Lycopene prevents 7-ketocholesterol-induced oxidative stress,cell cycle arrest and apoptosis in human macrophages

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10–25 μM) alone and in combination with lycopene (0.5–2 μM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, β-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than β-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.     

Base damage and single-strand break repair: mechanisms and functional significance of short- and long-patch repair subpathways

A large variety of DNA lesions induced by environmental agents or arising as an outcome of cellular metabolism are counteracted by a complex network of proteins that belong to the base excision repair/single strand break repair (BER/SSBR) processes. No matter whether the initial lesions are modified DNA bases or single-strand breaks with non-conventional termini these processes are completed either by replacement of a single (short-patch, SP) or more (long-patch, LP) nucleotides by different arrays of proteins. Here, the factors that are involved in the selection between SP- and LP-BER/SSBR are reviewed. The biological significance of these alternative subpathways is also presented as inferred from mutant mouse/cell models.     

The selective inhibition of inducible nitric oxide synthase prevents intestinal ischemia-reperfusion injury in mice.

Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.     

Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host.     

Paracoccidioidomycosis in people living with HIV/AIDS: A historical retrospective cohort study in a national reference center for infectious diseases,Rio de Janeiro,Brazil

Paracoccidioidomycosis (PCM) is one of the main endemic systemic mycoses in Latin America, usually occurring in rural areas. When PCM occurs simultaneously with underlying immunosuppressive conditions, it can present as an opportunistic disease. Between 2000 and 2017, literature reported around 200 PCM cases in people living with HIV/AIDS (PLWHA). To address research gaps on this co-infection and to study its possible temporal changes in the last decade, we performed an active co-infection case search on the HIV/AIDS and PCM cohorts from a Brazilian reference center database from 1989 to 2019. We found 20 PLWHA among 684 PCM patients (2.92%), predominantly male (70.0%) and urban workers (80.0%). The median age of patients was higher in the 2010–2019 decade (p = 0.006). The occurrence of PCM in PLWHA was lower when compared with other fungal diseases. Although 50.0% of the patients had already been diagnosed with HIV infection and presented CD4+ T cell counts greater than 200/mm³ at the time of PCM diagnosis, the suspicion of immunosuppression in the context of atypical and more severe clinical forms of PCM revealed the diagnosis of HIV infection in 35.0% of the patients. Two (10.0%) patients had an evolution compatible with immune reconstitution inflammatory syndrome (IRIS) after starting antiretroviral therapy (ART).We highlight the importance of considering a PCM diagnosis in PLWHA to prevent a late-onset treatment and progression to severe manifestations and unfavorable outcomes. In addition, HIV investigation is recommended in PCM patients, especially those with atypical and more severe clinical presentations.     

Corneal keratocytes retain neural crest progenitor cell properties

Corneal keratocytes have a remarkable ability to heal the cornea throughout life. Given their developmental origin from the cranial neural crest, we asked whether this regenerative ability was related to the stem cell-like properties of their neural crest precursors. To this end, we challenged corneal stromal keratocytes by injecting them into a new environment along cranial neural crest migratory pathways. The results show that injected stromal keratocytes change their phenotype, proliferate and migrate ventrally adjacent to host neural crest cells. They then contribute to the corneal endothelial and stromal layers, the musculature of the eye, mandibular process, blood vessels and cardiac cushion tissue of the host. However, they fail to form neurons in cranial ganglia or branchial arch cartilage, illustrating that they are at least partially restricted progenitors rather than stem cells. The data show that, even at late embryonic stages, corneal keratocytes are not terminally differentiated, but maintain plasticity and multipotentiality, contributing to non-neuronal cranial neural crest derivatives.     

Molecular study of genes involved in virulence regulatory pathways in Bacillus anthracis vaccine strain "Carbosap"

This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.     

Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis

A small percentage of pathologically obese subjects with fatty livers develop histological signs of necroinflammation and fibrosis, suggesting a variety of cofactors in the pathogenesis of obesity-related liver diseases including nonalcoholic steatohepatitis. Since several observations have linked bacterial endotoxins to liver damage, the aim of this study was to determine the effect of obesity on intestinal mucosal integrity and portal blood endotoxemia in two strains of obese mice: leptin-deficient (ob/ob) and hyperleptinemic (db/db) mice. Murine intestinal mucosal barrier function was assessed using a Ussing chamber, whereas ileum tight junction proteins were analyzed by immunocytochemistry and Western blot analysis. Circulating proinflammatory cytokines and portal blood endotoxin levels were measured by ELISA and the limulus test, respectively. The inflammatory and fibrogenic phenotype of murine hepatic stellate cells (HSCs) was determined by ELISA and quantitative RT-PCR. Ob/ob and db/db mice showed lower intestinal resistance, profoundly modified distribution of occludin and zonula occludens-1 in the intestinal mucosa, and higher circulating levels of inflammatory cytokines and portal endotoxemia compared with lean control mice. Moreover, HSCs isolated from ob/ob and db/db mice showed higher membrane CD14 mRNA levels and more pronounced lipopolysaccharide-induced proinflammatory and fibrogenic responses than HSCs from lean animals. In conclusion, genetically obese mice display enhanced intestinal permeability leading to increased portal endotoxemia that makes HSCs more sensitive to bacterial endotoxins. We suggest that in metabolic syndrome, patients may likewise have a greater intestinal mucosa permeability and increased lipopolysaccharide levels in portal blood that can contribute to the liver inflammatory damage.