Macrophages transmit human immunodeficiency virus type 1 products to CD4-negative cells: involvement of matrix metalloproteinase 9

It was previously reported that human immunodeficiency virus type 1 (HIV-1) spreads in CD4 lymphocytes through cell-to-cell transmission. Here we report that HIV-1-infected macrophages, but not lymphocytes, transmit HIV-1 products to CD4-negative cells of either epithelial, neuronal, or endothelial origin in the absence of overt HIV-1 infection. This phenomenon was detectable as early as 1 h after the start of cocultivation and depended on cell-to-cell contact but not on the release of viral particles from donor cells. Transfer of HIV-1 products occurred upon their polarization and colocalization within zones of cell-to-cell contact similar to virological synapses. Neither HIV-1 Env nor Nef expression was required but, interestingly, we found that an HIV-1-dependent increase in matrix metalloproteinase 9 production from donor cells significantly contributed to the cell-to-cell transmission of the viral products. The macrophage-driven transfer of HIV-1 products to diverse CD4-negative cell types may have a significant role in AIDS pathogenesis.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

Pregnancy Outcomes after a Mass Vaccination Campaign with an Oral Cholera Vaccine in Guinea: A Retrospective Cohort Study

Introduction

Since 2010, WHO has recommended oral cholera vaccines as an additional strategy for cholera control. During a cholera episode, pregnant women are at high risk of complications, and the risk of fetal death has been reported to be 2–36%. Due to a lack of safety data, pregnant women have been excluded from most cholera vaccination campaigns. In 2012, reactive campaigns using the bivalent killed whole-cell oral cholera vaccine (BivWC), included all people living in the targeted areas aged ≥1 year regardless of pregnancy status, were implemented in Guinea. We aimed to determine whether there was a difference in pregnancy outcomes between vaccinated and non-vaccinated pregnant women.

Methods and Findings

From 11 November to 4 December 2013, we conducted a retrospective cohort study in Boffa prefecture among women who were pregnant in 2012 during or after the vaccination campaign. The primary outcome was pregnancy loss, as reported by the mother, and fetal malformations, after clinical examination. Primary exposure was the intake of the BivWC vaccine (Shanchol) during pregnancy, as determined by a vaccination card or oral history. We compared the risk of pregnancy loss between vaccinated and non-vaccinated women through binomial regression analysis. A total of 2,494 pregnancies were included in the analysis. The crude incidence of pregnancy loss was 3.7% (95%CI 2.7–4.8) for fetuses exposed to BivWC vaccine and 2.6% (0.7–4.5) for non-exposed fetuses. The incidence of malformation was 0.6% (0.1–1.0) and 1.2% (0.0–2.5) in BivWC-exposed and non-exposed fetuses, respectively. In both crude and adjusted analyses, fetal exposure to BivWC was not significantly associated with pregnancy loss (adjusted risk ratio (aRR = 1.09 [95%CI: 0.5–2.25], p = 0.818) or malformations (aRR = 0.50 [95%CI: 0.13–1.91], p = 0.314).

Conclusions

In this large retrospective cohort study, we found no association between fetal exposure to BivWC and risk of pregnancy loss or malformation. Despite the weaknesses of a retrospective design, we can conclude that if a risk exists, it is very low. Additional prospective studies are warranted to add to the evidence base on OCV use during pregnancy. Pregnant women are particularly vulnerable during cholera episodes and should be included in vaccination campaigns when the risk of cholera is high, such as during outbreaks.     

The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells

Background

The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs), the knowledge of their effects on normal cells is of pivotal importance.

Design and Methods

We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs).

Results

Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase.

Conclusions

Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.     

Anthropogenic refugia ameliorate the severe climate-related decline of a montane mammal along its trailing edge

We conducted detailed resurveys of a montane mammal, Urocitellus beldingi, to examine the effects of climate change on persistence along the trailing edge of its range. Of 74 California sites where U. beldingi were historically recorded (1902-1966), 42 per cent were extirpated, with no evidence for colonization of previously unoccupied sites. Increases in both precipitation and temperature predicted site extirpations, potentially owing to snowcover loss. Surprisingly, human land-use change buffered climate change impacts, leading to increased persistence and abundance. Excluding human-modified sites, U. beldingi has shown an upslope range retraction of 255 m. Generalized additive models of past distribution were predictive of modern range contractions (AUC = 0.76) and projected extreme reductions (52% and 99%, respectively) of U. beldingi's southwestern range to 2080 climates (Hadley and CCCMA A2). Our study suggests the strong impacts of climate change on montane species at their trailing edge and how anthropogenic refugia may mitigate these effects.     

Quantification of gliadin levels to the picogram level by flow cytometry.

BACKGROUND: Celiac disease is a widely prevalent enteropathy caused by intolerance to gliadin, one of the gluten proteins. We developed two methods for the analysis of gliadin levels. Both methods use flow cytometry and rat antibodies against a 16-residue peptide of gliadin. The peptide is common to the alpha-, beta-, gamma-, and omega-gliadins. METHODS: In the one-site assay, the antigen (gliadin standard or food extract) was adsorbed on 3-mum latex particles. Sensitized particles were then incubated, in this order, with rat anti-gliadin peptide antibodies and anti-rat immunoglobulin G antibodies labeled with fluorescein isothiocyanate. In the two-site assay, the antigen was trapped on the latex particles by rat anti-gliadin antibodies and then measured by the same antibodies labeled with fluorescein. RESULTS: Detection limits were 1 ng/ml for the one-site assay and 10 pg/ml for the two-site assay. The two-site assay displayed gliadin at concentrations above the limit proposed by the Codex Alimentarius in 2 of 40 gluten-free products. CONCLUSION: There is a growing concern that gliadin, even when present in gluten-free foods within the limit fixed by the Codex Alimentarius, over the long term may become toxic to patients with celiac disease. The techniques described in this study provide an opportunity to further decrease the acceptable limit of gliadin in gluten-free foods.     

Identification and antibiotic susceptibility of bacterial isolates from probiotic products available in Italy

This study was carried out to assay the bacterial viability and the probable contamination of a range of probiotic products available in Italy and to test the susceptibility of the isolates. Eleven dried food supplements and five fermented functional foods were examined using different isolation media under standardized cultivation conditions. The identification was made by conventional phenotypic characteristics and biochemical tests. Among isolates from the probiotic products antibiotic susceptibility was detected using the E-test (ABBiodisk). Our results demonstrate that nine food supplements and two fermented foods claimed species which could not be isolated, whereas potential pathogens (i.e. Micromonas micros) were isolated. Lactobacilli displayed species-dependent antibiotic resistance. Atypical resistance occurred for penicillin in Lactobacillus acidophilus and Lactobacillus bulgaricus and for erythromycin in Lactobacillus lactis and Lactobacillus salivarius. A broad range of MICs was observed for cephalosporins and fluroquinolones. Aminoglycosides had poor activity against Lactobacillus isolates. Two of the four isolates of Bifidobacterium exhibited high resistance to trimethroprim/sulfametoxazole and to fluoroquinolones. Our results suggest that some probiotic products claim species that cannot always be isolated, and are sometimes contaminated by potential pathogens. Moreover, the probable transferable erythromycin or penicillin resistance among the lactobacilli isolated should be taken into account.     

Regulatory T cells are expanded by Teriparatide treatment in humans and mediate intermittent PTH‐induced bone anabolism in mice

Teriparatide is a bone anabolic treatment for osteoporosis, modeled in animals by intermittent PTH (iPTH) administration, but the cellular and molecular mechanisms of action of iPTH are largely unknown. Here, we show that Teriparatide and iPTH cause a ~two‐threefold increase in the number of regulatory T cells (Tregs) in humans and mice. Attesting in vivo relevance, blockade of the Treg increase in mice prevents the increase in bone formation and trabecular bone volume and structure induced by iPTH. Therefore, increasing the number of Tregs is a pivotal mechanism by which iPTH exerts its bone anabolic activity. Increasing Tregs pharmacologically may represent a novel bone anabolic therapy, while iPTH‐induced Treg increase may find applications in inflammatory conditions and transplant medicine.