Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio)

While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.     

Evaluation of a cervical cancer screening program based on HPV testing and LLETZ excision in a low resource setting

We conducted studies in Vanuatu to evaluate potential screening and treatment strategies to assist with control of cervical cancer. In a pilot study of 496 women, visual inspection and cytology were evaluated as screening tests for detection of CIN 2 or worse (CIN2+), observed in 21 of 206 subjects biopsied on the basis of abnormal visual inspection or cytology. Sensitivity of visual inspection with Lugol's Iodine for detection of CIN2+ on biopsy was 0.63, specificity was 0.32, and the positive predictive value was 0.09. For HSIL cytology, sensitivity was 0.99, specificity was 0.77, and the positive predictive value was 0.88. HSIL cytology was significantly more sensitive and had a significantly higher PPV for CIN 2+ than visual inspection (p<0.01). In a further study of 514 women, we compared testing for HR HPV and cytology as predictors of biopsy proven CIN 2+. Sensitivity of HSIL cytology for CIN2+ as established by loop excision of the cervix was 0.81, specificity was 0.94, and positive predictive value was 0.48. Sensitivity of a positive test for HR HPV for detection of CIN2+ was non-significantly different from cytology at 0.81, specificity was 0.94, and positive predictive value was 0.42. Combining the two tests gave a significantly lower sensitivity of 0.63, a specificity of 0.98, and a positive predictive value of 0.68. For women over 30 in a low resource setting without access to cytology, a single locally conducted test for high risk HPV with effective intervention could reduce cervical cancer risk as effectively as intervention based on cytology conducted in an accredited laboratory.     

A genome-wide approach to identifying novel-imprinted genes

Genomic imprinting is an epigenetic process in which the copy of a gene inherited from one parent (maternal or paternal) is consistently silenced or expressed at a significantly lower level than the copy from the other parent. In an effort to begin a systematic genome-wide screen for imprinted genes, we assayed differential allelic expression (DAE) at 3,877 bi-allelic protein-coding sites located in 2,625 human genes in 67 unrelated individuals using genotyping microarrays. We used the presence of both over- and under-expression of the reference allele compared to the alternate allele to identify candidate-imprinted genes. We found 61 genes with at least twofold DAE plus “flipping” of the more highly expressed allele between reference and alternate across heterozygous samples. Sixteen flipping genes were genotyped and assayed for DAE in an independent data set of lymphoblastoid cell lines from two CEPH pedigrees. We confirmed that PEG10 is paternally expressed, identified one gene (ZNF331) with multiple lines of data indicating it is imprinted, and predicted several additional imprinting candidate genes. Our findings suggest that there are at most several hundred genes in the human genome that are universally imprinted. With samples of mRNA from appropriate tissues and a collection of informative cSNPs, a genome-wide search using this methodology could expand the list of genes that undergo genomic imprinting in a tissue- or temporal-specific manner. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.     

HPV16-E7 Expression in Squamous Epithelium Creates a Local Immune Suppressive Environment via CCL2- and CCL5- Mediated Recruitment of Mast Cells

Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions.     

Ozone effects on visible foliar injury and growth of Fagus sylvatica and Viburnum lantana seedlings grown in monoculture or in mixture

Seedlings of Fagus sylvatica (beech) and Viburnum lantana (Viburnum) grown in monoculture and mixture were exposed to ambient and sub-ambient (charcoal-filtered) ozone concentrations in open-top chambers over the course of the 2003 and 2004 growing seasons at the WSL Lattecaldo open-top chamber facility in southern Switzerland. The aim of the study was to determine how the sensitivity to ozone in ambient air of these two species would differ between monocultures and mixtures in terms of growth and visible foliar injury development. Ambient ozone concentrations were consistently higher from the end of April to the middle of October in 2003 than in 2004 with seasonal peaks and means reaching 147 and 50 parts-per-billion (ppb) in 2003 compared to 124 and 40 ppb in 2004. Ambient AOT40 (ozone concentration accumulated over a threshold of 40 ppb during daylight hours with global radiation >50 W m^?2) values from the end of April to the middle of October reached 48.3 and 26.8 parts-per-billon hours (ppm h) in 2003 and 2004, respectively. In general, Viburnum was a stronger competitor than beech over the course of this 2-year study. Seedlings of Viburnum benefited from interspecific competition in terms of both height growth and above-ground biomass accumulation at the expense of beech seedlings, which showed significantly reduced growth in the mixture as compared to the monoculture. However, as this was only the case for Viburnum growing in the charcoal-filtered treatment, ozone seemed to counteract the beneficial effect of interspecific competition on above-ground biomass accumulation in Viburnum, while at the same time decreasing relative biomass allocation to roots. Foliar sensitivity of the two species was also altered under interspecific competition suggesting that results based on seedlings of single species grown in monocultures may significantly over- or under-estimate foliar sensitivity to ozone. These results demonstrate that competition is an important factor affecting plant responses to ozone stress, but the direction and severity of these effects depend on the interacting species.