A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.     

Young’s modulus of elasticity of Schlemm’s canal endothelial cells

Schlemm’s canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young’s modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young’s modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8–15 mm Hg). However, increasing intraocular pressure (22–30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.     

EGFR/Met association regulates EGFR TKI resistance in breast cancer

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.     

Low socioeconomic status is associated with prolonged times to assessment and treatment, sepsis and infectious death in pediatric Fever in el salvador

Background

Infection remains the most common cause of death from toxicity in children with cancer in low- and middle-income countries. Rapid administration of antibiotics when fever develops can prevent progression to sepsis and shock, and serves as an important indicator of the quality of care in children with acute lymphoblastic leukemia and acute myeloid leukemia. We analyzed factors associated with (1) Longer times from fever onset to hospital presentation/antibiotic treatment and (2) Sepsis and infection-related mortality.

Method

This prospective cohort study included children aged 0–16 years with newly diagnosed acute leukemia treated at Benjamin Bloom Hospital, San Salvador. We interviewed parents/caregivers within one month of diagnosis and at the onset of each new febrile episode. Times from initial fever to first antibiotic administration and occurrence of sepsis and infection-related mortality were documented.

Findings

Of 251 children enrolled, 215 had acute lymphoblastic leukemia (85.7%). Among 269 outpatient febrile episodes, median times from fever to deciding to seek medical care was 10.0 hours (interquartile range [IQR] 5.0–20.0), and from decision to seek care to first hospital visit was 1.8 hours (IQR 1.0–3.0). Forty-seven (17.5%) patients developed sepsis and 7 (2.6%) died of infection. Maternal illiteracy was associated with longer time from fever to decision to seek care (P = 0.029) and sepsis (odds ratio [OR] 3.06, 95% confidence interval [CI] 1.09–8.63; P = 0.034). More infectious deaths occurred in those with longer travel time to hospital (OR 1.36, 95% CI 1.03–1.81; P = 0.031) and in families with an annual household income InterpretationIlliteracy, poverty, and longer travel times are associated with delays in assessment and treatment of fever and with sepsis and infectious mortality in pediatric leukemia. Providing additional education to high-risk families and staying at a nearby guest house during periods of neutropenia may decrease sepsis and infectious mortality.     

5-oxo-6,8,11,14-eicosatetraenoic acid stimulates the release of the eosinophil survival factor granulocyte/macrophage colony-stimulating factor from monocytes

Allergic diseases such as asthma are characterized by tissue eosinophilia induced by the combined effects of chemoattractants and cytokines. Lipid mediators are a major class of endogenous chemoattractants, among which 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent for human eosinophils. In this study, we investigated the effects of 5-oxo-ETE on eosinophil survival by flow cytometry. We found that this compound could promote eosinophil survival in the presence of small numbers of contaminating monocytes, but not in their absence. The conditioned medium from monocytes treated for 24 h with 5-oxo-ETE also strongly promoted eosinophil survival, whereas the medium from vehicle-treated monocytes had no effect. An antibody against the granulocyte/macrophage colony-stimulating factor (GM-CSF) completely blocked the response of eosinophils to the conditioned medium from 5-oxo-ETE-treated monocytes, whereas an antibody against interleukin-5 had no effect. Furthermore, 5-oxo-ETE stimulated the release of GM-CSF from cultured monocytes in amounts compatible with eosinophil survival activity, with a maximal effect being observed after 24 h. This effect was concentration-dependent and could be observed at concentrations in the picomolar range. 5-Oxo-ETE and leukotriene B(4) had similar effects on GM-CSF release at low concentrations, but 5-oxo-ETE induced a much stronger response at concentrations of 10 nm or higher. This is the first report that 5-oxo-ETE can induce the release of any cytokine, suggesting that it could be an important mediator in allergic and other inflammatory diseases due both to its chemoattractant properties and to its potent effects on the synthesis of the survival factor GM-CSF.     

Strategy to design improved proteomic experiments based on statistical analyses of the chemical properties of identified peptides

Proteomics is an emerging field that uses many types of proteomic platforms however has few standardized procedures. Deciding which platform to use to perform large-scale proteomic studies is either based on personal preference or on so-called "figures of merit" such as dynamic range, resolution, and the limit of detection; these factors are often insufficient to predict the outcome of the experiment as the detection of peptides correlates to the chemical properties of each peptide. There is a need for a novel figure of merit that describes the overall performance of a platform based on measured output, which in proteomics is often a list of identified peptides. We report the development of such a figure of merit based on a predictive genetic algorithm. This algorithm takes into account the properties of the observed peptides such as length, hydrophobicity, and pI. Several large-scale studies that differed in sample type or platform were used to demonstrate the usefulness of the algorithm for improved experimental design. The figures that were obtained were clustered to find platforms that were biased in similar ways. Even though some platforms are different, they lead to the identification of similar peptide types and are thus redundant. The algorithm can thus be used as an exploratory tool to suggest a minimal number of complementary experiments in order to maximize experimental efficiency.     

L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)