Oral Administration of Coronavirus Spike Protein Provides Protection to Newborn Pigs When Challenged with PEDV.

To investigate whether oral administration of maize-produced S antigen can provide passive immunity to piglets against porcine epidemic diarrhea virus (PEDV), 16 pregnant sows were randomly assigned to one of four treatments: (1) injection of PEDV vaccine (INJ), (2) maize grain without S protein (CON), (3) maize grain containing low dose of S antigen (LOV) and (4) maize grain containing a high dose of S antigen (HOV). Vaccines were administered on days 57, 85 and 110 of gestation. Sows' serum and colostrum were collected at farrowing and milk on day 6 post-challenge to quantify neutralizing antibodies (NABs) and cytokines. Piglets were challenged with PEDV 3-5 d after farrowing, and severity of disease and mortality assessed on day 11 post-challenge. Disease severity was lower in LOV and INJ compared with HOV and CON, whereas the survival rate increased in piglets from LOV sows compared with HOV and CON (p ≤ 0.001). Higher titers of NABs and lower levels of cytokine granulocyte-macrophage colony-stimulating factor in sows' milk were positively correlated with piglet survivability (p ≤ 0.05). These data suggest that feeding S protein in corn to pregnant sows protects nursing piglets against PEDV.

Pre-Columbian fire management and control of climate-driven floodwaters over 3,500 years in southwestern Amazonia.

In landscapes that support economic and cultural activities, human communities actively manage environments and environmental change at a variety of spatial scales that complicate the effects of continental-scale climate. Here, we demonstrate how hydrological conditions were modified by humans against the backdrop of Holocene climate change in southwestern Amazonia. Paleoecological investigations (phytoliths, charcoal, pollen, diatoms) of two sediment cores extracted from within the same permanent wetland, ∼22 km apart, show a 1,500-y difference in when the intensification of land use and management occurred, including raised field agriculture, fire regime, and agroforestry. Although rising precipitation is well known during the mid to late Holocene, human actions manipulated climate-driven hydrological changes on the landscape, revealing differing histories of human landscape domestication. Environmental factors are unable to account for local differences without the mediation of human communities that transformed the region to its current savanna/forest/wetland mosaic beginning at least 3,500 y ago. Regional environmental variables did not drive the choices made by farmers and fishers, who shaped these local contexts to better manage resource extraction. The savannas we observe today were created in the post-European period, where their fire regime and structural diversity were shaped by cattle ranching.

Co-Administration of Injected and Oral Vaccine Candidates Elicits Improved Immune Responses over Either Route Alone.

Infectious diseases continue to be a significant cause of morbidity and mortality, and although efficacious vaccines are available for many diseases, some parenteral vaccines elicit little or no mucosal antibodies which can be a significant problem since mucosal tissue is the point of entry for 90% of pathogens. In order to provide protection for both serum and mucosal areas, we have tested a combinatorial approach of both parenteral and oral administration of antigens for diseases caused by a viral pathogen, Hepatitis B, and a fungal pathogen, Coccidioides. We demonstrate that co-administration by the parenteral and oral routes is a useful tool to increase the overall immune response. This can include achieving an immune response in tissues that are not elicited when using only one route of administration, providing a higher level of response that can lead to fewer required doses or possibly providing a better response for individuals that are considered poor or non-responders.

Oral delivery of wafers made from HBsAg-expressing maize germ induces long-term immunological systemic and mucosal responses.

BACKGROUND: The hepatitis B surface antigen (HBsAg) has been administered over the last 20 years as a parenteral vaccine against the hepatitis B virus (HBV). Despite high seroconversion rates, chronic infection rates are still high worldwide. Orally delivered vaccines provide a practical alternative to injected vaccines, potentially helping poorly responding populations and providing a viable alternative for populations in remote locations. Anamnestic responses are vital to establishing the efficacy of a given vaccine and have been assessed in this study using a plant-based oral delivery platform expressing HBsAg. METHODS: Long-term immunological memory was assessed in mice injected with a primary dose of Recombivax and boosted with orally-delivered HBsAg wafers, control wafers, or parenterally-delivered commercial vaccine (Recombivax). RESULTS: Mice boosted with HBsAg orally-administered wafers displayed sharp increases in mucosal IgA titers in fecal material and steep increases in serum IgA, whereas mice boosted with Recombivax showed no detectable levels of IgA in either fecal or serum samples following four boosting treatments. Long-term memory in the orally-treated mice was evidenced by sustained fecal IgA, and serum IgA, IgG, and mIU/mL over one year, while Recombivax-treated mice displayed sustained serum IgG and mIU/mL. Furthermore, sharp increases in these same antibodies were induced after re-boosting at 47 and 50 weeks post-primary injection. CONCLUSIONS: Orally-delivered vaccines can provide long-term immune responses mucosally and systemically. For sexually-transmitted diseases that can be acquired at mucosal surfaces, such as HBV, an oral delivery platform may provide added protection over a conventional parenterally administered vaccine.

Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation.

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

Supercritical fluid extraction provides an enhancement to the immune response for orally-delivered hepatitis B surface antigen.

The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 µg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.

The S100A6 calcium-binding protein regulates endothelial cell-cycle progression and senescence.

Endothelial cells regulate many aspects of vascular physiology, including vasculogenesis and angiogenesis. The S100 family of calcium-binding proteins regulates many aspects of cell function but their roles in vascular physiology are less well understood. Herein, we investigated the expression and function of S100-related family members in endothelial cells. Analysis of total endothelial mRNAs using a human gene chip array revealed significant gene expression of the S100 calcium-binding protein family members S100A6, S100A10, S100A11 and S100A13. We then examined the expression and functional properties of the major S100 family member, S100A6, in vascular endothelial cells. Comparison of primary and transformed human cells revealed significant differences in S100A6 protein levels in these cells. In primary human endothelial cells, S100A6 was present in both the nucleus and the cytoplasm. To assess the function of endothelial S100A6, we depleted protein levels using RNA interference and this caused increased cell-cycle arrest in the G2/M phase under different conditions. S100A6 depletion caused a decrease in both cyclin-dependent kinase 1 (CDK1) and phospho-CDK1 levels, which are essential for eukaryote cell-cycle progression. S100A6 depletion also decreased expression of CDK1, cyclin A1 (CCNA1) and cyclin B (CCNB1) genes with effects on cell-cycle progression. Depletion of endothelial S100A6 levels also elevated ß-galactosidase expression, which is an important hallmark of cellular senescence and exit from the mammalian cell cycle. We thus propose that S100A6 has an important role in regulating endothelial commitment to, and progression through, the cell cycle.

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A.

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.

A VE-cadherin-PAR3-α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A(2)α in endothelial cells.

Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell-cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA(2)α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA(2)α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA(2)α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not ß-catenin, regulates cPLA(2)α Golgi localization linked to cell confluence. Furthermore, cPLA(2)α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA(2)α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA(2)α-dependent endothelial tubule formation. Thus a VE-cadherin-PAR3-α-catenin adhesion complex regulates cPLA(2)α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.

Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen.

Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.

Persistence with prostaglandin agonist use with and without adjunctive therapy for glaucoma patients: a Canadian population-based analysis.

BACKGROUND: Glaucoma is one of the leading causes of blindness and visual disability. Few studies have examined persistence and adherence with topical medications in glaucoma patients. OBJECTIVE: The objective of this study was to compare patient persistence with prostaglandin agonist (PA) monotherapy versus with concomitant adjunctive therapy (AT) in Canada. METHODS: Data were obtained from the Québec prescription claims database. Persistence rates were determined for previously treated naive glaucoma patients at 1 year after their index date for use of any of the three available PAs (bimatoprost, latanoprost, and travoprost). Patients who had at least 334 days on their index PA were defined as being persistent during the analysis timeframe. Patient baseline demographics and persistence rates were reported. A logistic regression was used for comparing the PA and PA + AT groups, which incorporated baseline cofounders, such as age and sex, in the analyses. RESULTS: From an initial cohort of 28 534 patients, 14 893 were identified as naive to glaucoma therapy and had a PA as their index therapy. Of these, 11 197 (75.2%) continued to receive monotherapy and 3696 (24.8%) had an AT added to the PA; 59.0% were females, and the average age was 70.5 ± 11.3 years. Overall, at the end of the first year of therapy, 57.4% of patients were persistent on their index PA; however, a statistically significant difference was observed between the two subgroups, with 54.6% for those receiving PA monotherapy and 65.8% for those receiving PA + AT (p < 0.01) persistent with therapy. On average, 10.5 prescriptions per year were dispensed to persistent patients. CONCLUSIONS: In this Canadian population, persistence rates fall to approximately 60% at the end of the first year of therapy, with patients taking AT being more persistent. Similar persistence analyses are warranted on other populations, and would yield helpful data for conducting economic evaluations of non-persistence.

Assessing college-level learning difficulties and "at riskness" for learning disabilities and ADHD: development and validation of the learning difficulties assessment.

This article describes the development and validation of the Learning Difficulties Assessment (LDA), a normed and web-based survey that assesses perceived difficulties with reading, writing, spelling, mathematics, listening, concentration, memory, organizational skills, sense of control, and anxiety in college students. The LDA is designed to (a) map individual learning strengths and weaknesses, (b) provide users with a comparative sense of their academic skills, (c) integrate research in user-interface design to assist those with reading and learning challenges, and (d) identify individuals who may be at risk for learning disabilities and attention-deficit/hyperactivity disorder (ADHD) and who should thus be further assessed. Data from a large-scale 5-year study describing the instrument's validity as a screening tool for learning disabilities and ADHD are presented. This article also describes unique characteristics of the LDA including its user-interface design, normative characteristics, and use as a no-cost screening tool for identifying college students at risk for learning disorders and ADHD.

Scavenger receptors and their potential as therapeutic targets in the treatment of cardiovascular disease.

Scavenger receptors act as membrane-bound and soluble proteins that bind to macromolecular complexes and pathogens. This diverse supergroup of proteins mediates binding to modified lipoprotein particles which regulate the initiation and progression of atherosclerotic plaques. In vascular tissues, scavenger receptors are implicated in regulating intracellular signaling, lipid accumulation, foam cell development, and cellular apoptosis or necrosis linked to the pathophysiology of atherosclerosis. One approach is using gene therapy to modulate scavenger receptor function in atherosclerosis. Ectopic expression of membrane-bound scavenger receptors using viral vectors can modify lipid profiles and reduce the incidence of atherosclerosis. Alternatively, expression of soluble scavenger receptors can also block plaque initiation and progression. Inhibition of scavenger receptor expression using a combined gene therapy and RNA interference strategy also holds promise for long-term therapy. Here we review our current understanding of the gene delivery by viral vectors to cells and tissues in gene therapy strategies and its application to the modulation of scavenger receptor function in atherosclerosis.

Wild-type and Hupki (human p53 knock-in) murine embryonic fibroblasts: p53/ARF pathway disruption in spontaneous escape from senescence.

Research on cell senescence and immortalization of murine embryonic fibroblasts (MEFs) has revealed important clues about genetic control of senescence in humans. To investigate senescence and genetic alterations in the p53 pathway that lead to senescence bypass in culture, we compared the behavior of MEFs from wild-type mice with MEFs from Hupki mice, which harbor a humanized p53 gene. We found that humanizing the p53 gene in mice preserved major features of the MEF senescence/immortalization process. In both genotypes, a significant proportion of spontaneously arising cell lines had sustained either a p53 point mutation or p19/ARF biallelic deletion. The p53 mutations selected for during Hupki MEF immortalization have been found in human tumors and are classified in the yeast transactivation assay as transcriptionally defunct, suggesting that disabling this component of p53 activity is crucial in senescence bypass. Surprisingly, in spontaneously immortalized cell lines from both wild-type and Hupki MEFs, the predominant type of p53 mutation was a G to C transversion, rather than the G to T substitutions expected from the raised oxygen levels characteristic of standard culture conditions. Over half of the cell lines did not reveal evidence of p53 mutation or loss of p19/ARF and retained a robust wild-type p53 response to DNA damage, supporting the inference from senescence bypass screens that alternative genetic routes to immortalization occur.

The role of Cajal bodies in the expression of late phase adenovirus proteins.

Cajal bodies (CBs) are subnuclear structures involved in RNA metabolism. Here we show that, following infection of HeLa cells by adenovirus type 5 (Ad5), CBs fragment and form ordered structures, which we have termed "rosettes". Formation of CB rosettes was prevented by inhibition of viral DNA synthesis and preceded expression of the L4-33K protein. CB rosettes localised to the periphery of E2A-72K-containing replication centers and to the edges of ASF/SF2 and hnRNP A1 ring structures that demarcate sites of viral transcription and splicing. At later times of infection, CB rosettes were undetectable. Furthermore, knock-down of p80-coilin (the major structural protein of CBs) by RNA interference reduced the yield of infectious Ad5 and expression of the late proteins IIIa (from L1), hexon (from L3) and fiber (from L5), whereas the E2A-72K protein was unaffected. We conclude that CBs have an important role in the expression of adenovirus major late gene products.

Association Between Use of Proton Pump Inhibitors and a Clostridium difficile-Associated Disease Outbreak: Case-Control Study.

BACKGROUND: The use of proton pump inhibitors (PPIs) has been implicated as a potential contributor to the development of Clostridium difficile-associated disease (CDAD) because of the ability of these drugs to substantially reduce the bactericidal effect of gastric acid. This study focused on the impact of PPIs, among other known risk factors, during an outbreak of CDAD in a hospital setting. OBJECTIVES: The primary objective was to determine whether there was an association between current use of a PPI and the CDAD outbreak. Secondary objectives were to evaluate any correlations between the CDAD outbreak and past use of PPIs, use of antibiotics, diabetes mellitus, enteral feeding, cancer, gastrointestinal surgery, inflammatory bowel disease, and previous care or residence in an institutional setting. METHODS: A retrospective case-control study was conducted. One hundred and fifty cases of hospital-acquired Clostridium difficile were identified. Patients were individually matched to controls for age, sex, date of admission to hospital, and hospital unit. The groups were compared with respect to each exposure. RESULTS: Eight case patients could not be matched with suitable controls. Therefore, data from 142 cases and 142 controls were analyzed. There was no association between current use of a PPI and the CDAD outbreak (odds ratio [OR] 1.0, 95% confidence interval [CI] 0.99-1.01). Similarly, there was no correlation between the CDAD outbreak and diabetes, enteral feeding, cancer, gastrointestinal surgery, inflammatory bowel disease, or previous care or residence in an institution. However, the development of CDAD was positively associated with use of antibiotics within the 30 days preceding the infection (OR 12.0, 95% CI 4.0-35.7) and with past use of a PPI (OR 2.4, 95% CI 1.4-4.3). CONCLUSIONS: The development of CDAD during a hospital outbreak was associated with use of antibiotics and with past, not current, use of PPIs.

Ligand-stimulated VEGFR2 signaling is regulated by co-ordinated trafficking and proteolysis.

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.

Economic impact of the introduction of an acellular pertussis vaccine in Canada: a 6-year analysis.

BACKGROUND: Between July 1997 and April 1998, Canadian public health agencies switched from the whole cell vaccine to the acellular vaccine for pertussis immunization. The acellular vaccine provided better efficacy and fewer adverse events than the whole cell vaccine did. OBJECTIVE: To determine the economic impact of replacing the whole cell vaccine with an acellular vaccine in Canada. METHODS: A decision analytic model was developed comparing costs and outcomes of pertussis vaccination for Canadian children born in the years 1991-2004. Effectiveness was measured as number of avoided pertussis cases as well as the number of avoided hospital admissions. Incremental costs per avoided pertussis case and per avoided hospital admission were calculated for Ministry of Health (MoH) and societal (SOC) perspectives. Various one-way sensitivity analyses as well as a Monte Carlo simulation were performed by varying key model parameters. RESULTS: The switch in immunization programs resulted in an incremental cost to the MoH of CAD $108 per pertussis case avoided (CAD $0.96 per child-year). From the SOC perspective, there was a savings of CAD $184 per pertussis case avoided (CAD $0.13 per child-year). The one-way sensitivity analyses provided incremental cost-effective ratios (ICERs) ranging from an incremental cost of CAD $1034 per avoided pertussis case from the MoH perspective to a saving of CAD $1583 per avoided case from the SOC perspective. The Monte Carlo simulation confirmed the robustness of these results. CONCLUSIONS: Pertussis vaccination with AcE was cost-saving from the societal perspective and cost-effective from the Ministry of Health perspective.

VEGF-A-stimulated signalling in endothelial cells via a dual receptor tyrosine kinase system is dependent on co-ordinated trafficking and proteolysis.

The mammalian endothelium expresses two related but distinct receptor tyrosine kinases, VEGFR1 and VEGFR2 [VEGF (vascular endothelial growth factor) receptor 1 and 2], that regulate the vascular response to a key cytokine, VEGF-A. In the present review, we suggest a model for integrating the signals from these receptor tyrosine kinases by co-ordinating the spatial and temporal segregation of these membrane proteins linked to distinct signalling outputs associated with each intracellular location. Activation of pro-angiogenic VEGFR2 stimulates a programme of tyrosine phosphorylation, ubiquitination and proteolysis. This is linked to ESCRT (endosomal sorting complex required for transport)-mediated recognition of activated VEGFR2 and sorting in endosomes before arrival in lysosomes for terminal degradation. In addition, Rab GTPases regulate key events in VEGFR2 trafficking between the plasma membrane, early and late endosomes, with distinct roles for Rab4a, Rab5a and Rab7a. Manipulation of GTPase levels affects not only VEGFR2 activation and intracellular signalling, but also functional outputs such as VEGF-A-stimulated endothelial cell migration. In contrast, VEGFR1 displays stable Golgi localization that can be perturbed by cell stimuli that elevate cytosolic Ca(2+) ion levels. One model is that VEGFR1 translocates from the trans-Golgi network to the plasma membrane via a calcium-sensitive trafficking step. This allows rapid and preferential sequestration of VEGF-A by the higher-affinity VEGFR1, thus blocking further VEGFR2 activation. Recycling or degradation of VEGFR1 allows resensitization of the VEGFR2-dependent signalling pathway. Thus a dual VEGFR system with a built-in negative-feedback loop is utilized by endothelial cells to sense a key cytokine in vascular tissues.

Cost-of-illness study of type 2 diabetes mellitus in Colombia.

OBJECTIVE: To determine the per patient and overall cost of illness of type 2 diabetes mellitus (T2DM) in Colombia from Ministry of Health and societal perspectives. METHODS: A published Markov transition model was adapted for Colombia, using the clinical expertise of a Colombian endocrinologist. Transition probabilities for the model were derived from an international literature review. A model was run for a time horizon of 42 years. Direct resources (drugs, laboratory, medical, hospital, other health care) were identified and cost was ascertained by using national price lists, international health care guidelines, and other Colombian studies or data from other countries. Indirect costs (work time lost) were calculated by using the human capital approach. Annual and lifetime direct and indirect costs, in 2007 U.S. dollars with a 5% discount rate, were determined on a per patient basis and projected to the overall Colombian population. Costs were clustered according to treatments and outcomes. RESULTS: The estimated annual cost was $2.7 billion from the societal perspective and $921 million from the Ministry of Health perspective. The annual direct cost per patient was $288, and the indirect cost was $559 (total = $847). This cost was distributed across disease outcomes as follows: diabetes treatment (drugs), 47%; cardiac and coronary disease, 24%; stroke, 15%; amputation, 9%; nephropathy, 3%; retinopathy, 2%. Macrovascular complications made up 86% of the annual direct costs and 95% of the annual indirect costs of T2DM. CONCLUSIONS: We estimated the annual cost of T2DM for Colombia from societal, Ministry of Health, and Colombian Health System perspectives. We also estimated annual direct cost per patient and the cost of treating diabetes and macrovascular complications. The economic burden is substantial and comparable to results for other countries. The model showed a logical disease progression.