Circulating MR-proADM levels, as an indicator of endothelial dysfunction, for early risk stratification of mid-term mortality in COVID-19 patients.

OBJECTIVES: Thromboinflammation, resulting from a complex interaction between thrombocytopathy, coagulopathy, and endotheliopathy, contributes to increased mortality in COVID-19 patients. MR-proADM, as a surrogate of adrenomedullin system disruption, leading to endothelial damage, has been reported as a promising biomarker for short-term prognosis. We evaluated the role of MR-proADM in the mid-term mortality in COVID-19 patients. METHODS: A prospective, observational study enrolling COVID-19 patients from August to October 2020. A blood sample for laboratory test analysis was drawn on arrival in the emergency department. The primary endpoint was 90-day mortality. The area under the curve (AUC) and Cox regression analyses were used to assess discriminatory ability and association with the endpoint. RESULTS: A total of 359 patients were enrolled, and the 90-day mortality rate was 8.9%. ROC AUC for MR-proADM predicting 90-day mortality was 0.832. An optimal cutoff of 0.80 nmol/L showed a sensitivity of 96.9% and a specificity of 58.4%, with a negative predictive value of 99.5%. Circulating MR-proADM levels (inverse transformed), after adjusting by a propensity score including eleven potential confounders, were an independent predictor of 90-day mortality (HR: 0.162 [95% CI: 0.043-0.480]) CONCLUSIONS: Our data confirm that MR-proADM has a role in the mid-term prognosis of COVID-19 patients and might assist physicians with risk stratification.

Prognostic value of pro-adrenomedullin and NT-proBNP in patients referred from the emergency department with influenza syndrome. / Valor pronóstico de la proadrenomedulina y el NT-proBNP en los pacientes procedentes de urgencias con síndrome gripal.

OBJECTIVES: To assess the prognostic value of procalcitonin (PTC), C-reactive protein (CRP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and mid-regional pro-adrenomedullin (MR-proADM) in patients with influenza syndrome. MATERIAL AND METHODS: Prospective study in patients admitted from the emergency department with influenza syndrome. Biomarker concentrations were measured in the first 24 h after admission and a test for influenza. The results were analyzed for ability to predict a hospital stay longer than 7 days, intensive care unit admission, or in-hospital death. RESULTS: Ninety-eight patients were included; the prognosis of 44 (44.9%) was classified as poor. The areas under the receiving operator characteristic curve were 0.68 (95% CI, 0.56-0.80) for NT-proBNP, 0.73 (95% CI, 0.62-0.84) for MR-proADM, and nonsignificant for PCT and CRP. The following variables were independently associated with a poor prognosis: pneumonia (OR, 7.46 [95% CI, 2.08-26.73]; P=.002), heart failure (OR, 5.16 [95% CI, 1.35-19.74]; P=.016), and NT-proBNP > 580 pg/mL (OR, 4.68 [95% CI, 1.53-14.26]; P=.006). In the 53 patients with confirmed A(H1N1) influenza, only NT-proBNP was an independent predictor of prognosis (adjusted OR, 5.75 [95% CI, 1.46- 22.61]; P=.012). CONCLUSION: NT-proBNP and MR-proADM were the only biomarkers with prognostic value. Only NT-proBNP was a useful predictor in patients with confirmed influenza.

OBJETIVO: Analizar el valor pronóstico de la procalcitonina (PCT), la proteína C reactiva (PCR), el NT-proBNP y la región medial de la proadrenomedulina (MR-proADM) en pacientes hospitalizados con síndrome gripal. METODO: Estudio prospectivo realizado en pacientes hospitalizados desde urgencias por síndrome gripal. Se analizaron las concentraciones de biomarcadores en las primeras 24 h de ingreso y el test de gripe y se analizó su capacidad predictiva de mal pronóstico: estancia superior a 7 días, ingreso en unidad de cuidados intensivos o fallecimiento intrahospitalario. RESULTADOS: Se incluyeron 98 pacientes, 44 (44,9%) de ellos con mal pronóstico. Las áreas bajo la curva COR para mal pronóstico fueron de 0,68 (IC 95% 0,56-0,80) para NT-proBNP y de 0,73 (IC 95% 0,62-0,84) para la MRproADM, y no significativas para PCT y PCR. Las variables asociadas independientemente con mal pronóstico fueron: neumonía (OR 7,46 [IC 95% 2,08-26,73]; p = 0,002), insuficiencia cardiaca (OR 5,16 [IC 95% 1,35-19,74]; p = 0,016) y NT-proBNP > 580 pg/ml (OR 4,68 [IC 95% 1,53-14,26]; p = 0,006). En los 53 pacientes con gripe A(H1N1) confirmada, solo el NT-proBNP tuvo un valor pronóstico independiente (OR ajustado 5,75 [IC 95% 1,46-22,61]; p = 0,012). CONCLUSIONES: En pacientes con síndrome gripal, el NT-proBNP y la MR-proADM fueron los únicos biomarcadores con valor pronóstico, y solo el primero de ellos mantuvo esta asociación en pacientes con gripe confirmada.

Valor pronóstico de la proadrenomedulina y el NT-proBNP en los pacientes procedentes de urgencias con síndrome gripal / Prognostic value of pro-adrenomedullin and NT-proBNP in patients referred from the emergency department with influenza syndrome

Objetivos: Analizar el valor pronóstico de la procalcitonina (PCT), la proteína C reactiva (PCR), el NT-proBNP y la región medial de la proadrenomedulina (MR-proADM) en pacientes hospitalizados con síndrome gripal. Método: Estudio prospectivo realizado en pacientes hospitalizados desde urgencias por síndrome gripal. Se analizaron las concentraciones de biomarcadores en las primeras 24 h de ingreso y el test de gripe y se analizó su capacidad predictiva de mal pronóstico: estancia superior a 7 días, ingreso en unidad de cuidados intensivos o fallecimiento intrahospitalario. Resultados: Se incluyeron 98 pacientes, 44 (44,9%) de ellos con mal pronóstico. Las áreas bajo la curva COR para mal pronóstico fueron de 0,68 (IC 95% 0,56-0,80) para NT-proBNP y de 0,73 (IC 95% 0,62-0,84) para la MRproADM, y no significativas para PCT y PCR. Las variables asociadas independientemente con mal pronóstico fueron: neumonía (OR 7,46 [IC 95% 2,08-26,73]; p = 0,002), insuficiencia cardiaca (OR 5,16 [IC 95% 1,35-19,74]; p = 0,016) y NT-proBNP > 580 pg/ml (OR 4,68 [IC 95% 1,53-14,26]; p = 0,006). En los 53 pacientes con gripe A(H1N1) confirmada, solo el NT-proBNP tuvo un valor pronóstico independiente (OR ajustado 5,75 [IC 95% 1,46-22,61]; p = 0,012). Conclusiones: En pacientes con síndrome gripal, el NT-proBNP y la MR-proADM fueron los únicos biomarcadores con valor pronóstico, y solo el primero de ellos mantuvo esta asociación en pacientes con gripe confirmada

Objectives: To assess the prognostic value of procalcitonin (PTC), C-reactive protein (CRP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and mid-regional pro-adrenomedullin (MR-proADM) in patients with influenza syndrome. Methods: Prospective study in patients admitted from the emergency department with influenza syndrome. Biomarker concentrations were measured in the first 24 h after admission and a test for influenza. The results were analyzed for ability to predict a hospital stay longer than 7 days, intensive care unit admission, or in-hospital death. Results: Ninety-eight patients were included; the prognosis of 44 (44.9%) was classified as poor. The areas under the receiving operator characteristic curve were 0.68 (95% CI, 0.56-0.80) for NT-proBNP, 0.73 (95% CI, 0.62-0.84) for MR-proADM, and nonsignificant for PCT and CRP. The following variables were independently associated with a poor prognosis: pneumonia (OR, 7.46 [95% CI, 2.08-26.73]; P=.002), heart failure (OR, 5.16 [95% CI, 1.35-19.74]; P=.016), and NT-proBNP > 580 pg/mL (OR, 4.68 [95% CI, 1.53-14.26]; P=.006). In the 53 patients with confirmed A(H1N1) influenza, only NT-proBNP was an independent predictor of prognosis (adjusted OR, 5.75 [95% CI, 1.4622.61]; P=.012). Conclusions: NT-proBNP and MR-proADM were the only biomarkers with prognostic value. Only NT-proBNP was a useful predictor in patients with confirmed influenza

Dock10 regulates CD23 expression and sustains B-cell lymphopoiesis in secondary lymphoid tissue.

Dock10, a guanine nucleotide exchange factor for the Rho GTPases Rac1 and Cdc42, affects cell morphology, membrane protrusive activity, and cell movement. Dock10 is prominently expressed in lymphoid tissue and upregulated by IL-4 in B cells. To investigate the physiological role of Dock10, WT mice and Dock10 KO mice were used. KO mice showed decreased numbers of B cells in spleen, both follicular B cells and marginal zone B cells, and in peripheral blood, but not in bone marrow. The antiapoptotic effect of IL-4 in vitro, the migratory response to CXCL13 or CCL21 in vitro, and the whole genome expression profile were intact in spleen B cells from KO mice. CD23, the low-affinity receptor for immunoglobulin E, was overexpressed on follicular B cells from KO mice, suggesting that Dock10 negatively regulates membrane CD23 expression. Negative regulation of CD23 expression by Dock10 could play a role in B cell maturation and function.

IL-4 Up-Regulates MiR-21 and the MiRNAs Hosted in the CLCN5 Gene in Chronic Lymphocytic Leukemia.

Interleukin 4 (IL-4) induces B-cell differentiation and survival of chronic lymphocytic leukemia (CLL) cells. MicroRNAs (miRNAs) regulate mRNA and protein expression, and several miRNAs, deregulated in CLL, might play roles as oncogenes or tumor suppressors. We have studied the miRNA profile of CLL, and its response to IL-4, by oligonucleotide microarrays, resulting in the detection of a set of 129 mature miRNAs consistently expressed in CLL, which included 41 differentially expressed compared to normal B cells (NBC), and 6 significantly underexpressed in ZAP-70 positive patients. IL-4 stimulation brought about up-regulation of the 5p and 3p mature variants of the miR-21 gene, which maps immediately downstream to the VMP1 gene, and of the mature forms generated from the miR-362 (3p and 5p), miR-500a (3p), miR-502 (3p), and miR-532 (3p and 5p) genes, which map within the third intron of the CLCN5 gene. Both genes are in turn regulated by IL-4, suggesting that these miRNAs were regulated by IL-4 as passengers from their carrier genes. Their levels of up-regulation by IL-4 significantly correlated with cytoprotection. MiR-21 has been reported to be leukemogenic, associated to bad prognosis in CLL, and the miRNA more frequently overexpressed in human cancer. Up-regulation by IL-4 of miR-21 and the miRNAs hosted in the CLCN5 locus may contribute to evasion of apoptosis of CLL cells. These findings indicate that the IL-4 pathway and the miRNAs induced by IL-4 are promising targets for the development of novel therapies in CLL.

Dock10, a Cdc42 and Rac1 GEF, induces loss of elongation, filopodia, and ruffles in cervical cancer epithelial HeLa cells.

Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells.

The gene expression response of chronic lymphocytic leukemia cells to IL-4 is specific, depends on ZAP-70 status and is differentially affected by an NFκB inhibitor.

Interleukin 4 (IL-4), an essential mediator of B cell development, plays a role in survival of chronic lymphocytic leukemia (CLL) cells. To obtain new insights into the function of the IL-4 pathway in CLL, we analyzed the gene expression response to IL-4 in CLL and in normal B cells (NBC) by oligonucleotide microarrays, resulting in the identification of 232 non-redundant entities in CLL and 146 in NBC (95 common, 283 altogether), of which 189 were well-defined genes in CLL and 123 in NBC (83 common, 229 altogether) (p<0.05, 2-fold cut-off). To the best of our knowledge, most of them were novel IL-4 targets for CLL (98%), B cells of any source (83%), or any cell type (70%). Responses were significantly higher for 54 and 11 genes in CLL and NBC compared to each other, respectively. In CLL, ZAP-70 status had an impact on IL-4 response, since different sets of IL-4 targets correlated positively or negatively with baseline expression of ZAP-70. In addition, the NFκB inhibitor 6-Amino-4-(4-phenoxyphenethylamino)quinazoline, which reversed the anti-apoptotic effect of IL-4, preferentially blocked the response of genes positively correlated with ZAP-70 (e.g. CCR2, SUSD2), but enhanced the response of genes negatively correlated with ZAP-70 (e.g. AUH, BCL6, LY75, NFIL3). Dissection of the gene expression response to IL-4 in CLL and NBC contributes to the understanding of the anti-apoptotic response. Initial evidence of a connection between ZAP-70 and NFκB supports further exploration of targeting NFκB in the context of the assessment of inhibition of the IL-4 pathway as a therapeutic strategy in CLL, especially in patients expressing bad prognostic markers.

Human and mouse DOCK10 splicing isoforms with alternative first coding exon usage are differentially expressed in T and B lymphocytes.

DOCK10 is a member of the dedicator of cytokinesis (DOCK) family of Rho GTPase activators preferentially expressed in lymphocytes. In this paper, we analyzed DOCK10 mRNA diversity produced because of alternative splicing. Alternative first coding exon usage led to 2 main protein-coding transcripts, DOCK10.1 and DOCK10.2. Full-length cDNA clones of both isoforms were obtained from both normal human peripheral blood mononuclear cells and mouse spleen for the first time for human DOCK10.1, mouse DOCK10.1, and mouse DOCK10.2. Human and mouse DOCK10.1 clones corresponded to the protein coding assemblies provided by the National Center for Biotechnology Information as Reference Sequences for DOCK10. Our analysis especially focused on human cDNA clones, of which 63% were alternatively spliced forms involving diverse exons and introns. DOCK10.1 expression was enriched in normal T cells, and DOCK10.2 expression was enriched in normal B cells and chronic lymphocytic leukemia (CLL) B cells. Both isoforms were upregulated in response to interleukin-4 in B cells, both normal and CLL, but not in T cells. Our data suggest that cell-specific mechanisms regulate expression of the alternative first exon variants of DOCK10 in vertebrates.

Dock10, a novel CZH protein selectively induced by interleukin-4 in human B lymphocytes.

The Dock or CZH proteins are a family of activators for Rho GTPase proteins. The Zizimin subfamily is composed of three members: Dock9, Dock10, and Dock11. We have identified DOCK10 as an interleukin-4 (IL4)-inducible gene in chronic lymphocytic leukemias (CLLs). Subsequently, we have obtained the full-length cDNA sequence, which encodes a 2180 amino acid protein. Dock9 (2069 amino acids) and Dock11 (2073 amino acids) share more identity between them (58%) than with Dock10 (52% and 50%, respectively). Among normal human tissues, DOCK10 and DOCK11 mRNAs were mainly expressed in peripheral blood (PB) leukocytes. Dock10 protein was expressed at similar levels in normal PB-B and PB-T cells. Dock10 protein levels were heterogeneous in CLLs. IL4 consistently increased Dock10 mRNA and protein levels in CLL and normal PB-B cells. In contrast, IL4 did not affect the levels of Dock10 expression in normal PB-T cells. IL4 neither increased DOCK9 or DOCK11 mRNA levels in CLL cells. Dock10 protein distributed in the cytoplasm and nucleus of CLL cells, and IL4 increased its expression in both cellular compartments. The rapid and distinctive induction of Dock10 expression by IL4 in CLL and normal PB-B cells suggests a role for Dock10 in IL4-induced B-cell activation. Dock10 could represent a point of convergence for IL4 signalling and small Rho GTPase function in B cells.