Mosaic HIV-1 vaccination induces anti-viral CD8+ T cell functionality in the phase 1/2a clinical trial APPROACH.

IMPORTANCE: The functionality of CD8+ T cells against human immunodeficiency virus-1 (HIV-1) antigens is indicative of HIV-progression in both animal models and people living with HIV. It is, therefore, of interest to assess CD8+ T cell responses in a prophylactic vaccination setting, as this may be an important component of the immune system that inhibits HIV-1 replication. T cell responses induced by the adenovirus serotype 26 (Ad26) mosaic vaccine regimen were assessed previously by IFN-γ ELISpot and flow cytometric assays, yet these assays only measure cytokine production but not the capacity of CD8+ T cells to inhibit replication of HIV-1. In this study, we demonstrate direct anti-viral function of the clinical Ad26 mosaic vaccine regimen through ex vivo inhibition of replication of diverse clades of HIV-1 isolates in the participant's own CD4+ T cells.

Ceefourin-1, a MRP4/ABCC4 inhibitor, induces apoptosis in AML cells enhanced by histamine.

BACKGROUND: Ceefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. METHODS: U937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. RESULTS: Ceefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. CONCLUSIONS: These results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine. GENERAL SIGNIFICANCE: This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.

RUNX1 Is Regulated by Androgen Receptor to Promote Cancer Stem Markers and Chemotherapy Resistance in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which no effective targeted therapies are available. Growing evidence suggests that chemotherapy-resistant cancer cells with stem-like properties (CSC) may repopulate the tumor. The androgen receptor (AR) is expressed in up to 50% of TNBCs, and AR inhibition decreases CSC and tumor initiation. Runt-related transcription factor 1 (RUNX1) correlates with poor prognosis in TNBC and is regulated by the AR in prostate cancer. Our group has shown that RUNX1 promotes TNBC cell migration and regulates tumor gene expression. We hypothesized that RUNX1 is regulated by the AR and that both may work together in TNBC CSC to promote disease recurrence following chemotherapy. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments in MDA-MB-453 revealed AR binding to RUNX1 regulatory regions. RUNX1 expression is upregulated by dihydrotestosterone (DHT) in MDA-MB-453 and in an AR+-TNBC HCI-009 patient-derived xenograft (PDX) tumors (p < 0.05). RUNX1 is increased in a CSC-like experimental model in MDA-MB-453 and SUM-159PT cells (p < 0.05). Inhibition of RUNX1 transcriptional activity reduced the expression of CSC markers. Interestingly, RUNX1 inhibition reduced cell viability and enhanced paclitaxel and enzalutamide sensitivity. Targeting RUNX1 may be an attractive strategy to potentiate the anti-tumor effects of AR inhibition, specifically in the slow-growing CSC-like populations that resist chemotherapy which lead to metastatic disease.

Atezolizumab Plus Bevacizumab as First-line Treatment for Patients With Metastatic Nonsquamous Non-Small Cell Lung Cancer With High Tumor Mutation Burden: A Nonrandomized Controlled Trial.

Importance: Antiangiogenic drug combinations with anti-programmed cell death 1 protein and anti-programmed cell death 1 ligand 1 (PD-L1) agents are a novel treatment option for lung cancer. However, survival remains limited, and the activity of these combinations for tumors with high tumor mutation burden (TMB) is unknown. Objective: To assess the clinical benefits and safety of atezolizumab plus bevacizumab for patients with high-TMB advanced nonsquamous non-small cell lung cancer (NSCLC). Design, Setting, and Participants: This multicenter, single-arm, open-label, phase 2 nonrandomized controlled trial (Atezolizumab Plus Bevacizumab in First-Line NSCLC Patients [TELMA]) included treatment-naive patients aged 18 years or older with confirmed stage IIIB-IV nonsquamous NSCLC with TMB of 10 or more mutations/megabase and no EGFR, ALK, STK11, MDM2, or ROS1 alterations. From May 2019 through January 2021, patients were assessed at 13 sites in Spain, with follow-up until February 28, 2022. Interventions: Participants were given atezolizumab, 1200 mg, plus bevacizumab, 15 mg/kg, on day 1 of each 21-day cycle. Treatment was continued until documented disease progression, unacceptable toxic effects, patient withdrawal, investigator decision, or death. Main Outcomes and Measures: The primary end point was 12-month progression-free survival (PFS) rate (according to Response Evaluation Criteria in Solid Tumours, version 1.1 criteria); PFS was defined as the time from enrollment to disease progression or death. Adverse events were monitored according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. Results: A total of 307 patients were assessed for trial eligibility, of whom 266 were ineligible for enrollment. Of the 41 patients enrolled, 3 did not fulfill all inclusion criteria and were excluded. The remaining 38 patients (28 [73.7%] male; mean [SD] age, 63.7 [8.3] years) constituted the per-protocol population. The 12-month PFS rate was 51.3% (95% CI, 34.2%-66.0%), which met the primary end point. The 12-month overall survival (OS) rate was 72.0% (95% CI, 54.1%-83.9%). The median PFS was 13.0 months (95% CI, 7.9-18.0 months), and the median OS was not reached. Of the 38 patients, 16 (42.1%) achieved an objective response and 30 (78.9%) achieved disease control. The median time to response was 2.8 months (IQR, 2.8-3.58 months), with a median duration of response of 11.7 months (range, 3.57-22.4 months; the response was ongoing at cutoff). Of 16 responses, 8 (50.0%) were ongoing. Most adverse events were grade 1 or 2. For atezolizumab, the most common adverse events were fatigue (6 [15.8%]) and pruritus (6 [15.8%]). For bevacizumab, they were hypertension (10 [26.3%]) and proteinuria (4 [10.5%]). Drug discontinuation occurred in 2 patients receiving atezolizumab (5.3%) and 3 patients receiving bevacizumab (7.9%). PD-L1 levels were not associated with response, PFS, or OS. Conclusions and Relevance: These findings suggest that atezolizumab with bevacizumab is a potential treatment for high-TMB nonsquamous NSCLC. Trial Registration: ClinicalTrials.gov Identifier: NCT03836066.

Real-world data with afatinib in Spanish patients with treatment-naïve non-small-cell lung cancer harboring exon 19 deletions in epidermal growth factor receptor (Del19 EGFR): Clinical experience of the Galician Lung Cancer Group.

BACKGROUND: In clinical studies, first-line afatinib demonstrated efficacy in Del19-EGFR NSCLC. MATERIALS AND METHODS: This prospective, non-interventional study assessed efficacy and safety of first-line afatinib in patients with advanced/metastatic NSCLC with Del19-EGFR from Galicia (Spain), with a preplanned analysis by age (<70 vs ≥70 years). RESULTS: Median age of 46 patients enrolled was 69.5 years (range 37-87). The objective response rate (ORR) was 78.2%, with median progression-free survival (PFS) of 20.5 months (95% CI 12.7, 28.3) and median overall survival (OS) of 37.5 months (95% CI 19.2-55.8). Outcomes by age (<70 vs ≥70 years) were ORR of 82.6% vs 73.9%, median PFS of 20.2 months (95% CI 14.8-25.6) vs 24.1 (9.8-38.3), and median OS of 45.1 months (95% CI, 17.0-73.1) vs 33.9 (28.7-39.1), respectively. Median treatment duration was 17.2 months (range 0.4-64.1) with 11 patients still on treatment; 14 patients received osimertinib at discontinuation due to T790M. Grade 3 adverse events included mucositis (n = 7, 15.2%), skin toxicity (n = 9, 19.6%), and diarrhea (n = 6, 13.0%) that were manageable with dose reductions. The afatinib dose was reduced in 31 patients (67.4%) and treatment was discontinued in 8 patients (17.4%) due to adverse events. By age (<70 vs ≥70 years), afatinib was dose-reduced in 13 (56.5%) vs 18 patients (78.3%) and discontinued in 3 (13.0%) vs 5 patients (21.7%), respectively. CONCLUSIONS: PFS in our patients was longer than reported in clinical studies with similar response rates and toxicity, even in older patients, reflecting a good risk-benefit from afatinib in patients with Del19-EGFR NSCLC. MICROABSTRACT: This real-world study of first-line afatinib in Caucasian patients with Del19 EGFR NSCLC reported durable efficacy and showed that older patients (> 70 years) benefitted from afatinib as much as younger patients. The safety profile of afatinib was as expected, albeit more dose reductions in older patients. Afatinib may be an option for patients with Del19 EGFR NSCLC, even in those who are older.

Comparison of non-invasive fibrosis scores to predict increased liver stiffness in the general population with unknown liver disease: Searching for the primary physician's best friend.

BACKGROUND: Serum-based scores (SBS) appear to be a high applicability strategy for assessment of liver fibrosis in primary care. Aim of the study was to evaluate their performance to detect ≥F2 in a general population and to design a highly-applicable strategy for screening. METHODS: prospective population-based cohort study in randomly identified subjects, aged 40-70y. Medical history, blood tests and elastography were obtained, ≥F2 was determined by using LSM cutoff ≥9.2/7.8 kPa for M/XL probe and SBS diagnostic accuracies were evaluated. RESULTS: 986 patients were analyzed. LSM prevalence estimate suggestive of ≥F2 was 1.9% and Metabolic Sindrome (MS) (OR 3.4, 1.3-9.0;p = 0.01), was the only factor independently associated with ≥F2, with increasing prevalence according to the number of criteria (0 criterion:0%,1:0.3%,2:2.8%,3:2.4%,4:6.9%,5:14.3%;p<0.001). FLI and NFS were the two best-performing tests in the cross-sectional study, with AUROCs for ≥F2 of 0.89 (95%CI,0.84- 0.95) and 0.82 (95%CI,0.74-0.90), respectively. Predefined cutoff for FLI≥60 (Sn89.5%, Sp72.1%, NPV99.9%) and NFS≥-1.455 (Sn83.3%, Sp68%, NPV99.6%) showed adequate diagnostic accuracy. Based on these findings, a 3- step algorithm strategy to detect liver fibrosis in the community setting is proposed (Sn84.2%, Sp75.2%, NVP99.6%). CONCLUSIONS: A staged risk-stratification model improves the detection of ≥F2 in the community setting, while reducing unnecessary referrals.

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling.

G protein-coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2-mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates' ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation-independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

Antihistamines Potentiate Dexamethasone Anti-Inflammatory Effects. Impact on Glucocorticoid Receptor-Mediated Expression of Inflammation-Related Genes.

Antihistamines and glucocorticoids (GCs) are often used together in the clinic to treat several inflammation-related situations. Although there is no rationale for this association, clinical practice has assumed that, due to their concomitant anti-inflammatory effects, there should be an intrinsic benefit to their co-administration. In this work, we evaluated the effects of the co-treatment of several antihistamines on dexamethasone-induced glucocorticoid receptor transcriptional activity on the expression of various inflammation-related genes in A549 and U937 cell lines. Our results show that all antihistamines potentiate GCs' anti-inflammatory effects, presenting ligand-, cell- and gene-dependent effects. Given that treatment with GCs has strong adverse effects, particularly on bone metabolism, we also examined the impact of antihistamine co-treatment on the expression of bone metabolism markers. Using MC3T3-E1 pre-osteoblastic cells, we observed that, though the antihistamine azelastine reduces the expression of dexamethasone-induced bone loss molecular markers, it potentiates osteoblast apoptosis. Our results suggest that the synergistic effect could contribute to reducing GC clinical doses, ineffective by itself but effective in combination with an antihistamine. This could result in a therapeutic advantage, as the addition of an antihistamine may reinforce the wanted effects of GCs, while related adverse effects could be diminished or at least mitigated. By modulating the patterns of gene activation/repression mediated by GR, antihistamines could enhance only the desired effects of GCs, allowing their effective dose to be reduced. Further research is needed to correctly determine the clinical scope, benefits, and potential risks of this therapeutic strategy.

Non-malignant portal vein thrombosis in a cohort of cirrhotic patients: Incidence and risk factors.

AIM: Non-malignant portal vein thrombosis (PVT) is a complication of liver cirrhosis. The aim of this study was to evaluate the annual incidence of PVT and related risk factors. METHODS: We retrospectively reviewed clinical, laboratory, and radiological data collected prospectively from September 2016 to September 2017. A follow-up of 36 months was performed in a subset of patients to determine the cumulative incidence of PVT and related complications. RESULTS: The study included 567 patients. The incidence of PVT at 12, 24, and 36 months was 3.7%, 0.8%, and 1.4%, respectively. Patients with PVT were compared with patients without PVT, and showed differences in albumin (p = 0.04), aspartate aminotransferase (p = 0.04), hemoglobin (p = 0.01), and prothrombin activity (p = 0.01). The presence of hydropic decompensation (57.1% vs. 30.1%; p 0.004), gastroesophageal varices (76.2% vs. 39.5%; p = 0.05), variceal bleeding (52.4% vs. 22.7%; p < 0.001), hepatic encephalopathy (38.1% vs. 9.9%; p = 0.01), spontaneous bacterial peritonitis (9.5% vs. 1.7%; p < 0.001), and use of beta-blockers (71.4% vs. 27.7%; p < 0.001) were significantly associated. In the multivariate analysis, use of beta-blockers and hepatic encephalopathy appeared as risk factors, and high albumin levels a protective factor. CONCLUSIONS: The incidence of PVT was 3.7%. Beta-blockers and hepatic encephalopathy were risks factors. High albumin levels were a protective factor.

MRP4/ABCC4 expression is regulated by histamine in acute myeloid leukemia cells, determining cAMP efflux.

Intracellular cAMP (i-cAMP) levels play an important role in acute myeloid leukemia (AML) cell proliferation and differentiation. Its levels are the result of cAMP production, degradation, and exclusion. We have previously described histamine H2 receptors and MRP4/ABCC4 as two potential targets for AML therapy. Acting through histamine H2 receptors, histamine increases cAMP production/synthesis, while MRP4/ABCC4 is responsible for the exclusion of this cyclic nucleotide. In this study, we show that histamine treatment induces MRP4/ABCC4 expression, augmenting cAMP efflux, and that histamine, in combination with MRP inhibitors, is able to reduce AML cell proliferation. Histamine, through histamine H2 receptor, increases i-cAMP levels and induces MRP4 transcript and protein levels in U937, KG1a, and HL-60 cells. Moreover, histamine induces MRP4 promoter activity in HEK293T cells transfected with histamine H2 receptor (HEK293T-H2 R). Our results support that the cAMP/Epac-PKA pathway, and not MEK/ERK nor PI3K/AKT signaling cascades, is involved in histamine-mediated upregulation of MRP4 levels. Finally, the addition of histamine potentiates the inhibition of U937, KG1a, and HL-60 cell proliferation induced by MRP4 inhibitors. Our data highlight that the use of a poly-pharmacological approach aimed at different molecular targets would be beneficial in AML treatment.

Construcciones de juventud y trayectorias militantes católicas contemporáneas en parroquias del Gran Buenos Aires / Constructions of youth and contemporary militant trajectories in parishes in Gran Buenos Aires (Argentina) / Construções juvenis e trajetórias militantes contemporâneas nas paróquias da grande Buenos Aires (Argentina)

Resumen (analítico) El artículo analiza las construcciones de juventud presentes en la Acción Católica Argentina y sus vínculos con trayectorias de jóvenes militantes insertos en parroquias del Gran Buenos Aires. Para el abordaje metodológico cualitativo se utilizan los siguientes materiales de campo, producidos entre 2016 y 2018: entrevistas en profundidad realizadas a jóvenes católicos, registros de observación participante en parroquias y eventos nacionales de Acción Católica Argentina y análisis de documentos institucionales. Dentro de la Acción Católica Argentina, se identifican tensiones entre las definiciones de juventud producidas por la institución y las trayectorias juveniles. Los jóvenes experimentan modos de «ser¼ y de «vivir¼ la juventud distintos a los propuestos por la Acción Católica Argentina, acordes con los cambios sociales y culturales que se produjeron en Argentina en las últimas décadas.

Abstract (analytical) This article analyzes the constructions of youth produced in the Acción Católica Argentina (Argentinian Catholic Action) organization and their link with the trajectories of militant youth living in parishes of Gran Buenos Aires. Field materials produced between 2016 and 2018 are analyzed using a qualitative methodological approach that consists of: in-depth interviews with Catholic youth; participant observation records from parish and national Argentinian Catholic Action events; and the analysis of institutional documents. In Argentinian Catholic Action, tensions were identified between the definitions of youth produced by the institution and young people's own trajectories. Young people experience ways of «being¼ and «living¼ that are different from those proposed by the Argentinian Catholic Action and that respond to the social and cultural changes produced in Argentine society in recent decades.

Resumo (analítico) O artigo analisa as construções de jovens presentes na Acción Católica Argentina seus vínculos com as trajetórias de jovens militantes inseridos nas paróquias da Gran Buenos Aires. Para a abordagem metodológica qualitativo, são utilizados materiais de campo produzidos entre 2016 e 2018, que consistem em entrevistas em profundidade com jovens católicos; registros de observação participante nas paróquias e eventos nacionais da Acción Católica Argentina e análise de documentos institucionais. Na Acción Católica Argentina, são identificadas tensões entre as definições de juventude produzidas pela instituição e as trajetórias da juventude. Os jovens experimentam maneiras de «ser¼ e «viver¼ a juventude diferentes dos propostos pela Acción Católica Argentina de acordo com as mudanças sociais e culturais produzidas na Argentina nas últimas décadas.

Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs.

Matrix metalloproteinases (MMPs) degrade several ECM components and are crucial modulators of cell invasion and tissue organization. Although much has been reported about their function in remodeling ECM in health and disease, their trafficking across the Golgi apparatus remains poorly understood. Here we report that the cis-Golgi protein nucleobindin-1 (NUCB1) is critical for MMP2 and MT1-MMP trafficking along the Golgi apparatus. This process is Ca2+-dependent and is required for invasive MDA-MB-231 cell migration as well as for gelatin degradation in primary human macrophages. Our findings emphasize the importance of NUCB1 as an essential component of MMP transport and its overall impact on ECM remodeling.

Metas lipídicas en pacientes diabéticos. Implicaciones clínicas luego de aplicar una nueva fórmula para el cálculo del colesterol-LDL / Lipid Goals in Diabetic Patients. Clinical Implications after Application of a New Formula for LDL-cholesterol Calculation

RESUMEN Introducción: Existen claras recomendaciones para el manejo lipídico en los diabéticos. Una nueva fórmula para el cálculo del C-LDL mejoraría la imprecisión de la fórmula de Friedewald. Objetivos: Analizar el uso de estatinas y el cumplimiento de las metas lipídicas en pacientes diabéticos, evaluando las consecuencias de aplicar una nueva fórmula para el cálculo del C-LDL. Métodos: Estudio descriptivo, transversal y multicéntrico. Se incluyeron diabéticos tipo 2 mayores de 18 años. El C-LDL se calculó con la fórmula clásica (Friedewald) y la nueva fórmula. Se siguieron las recomendaciones del documento de posición para el uso adecuado de estatinas (Sociedad Argentina de Cardiología). Resultados: Se incluyeron 528 pacientes. En prevención secundaria, el 77,2% recibió estatinas (23,4% alta intensidad). El 36,6% y el 36,0% alcanzaron la meta de C-LDL menor a 70 mg/dL y de C-noHDL inferior a 100 mg/dL, respectivamente. El 20,8% de los pacientes con un C-LDL menor de 70 mg/dL (Friedewald) salió de meta al aplicar la nueva fórmula. En los pacientes en prevención primaria con factores de riesgo o daño de órgano blanco, el 62,2% recibió estatinas (14,7% alta intensidad). El 20,9% y el 20,4% alcanzaron la meta de C-LDL menor a 70 mg/dL y de C-noHDL inferior a 100 mg/dL. El 27,7% de los pacientes con un C-LDL menor de 70 mg/dL (Friedewald) salió de meta al aplicar la nueva fórmula. A mayor nivel de triglicéridos, más pacientes salieron de meta de C-LDL con la nueva fórmula. Conclusión: El cumplimiento de las metas lipídicas y el uso adecuado de estatinas en esta población fue deficiente. Aplicar la nueva fórmula de C-LDL optimizó la evaluación de estos pacientes.

ABSTRACT Background: There are clear recommendations for lipid management in diabetic patients. A new formula for the calculation of LDL-cholesterol (LDL-C) would improve the inaccuracy of the Friedewald formula. Objectives: The aim of this study was to analyze the use of statins and the fulfillment of lipid goals in diabetic patients, evaluating the consequences of applying a new formula for LDL-C calculation. Methods: This was a descriptive, cross-sectional, multicenter study including type 2 diabetic patients over 18 years of age. LDL-C was calculated using the classic Friedewald formula and the new formula. Recommendations of the position document for the appropriate use of statins from the Argentine Society of Cardiology were followed. Results: A total of 528 patients were included in the study. In secondary prevention, 77.2% of patients received statins (23.4% high-intensity statins) and 36.6% and 36.0% of these patients achieved the goals of LDL-C below 70% mg/dl and non-HDL-C below 100 mg/dl, respectively. In 20.8% of patients with LDL-C below 70 mg/dl according to the Friedewald formula, this goal was not attained when the new formula was applied. In primary prevention, 62.2% patients with risk factors or white organ damage received statins (14.7% high-intensity statins) and 20.9% and 20.4% achieved the goals of LDL-C below 70% mg/dl and non-HDL-C below 100 mg/dl. In 27.7% of patients with LDL-C below 70 mg/dl using the Friedewald formula, this goal was not reached when applying the new formula. More patients did not achieve the LDL-C goal with the new formula when the triglyceride level was higher. Conclusion: In this population, the appropriate use of statins and the fulfillment of lipid goals were poor. Applying the new LDL-C formula optimized the evaluation of these patients.

Number and function of circulatory helper innate lymphoid cells are unaffected by immunosuppressive drugs used in solid organ recipients - a single centre cohort study.

In transplanted intestines, depletion of T cells together with long-term persistence of ILC is observed, suggesting ILC insensitivity to immunosuppressive drugs. To further analyze helper ILC (hILC) apparent resistance to therapy, cytotoxic ILC (NK cells), hILC subsets (ILC1, ILC2, and ILC precursors (ILCP)), and their signature cytokines (IFNγ, IL4 + IL13, and IL22) were analyzed in peripheral blood of kidney and liver transplant recipients. Early after transplantation (posTx), transplanted patients showed significantly lower Lin + and NK cells, whereas total hILC, ILC1, ILC2, and ILCP numbers were similar in patients and controls. Between paired pre- and posTx samples, Lin + cell and NK cell counts significantly decreased, whereas all three hILC counts and their cytokine production remained similar. ILC1, ILC2, and ILCP numbers were also similar in patients under thymoglobulin or basiliximab (BAS), patients without induction (only maintenance therapy) and controls. hILC showed lower TMG binding comparing to Lin + cells, reduced expression of CD25 (BAS target), and diminished calcineurin activity with undetectable calcineurin and FKBP12 (tacrolimus target). hILC counts were not related to delayed graft function or biopsy-proven acute rejection. Thus, hILC remain stable early after transplantation and seem unaffected by immunosuppressors, which may be related to reduced targets expression and low calcineurin activity.

Hyperinsulinemic hypoglycemia caused by nesidioblastosis associated with non-surgical weight loss.

INTRODUCCIÓN: La nesidioblastosis es una enfermedad poco frecuente en los adultos caracterizada por hiperplasia de los islotes pancreáticos, produciendo hipoglucemia por incremento de la producción de insulina. Su patogenia es poco conocida. Clínicamente se ha descrito un cuadro de hipoglucemia con valores elevados de insulina y de péptido C sin ninguna lesión tumoral pancreática detectable. El diagnóstico final es histopatológico. CASO CLÍNICO: Mujer de 36 años que cursa con episodios recurrentes de hipoglucemia sintomática de predominio posprandial, asociada a elevación de los valores sanguíneos de insulina y péptido C. Los estudios de imágenes (tomografía computarizada trifásica, resonancia magnética, ecoendoscopia y octreoscan) no mostraron lesiones compatibles con insulinoma. Se le realizó estimulación intraarterial con calcio, que mostró un incremento de la insulinemia en más de un segmento pancreático posestimulación. La paciente fue sometida a pancreatectomía corporocaudal, cuya evaluación histopatológica informó de nesidioblastosis. CONCLUSIONES: La nesidioblastosis representa menos del 5% de los casos de hipoglucemia hiperinsulinémica en los adultos, por lo que debe considerarse como diagnóstico -diferencial en estos pacientes; su diagnóstico es difícil y requiere confirmación histopatológica. BACKGROUND: Nesidioblastosis is a rare disease in adults caused by pancreatic islet hyperplasia, producing hypoglycemia due to an increase in insulin production. The pathogenesis is poorly understood, however a clinical picture characterized by hypoglycemia with high levels of insulin and C-peptide without any detectable pancreatic lesion has been described, its final diagnosis is histological. CASE REPORT: We report a case of a 36-year-old female patient who presents with recurrent episodes of hypoglycemia symptomatic predominantly postprandial, associated with insulin and C-peptide elevation. Images were made (triphasic computed tomography, magnetic resonance imaging, echoendoscopy and octreoscan), being negative for insulinoma. Subsequent tests included calcium stimulation of the pancreas, revealing marked insulin release in more than one pancreatic segment. The patient was subjected to distal pancreatectomy confirming in the histological study the diagnosis of nesidioblastosis. CONCLUSIONS: Nesidioblastosis accounts for less than 5% of the cases of cases of hyperinsulinemic hypoglycemia in adult patients, should be considered as a differential diagnosis in patients with persistent hypoglycemia, the diagnosis is difficult and usually requires histological confirmation.

BCL2L10 Is Overexpressed in Melanoma Downstream of STAT3 and Promotes Cisplatin and ABT-737 Resistance.

The anti-apoptotic proteins from the Bcl-2 family are important therapeutic targets since they convey resistance to anticancer regimens. Despite the suspected functional redundancy among the six proteins of this subfamily, both basic studies and therapeutic approaches have focused mainly on BCL2, Bcl-xL, and MCL1. The role of BCL2L10, another member of this group, has been poorly studied in cancer and never has been in melanoma. We describe here that BCL2L10 is abundantly and frequently expressed both in melanoma cell lines and tumor samples. We established that BCL2L10 expression is driven by STAT3-mediated transcription, and by using reporter assays, site-directed mutagenesis, and ChIP analysis, we identified the functional STAT3 responsive elements in the BCL2L10 promoter. BCL2L10 is a pro-survival factor in melanoma since its expression reduced the cytotoxic effects of cisplatin, dacarbazine, and ABT-737 (a BCL2, Bcl-xL, and Bcl-w inhibitor). Meanwhile, both genetic and pharmacological inhibition of BCL2L10 sensitized melanoma cells to cisplatin and ABT-737. Finally, BCL2L10 inhibited the cell death upon combination treatments of PLX-4032, a BRAF inhibitor, with ABT-737 or cisplatin. In summary, we determined that BCL2L10 is expressed in melanoma and contributes to cell survival. Hence, targeting BCL2L10 may enhance the clinical efficacy of other therapies for malignant melanoma.

An Autocrine Wnt5a Loop Promotes NF-κB Pathway Activation and Cytokine/Chemokine Secretion in Melanoma.

Wnt5a signaling has been implicated in the progression of cancer by regulating multiple cellular processes, largely migration and invasion, epithelial-mesenchymal transition (EMT), and metastasis. Since Wnt5a signaling has also been involved in inflammatory processes in infectious and inflammatory diseases, we addressed the role of Wnt5a in regulating NF-κB, a pivotal mediator of inflammatory responses, in the context of cancer. The treatment of melanoma cells with Wnt5a induced phosphorylation of the NF-κB subunit p65 as well as IKK phosphorylation and IκB degradation. By using cDNA overexpression, RNA interference, and dominant negative mutants we determined that ROR1, Dvl2, and Akt (from the Wnt5a pathway) and TRAF2 and RIP (from the NF-κB pathway) are required for the Wnt5a/NF-κB crosstalk. Wnt5a also induced p65 nuclear translocation and increased NF-κB activity as evidenced by reporter assays and a NF-κB-specific upregulation of RelB, Bcl-2, and Cyclin D1. Further, stimulation of melanoma cells with Wnt5a increased the secretion of cytokines and chemokines, including IL-6, IL-8, IL-11, and IL-6 soluble receptor, MCP-1, and TNF soluble receptor I. The inhibition of endogenous Wnt5a demonstrated that an autocrine Wnt5a loop is a major regulator of the NF-κB pathway in melanoma. Taken together, these results indicate that Wnt5a activates the NF-κB pathway and has an immunomodulatory effect on melanoma through the secretion of cytokines and chemokines.

Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening.

AIMS: G protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. MATERIALS AND METHODS: We performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549 cells. GRK2(45-178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. KEY FINDINGS: We identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SIGNIFICANCE: We obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.

Identification of MRP4/ABCC4 as a Target for Reducing the Proliferation of Pancreatic Ductal Adenocarcinoma Cells by Modulating the cAMP Efflux.

Pancreatic cancer is one of the most lethal types of tumors with no effective therapy available; is currently the third leading cause of cancer in developed countries; and is predicted to become the second deadliest cancer in the United States by 2030. Due to the marginal benefits of current standard chemotherapy, the identification of new therapeutic targets is greatly required. Considering that cAMP pathway is commonly activated in pancreatic ductal adenocarcinoma (PDAC) and its premalignant lesions, we aim to investigate the multidrug resistance-associated protein 4 (MRP4)-dependent cAMP extrusion process as a cause of increased cell proliferation in human PDAC cell lines. Our results from in silico analysis indicate that MRP4 expression may influence PDAC patient outcome; thus, high MRP4 levels could be indicators of poor survival. In addition, we performed in vitro experiments and identified an association between higher MRP4 expression levels and more undifferentiated and malignant models of PDAC and cAMP extrusion capacity. We studied the antiproliferative effect and the overall cAMP response of three MRP4 inhibitors, probenecid, MK571, and ceefourin-1 in PDAC in vitro models. Moreover, MRP4-specific silencing in PANC-1 cells reduced cell proliferation (P < 0.05), whereas MRP4 overexpression in BxPC-3 cells significantly incremented their growth rate in culture (P < 0.05). MRP4 pharmacological inhibition or silencing abrogated cell proliferation through the activation of the cAMP/Epac/Rap1 signaling pathway. Also, extracellular cAMP reverted the antiproliferative effect of MRP4 blockade. Our data highlight the MRP4-dependent cAMP extrusion process as a key participant in cell proliferation, indicating that MRP4 could be an exploitable therapeutic target for PDAC. SIGNIFICANCE STATEMENT: ABCC4/MRP4 is the main transporter responsible for cAMP efflux. In this work, we show that MRP4 expression may influence PDAC patient outcome and identify an association between higher MRP4 expression levels and more undifferentiated and malignant in vitro models of PDAC. Findings prove the involvement of MRP4 in PDAC cell proliferation through a novel extracellular cAMP mitogenic pathway and further support MRP4 inhibition as a promising therapeutic strategy for PDAC treatment.

Comparative study of overweight and obese patients with nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in the western world. Although NAFLD prevalence is higher in patients with a BMI > 25 kg /m2, it is unclear if there are differences between overweight and obese patients. The associated biochemical, dietary and genetic parameters were compared between overweight and obese patients with NAFLD. METHODS: patients with biopsy-proven NAFLD (n = 203) were enrolled in a cross-sectional study. The MEDAS questionnaire was used to assess adherence to the Mediterranean diet. Biochemical, anthropometrical parameters and the I148M variant (rs738409) of the PNPLA3 gene and rs180069 of the TNF-α gene were evaluated. RESULTS: overweight patients had higher serum adiponectin levels (22.5 ± 21.9 vs 11.2 ± 18.1 ng/ml; p < 0.05) and lower resistin (3.3 ± 1.7 vs 8.1 ± 8 ng/ml; p < 0.001) and leptin concentrations (22.9 ± 21.9 vs 55.8 ± 45 ng/ml; p < 0.001) than obese patients. Non-alcoholic steatohepatitis (NASH) was more frequent in the obese group (59.3% vs 41.3%; p = 0.02). The multivariate analysis showed adherence to the Mediterranean diet to be an independent protective factor for NASH and liver fibrosis in overweight patients (OR 0.7, 95% CI 0.5-0.8). CONCLUSIONS: NASH was more prevalent in obese patients than in overweight subjects. HOMA-IR and adherence to the Mediterranean diet provided protection against fibrosis in overweight patients. Adherence to the Mediterranean diet was the only independent factor associated with NASH in these patients.