Respiratory and Systemic Effects of LASSBio596 Plus Surfactant in Experimental Acute Respiratory Distress Syndrome.

BACKGROUND/AIMS: Exogenous surfactant has been proposed as adjunctive therapy for acute respiratory distress syndrome (ARDS), but it is inactivated by different factors present in the alveolar space. We hypothesized that co-administration of LASSBio596, a molecule with significant anti-inflammatory properties, and exogenous surfactant could reduce lung inflammation, thus enabling the surfactant to reduce edema and improve lung function, in experimental ARDS. METHODS: ARDS was induced by cecal ligation and puncture surgery in BALB/c mice. A sham-operated group was used as control (CTRL). After surgery (6 hours), CTRL and ARDS animals were assigned to receive: (1) sterile saline solution; (2) LASSBio596; (3) exogenous surfactant or (4) LASSBio596 plus exogenous surfactant (n = 22/group). RESULTS: Regardless of exogenous surfactant administration, LASSBio596 improved survival rate and reduced collagen fiber content, total number of cells and neutrophils in PLF and blood, cell apoptosis, protein content in BALF, and urea and creatinine levels. LASSBio596 plus surfactant yielded all of the aforementioned beneficial effects, as well as increased BALF lipid content and reduced surface tension. CONCLUSION: LASSBio596 exhibited major anti-inflammatory and anti-fibrogenic effects in experimental sepsis-induced ARDS. Its association with surfactant may provide further advantages, potentially by reducing surface tension.

Effects of citric acid on cultured human osteoblastic cells.

We investigated the effects of citric acid (CA) on cultured human osteoblastic (HOB) cells by evaluating cell adhesion, proliferation, and cytotoxicity. (3)H-Thymidine-labeled HOB cells were incubated in culture medium supplemented or not with 4%, 6%, 8%, or 10% CA for 1 minute. After incubation, cell morphology was evaluated by Nomarski interferential light microscopy, cell proliferation was accessed by measurements of (3)H-thymidine associated to the cells, and cell lysis was monitored by measuring the amount of (3)H-thymidine released by cells. We observed that most of the CA-treated cells presented numerous atypical vacuoles, and such cells were also highly polymorphic, exhibiting round-shaped cells. Nonetheless, CA at all concentrations assayed did not yield cytotoxicity as measured by (3)H-containing DNA release, although significant decrease in cell proliferation was observed (P > .05). Furthermore, cells which were treated with CA at the lowest concentration assayed (4%) restored normal proliferation rates 3 days after treatment.

Cysteine peptidase expression in Trichomonas vaginalis isolates displaying high- and low-virulence phenotypes.

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.

Biological characterization of a clinical and an environmental isolate of Acanthamoeba polyphaga: analysis of relevant parameters to decode pathogenicity.

Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.

Signal transduction in human endothelial cells induced by their interaction with group B Streptococci.

The molecular mechanisms underlying entry of group B Streptococci (GBS) into human endothelial cells are not yet fully understood. This study is centered on the triggering of signaling cascade in human umbilical vein endothelial cells (HUVEC) during their interaction with different GBS serotypes/strains (type III: 80340-vagina and 90356-CSF and type V: 88641-vagina and 90186-blood). We have shown that the analyzed microorganisms adhere to HUVEC, but only those of the strains 90356-CSF, 88641-vagina and 90186-blood presented intracellular viability. Activation of PKC directly increased F-actin content and organization into stress fibers, and increased intracellular viability of GBS-III microorganisms. PKA inhibitor seems to promote surveillance of GBS type V microorganisms within HUVEC. These studies indicate that different molecules present at the cell surface of the GBS might induce different responses to HUVEC, interfering with the recruitment of cortical actin filaments.

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates.

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.