Faculty Bibliography

The central nervous system areas displaying the highest structural and functional complexity correspond to the so called cortices, i.e., concentric alternating neuronal and fibrous layers. Corticogenesis, i.e., the development of the cortical organization, depends on the temporal-spatial organization of several developmental events: (a) the duration of the proliferative phase of the neuroepithelium, (b) the relative duration of symmetric (expansive) versus asymmetric (neuronogenic) sub phases, (c) the spatial organization of each kind of cell division, (e) the time of determination and cell cycle exit and (f) the time of onset of the post-mitotic neuronal migration and (g) the time of onset of the neuronal structural and functional differentiation. The first five events depend on molecular mechanisms that perform a fine tuning of the proliferative activity. Changes in any of them significantly influence the cortical size or volume (tangential expansion and radial thickness), morphology, architecture and also impact on neuritogenesis and synaptogenesis affecting the cortical wiring. This paper integrates information, obtained in several species, on the developmental roles of cell proliferation in the development of the optic tectum (OT) cortex, a multilayered associative area of the dorsal (alar) midbrain. The present review (1) compiles relevant information on the temporal and spatial organization of cell proliferation in different species (fish, amphibians, birds, and mammals), (2) revises the main molecular events involved in the isthmic organizer (IsO) determination and localization, (3) describes how the patterning installed by IsO is translated into spatially organized neural stem cell proliferation (i.e., by means of growth factors, receptors, transcription factors, signaling pathways, etc.) and (4) describes the morpho- and histogenetic effect of a spatially organized cell proliferation in the above mentioned species. A brief section on the OT evolution is also included. This section considers how the differential operation of cell proliferation could explain differences among species.

BACKGROUND/AIMS/OBJECTIVE:The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS:We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS:Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION/CONCLUSIONS:Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.

BACKGROUND: In 2007, the World Health Organization (WHO) recommended scaling up voluntary medical male circumcision (VMMC) in priority countries with high HIV prevalence and low male circumcision (MC) prevalence. According to the Joint United Nations Programme on HIV/AIDS (UNAIDS), an estimated 5.8 million males had undergone VMMC by the end of 2013. Implementation experience has raised questions about the need to refocus VMMC programs on specific subpopulations for the greatest epidemiological impact and programmatic effectiveness. As Malawi prepared its national operational plan for VMMC, it sought to examine the impacts of focusing on specific subpopulations by age and region. METHODS: We used the Decision Makers' Program Planning Toolkit, Version 2.0, to study the impact of scaling up VMMC to different target populations of Malawi. National MC prevalence by age group from the 2010 Demographic and Health Survey was scaled according to the MC prevalence for each district and then halved, to adjust for over-reporting of circumcision. In-country stakeholders advised a VMMC unit cost of $100, based on implementation experience. We derived a cost of $451 per patient-year for antiretroviral therapy from costs collected as part of a strategic planning exercise previously conducted in- country by UNAIDS. RESULTS: Over a fifteen-year period, circumcising males ages 10-29 would avert 75% of HIV infections, and circumcising males ages 10-34 would avert 88% of infections, compared to the current strategy of circumcising males ages 15-49. The Ministry of Health's South West and South East health zones had the lowest cost per HIV infection averted. Moreover, VMMC met WHO's definition of cost-effectiveness (that is, the cost per disability-adjusted life-year [DALY] saved was less than three times the per capita gross domestic product) in all health zones except Central East. Comparing urban versus rural areas in the country, we found that circumcising men in urban areas would be both cost-effective and cost-saving, with a VMMC cost per DALY saved of $120 USD and with 15 years of VMMC implementation resulting in lifetime HIV treatment costs savings of $331 million USD. CONCLUSIONS: Based on the age analyses and programmatic experience, Malawi's VMMC operational plan focuses on males ages 10-34 in all districts in the South East and South West zones, as well as Lilongwe (an urban district in the Central zone). This plan covers 14 of the 28 districts in the country.

Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet beta cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8(+) T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8(+) T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158-166 and 282-290) and one in a non-beta cell protein, dopamine beta-hydroxylase (aa 233-241). Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DbetaH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DbetaH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1alpha) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1alpha is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1alpha degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1alpha as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFalpha, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 +/- 0.45 days vs. 19 +/- 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1alpha and upregulation of pro-angiogenic genes downstream of HIF-1alpha, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Human colon cancers commonly harbor loss of function mutations in APC, a repressor of the canonical WNT pathway, thus leading to hyperactive WNT-TCF signaling. Re-establishment of Apc function in mice, engineered to conditionally repress Apc through RNAi, resolve the intestinal tumors formed due to hyperactivated Wnt-Tcf signaling. These and other results have prompted the search for specific WNT pathway antagonists as therapeutics for clinically problematic human colon cancers and associated metastases, which remain largely incurable. This widely accepted view seems at odds with a number of findings using patient-derived material: Canonical TCF targets are repressed, instead of being hyperactivated, in advanced colon cancers, and repression of TCF function does not generally result in tumor regression in xenografts. The results of a number of genetic mouse studies have also suggested that canonical WNT-TCF signaling drives metastases, but direct in vivo tests are lacking, and, surprisingly, TCF repression can enhance directly seeded metastatic growth. Here we have addressed the abilities of enhanced and blocked WNT-TCF signaling to alter tumor growth and distant metastases using xenografts of advanced human colon cancers in mice. We find that endogenous WNT-TCF signaling is mostly anti-metastatic since downregulation of TCF function with dnTCF generally enhances metastatic spread. Consistently, elevating the level of WNT signaling, by increasing the levels of WNT ligands, is not generally pro-metastatic. Our present and previous data reveal a heterogeneous response to modulating WNT-TCF signaling in human cancer cells. Nevertheless, the findings that a fraction of colon cancers tested require WNT-TCF signaling for tumor growth but all respond to repressed signaling by increasing metastases beg for a reevaluation of the goal of blocking WNT-TCF signaling to universally treat colon cancers. Our data suggest that WNT-TCF blockade may be effective in inhibiting tumor growth in only a subset of cases but will generally boost metastases.