Junctional adhesion molecule-C regulates vascular endothelial permeability by modulating VE-cadherin-mediated cell-cell contacts

We recently reported that junctional adhesion molecule (JAM)-C plays a role in leukocyte transendothelial migration. Here, the role of JAM-C in vascular permeability was investigated in vitro and in vivo. As opposed to macrovascular endothelial cells that constitutively expressed JAM-C in cell-cell contacts, in quiescent microvascular endothelial cells, JAM-C localized mainly intracellularly, and was recruited to junctions upon short-term stimulation with vascular endothelial growth factor (VEGF) or histamine. Strikingly, disruption of JAM-C function decreased basal permeability and prevented the VEGF- and histamine-induced increases in human dermal microvascular endothelial cell permeability in vitro and skin permeability in mice. Permeability increases are essential in angiogenesis, and JAM-C blockade reduced hyperpermeability and neovascularization in hypoxia-induced retinal angiogenesis in mice. The underlying mechanisms of the JAM-C-mediated increase in endothelial permeability were studied. JAM-C was essential for the regulation of endothelial actomyosin, as revealed by decreased F-actin, reduced myosin light chain phosphorylation, and actin stress fiber formation due to JAM-C knockdown. Moreover, the loss of JAM-C expression resulted in stabilization of VE-cadherin-mediated interendothelial adhesion in a manner dependent on the small GTPase Rap1. Together, through modulation of endothelial contractility and VE-cadherin-mediated adhesion, JAM-C helps to regulate vascular permeability and pathologic angiogenesis.     

CD16+ monocytes exposed to HIV promote highly efficient viral replication upon differentiation into macrophages and interaction with T cells

The CD16+ subset of monocytes is dramatically expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unknown. Here, we demonstrate that CD16+ but not CD16- monocytes promote high levels of HIV replication upon differentiation into macrophages and interaction with T cells. Conjugates formed between CD16+ monocyte-derived macrophages and T cells are major sites of viral replication. Furthermore, similar monocyte-T cell conjugates detected in peripheral blood of HIV-infected patients harbor HIV DNA. Thus, expansion of CD16+ monocytes during HIV infection and their subsequent recruitment into tissues such as lymph nodes, brain, and intestine may contribute to HIV dissemination and establishment of productive infection in T cells.     

The noncellular reduction of MTT tetrazolium salt by TiO2 nanoparticles and its implications for cytotoxicity assays

We report results of noncellular tests, revealing the occurrence of photocatalytic interactions between titanium dioxide (TiO₂, titania) nanoparticles and the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] cytotoxicity indicator. These interactions induce the reduction of MTT and formation of purple formazan under biologically relevant conditions. Classical MTT assays have been performed to evaluate the production of formazan in DMEM-F12 and RPMI-1640 cell culture media (containing 10% fetal bovine serum-FBS) treated with Degussa-P25 TiO₂ nanoparticles, in the absence of cells. The colorimetric determinations revealed the noncellular MTT to formazan transformation induced by TiO₂ nanoparticles, under conditions commonly used for in vitro cytotoxicity testing of nanomaterials. The formazan precipitation was found to be proportional to the TiO₂ concentration, being enhanced under laboratory daylight exposure. The photocatalytic nature of the studied effect was assessed under UV irradiation at 365 nm. The biological significance of the reported reaction was established with respect to cellular reference experiments performed on V79-4, HeLa and B16 cell lines. The results show false viability increases with up to 14% (for TiO₂ concentrations generally higher than 50 μg/ml), induced by the TiO₂–MTT reaction. This type of artifacts may lead to underestimated toxicity or false proliferation results.     

The relationship between gastroesophageal reflux disease and recurrent wheezing in children

Gastroesophageal reflux disease occurs when gastric contents flow back into the esophagus and produce symptoms. Recurrent wheezing affects the quality of life for the patient and family. The association of gastroesophageal reflux with recurrent wheezing is suggested by different studies. The purpose of this study was to explore this relationship and to evaluate the outcome after appropriate treatment.A retrospective study on 85 children with recurrent wheezing, admitted in a pediatric gastroenterology regional center in Romania was performed. 24-hour continuous esophageal pH monitoring was used to evaluate the presence of gastroesophageal reflux and the results were interpreted using the Boix Ochoa score. All patients with positive score received treatment with proton pump inhibitors and they were evaluated again after 2 months.Gastroesophageal reflux was present in 71 children (83.5%), while 14 (16.5%) had a negative score, with a statistic significance (χ² = 6.88, P = .0086, 95% confidence interval). After 2 months treatment with proton pump inhibitors, the Boix Ochoa score was still positive in 15 patients (21.13%).Recurrent wheezing is a solid reason for evaluating the presence of gastroesophageal reflux by 24-hour continuous esophageal pH-metry. Adequate treatment of gastroesophageal reflux solves also the recurrent wheezing in the majority of patients.     

Effects of the tyrosine-kinase inhibitor geldanamycin on ligand-induced HER-2/NEU activation,receptor expression and proliferation of HER-2-positive malignant cell lines

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2⁺ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [³H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC₅₀ 2nM) and MCF7 (IC₅₀ 20nM), while OVCAR3 was only moderately sensitive (IC₅₀ 2μM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced. Int. J. Cancer, 70:221–229, 1997. © 1997 Wiley-Liss, Inc.     

Coreceptors and HIV-1 Pathogenesis

The major HIV-1 coreceptors, CCR5 and CXCR4, mediate virus entry into CD4+ cells and are therefore a critical component of the HIV-1 life cycle. Alterations in coreceptor preference as well as the efficiency and mechanism of interaction between HIV-1 and CCR5 and/or CXCR4 has a significant influence on viral tropism, progression of disease, and response to coreceptor antagonists. In addition, these alterations influence the susceptibility of CD4+ T-cell, monocyte, and dendritic cell subsets to infection and therefore, are important for several facets of HIV-1 pathogenesis including the establishment of latent reservoirs, trafficking, and transmission. This review highlights recent literature that has advanced our understanding of the role of coreceptors in HIV-1 pathogenesis.     

Maternal α-linolenic acid availability during gestation and lactation alters the postnatal hippocampal development in the mouse offspring

The availability of ω-3 polyunsaturated fatty acids is essential for perinatal brain development. While the roles of docosahexaenoic acid (the most abundant ω-3 species) were extensively described, less is known about the role of α-linolenic acid (ALA), which is the initial molecular species undergoing elongation and desaturation within the ω-3 pathways. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in hippocampal development (dentate gyrus) in the mouse male offspring, at the end of lactation (postnatal day 19, P19). Postnatal ALA supplementation increased cell proliferation (36% more proliferating cells compared to a control group) and early neuronal differentiation, while postnatal ALA deficiency increased cellular apoptosis within the dentate gyrus of suckling pups (61% more apoptotic cells compared to a control group). However, maternal ALA deficiency during gestation prevented the increased neurogenesis induced by postnatal supplementation. Fatty acid analysis revealed that ALA supplementation increased the concentration of the ω-3 species in the maternal liver and serum, but not in the brain of the offspring, excepting for ALA itself. Interestingly, ALA supplementation also increased the concentration of dihomo γ-linolenic acid (a ω-6 species) in the P19 brains, but not in maternal livers or serum. In conclusion, postnatal ALA supplementation enhances neurogenesis in the dentate gyrus of the offspring at postnatal day 19, but its beneficial effects are offset by maternal ALA deficiency during gestation. These results suggest that ALA is required in both fetal and postnatal stages of brain development.