Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.     

Biophysical Model of Ion Transport across Human Respiratory Epithelia Allows Quantification of Ion Permeabilities

Lung health and normal mucus clearance depend on adequate hydration of airway surfaces. Because transepithelial osmotic gradients drive water flows, sufficient hydration of the airway surface liquid depends on a balance between ion secretion and absorption by respiratory epithelia. In vitro experiments using cultures of primary human nasal epithelia and human bronchial epithelia have established many of the biophysical processes involved in airway surface liquid homeostasis. Most experimental studies, however, have focused on the apical membrane, despite the fact that ion transport across respiratory epithelia involves both cellular and paracellular pathways. In fact, the ion permeabilities of the basolateral membrane and paracellular pathway remain largely unknown. Here we use a biophysical model for water and ion transport to quantify ion permeabilities of all pathways (apical, basolateral, paracellular) in human nasal epithelia cultures using experimental (Ussing Chamber and microelectrode) data reported in the literature. We derive analytical formulas for the steady-state short-circuit current and membrane potential, which are for polarized epithelia the equivalent of the Goldman-Hodgkin-Katz equation for single isolated cells. These relations allow parameter estimation to be performed efficiently. By providing a method to quantify all the ion permeabilities of respiratory epithelia, the model may aid us in understanding the physiology that regulates normal airway surface hydration.     

Nicotine Inhibits Memory CTL Programming

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.     

Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology–Genomic Integration Analysis

Background

The intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters.

Methods and Findings

We developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10⁻⁴, hazard ratio = 1.47, 95% CI 1.17–1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26–2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size.

Conclusions

To our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells.     

Inclusion of Dominance Effects in the Multivariate GBLUP Model

New proposals for models and applications of prediction processes with data on molecular markers may help reduce the financial costs of and identify superior genotypes in maize breeding programs. Studies evaluating Genomic Best Linear Unbiased Prediction (GBLUP) models including dominance effects have not been performed in the univariate and multivariate context in the data analysis of this crop. A single cross hybrid construction procedure was performed in this study using phenotypic data and actual molecular markers of 4,091 maize lines from the public database Panzea. A total of 400 simple hybrids resulting from this process were analyzed using the univariate and multivariate GBLUP model considering only additive effects additive plus dominance effects. Historic heritability scenarios of five traits and other genetic architecture settings were used to compare models, evaluating the predictive ability and estimation of variance components. Marginal differences were detected between the multivariate and univariate models. The main explanation for the small discrepancy between models is the low- to moderate-magnitude correlations between the traits studied and moderate heritabilities. These conditions do not favor the advantages of multivariate analysis. The inclusion of dominance effects in the models was an efficient strategy to improve the predictive ability and estimation quality of variance components.     

GPS Tracking of Free-Ranging Pigs to Evaluate Ring Strategies for the Control of Cysticercosis/Taeniasis in Peru

Background

Taenia solium, a parasitic cestode that affects humans and pigs, is the leading cause of preventable epilepsy in the developing world. T. solium eggs are released into the environment through the stool of humans infected with an adult intestinal tapeworm (a condition called taeniasis), and cause cysticercosis when ingested by pigs or other humans. A control strategy to intervene within high-risk foci in endemic communities has been proposed as an alternative to mass antihelminthic treatment. In this ring strategy, antihelminthic treatment is targeted to humans and pigs residing within a 100 meter radius of a pig heavily-infected with cysticercosis. Our aim was to describe the roaming ranges of pigs in this region, and to evaluate whether the 100 meter radius rings encompass areas where risk factors for T. solium transmission, such as open human defecation and dense pig activity, are concentrated.

Methodology/Principal Findings

In this study, we used Global Positioning System (GPS) devices to track pig roaming ranges in two rural villages of northern Peru. We selected 41 pigs from two villages to participate in a 48-hour tracking period. Additionally, we surveyed all households to record the locations of open human defecation areas. We found that pigs spent a median of 82.8% (IQR: 73.5, 94.4) of their time roaming within 100 meters of their homes. The size of home ranges varied significantly by pig age, and 93% of the total time spent interacting with open human defecation areas occurred within 100 meters of pig residences.

Conclusions/Significance

These results indicate that 100 meter radius rings around heavily-infected pigs adequately capture the average pig’s roaming area (i.e., home range) and represent an area where the great majority of exposure to human feces occurs.     

In Vitro Study of Taenia solium Postoncospheral Form

Background

The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages.

Methodology/Principal Findings

T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development–days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15–30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages.

Conclusions/Significance

This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite’s immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.     

Growth response of cultured larvae of silver therapon Leiopotherapon plumbeus (Kner, 1864) in outdoor tanks in relation to fertilizer type and fish density

This study evaluated the effects of fertilizer type and fish density on early growth and survival of silver therapon Leiopotherapon plumbeus (Kner, 1864) larvae reared in outdoor tanks. In the first experiment, larvae (1.92 ± 0.09 mm total length) were stocked into nine, 4 m³ tanks at an initial density of 0.5 larvae L^?1 and reared for 42 days at an ambient temperature of 28.8–30.7°C. Three treatments with three replicates each were compared: organic (chicken manure, OF) or inorganic fertilizers (ammonium phosphate, IF) applied once every 2 weeks, and the unfertilized (NF) tanks serving as the control group. Water quality, zooplankton densities, survival or growth of L. plumbeus larvae did not vary significantly in either fertilized or unfertilized tanks. Fertilization resulted in elevated nutrient concentrations, which did affect survival (2.10%–6.07%) of the fish larvae. In the second experiment, larvae were stocked at densities of 0.4 or 0.6 larvae L^?1 in tanks fertilized at 4–5 days interval with OF and IF for 30 days. Growth performance of L. plumbeus larvae was affected by fish density, with significantly larger (20.04 ± 2.65 mm in total length) and higher specific growth rate (SGR; 6.97 ± 0.48% day^?1) at 0.4 larvae L^?1 than at 0.6 L^?1. Fry production did not vary significantly between fish density treatment groups given the same fertilizer types, but survival rates were improved at 0.4 L^?1. Together, production of L. plumbeus larvae in outdoor tanks can be optimized at a lower stocking density, regardless of the type of fertilizer used.