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Objectives:To identify the risk factors of neonatal sepsisduring the period from January 2017 to December 2019. Methods:A cross-sectional, retrospective study, observational, descriptive; the record of 106 newborns diagnosed with sepsis were reviewed. We excluded 15 were included in the data collection, diagnosis of early and late neonatal sepsis, maternal age, number of gestation, prenatal care, premature birth, diagnosis of UTI diagnosis of chorioamnionitis, premature rupture of membrane (s RPM ), sex product, invasive procedures, product weight and route of obtaining the product. Results:Of the 1401 patients obtained alive, 191 obtained diagnosis of neonatal sepsis and an incidence of 6.49% of early neonatal sepsis was obtained. The main risk factor associated with early neonatal sepsis has been the way of obtaining caesarean with 52.74%, the low birth weight with 32.96% and multiparity an incidence of 34.06% and there were no significant differences between sexes as the man / femalerelationship was 1.02.However if a significant importance in the delivery / caesarean relationship was obtained0.89. There was only one death recorded. Conclusions:The main risk factors associated with early neonatal sepsis are obtained via caesarean section, low birth weight and multiparity, male sex was the most affected but with no significant differences, the IVU, PROM, maternal age less to 18 years and the lack of prenatal care they had similar incidence. Only one late neonatal sepsis and death was obtained. I do not chorioamnionitis data was obtained.

The benign prostatic hyperplasia (BPH) is a (noncancerous) malignant growth in the size of the prostate. This enlargement of the prostate gland is produced by a relative increase in estrogen (female hormones) on testosterone (male hormone), which appears in men with age. Objective:To determine the relationship of PSA, with the size of the prostate in patients with benign prostatic hyperplasia admitted to General Hospital of Escarcega, Campeche Dr. Janel Aguilar during the period from August 2019 to January 2020. Method:patients were studied with the diagnosis of benign prostatic hyperplasia 50 years of age and older and excluded those who had other diseases that may alter prostate specific antigen, subsequently classified by age determined through PSA ultrasound prostate-specific and the average size of the prostate in grams and the average PSA for each age range and the relationship between them, while describes how it affects their self-esteem was determined. Results:Of the patients studied, 22 belong to the range of 50 to 59 years old (51.16%), 14 to range from 60 to 69 years old (32.55%) and 7 to range from 70 to 79 years old ( 16.27%). 44.1% of patients presented a prostate of 50 grams, the 51.16% prostates 60 grams and 4.6% a prostate gland of 70 grams. The average size of the prostate in grams was 50 grams in the range 50 to 59 years old, 60 g in the range 60 to 69 years old and 60 grams in the range of 70 to 79 years, and the average value of PSA was 5.5 ng / ml, 6 ng / ml and 7.5 ng / ml respectively. In the range 50 to 59 years of age every gram prostate equivalent to 0.11 ng / ml of PSA in the range 60 to 69 years of age every gram prostate equivalent to 0.1 ng / ml of PSA and the range 70 to 79 years old every gram of prostate equivalent to 0.125 ng / ml of PSA. Similarly we find that the entire population 72% have low self-esteem and problems with its image. Conclusions:In our environment in the group of 50-59 years old is the highest number of patients with benign prostatic hyperplasia.

After laparoscopic sleeve gastrectomy (LSG), several studies have reported an increase in the incidence of gastroesophageal reflux (GERD). The etiopathogenesis of GERD post-LSG is multifactorial, and hiatal hernia (HH) is one of them. The primary objective was to measure the incidence of de novo HH post-LSG. The secondary objectives were to relate the presence of HH with GERD, the chronic use of proton pump inhibitors (PPI), and the time elapsed from LSG.A surgical evaluation of the crura after LSG was performed. A retrospective cohort study of 74 consecutive patients with history of LSG submitted to an intra-abdominal surgery that allowed the evaluation of the crura.Of a total of 74 patients, 51 were included. At the time of surgery, 37 patients (72.5%) had a HH; 24 patients (47.1%) had GERD, and 23 patients (45.1%) were frequently using PPI. When patients with HH and those without HH were compared, GERD was observed in 56.8% versus 21.4% (p = 0.01) and frequent consumption on PPI was found in 54.1% versus 21.4% (p = 0.02). According to the data of LSG, with a follow-up of 18 months, 60% presented HH; meanwhile, with a follow-up of 18 months, 84.6% presented HH (p = 0.02).Patients submitted to LSG showed a high incidence of de novo HH. HH was associated with a higher incidence of GERD and PPI dependence. The longer the time elapsed from the LSG, the greater the incidence of HH.

Left ventricular aneurysms (LVA) occur after an infarcted area of the myocardium necrotizes, fibroses, and expands, forming a dyskinetic cavity. Most ventricular aneurysms are asymptomatic and go unrecognized unless found incidentally. Symptoms commonly reported include angina, heart failure, syncope, and even sudden cardiac death. Late complications from left ventricular aneurysms are infrequently reported. This case reports an elderly woman who presented with new-onset angina from an expanding 18-year-old true left ventricular aneurysm that was successfully treated with surgical repair.

Objective The primary objective of this research was to explore the link between sleep and flare pain associated with chikungunya virus (CHIKV) infection. The secondary objective was to investigate if cytokines and T regulatory (Treg) cells have an influence on this relationship. Methods A cross-sectional study was performed using data collected in Barranquilla, Colombia, which enrolled patients with and without chronic arthritis with a history of chikungunya infection. Flare severity was measured by a version of the Outcome Measures in Rheumatoid Arthritis Clinical Trials (OMERACT) flare questionnaire adapted for CHIKV arthritis, including metrics for pain, difficulty with physical activity, fatigue, stiffness and difficulty maintaining social activities due to arthritis that contribute to flare severity. In addition, four sleep disturbance items, five inflammatory cytokine levels, four anti-inflammatory cytokine levels, and six Treg levels were measured. Then, multivariable linear regression models were used to test the direct and indirect effects of flare-pain on sleep disturbance, and to determine whether this relationship was mediated by cytokines or Tregs. Finally, the SAS CALIS procedure was used to test path models showing possible causal effects with mediators and confounds. Results The analysis showed that sleep disturbance is positively correlated with CHIKV arthritis flare pain, and that it is a significant predictor of flare severity after adjusting for demographic variables, cytokine, and T cell levels. Further, neither T cells nor cytokines mediate the pain/sleep relationship in CHIKV arthritis. Conclusion There is a strong association between sleep disturbance and arthritis flare pain and severity; however, this relationship is not mediated by cytokines or T cells. Since this study is unable to determine causation, further research is needed to determine the mechanism underlying the relationship between sleep disturbances and CHIKV arthritis flares.

Point of care ultrasound has emerged as useful tool in cardiac arrest situations in adult population. Despite of these, there is not a protocol for cardiopulmonary resuscitation in the delivery room for newborns. We describe two case were ultrasound helped to rule out the cardiopulmonary arrest case and we propose and algorithm were ultrasound is integrated in the newborn resuscitation and may help to diagnose the cardiac arrest cause.

Objectives To correlate the ultrasound (US) measurements of the median nerve cross-sectional area (CSA) and the measurements of its stiffness by shear wave elastography (SWE) with the severity grade of carpal tunnel syndrome (CTS) using electrodiagnostic testing (EDT) and to determine the cutoff points for CSA and SWE measurements to allow us to discriminate patients with moderate and severe CTS from those with mild or negative EDT findings. Methods Seventy-three patients with 105 hands with a clinical suspicion of CTS were studied with US and SWE. We measured the median nerve CSA and elasticity (E) at the tunnel inlet (CSAu and Eu), in the quadratus pronator (CSAo and Eo), and the differences (CSAu - CSAo and Eu - Eo). Results The nerve area and stiffness increased according to the EDT severity of CTS; the CSA increased proportionally as CTS increased from negative to severe according to EDT, and the stiffness was not different between patients with negative and mild EDT findings but was higher in patients with moderate and severe EDT findings versus negative and mild EDT findings. The cutoff points of a CSAu of 14 mm2 or greater and an Eu - Eo of 57 kPa or greater together allowed the discrimination of moderate and severe CTS from the rest. Conclusions The joint use of US and SWE is an alternative to EDT in the clinical management and treatment of patients with a clinical suspicion of CTS.

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

Information within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. Here we introduce SynTagMA to tag active synapses in a user-defined time window. Upon 395–405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior., Calcium imaging has been used to visualize the activity of individual synapses, but cannot be scaled up to monitor thousands of synapses in tissue. Here, the authors present genetic tools that can be photoconverted from green to red to create a map of active synapses.

The autosomal dominant polycystic kidney disease (ADPKD) accounts for one out of 400-1000 live births, being a hereditary disorder with cystic and noncystic manifestations as well as extrarenal involvement. The pericardial effusion (PE) in the context of a patient with ADPKD is complex, and it is not entirely defined. Several theories have been proposed. The most accepted, so far, is linked to mutations in the PKD1 gene which can entail an abnormal production of matrix components, matrix-degrading enzymes, and inhibitors of metalloproteinases, and defects in connective tissue which would lead to an abnormal distensibility of the connective tissue. We report the case of a 35-year-old female Moroccan patient with the diagnosis of ADPKD associated with arterial hypertension who came into the Emergency Department with lower abdominal pain lasting for five days being diagnosed as salpingitis. Abdominal computed tomography scan with contrast showed both kidneys with several cystic images with a thin wall. A transthoracic echocardiogram revealed the presence of moderate PE more in the anterior aspect. A greater set of standard tests to rule out collagen vascular disease, rheumatoid diseases, autoimmune disorders, and malignancies was ordered. These tests yielded no abnormality. The association of ADPKD with PE is rare. The awareness of this connection by the emergency physicians is key to prevent misplaced concern.

CD8 T cells are the main effectors of the adaptive immune system for T helper (Th) 1 or cytotoxic type of responses. In several types of cancer, CD8 lymphocyte infiltrates correlate with good prognosis. CD8 T cells can destroy directly transformed cells and secrete proinflammatory cytokines as IFN-γ and TNF-α, favoring the recruitment and activity of other effectors of antitumoral immunity such as M1 macrophages and NK cells. Hence, in the past few years the potential for the use of adaptive immune system as a base for immunotherapies has expanded enormously and it has focused in CD8 T cells activity. Chimeric antigen receptor (CAR)-T cell technology is one of those striking findings with remarkable successes in the treatment of refractory and/or relapse (r/r) B cell acute lymphoblastic leukemia (B-ALL). The duration of the event-free survival and the rates of overall survival overpass the previously existent treatments prompting to its proposal as therapy for less advanced stages of disease. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTPN2, enhance proinflammatory type I interferon signaling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling. Very recently other groups have linked PTPN2 deficiency has with enhanced CD8 antitumoral responses. Unpublished observations from our laboratory using PTPN1/PTPN2 dual specific small molecule inhibitors suggested a role for both phosphatases in the enhancement of CD8 T cell cytotoxic activity. Following this lead, we decided to develop a genetic model to study the contribution of each enzyme to the CD8 T cell ability to respond to antigenic cues. Indeed, we found that a combined total or partial reduction of these genes increased the cytotoxic activity and IFN-γ secretion of CD8 T cells, underscoring synergistic but non-redundant mechanisms of action. The reduced activity of these phosphatases correlated with an increase in the tonic signals mediated by the JAK-STAT axis, particularly STAT 3 and STAT5a. As consequence, cells are sensitized to respond to type 1 and type 2 interferons. Transcriptomic analysis of these enhanced effector cells correlated their functional state with an increased expression, at transcript and protein level, several CD8 relevant transcription factors including of IRF4, Myb and the Basic leucine zipper transcriptional factor ATF-like 3 (BatF3). Henceforth, our results suggest that small molecule inhibitors with dual specificity for PTP1B and TC-PTP are a powerful therapeutic tool for potentiating CTL cytotoxic responses and CTL based immunotherapies as CAR-T cells. Citation Format: Luis Alberto Perez-Quintero, Yevgen Zolotarov, Zuzet Martinez-Cardoba, Chu Han Feng, Alexandre Poirier, Kelly Anne Pike, Jean-Sebastien Delisle, Michel L. Tremblay. Concomitant reduction of PTPN1/PTPN2 activity synergistically enhanced antitumoral effector function of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG16.

T-cell protein tyrosine phosphatase (TC-PTP) has a critical role in the development of the immune system and has been identified as a negative regulator of inflammation. Single-nucleotide polymorphisms in the TC-PTP locus have been associated with increased susceptibility to inflammatory bowel diseases (IBDs) in patients. To further understand how TC-PTP is related to IBDs, we investigated the role of TC-PTP in maintaining the intestinal epithelial barrier using an in vivo genetic approach. Intestinal epithelial cell (IEC)-specific deletion of TC-PTP was achieved in a mouse model at steady state and in the context of dextran sulphate sodium (DSS)-induced colitis. Knockout (KO) of TC-PTP in IECs did not result in an altered intestinal barrier. However, upon DSS treatment, IEC-specific TC-PTP KO mice displayed a more severe colitis phenotype with a corresponding increase in the immune response and inflammatory cytokine profile. The absence of TC-PTP caused an altered turnover of IECs, which is further explained by the role of the tyrosine phosphatase in colonic stem cell (CoSC) proliferation. Our results suggest a novel role for TC-PTP in regulating the homeostasis of CoSC proliferation. This supports the protective function of TC-PTP against IBDs, independently of its previously demonstrated role in intestinal immunity.

The present study was performed to evaluate the Ca v 1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Ca v 1.2 and Ca v 1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Ca v 1.2 and Ca v 1.3 subtypes, but not Ca v 1.1 or Ca v 1.4. Electrophysiological experiments were conducted to investigate the contribution of Ca v 1 channels to the exocytotic process and cell excitability. Ca v 1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3 μM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200 ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (−34 mV), which elicits 10.4 mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Ca v 1 channels in exocytosis and cell excitability.

Information within the brain travels from neuron to neuron across synapses. At any given moment, only a few synapses within billions will be active and are thought to transmit key information about the environment, a behavior being executed or memory being recalled. Here we present SynTagMA, which marks active synapses within a ~2 s time window. Upon violet illumination, the genetically expressed tag converts from green to red fluorescence if bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we developed an analysis program that automatically identifies and tracks the fluorescence of thousands of individual synapses in tissue. Together, these tools provide a high throughput method for repeatedly mapping active synapses in vitro and in vivo.

SECCION IV. Diagnóstico de los casos de infección por SARS-CoV-2/COVID-19

Background and study aims: Hereditary diffuse gastric cancer (HGGC), an autosomal dominant tumor-syndrome, accounts for 1 % to 3 % of gastric cancers worldwide. Presumably 30 % to 40 % of all patients fulfilling the clinical guidelines for HDGC are carriers of a pathogenic mutation in the CDH1 gene. Patients often show multiple foci of signet ring cell carcinoma at early age and are advised to undergo prophylactic total gastrectomy (PTG). Our aim was to improve the endoscopic detection of HDGC by using an enhanced endoscopic protocol. Patient and methods: Patients with a proven CDH1 germline mutation identified in our institute were prospectively included. Patients were advised to undergo PTG and offered a baseline endoscopic examination prior surgery. Examination was performed by using high-resolution white-light endoscopy and pan-gastric chromoendoscopy with indigo carmine as dye combined with targeted and multiple random biopsies assessed by an expert histopathologist. Postoperative histopathology was compared with results from endoscopic biopsies. Results: Between September 2012 and November 2014 8 patients with a proven CDH1 germline mutation were included. We conducted 44 targeted (6.3/patient) and 225 random (32.1/patient) biopsies in 7 patients. We detected 1 gastric cancer by random biopsy (14 %). All other examinations showed no signs of cancer. Histopathology of gastrectomy specimen revealed multiple foci of gastric carcinoma in 6 patients (86 %) with a total number of 27 cancer foci. Conclusions: Examination with targeted and random biopsies combined with chromoendoscopy is not able to detect small foci of gastric cancer in CDH1 mutation carriers. Therefore PTG is advocated in these patients.

Author(s): Okhunov, Zhamshid; Yoon, Renai; Perez Lanzac, Alberto; Sgroi, Michael; Lau, Wei Ling; Del Junco, Michael; Ordon, Michael; Drysch, Austin; Hwang, Christina; Vernez, Simone L; Fujitani, Roy; Kabutey, Nii-Kabu; Kalantar-Zadeh, Kamyar; Landman, Jaime | Abstract: ObjectivesWe attempted to create a surgical model to evaluate the retroperitoneal space for the ability to transfer solutes through the retroperitoneal membrane. Our dual objectives were to develop a technique to assess the feasibility of retroperitoneal dialysis (RPD) in a porcine model.MethodsWe incorporated two 35-kg Yorkshire pigs for this pilot study. In the first animal, we clamped renal vessels laparoscopically. In the second animal, we embolized renal arteries. In both animals, we dilated the retroperitoneal space bilaterally and deployed dialysis catheters. We measured serum creatinine (Cr), urea, and electrolytes at baseline 6 hours before the dialysis and every 4 hours after.ResultsWe successfully created retroperitoneal spaces bilaterally and deployed dialysis catheters in both animals. In the first animal, dialysate and plasma Cr ratio (D/P) on the left and right side were 0.43 and 0.3, respectively. Cr clearance by 40 minutes of dialysis treatment was 6.3 mL/min. The ratio of dialysate glucose at 4 hours dwell time to dialysate glucose at 0 dwell time (D/D0) for left/rights sides were 0.02 and 0.02, respectively. kt/Vurea was 0.43. In the second animal, D/P Cr for left/right sides were 0.34 and 0.33, respectively. kt/Vurea was 0.17. We euthanized the pigs due to fluid collection in the peritoneal space and rapid increase of serum Cr, urea, and electrolytes.ConclusionsWe demonstrated the feasibility of creation of a functionally anephric porcine model with successful development of retroperitoneal spaces using balloon inflation. Notwithstanding minimal clearance and limited diffusion capacity in this experiment, additional studies are needed to examine potential use of retroperitoneal space for peritoneal dialysis.

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.

Bivalirudin is an alternative to unfractionated heparin (UFH) anticoagulation during percutaneous coronary intervention. Previously, we have reported clinical benefit on major bleeding in favor of bivalirudin compared with UFH monotherapy but inconclusive results on mortality. Controversial data have been reported in the last 2 years. We conducted an updated meta-analysis including randomized trials and observational studies, which evaluated ischemic and bleeding outcomes for bivalirudin compared with UFH-only during percutaneous coronary intervention. We included 18 observational studies and 12 randomized trials published from 2003 to 2015. Primary outcomes were major adverse cardiovascular events within 30 days including death, myocardial infarction, and urgent revascularization and stent thrombosis, major bleeding, and transfusion. Overall, we found a significant risk reduction with bivalirudin for major bleeding (odds ratio [OR] 0.59, 95% confidence interval [CI] 0.49 to 0.71, p0.0001) and for transfusion (OR 0.79, 95% CI 0.66 to 0.95, p = 0.01) and similar risk for major adverse cardiovascular events (OR 0.98, 95% CI 0.86 to 1.12, p = 0.80). However, there was a substantial increased risk of stent thrombosis associated with bivalirudin (OR 1.52, 95% CI 1.11 to 2.08, p = 0.009). No impact on mortality was found. Meta-regression analyses on major bleeding suggested that bivalirudin was more effective than UFH at doses60 IU/kg and independent of radial access. In conclusion, compared with UFH monotherapy, bivalirudin remains associated with less bleeding risk but higher stent thrombosis risk. Further study remains required to define its role in current antithrombotic armamentarium.

3138 Background: Clinical effective multi-antigen active immunotherapy for colorectal cancer (CRC) is still limited and most studies have failed. We consider this is because the targets in some of the studies are not oncogenic drivers. This is especially important for patients that progressed to standard of care treatment. Also, most of the studies with Immune checkpoint inhibitors (ICH) have failed in CRC but potentially may impact and provide better outcomes if used as combination therapies. We treated CRC patients with progressive disease in a pilot study n = 15 and found clinical responses that correlate with the CD8, delayed-type hypersensitivity (DTH) and Th1 parameters. After patients achieved remission we used the same peptides to prevent relapse with clinical and statistics significance. Methods: N = 15 CRC patients were enrolled in this pilot trial after approval for the ethic IRB committee from CICS Mexico. Patients were treated with an intradermal vaccine every week for four weeks, every two weeks four times in axillary and inguinal lymph nodes (LN) areas, and finally subcutaneously every month six times in the sites with tumor activity. Previously to the treatment we perform Granzyme B ELISPOT, ELISA, DTH and CT scan as initial controls. We delivered intradermal four peptides from sixteen peptides predicted from four proteins such as Fascin-1, Ape-1, VCP and RCAS1. Results: 86% of the patients had an objective clinical responses and 14% stable disease. All the patients had a correlation with the immunological assays as following. 58% of patients had an increased in the CD8 cells demonstrated by Granzyme B; DTH reactions were gradually increasing and by the first month of treatment the DTH were positive for Fascin-1 (78%), Ape-1 (85%), VCP (95%) and RCAS1 (83%) peptides. The more immunogenic peptides by ELISA were Fascin-1 A (P = 0.001), Fascin-1 D (P = 0.005), Ape-1 C (p = 0.001), VCP A (P = 0.003), RCAS1 A (p = 0.001) and RCAS1 B (P = 0.01). Conclusions: The treatment was effective with CR and SD responses. Once we validate this data we are planning a clinical study combining the treatment with low dose of ICH. Currently all the patients with CR are now under relapse prevention. We are still treating the patients with SD until disease progression. Importantly we believe that this data may impact the PFS and OS in CRC patients who have Karnoksky > to 80% despite progressive disease to standard of care.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue. ispartof: NATURE COMMUNICATIONS vol:9 issue:1 ispartof: location:England status: published

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft-associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC-MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 (http://proteomecentral.proteomexchange.org/dataset/PXD002365).

This article reviews antithrombotic strategies for percutaneous coronary interventions according to the access site and the current evidence with the aim of limiting ischemic complications and preventing radial artery occlusion (RAO). Prevention of RAO should be part of the quality control of any radial program. The incidence of RAO postcatheterization and interventions should be determined initially using the echo-duplex and then frequently assessed using the more cost-effective pulse oximetry technique. Any evidence of higher risk of RAO should prompt internal analysis and multidisciplinary mechanisms to be put in place.

BackgroundLower esophageal sphincter (LES) electrical stimulation therapy (EST) has been shown to improve outcome in gastroesophageal reflux disease (GERD) patients at 1 year. The aim of this open-label extension trial (NCT01578642) was to study the 2-year safety and efficacy of LES-EST in GERD patients.MethodsGERD patients responsive partially to proton pump inhibitors (PPI) with off-PPI GERD health-related quality of life (HRQL) of ≥20, 24-hour esophageal pH ≤4.0 for >5% of the time, hiatal hernia ≤3 cm, and esophagitis LA grade C or lower participated in this trial. Bipolar stitch electrodes and a pulse generator (EndoStim BV, The Hague, The Netherlands) were implanted laparoscopically. LES-EST at 20 Hz, 215 μs, 3-8 mAmp was delivered over 30-minute sessions, 6-12 sessions per day, starting on day 1 after implantation. Patients were evaluated using GERD-HRQL, symptom diaries, Short Form-12, and esophageal pH testing at regular intervals. Stimulation sessions were optimized based on residual symptoms and esophageal pH at follow-up.ResultsTwenty-five patients (mean age [SD] = 52 [12] years; 14 men) were implanted successfully; 23 patients participated in the 2-year extension trial, and 21 completed their 2-year evaluation. At 2 years, there was improvement in their median GERD-HRQL on LES-EST compared with both their on-PPI (9 vs 0; P = .001) and off-PPI (23.5 vs 0; P

Brain activity requires a flux of glucose to active regions to sustain increased metabolic demands. Insulin, the main regulator of glucose handling in the body, has been traditionally considered not to intervene in this process. However, we now report that insulin modulates brain glucose metabolism by acting on astrocytes in concert with IGF-I. The cooperation of insulin and IGF-I is needed to recover neuronal activity after hypoglycemia. Analysis of underlying mechanisms show that the combined action of IGF-I and insulin synergistically stimulates a mitogen-activated protein kinase/protein kinase D pathway resulting in translocation of GLUT1 to the cell membrane through multiple protein-protein interactions involving the scaffolding protein GAIP-interacting protein C terminus and the GTPase RAC1. Our observations identify insulin-like peptides as physiological modulators of brain glucose handling, providing further support to consider the brain as a target organ in diabetes., This work was funded by MINECO (Spain) grants SAF2010-60051 and SAF2013-40710-R, and by CIBERNED. E.H.-G. was partially funded by a fellowship from ColFuturo and CIBERNED. T.I. is funded by MINECO (SAF2014-52737-P). T.I. and I.T.A. are funded by CIBERNED and Comunidad de Madrid, Spain (P2010/BMD-2331-Neurodegmodels).

To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (β-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.

Background Intensity-modulated radiation therapy (IMRT) uses solid compensators or multileaf collimators to modulate the intensity of radiation in each field, delivering highly conformal dose distributions. This technique allows treating volumes with concave shapes when the target is close to a critical structure. The movement of multileaf collimator under computer control can modulate the dose in 3 main ways: IMRT with static field with segments, IMRT with dynamic delivery, and IMRT rotational therapy. Volumetric modulated arc therapy (VMAT) is a novel radiation technique that creates conformal distributions with variable gantry speed, dynamic movements of multileaf collimator, and variations in dose rate. The purpose of this study was to review the basis of VMAT, highlighting the differences with other IMRT techniques. © 2014 Wiley Periodicals, Inc. Head Neck 37: 909–914, 2015

The autonomic nervous supply to the kidneys is involved in the development of several diseases including hypertension. The neural distribution at the segmental vessels and intrarenal vasculature has not been well characterized. Thus, we evaluated the autonomic nerve distribution from the great vessels to the renal cortex in a cadaveric model.We performed a detailed anatomical nerve dissection from the inferior mesenteric artery to the renal operculum in 2 human cadaveric torsos. Autonomic nerve fibers were verified by dissecting the greater splanchnic, sympathetic trunk and ganglia. We then systematically cross-sectioned the kidneys in 12, 1 mm slices across 3.6 cm, and stained the slices for histopathological analysis of neural tissue in relation to segmental arteries and other anatomical landmarks. Advanced reconstructive software was used to create a 3-dimensional computer image.Autonomic nerve fibers are located almost exclusively anteriorly on the main renal arteries and segmental arteries, and are absent from veins. Histopathology revealed that the intrarenal nerves continued to track exclusively with the arteries but were more circumferentially distributed. There is minimal nerve tissue around the veins. Many nerves were within a few millimeters of the renal collecting system.The autonomic nerves supplying the kidney maintain their distribution almost exclusively along the anterior surface of arteries as they pass from the aorta to the segmental arteries. Once inside the renal parenchyma, the nerves are circumferentially distributed around the renal arteries and are in close proximity to the renal collecting system.

e17079 Background: There are several reports of increased of SUV in the lymph nodes of cancer patients after immune manipulation such as the administration of vaccines and checkpoint inhibitors. However the clinical importance of these phenomena is poorly understood. The administration of two immunogenic drugs and eight peptides in HGSOC induced atypical bright zones in multiple lymph nodes and, in areas with microscopic disease where potentially we can have cancer stem cells (CSC’s). Methods: We enrolled N = 10 HGSOC after the local IRB ethic committee approval. All the patients had 3 recurrences with mPFS = 8 months of the third relapse. The last FDG-PET scan was used as basal image for the study. Blood was collected for cellular and humoral immunology analysis such as Granzyme B ELISPOT and ELISA. Tissue was used for IHC analysis for the evaluation of CD8, FOXP3, Th1, and proteins related with CSC’s. The patients received 50 mg of oxaliplatin and 30 mg of doxorubicin total dose every week four times simultaneously with multi peptide immunotherapy. A second FDG-PET scan was performed after the termination of the treatment. Results: After the treatment 100% of the patients showed intense atypical bright uptake areas in lymph nodes and in the peritoneum. There was a correlation between the brightness intensity (SUV), and the Granzyme B production (p = 0.001). We found multiple bright small lesions in the peritoneum that were not visible before treatment. We demonstrated the presence of proteins related with CSC’s in the original tumor tissue and an antibody immune response against EGFR (P = 0.005), Ape-1 (p = 0.003) and Bcl-2 (p = 0.05). Conclusions: Immunotherapy may produce hypermetabolic activity that could lead to overtreatment when evaluated by FDG-PET in HGSOC. Vaccination may activate lymph nodes and anti-cancer immune interventions may also activate CD8 and Th1 cells able to accumulate in CSC’s clusters and increased notably the SUV, which may lead to false relapse interpretation by the radiologists and oncologists.

There are many risk factors for developing hypertension. In the XXI century, smarter ways to investigate are needed, so preventing the turning of an adolescent into a hypertensive adult must be a priority. Objective: The aim of this paper is to predict the risk of high blood pressure onset in

Mounting a T helper 1 (Th1) type of response is required for the successful priming of antigen specific CD8 T-cells which ultimately lead to effective antitumoral responses. Several cellular components of the immune system participate of the Th1 decision-making process. They include innate cells sensing the transformed cells, mainly dendritic cells (DCs), and the further involvement of antigen specific CD4 T-cells promoting direct activation of CD8 T-cells through cross-presentation (1). The correct stimulation of DCs by type I interferons and molecular pattern sensors as STING is required to drive their differentiation to conventional DC 1 (cDC1) which through the secretion of large amounts of IL-12 and the expression of CD40 skew the CD4 T-cells differentiation to Th1 (2). However, this system is sensitive to modifications of the tumor cytokine microenvironmen,t leading to failure. Adoptive cell transfer (ACT) cancer immunotherapies are promising treatments for advanced malignancies. These therapies attempt to supply key cellular actors missing and induce proper antitumoral immunity. They include mainly the in vitro modification of autologous cells, from DCs loaded with tumor antigens to the expression of chimeric antigen receptor (CAR) on T and NK cells. Constant development of these technologies has given successful results as clinical trials have already showed up to 90 % of complete remission in relapsed/refractory B cell acute lymphoid leukemia (B-ALL) (3), treatment now approved for commercial use. However, there is still lack of consistency in the responses obtained from different individuals and in different trials. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTP-N2, enhance proinflammatory type I interferon signalling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling (4, 5). Specifically, we have demonstrated that partial inhibition of these phosphatases in immune cells enhances the secretion of IL-12p70 by DCs[6] and increases the cytotoxic activity of antigen specific CD8 T-cells [Pérez-Quintero LA, Tremblay ML, unpublished results]. Moreover, the partial inhibition of these phosphatases in CD8 T-cells enhance the acquisition of a central memory (Tcm) phenotype, which has been found to be associated with better remission in CAR-T-cells therapies. Nevertheless, alternative methods to enrich this phenotype, as cytokine cocktails, have proven expensive and ineffective. Having this in mind, we propose here the use of small-molecule inhibitors specific for PTPN1 and PTPN2 as a simple and cost-effective method to enhance antitumoral responses. By treating ex vivo the cellular products in animal models for DCs and CD8 ACT therapies we show, as a proof of concept, a marked improvement on the reduction of tumor burden and remission, with the late goal of translating these findings into a clinical setup. References: 1. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor microenvironment. Nature Immunology 2013;14:1014. 2. Theisen D,Murphy K. The role of cDC1s in vivo: CD8 T-cell priming through cross-presentation [version 1]. 2017;6. 3. Oluwole OO,Davila ML. At The Bedside: Clinical review of chimeric antigen receptor (CAR) T-cell therapy for B cell malignancies. Journal of Leukocyte Biology 2016. 4. Pike KA, et al. Protein tyrosine phosphatase 1B is a regulator of the interleukin-10–induced transcriptional program in macrophages. Science Signaling 2014;7(324): ra43-ra43. 5. Pike KA, et al. TC-PTP regulates the IL-7 transcriptional response during murine early T-cell development. Scientific Reports 2017;7(1):13275. 6. Penafuerte C, et al. Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling. OncoImmunology 2017;6(6):e1321185. Citation Format: Luis Alberto Perez-Quintero, Kelly Anne Pike, Penafuerte Claudia, Michel Tremblay. Enhancing antitumoral activity of cell-based immunotherapies by modulating the JAK-STAT axis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B035.

To assess the reliability of the S.T.O.N.E. (stone size [S], tract length [T], obstruction [O], number of involved calices [N], and essence or stone density [E]) nephrolithometry scoring system by testing its reproducibility between different observers.Preoperative images of 58 patients who underwent percutaneous nephrolithotomy (PCNL) were reviewed. Medical students, urology residents, one fellow, and a urology attending independently reviewed all images and scored the renal stones. Interobserver reliabilities of the total score for all categories and each component were evaluated by the intraclass correlation (ICC) and a κ coefficient.The interobserver reliability for the total score demonstrated high correlations for all components and total score (ICC=S, T, O, N, E and total 0.80, 0.97, 0.89, 0.84, 0.91, and 0.87, respectively). κ rates for individual components between two medical students were 0.36, 1, 0.31, 0.45, 0.33, and 0.30 for the S, T, O, N, E components and total score, respectively. κ values between the two urology residents were 0.71, 1, 0.92, 0.79, 0.93, and 0.67 for S, T, O, N, E components and total score, respectively. κ values between the urology fellow and an attending physician were 0.95, 1, 0.88, 0.94, 0.89, and 0.87 for S, T, O, N, E components and total score, respectively. P value for all the scoring components was0.05, indicating that the estimated κ was not a result of chance.The S.T.O.N.E. nephrolithometry has excellent interobserver reliability. Quantifying the S and N metrics was the most challenging and least reliable. Standardized protocols to measure these components should be considered to improve accuracy and reproducibility of the scoring system.

The late-stage clinical course, histopathologic analysis, and surgical salvage procedures after eventful cosmetic injection of hydroxyethylmethacrylate particles suspended in hyaluronic acid (DermaLive, marketed through Dermatech, Paris, France and Novamedical GmbH, Langenfeld, Germany) have been studied. The study was designed to present modified treatment guidelines, report results, and draw conclusions regarding the future management of patients with late-stage DermaLive complications.The study consulted and reviewed 21 patients with late-stage complications after facial implantation of DermaLive. Histology samples from excisional biopsies followed by radical surgical excision procedures were obtained and analyzed for nine patients.The treated zones included nasolabial folds, forehead and glabella, lips, and the perioral and zygoma regions. The late-onset period between injection and first symptoms averaged 17.5 months. Lesions were mostly symptomatic, with discomfort, pain, swelling, and edema. Clinical aspects included bulging, palpable nodules, erythema, and scar formation. A positive correlation between patient age and the delay period was found. All surgically treated patients recovered well. The initial conservative measures achieved only partial success, whereas all nine surgically treated patients (43 %) demonstrated complete relief of their clinical symptoms.Soft tissue augmentation with DermaLive led to a series of severe delayed adverse reactions. Sclerosing DermaLive granulomas demonstrated low success rates with conservative measures, whereas early radical surgical excision achieved good results.This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

One of the clinical situations linked to the use of warfarin is the skin necrosis occurring in approximately 0.01 to 0.1% of all patients receiving warfarin. Typically, lesions develop during the first days after initiation of warfarin therapy (usually around the tenth day) and are often associated with the administration of a loading dose. The pathophysiological mechanisms for warfarin-induced skin necrosis, despite several theories, remain uncertain. For the diagnosis, along with a high degree of suspicion, a rapid recognition and management is required.

Nowadays, risk prediction is being focused as a key element in identifying risk factors for cardiovascular disease (CVD).1 The publication of the Third Report of the National Cholesterol Education Program's Adult Treatment Panel (ATP-III)2 in 2001, among other work,3 was paramount for risk prediction becoming the cornerstone in the field of CVD prevention, particularly in adulthood. However, considering the title of the present publication, the author puts forward the following question: where do we stand in relation to the prediction of CVD that is focused on the early stages of life, i.e., childhood or adolescence? An extensive search on the subject in Medline yielded little evidence to suggest the development of models aimed to predict CVD in adulthood on the basis of cardiovascular risks in the early stages of life. Nevertheless, prediction of cardiovascular risks since childhood and adolescence is essential and constitutes a key issue in cardiovascular epidemiology. There are conditions such as arterial hypertension (HTN) for which the former statement can be fully justified, taking into consideration its increasing prevalence in the early stages of life.4 The author of this letter published one scientific paper,5 where two risk scores were proposed to assess the likelihood of hypertension in adulthood after analyzing cardiac risk factors in Cuban adolescents. It is thus justifiable to give a wake-up call to the medical community to go to the root of the problem (childhood and adolescence) where the genesis of hypertension occurs. A management strategy should then be adopted to prevent the increased chances of developing hypertension in adulthood. This can also apply to other conditions such as familial hyperlipidemia, diabetes and obesity, involving the broad class of CVD. This demonstrates the importance of developing a new model of CVD risk factors to predict CVD from childhood and adolescence. The benefit will be far less when addressing these risk factors once damage is already established. I strongly believe that the correct identification and management of risks in terms of prediction is decisive in preventing clinical vascular events in adult life. Keywords: prediction, cardiovascular disease

Myopodin is a cytoskeleton protein that shuttles to the nucleus depending on the cellular differentiation and stress. It has shown tumor suppressor functions. Myopodin methylation status was useful for staging bladder and colon tumors and predicting clinical outcome. To our knowledge, myopodin has not been tested in kidney cancer to date. The purpose of this study was to evaluate whether myopodin methylation status could be clinically useful in renal cancer (1) as a prognostic biomarker and 2) as a predictive factor of response to antiangiogenic therapy in patients with metastatic disease. Methylation-specific polymerase chain reactions (MS-PCR) were used to evaluate myopodin methylation in 88 kidney tumors. These belonged to patients with localized disease and no evidence of disease during follow-up (n = 25) (group 1), and 63 patients under antiangiogenic therapy (sunitinib, sorafenib, pazopanib, and temsirolimus), from which group 2 had non-metastatic disease at diagnosis (n = 32), and group 3 showed metastatic disease at diagnosis (n = 31). Univariate and multivariate Cox analyses were utilized to assess outcome and response to antiangiogenic agents taking progression, disease-specific survival, and overall survival as clinical endpoints. Myopodin was methylated in 50 out of the 88 kidney tumors (56.8 %). Among the 88 cases analyzed, 10 of them recurred (11.4 %), 51 progressed (57.9 %), and 40 died of disease (45.4 %). Myopodin methylation status correlated to MSKCC Risk score (p = 0.050) and the presence of distant metastasis (p = 0.039). Taking all patients, an unmethylated myopodin identified patients with shorter progression-free survival, disease-specific survival, and overall survival. Using also in univariate and multivariate models, an unmethylated myopodin predicted response to antiangiogenic therapy (groups 2 and 3) using progression-free survival, disease-specific, and overall survival as clinical endpoints. Myopodin was revealed hypermethylated in kidney cancer. Myopodin methylation status identified which patients showed a more aggressive clinical behavior and predicted antiangiogenic response. These observations support the clinical utility of an unmethylated myopodin as a prognostic and predictive biomarker in kidney cancer.

Background Electrical stimulation of the lower esophageal sphincter (LES) improves LES pressure without interfering with LES relaxation. The aim of this open-label pilot trial was to evaluate the safety and efficacy of long-term LES stimulation using a permanently implanted LES stimulator in patients with gastroesophageal reflux disease (GERD). Methods GERD patients who were at least partially responsive to proton pump inhibitors (PPI) with abnormal esophageal pH, hiatal hernia ≤3 cm, and esophagitis ≤LA grade C were included. Bipolar stitch electrodes were placed in the LES and an IPG was placed in a subcutaneous pocket. Electrical stimulation was delivered at 20 Hz, 215 μs, 3–8 mA in 30 min sessions. The number and timing of sessions was tailored to each patient’s GERD profile. Patients were evaluated using GERD-HRQL, daily symptom and medication diaries, SF-12, esophageal pH, and high-resolution manometry. Results 24 patients (mean age = 53 years, SD = 12 years; 14 men) were implanted; 23 completed their 6-month evaluation. Median GERD-HRQL scores at 6 months was 2.0 (IQR = 0–5.5) and was significantly better than both baseline on-PPI [9.0 (range = 6.0–10.0); p p p p Conclusions Electrical stimulation of the LES is safe and effective for treating GERD. There is a significant and sustained improvement in GERD symptoms, esophageal pH, and reduction in PPI usage without any side effects with the therapy. Furthermore, the therapy can be optimized to address an individual patient’s disease.

Chromaffin cells have been widely used to study neurosecretion since they exhibit similar calcium dependence of several exocytotic steps as synaptic terminals do, but having the enormous advantage of being neither as small or fast as neurons, nor as slow as endocrine cells. In the present study, secretion associated to experimental measurements of the exocytotic dynamics in human chromaffin cells of the adrenal gland was simulated by using a model that combines stochastic and deterministic approaches for short and longer depolarizing pulses, respectively. Experimental data were recorded from human chromaffin cells, obtained from healthy organ donors, using the perforated patch configuration of the patch-clamp technique. We have found that in human chromaffin cells, secretion would be mainly managed by small pools of non-equally fusion competent vesicles, slowly refilled over time. Fast secretion evoked by brief pulses can be predicted only when 75% of one of these pools (the "ready releasable pool" of vesicles, abbreviated as RRP) are co-localized to Ca2?+? channels, indicating an immediately releasable pool in the range reported for isolated cells of bovine and rat (Alvarez and Marengo, J Neurochem 116:155---163, 2011). The need for spatial correlation and close proximity of vesicles to Ca2?+? channels suggests that in human chromaffin cells there is a tight control of those releasable vesicles available for fast secretion.

BACKGROUND AND PURPOSE Expression of α7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells. EXPERIMENTAL APPROACH To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors. KEY RESULTS The nicotinic current provoked by 300 µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1 µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10 nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10 mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4 ms rise time) and inactivated component (8.5 ± 0.4 ms time constant) (termed α7), which was blocked by α-Bgtx or MLA, followed by a slower component (non-α7). α7 nAChR currents were dissected by blocking the non-α7 nAChR current component of the ACh and choline response with the α6* nAChR blocker α-conotoxin (α-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an α7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. α7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1 mM) and PNU-120596 (10 µM), potentiated responses that were blocked by α-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited α7 nAChR currents, using choline in the presence of α-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists. CONCLUSIONS AND IMPLICATIONS Our electrophysiological recordings of pure α7 nAChR currents elicited by rapid application of agonists demonstrated that functional α7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.

J. Neurochem. (2011) 116, 105–121. Abstract This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3−/−) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3−/− cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3−/− cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3−/− cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3−/− cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below −10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.

Angiosarcomas are a rare type of malignant vascular tumour characterised by proliferation of tumour cells with vascular endothelial features. These tumours are usually very aggressive and overall mortality is very high. Pulmonary angiosarcomas are usually secondary tumours, and only a few primary cases have been reported. We present a case of a 56 year-old male with persistent haemoptysis secondary to lung epithelioid angiosarcoma with pleural metastases.