Interferon Regulatory Factor (IRF)-1 Is a Master Regulator of the Cross Talk between Macrophages and L929 Fibrosarcoma Cells for Nitric Oxide Dependent Tumoricidal Activity

Macrophage tumoricidal activity relies, mainly, on the release of Tumor Necrosis Factor alpha (TNFα) and/or on reactive oxygen or nitrogen intermediates. In the present work, we investigated the cytotoxic activity of resident peritoneal macrophages against L929 fibrosarcoma cell line in vitro and in vivo. Resident macrophages lysed L929 cells in a mechanism independent of TNFα and cell-to-cell contact. The cytotoxic activity was largely dependent on nitric oxide (NO) release since treatment with L-NAME (NOS inhibitor) inhibited L929 cells killing. Macrophages from mice with targeted deletion of inducible NO synthase (iNOS) together with L929 cells produced less NO and displayed lower, but still significant, tumoricidal activity. Notably, NO production and tumor lysis were abolished in co-cultures with macrophages deficient in Interferon Regulatory Factor, IRF-1. Importantly, the in vitro findings were reproduced in vivo as IRF-1 deficient animals inoculated i.p with L929 cells were extremely susceptible to tumor growth and their macrophages did not produce NO, while WT mice killed L929 tumor cells and their macrophages produced high levels of NO. Our results indicate that IRF-1 is a master regulator of bi-directional interaction between macrophages and tumor cells. Overall, IRF-1 was essential for NO production by co-cultures and macrophage tumoricidal activity in vitro as well as for the control of tumor growth in vivo.     

CXCL12 rs1801157 polymorphism and expression in peripheral blood from breast cancer patients

The role of chemokines has been extensively analyzed both in cancer risk and tumor progression. Among different cytokines, CXCR4 and its ligand CXCL12 have been recently subjected to a closer examination. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/SDF1-3'A) in the CXCL12 gene and the relative expression of mRNA CXCL12 in peripheral blood were assessed in breast cancer patients, since the chemokine CXCL12 and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using MspI restriction enzyme and the expression analyses by quantitative RT-PCR. No difference in GG genotype and allele A carrier frequencies were observed between breast cancer patients and healthy blood donors and nor when CXCL12 mRNA expression was assessed among patients with different tumor stages. However a significant difference was observed when CXCL12 mRNA relative expression was analyzed in breast cancer patients in accordance to the presence or absence of the CXCL12 rs1801157 allele A. Allele A breast cancer patients presented a mRNA CXCL12 expression about 2.1-fold smaller than GG breast cancer patients. Estrogen positive patients presenting CXCL12 allele A presented a significantly lower expression of CXCL12 in peripheral blood (p=0.039) than GG hormone positive patients. Our findings demonstrated that allele A is associated with low expression of CXCL12 in the peripheral blood from ER-positive breast cancer patients, which suggests implications on breast cancer clinical outcome.     

Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

Mind the gap: non‐biological processes contributing to soil CO2 efflux

Widespread recognition of the importance of soil CO₂ efflux as a major source of CO₂ to the atmosphere has led to active research. A large soil respiration database and recent reviews have compiled data, methods, and current challenges. This study highlights some deficiencies for a proper understanding of soil CO₂ efflux focusing on processes of soil CO₂ production and transport that have not received enough attention in the current soil respiration literature. It has mostly been assumed that soil CO₂ efflux is the result of biological processes (i.e. soil respiration), but recent studies demonstrate that pedochemical and geological processes, such as geothermal and volcanic CO₂ degassing, are potentially important in some areas. Besides the microbial decomposition of litter, solar radiation is responsible for photodegradation or photochemical degradation of litter. Diffusion is considered to be the main mechanism of CO₂ transport in the soil, but changes in atmospheric pressure and thermal convection may also be important mechanisms driving soil CO₂ efflux greater than diffusion under certain conditions. Lateral fluxes of carbon as dissolved organic and inorganic carbon occur and may cause an underestimation of soil CO₂ efflux. Traditionally soil CO₂ efflux has been measured with accumulation chambers assuming that the main transport mechanism is diffusion. New techniques are available such as improved automated chambers, CO₂ concentration profiles and isotopic techniques that may help to elucidate the sources of carbon from soils. We need to develop specific and standardized methods for different CO₂ sources to quantify this flux on a global scale. Biogeochemical models should include biological and non‐biological CO₂ production processes before we can predict the response of soil CO₂ efflux to climate change. Improving our understanding of the processes involved in soil CO₂ efflux should be a research priority given the importance of this flux in the global carbon budget.     

A Computational,Tissue-Realistic Model of Pressure Ulcer Formation in Individuals with Spinal Cord Injury

People with spinal cord injury (SCI) are predisposed to pressure ulcers (PU). PU remain a significant burden in cost of care and quality of life despite improved mechanistic understanding and advanced interventions. An agent-based model (ABM) of ischemia/reperfusion-induced inflammation and PU (the PUABM) was created, calibrated to serial images of post-SCI PU, and used to investigate potential treatments in silico. Tissue-level features of the PUABM recapitulated visual patterns of ulcer formation in individuals with SCI. These morphological features, along with simulated cell counts and mediator concentrations, suggested that the influence of inflammatory dynamics caused simulations to be committed to “better” vs. “worse” outcomes by 4 days of simulated time and prior to ulcer formation. Sensitivity analysis of model parameters suggested that increasing oxygen availability would reduce PU incidence. Using the PUABM, in silico trials of anti-inflammatory treatments such as corticosteroids and a neutralizing antibody targeted at Damage-Associated Molecular Pattern molecules (DAMPs) suggested that, at best, early application at a sufficiently high dose could attenuate local inflammation and reduce pressure-associated tissue damage, but could not reduce PU incidence. The PUABM thus shows promise as an adjunct for mechanistic understanding, diagnosis, and design of therapies in the setting of PU.     

Development and morphology of the gynoecium and nutlet in two South-American Bulbostylis (Cyperaceae) species

The anatomy and ontogeny of the gynoecium and nutlets of two Bulbostylis species with different micro-morphology, Bulbostylis capillaris sensu Barros and B. major, were analyzed. The specific aim of this work was to identify which part of the pericarp determines the differences in the nutlet surface between these two species. We found that pre-fertilization development is the same in both species, with differences between species appearing only after fertilization. In the nutlet of B. major, the exocarp forms a tuberculate primary sculpture that has nipple-like protuberances consisting of one conoidal silica body per cell, whereas the secondary sculpture is micro-granulose and is constituted by the cuticle. In the nutlet of B. capillaris, the primary sculpture is granulose and is formed by the presence of starch granules in the exocarp that do not degrade, whereas the secondary sculpture is smooth.     

A new role for the p85-phosphatidylinositol 3-kinase regulatory subunit linking FRAP to p70 S6 kinase activation.

The serine/threonine kinase p70 S6 kinase (p70S6K) phosphorylates the 40 S ribosomal protein S6, modulating the translation of an mRNA subset that encodes ribosomal proteins and translation elongation factors. p70S6K is activated in response to mitogenic stimuli and is required for progression through the G(1) phase of the cell cycle and for cell growth. Activation of p70S6K is regulated by phosphorylation of seven different residues distributed throughout the protein, a subset of which depends on the activity of p85/p110 phosphatidylinositol 3-kinase (PI3K); in fact, the phosphorylation status of Thr(229) and Thr(389) is intimately linked to PI3K activity. In the full-length enzyme, however, these sites are also acutely sensitive to the action of FKBP 12-rapamycin-associated protein (FRAP). The mechanism by which PI3K and FRAP cooperate to induce p70S6K activation remains unclear. Here we show that the p85 regulatory subunit of PI3K also controls p70S6K activation by mediating formation of a ternary complex with p70S6K and FRAP. The p85 C-terminal SH2 domain is responsible for p85 coupling to p70S6K and FRAP, because deletion of the C-terminal SH2 domain inhibits complex formation and impairs p70S6K activation by PI3K. Formation of this complex is not required for activation of a FRAP-independent form of p70S6K, however, underscoring the role of p85 in regulating FRAP-dependent p70S6K activation. These studies thus show that, in addition to the contribution of PI3K activity, the p85 regulatory subunit plays a critical role in p70S6K activation.     

Early population differentiation in extinct aborigines from Tierra del Fuego-Patagonia: ancient mtDNA sequences and Y-chromosome STR characterization

Ancient mtDNA was successfully recovered from 24 skeletal samples of a total of 60 ancient individuals from Patagonia-Tierra del Fuego, dated to 100-400 years BP, for which consistent amplifications and two-strand sequences were obtained. Y-chromosome STRs (DYS434, DYS437, DYS439, DYS393, DYS391, DYS390, DYS19, DYS389I, DYS389II, and DYS388) and the biallelic system DYS199 were also amplified, Y-STR alleles could be characterized in nine cases, with an average of 4.1 loci per sample correctly typed. In two samples of the same ethnic group (Aonikenk), an identical and complete eight-loci haplotype was recovered. The DYS199 biallelic system was used as a control of contamination by modern DNA and, along with DYS19, as a marker of American origin. The analysis of both mtDNA and Y-STRs revealed DNA from Amerindian ancestry. The observed polymorphisms are consistent with the hypothesis that the ancient Fuegians are close to populations from south-central Chile and Argentina, but their high nucleotide diversity and the frequency of single lineages strongly support early genetic differentiation of the Fuegians through combined processes of population bottleneck, isolation, and/or migration, followed by strong genetic drift. This suggests an early genetic diversification of the Fuegians right after their arrival at the southernmost extreme of South America.