SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1 and PDGF

Liver fibrosis is an active process that involves changes in cell-cell and cell-extracellular matrix (ECM) interaction. Secreted protein, acidic and rich in cysteine (SPARC) is an ECM protein with many biological functions that is overexpressed in cirrhotic livers and upregulated in activated hepatic stellate cells (aHSCs). We have recently shown that SPARC downregulation ameliorates liver fibrosis in vivo. To uncover the cellular mechanisms involved, we have specifically knocked down SPARC in two aHSC lines [the CFSC-2G (rat) and the LX-2 (human)] and in primary cultured rat aHSCs. Transient downregulation of SPARC in hepatic stellate cells (HSCs) did not affect their proliferation and had only minor effects on apoptosis. However, SPARC knockdown increased HSC adhesion to fibronectin and significantly decreased their migration toward PDFG-BB and TGF-β(1). Interestingly, TGF-β(1) secretion by HSCs was reduced following SPARC small interfering RNA (siRNA) treatment, and preincubation with TGF-β(1) restored the migratory capacity of SPARC siRNA-treated cells through mechanisms partially independent from TGF-β(1)-mediated induction of SPARC expression; thus SPARC knockdown seems to exert its effects on HSCs partially through modulation of TGF-β(1) expression levels. Importantly, collagen-I mRNA expression was reduced in SPARC siRNA-transfected HSCs. Consistent with previous results, SPARC knockdown in aHSCs was associated with altered F-actin expression patterns and deregulation of key ECM and cell adhesion molecules, i.e., downregulation of N-cadherin and upregulation of E-cadherin. Our data together suggest that the upregulation of SPARC previously reported for aHSCs partially mediates profibrogenic activities of TGF-β(1) and PDGF-BB and identify SPARC as a potential therapeutic target for liver fibrosis.     

Combinations of host biomarkers predict mortality among Ugandan children with severe malaria: a retrospective case-control study

Background

Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria.

Methodology/Findings

Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1–99.9) sensitivity and 88.8% (79.7–94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%).

Conclusions

We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria.     

Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Renal safety of a tenofovir-containing first line regimen: experience from an antiretroviral cohort in rural Lesotho

Introduction

Current guidelines contraindicate TDF use when creatinine clearance (CrCl) falls below 50 ml/min. We report prevalence of abnormal renal function at baseline and factors associated with abnormal renal function from a community cohort in Lesotho.

Methods

We calculated changes in CrCl from baseline for patients initiated on TDF at 6 and 12 months and the proportion of patients initiated on TDF who developed renal impairment. Screening algorithms were developed using risk factors determined by multivariate analysis.

Results

Among 933 adults for whom baseline creatinine was available, 176 (18.9%) presented with a baseline CrCl <50 ml/min. Renal function improved during follow-up. 19 patients who developed renal toxicity during follow up remained on TDF; renal function improved (CrCl≥50 ml/min) in all but 3 of these patients. Among 15 patients with a baseline CrCl <50 ml/min were started in error, none developed severe renal impairment.

Conclusion

In this setting TDF-associated renal toxicity is rare and mainly transient. Further studies to assess TDF safety at lower CrCl thresholds are warranted.     

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

Bicaudal‐D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule‐based motors is essential for cell organisation and function. The Bicaudal‐D (BicD) protein participates in the transport of a subset of cargoes by the minus‐end‐directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co‐precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin‐mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high‐frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin‐associated trafficking processes and show a novel requirement for microtubule‐based motor transport in the synaptic vesicle cycle.     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.