surrounded by a starch sheath. These structures, which contain most if not all of the Ribulose-1,5-bisphosphate carboxylase oxygenase in several algal species that have been studied closely, are newly discovered for this species.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

BACKGROUND: In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. RESULTS: The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. CONCLUSIONS: This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

Targeted mutagenesis in pathogenic Leptospira species: disruption of the LigB gene does not affect virulence in animal models of leptospirosis.

The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spc(r)) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.

The OmpA-like protein Loa22 is essential for leptospiral virulence.

Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.

Virulent Mycobacterium fortuitum restricts NO production by a gamma interferon-activated J774 cell line and phagosome-lysosome fusion.

The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mphi) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-gamma) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-gamma-stimulated Mphi. Indeed, the amount of IFN-gamma-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 micro M) compared to SmTr infection (3.9 to 4.8 micro M) (P = 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-gamma-induced NO production by Mphi was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-DL-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-gamma-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes.