In vitro development and characterization of a manatee bronchial cell line

In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis.     

Doubling protected land area may be inefficient at preserving the extent of undeveloped land and could cause substantial regional shifts in land use

Projection of land use and land-cover change is highly uncertain yet drives critical estimates of carbon emissions, climate change, and food and bioenergy production. We use new, spatially explicit land availability data in conjunction with a model sensitivity analysis to estimate the effects of additional land protection on land use and land cover. The land availability data include protected land and agricultural suitability and is incorporated into the Moirai land data system for initializing the Global Change Analysis Model. Overall, decreasing land availability is relatively inefficient at preserving undeveloped land while having considerable regional land-use impacts. Current amounts of protected area have little effect on land and crop production estimates, but including the spatial distribution of unsuitable (i.e., unavailable) land dramatically shifts bioenergy production from high northern latitudes to the rest of the world, compared with uniform availability. This highlights the importance of spatial heterogeneity in understanding and managing land change. Approximately doubling the current protected area to emulate a 30% protected area target may avoid land conversion by 2050 of less than half the newly protected extent while reducing bioenergy feedstock land by 10.4% and cropland and grazed pasture by over 3%. Regional bioenergy land may be reduced (increased) by up to 46% (36%), cropland reduced by up to 61%, pasture reduced by up to 100%, and harvested forest reduced by up to 35%. Only a few regions show notable gains in some undeveloped land types of up to 36%. Half of the regions can reach the target using only unsuitable land, which would minimize impacts on agriculture but may not meet conservation goals. Rather than focusing on an area target, a more robust approach may be to carefully select newly protected land to meet well-defined conservation goals while minimizing impacts to agriculture.     

Opposing roles for CD34 in B16 melanoma tumor growth alter early stage vasculature and late stage immune cell infiltration

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.     

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.     

Anti-CD70 Immunocytokines for Exploitation of Interferon-γ-Induced RIP1-Dependent Necrosis in Renal Cell Carcinoma

Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel ‘immunocytokine’ chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC.     

SALT TOLERANCE WITHIN A TYPHA POPULATION

Mc Millan , Calvin . (U. Texas, Austin.) Salt tolerance within a Typha population. Amer. Jour. Bot. 46(7): 521–526. Illus. 1959.—Typha in a disturbed salt flat near Lincoln, Nebraska, provided material for an examination of population dynamics. Within the population, clones of T. angustifolia L. tended to occupy the drier sites and those of T. latifolia L. occupied the sites of greater moisture probability. Clones of intermediate morphological characteristics were distributed with both T. angustifolia and T. latifolia. Rhizomes taken from the clones were grown in various NaCl solutions in the greenhouse. Results indicated greatest salt tolerance by T. angustifolia and least salt tolerance by T. latifolia. The intermediate, probably hybrid, clones were intermediate in salt tolerance. Seeds of the 3 clone-types germinated over the same range of salt concentration. The seeds of all 3 types withstood 4 months submergence in a 2% salt solution and germinated upon being returned to tap water. In the salt flat habitat, the clones of T. latifolia were not vigorous during the years 1956–1957 and many died or were reduced considerably in area of occupancy. The clones of T. angustifolia remained vigorous and flowered over the same period. The intermediate clones were vigorous and increased their coverage, primarily in areas that were occupied prior to 1956 by T. latifolia. The spatial adjustments within the population probably resulted from the selective action of increased salt concentration accompanying the drier conditions of 1956 and 1957.     

Efficacy and safety of a new vaginal contraceptive antimicrobial formulation containing high molecular weight poly(sodium 4-styrenesulfonate)

Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC(50) = 5.3 microg/ml) and acrosin (IC(50) = 0.3 microg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 microg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC(50)= 16 microg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC(50) = 1.3 and 1.0 microg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC(50) < 1.0 gel/ml) and Chlamydia trachomatis (IC(50) = 1.2 microg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.