HER2 in high-risk rectal cancer patients treated in EXPERT-C, a randomized phase II trial of neoadjuvant capecitabine and oxaliplatin (CAPOX) and chemoradiotherapy (CRT) with or without cetuximab.

BACKGROUND: HER2 is an established therapeutic target in breast and gastric cancers. The role of HER2 in rectal cancer is unclear, as conflicting data on the prevalence of HER2 expression in this disease have been reported. We evaluated the prevalence of HER2 and its impact on the outcome of high-risk rectal cancer patients treated with neoadjuvant CAPOX and CRT±cetuximab in the EXPERT-C trial. PATIENTS AND METHODS: Eligible patients with available tumour tissue for HER2 analysis were included. HER2 expression was determined by immunohistochemistry (IHC) in pre-treatment biopsies and/or surgical specimens (score 0-3+). Immunostaining was scored according to the consensus panel recommendations on HER2 scoring for gastric cancer. Tumours with equivocal IHC result (2+) were tested for HER2 amplification by D-ISH. Tumours with IHC 3+ or D-ISH ratio ≥2.0 were classified as HER2+. The impact of HER2 on primary and secondary end points of the study was analysed. RESULTS: Of 164 eligible study patients, 104 (63%) biopsy and 114 (69%) surgical specimens were available for analysis. Only 3 of 104 (2.9%) and 3 of 114 (2.6%) were HER2+, respectively. In 77 patients with paired specimens, concordance for HER2 status was found in 74 (96%). Overall, 141 patients were assessable for HER2 and 6 out of 141 (4.3%) had HER2 overexpression and/or amplification. The median follow-up was 58.6 months. HER2 was not associated with a difference in the outcome for any of the study end points, including in the subset of 90 KRAS/BRAF wild-type patients treated±cetuximab. CONCLUSIONS: Based on the low prevalence of expression as recorded in the EXPERT-C trial, HER2 does not appear to represent a useful therapeutic target in high-risk rectal cancer. However, the role of HER2 as a potential predictive biomarker of resistance to anti-EGFR-based treatments and a therapeutic target in anti-EGFR refractory metastatic colorectal cancer (CRC) warrants further investigation. TRIAL REGISTRATION: ISRCTN Register: 99828560.

A Palatable Hyperlipidic Diet Causes Obesity and Affects Brain Glucose Metabolism in Rats

Background We have previously shown that either the continuous intake of a palatable hyperlipidic diet (H) or the alternation of chow (C) and an H diet (CH regimen) induced obesity in rats. Here, we investigated whether the time of the start and duration of these feeding regimens are relevant and whether they affect brain glucose metabolism. Methods Male Wistar rats received C, H, or CH diets during various periods of their life spans: days 30-60, days 30-90, or days 60-90. Experiments were performed the 60th or the 90th day of life. Rats were killed by decapitation. The glucose, insulin, leptin plasma concentration, and lipid content of the carcasses were determined. The brain was sliced and incubated with or without insulin for the analysis of glucose uptake, oxidation, and the conversion of [1-14C]-glucose to lipids. Results The relative carcass lipid content increased in all of the H and CH groups, and the H30-60 and H30-90 groups had the highest levels. Groups H30-60, H30-90, CH30-60, and CH30-90 exhibited a higher serum glucose level. Serum leptin increased in all H groups and in the CH60-90 and CH30-90 groups. Serum insulin was elevated in the H30-60, H60-90, CH60-90, CH30-90 groups. Basal brain glucose consumption and hypothalamic insulin receptor density were lower only in the CH30-60 group. The rate of brain lipogenesis was increased in the H30-90 and CH30-90 groups. Conclusion These findings indicate that both H and CH diet regimens increased body adiposity independent treatment and the age at which treatment was started, whereas these diets caused hyperglycemia and affected brain metabolism when started at an early age.

Hydrogenated Fat Diet Intake during Pregnancy and Lactation Modifies the PAI-1 Gene Expression in White Adipose Tissue of Offspring in Adult Life

We examine whether feeding pregnant and lactating rats hydrogenated fats rich in trans fatty acids modifies the plasma lipid profiles and the expression of adipokines involved with insulin resistance and cardiovascular disease in their 90-day-old offspring. Pregnant and lactating Wistar rats were fed with either a control diet (C group) or one enriched with hydrogenated vegetable fat (T group). Upon weaning, the male pups were sorted into four groups: CC, mothers were receiving C and pups were kept on C; CT, mothers were receiving C and pups were fed with T; TT, mothers were receiving T and pups were kept on T; TC, mothers were receiving T and pups were fed with C. Pups' food intake and body weight were quantified weekly and the pups were killed at day 90 of life by decapitation. Blood and carcass as well as retroperitoneal, epididymal, and subcutaneous white adipose tissues were collected. Food intake and body weight were lower in TC and TT, and metabolic efficiency was reduced in TT. Offspring of TT and TC rats had increased white adipose tissue PAI-1 gene expression. Insulin receptor was higher in TT than other groups. Ingestion of hydrogenated vegetable fat by the mother during gestation and lactation could promote deleterious consequences, even after the withdrawal of the causal factor.

Le Bisphénol A dans la prééclampsie

Résumé : La prééclampsie (PE) est un désordre de la grossesse caractérisée par une dysfonction endothéliale faisant en sorte que l’endothélium devient moins sensible aux signaux de vasodilatation. La réponse provoquée par la liaison de la sérotonine au sous-type de récepteur S[indice inférieur 2] entraîne la libération de molécules aux propriétés vasoconstrictrices, qui, par une boucle de rétroaction positive, entraîne la libération de davantage de sérotonine par les plaquettes. Cette boucle amplifie la réponse et contribue ainsi à l’hypertension présente chez les femmes ayant une PE. Précédemment, il a été démontré par notre laboratoire que le Bisphénol A (BPA) s’accumulait davantage dans le placenta des femmes avec PE en comparaison aux femmes normotensives. Cette accumulation pourrait découler d’une perturbation de sa métabolisation qui impliquerait notamment la β-glucuronidase (GUSB). Des études chez les animaux ont quant à elles démontré que le BPA pouvait inhiber l’activité de la monoamine oxydase (MAO) à forte dose. Nous avons étudié l’effet du BPA à faible concentration (10 ng/ml) sur la MAO-A des cellules placentaires et démontré que le BPA inhibait la MAO-A de façon significative sans affecter son expression protéique. Afin d’expliquer l’accumulation particulière du BPA chez les femmes PE, nous avons comparé l’activité spécifique et l’expression protéique de la β-glucuronidase (GUSB) placentaire en utilisant un devis cas-témoins. Une tendance non significative suggère que la GUSB pourrait partiellement contribuer à l’accumulation du BPA chez les femmes PE. Nous avons étudié la relation entre la concentration sérique maternelle de BPA et la concentration à laquelle le fœtus est exposé par régression linéaire et corrélation de Spearman. Un tel modèle ne pourrait être utilisé pour déterminer de façon quantitative l’exposition fœtale. En revanche, en vue de la forte corrélation entre ces deux variables, une haute concentration sérique maternelle de BPA devrait se refléter par une haute exposition fœtale. Cette corrélation implique aussi que le métabolisme placentaire ne joue pas un rôle significatif dans la protection du fœtus. Le BPA pourrait ainsi contribuer à l’hypertension chez les femmes PE présentant une dysfonction endothéliale en inhibant la MAO-A et ainsi, favorisant la hausse de sérotonine circulante. Cette étude suggère les bases d’un mécanisme par lequel le BPA s’accumulerait davantage chez les femmes PE et affecterait ainsi la MAO-A placentaire et potentiellement, la MAO-A fœtale vu ses propriétés physico-chimiques.

Infecciones urinarias y trasplante renal: Factores de riesgo y modelo predictivo

Introducción: Las infecciones del tracto urinario (ITU) representan la causa más frecuente de infección después del trasplante renal. Identificar los factores de riesgo de las ITU e intentar establecer un modelo para predecirlas podría evitar la aparición de las mismas o su recurrencia. Objetivos: El objetivo principal de este trabajo fue analizar la frecuencia de ITU en pacientes trasplantados renales en el primer año postrasplante, así como los factores de riesgo más importantes de las mismas, con el fin de establecer un modelo predictivo (nomograma). Los objetivos secundarios fueron los siguientes: analizar la frecuencia de ITU durante el ingreso, tras el alta y un mes postrasplante, así como los factores de riesgo relacionados y establecer modelos predictivos para cada uno de estos periodos; analizar la frecuencia de ITU recurrentes y recidivantes; analizar la tasa de rehospitalizaciones secundarias a ITU; analizar la relación de ITU con la supervivencia del injerto y del paciente. Métodos: En este trabajo desarrollamos un estudio analítico, observacional, retrospectivo de cohorte, en el que se recogieron datos de pacientes trasplantados renales de forma consecutiva, desde el 1 de enero de 2012 hasta el 31 de julio de 2014, en el Hospital Universitario Regional de Málaga. Analizamos la incidencia de ITU durante el primer año postrasplante, así como otras variables clínicas. El análisis comparativo de grupos se realizó mediante el test de t-Student y Chi-cuadrado. Para el análisis de supervivencia se aplicó el método de Kaplan-Meier. Se realizó un análisis de regresión logística para determinar los factores de riesgo asociados a las ITU que ocurrieron durante el ingreso. Se estimaron modelos de riesgo proporcionales por el método de regresión de Cox para analizar los factores de riesgo de las ITU que se presentaron en el primer año postrasplante, después del alta y un mes tras la cirugía. A partir de estos modelos de regresión se realizó un nomograma para calcular la probabilidad de ITU en cada grupo (ITU en el primer año postrasplante, durante el ingreso, tras el alta y un mes postrasplante), así como la curva ROC correspondiente. Se utilizó el modelo “hurdle” para estudiar los factores de riesgo asociados al número de ITU en cada paciente. El tratamiento estadístico se realizó con el programa SPSS 15.0 y con el ALcEst 1.9.26. Resultados: En este trabajo estudiamos datos de 322 pacientes trasplantados renales, el 66.1% varones, con una media de edad de 52.2 ± 13.2 años. La frecuencia de ITU durante el primer año fue del 46.27%. Dada la correlación existente entre función retrasada del injerto (FRI) y tiempo en diálisis, realizamos dos modelos de regresión de Cox multivariantes. En el primer modelo incluimos el sexo femenino, la edad del receptor y la FRI (HR 1.73, IC 95% 1.24-2.40, p<0.001; HR 1.01, IC 1-1.03, p<0.05; HR 1.6, IC 1.14-2.22, p<0.01, respectivamente). En el segundo modelo incluimos el sexo femenino, la edad del receptor y el tiempo en diálisis (HR 1.79, IC 1.29–2.48, p<0.001; HR 1.02, IC 1–1.03, p<0.01; HR 1, IC 0.99–1.01, p=0.054, respectivamente). La frecuencia de ITU durante el ingreso fue del 15.21%, y los factores de riesgo más importantes analizados mediante regresión logística fueron la edad del donante, el tiempo en diálisis, la FRI y la retirada de la sonda vesical después de siete días (OR 1.03, IC 1–1.05, p<0.05; OR 1.01, IC 1-1.02, p=0.13; OR 2.64, IC 1.33–5.28, p<0.01; OR 3.55, IC 1.85–6.93, p<0.001, respectivamente). La frecuencia de ITU tras el alta fue del 37.57%. En este caso, mediante regresión de Cox, las variables más significativas fueron el sexo femenino y la edad del receptor (HR 2.1, IC 1.46-3.03, p<0.001; HR 1.02, IC 1-1.03, p<0.01, respectivamente). La frecuencia de ITU un mes postrasplante fue del 33.85%. Los factores más significativos para este periodo, mediante regresión de Cox, fueron el sexo femenino, la edad del receptor, el tiempo en diálisis y la presencia de ITU previa (HR 2.17, IC 1.48–3.17, p<0.001; HR 1.02, IC 1–1.03, p<0.01; HR 1, IC 0.99–1.01, p=0.32; HR 2.71, IC 1.83–4.02, p<0.001, respectivamente). Desarrollamos nomogramas para cada periodo, con sus correspondientes curvas ROC: en el primer año postrasplante (ABC=0.60 para el primer modelo, ABC=0.59 para el segundo modelo), durante el ingreso (ABC=0.74), tras el alta (ABC=0.59) y un mes después del trasplante (ABC=0.66). Los factores de riesgo más significativos relacionados con el número de ITU en cada paciente, mediante el modelo Hurdle, fueron el sexo masculino, la diálisis peritoneal y la terapia con inducción, principalmente con Timoglobulina (p=0.068; p<0.05; p<0.05, respectivamente). La frecuencia de ITU recurrente fue del 4.03%, y recidivante del 20.49%. Fueron necesarios 77 ingresos por pielonefritis, y en 12 de estos ingresos se presentó sepsis urológica. No encontramos diferencias en la supervivencia de pacientes y de injertos entre los grupos con y sin ITU, sin embargo sí se apreciaron diferencias entre ambos grupos en el filtrado glomerular, aunque esta diferencia se perdió en el análisis multivariante. Conclusiones: La incidencia de ITU durante el primer año postrasplante es elevada. Los factores de riesgo más significativos para las ITU en los primeros doce meses postrasplante son el sexo femenino, la edad del receptor, la FRI y el tiempo en diálisis. Para las ITU que ocurren durante el ingreso, los factores más importantes son la edad del donante, el tiempo en diálisis, la FRI y la retirada de la sonda después de siete días. Las variables más significativas para las ITU que se presentan tras el alta, son el sexo femenino y la edad del receptor, y para las ITU que ocurren un mes postraplante también resulta significativo la presencia de alguna ITU previa. Los receptores de trasplante renal de sexo masculino, aquellos procedentes de diálisis peritoneal y los que han recibido terapia de inducción, tienen más posibilidades de presentar un mayor número de ITU durante el primer año postrasplante.

Purification and biological effects of a C-type lectin isolated from Bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 mu g/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Threshold values of sagittal abdominal diameter for the detection of cardio-metabolic risk factors in Northeastern Mexico: a cross-sectional study

Background: The use of sagittal abdominal diameter (SAD) has been proposed for screening cardio-metabolic risk factors; however, its accuracy can be influenced by the choice of thresholds values. Aim: To determine the SAD threshold values for cardio-metabolic risk factors in Mexican adults; to assess whether parallel and serial SAD testing can improve waist circumference (WC) sensitivity and specificity; and to analyze the effect of considering SAD along with WC and body mass index (BMI) in detecting cardio-metabolic risk. Methods: This cross-sectional study was conducted during 2012-2014 in Northeast Mexico (n = 269). Data on anthropometric, clinical, and biochemical measurements were collected. Sex-adjusted receiver-operating characteristic curves (ROC) were obtained using hypertension, dysglycemia, dyslipidemia and insulin resistance as individual outcomes and metabolic syndrome as a composite outcome. Age-adjusted odds ratios and 95% confidence intervals (CI) were estimated using logistic regression. Results: The threshold value for SAD with acceptable combination of sensitivity and specificity was 24.6 cm in men and 22.5 cm in women. Parallel SAD testing improved WC sensitivity and serial testing improved WC specificity. The co-occurrence of high WC/high SAD increased the risk for insulin resistance by 2.4-fold (95% CI: 1.1-5.3), high BMI/high SAD by 4.3-fold (95% CI: 1.7-11.9) and SAD alone by 2.2-fold (95% CI: 1.2.-4.2). Conclusions: The use of SAD together with traditional obesity indices such as WC and BMI has advantages over using either of these indices alone. SAD may be a powerful screening tool for interventions for high-risk individuals.

Effects of fibrosis morphology on reentrant ventricular tachycardia inducibility and simulation fidelity in patient-derived models

Myocardial fibrosis detected via delayed-enhanced magnetic resonance imaging (MRI) has been shown to be a strong indicator for ventricular tachycardia (VT) inducibility. However, little is known regarding how inducibility is affected by the details of the fibrosis extent, morphology, and border zone configuration. The objective of this article is to systematically study the arrhythmogenic effects of fibrosis geometry and extent, specifically on VT inducibility and maintenance. We present a set of methods for constructing patient-specific computational models of human ventricles using in vivo MRI data for patients suffering from hypertension, hypercholesterolemia, and chronic myocardial infarction. Additional synthesized models with morphologically varied extents of fibrosis and gray zone (GZ) distribution were derived to study the alterations in the arrhythmia induction and reentry patterns. Detailed electrophysiological simulations demonstrated that (1) VT morphology was highly dependent on the extent of fibrosis, which acts as a structural substrate, (2) reentry tended to be anchored to the fibrosis edges and showed transmural conduction of activations through narrow channels formed within fibrosis, and (3) increasing the extent of GZ within fibrosis tended to destabilize the structural reentry sites and aggravate the VT as compared to fibrotic regions of the same size and shape but with lower or no GZ. The approach and findings represent a significant step toward patient-specific cardiac modeling as a reliable tool for VT prediction and management of the patient. Sensitivities to approximation nuances in the modeling of structural pathology by image-based reconstruction techniques are also implicated.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.

Análisis de asociación de los genes receptor de rianodina (RYR-1) e insulin-like growth factor-2 (IGF-2) con caracteres productivos en una población porcina de tipo comercial en el estado de Nuevo León, México

Tesis (Maestría en Ciencias Veterinarias) U.A.N.L., 2006.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic beta-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic beta-cells is still to be clearly established. We investigated whether TLR4 is present in beta-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results: CD11b positive macrophages were practically absent from isolated human islets obtained from nondiabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to beta-cells. A significant loss of cell viability was observed in these beta-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in beta-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic beta-cells.