Assessment of aggressive bladder cancer mutations in plasma cell-free DNA.

Background and aims: The spatial and temporal genetic heterogeneity of bladder cancer (BC) makes challenging to find specific drivers of metastatic disease, thus preventing to determine those BC patients at high risk of tumor progression. Our aim was to identify DNA mutations providing aggressive behavior to bladder tumors and analyze them in patients' cell-free DNA (cfDNA) during their follow-up after radical cystectomy (RC) in order to monitor tumor evolution. Methods: Six BC patients who underwent RC and presented disease progression during their follow-up were included. Next-generation sequencing was used to determine somatic mutations in several primary tumor and metastatic specimens from each patient. Shared DNA mutations between primary bladder tumor and metastatic sites were identified in cfDNA samples through droplet digital PCR. Results: Besides BC genetic heterogeneity, specific mutations in at least one of these genes -TERT, ATM, RB1, and FGFR3- were found in primary tumors and their metastases in all patients. These mutations were also identified in the patients' cfDNA at different follow-up time points. Additionally, the dynamic changes of these mutations in cfDNA allowed us to determine tumor evolution in response to treatment. Conclusion: The analysis of BC mutations associated with poor prognosis in plasma cfDNA could be a valuable tool to monitor tumor evolution, thus improving the clinical management of BC patients.

α-Hispanolol Induces Apoptosis and Suppresses Migration and Invasion of Glioblastoma Cells Likely via Downregulation of MMP-2/9 Expression and p38MAPK Attenuation.

α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G1 population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.

[Characteristics of asthma cases reported as a suspected occupational disease in Navarra. Period 2010-2015]. / Características de los casos de asma notificados como sospecha de enfermedad profesional en Navarra. Años 2010-2015.

OBJECTIVE: Although, occupational asthma (OA) causes great work disability, due to its diagnostic complexity, it is difficult to have it recognized by the official registration systems. To improve its detection, suspected systems have been developed. After years of experience in Navarre, we set out to characterize the suspected cases and compare them with those declared as an occupational disease (OD). METHODS: We identified the cases of OA reported as sentinel events in the Epidemiological Surveillance Program in Occupational Health in Navarra from January 2010 to December 2015 and the cases declared as OD (asthma). The population under study was the Navarra employed population (357,200 on average). In the cases with consent, the clinical and labor characteristics were compared with those of OD. Using the SPSS Statistics® 20v computer program, mean differences were calculated by the T test for independent samples and distribution percentage differences by Chi-square test, Fisher's exact test and nonparametric tests when application conditions were not satisfied. As an indicator of notification, the accumulated incidence per 100,000 workers was calculated for each year of program monitoring. RESULTS: In the period, 79 sentinel events were reported (in 22 the investigation was concluded) and 50 cases of OD (24% relapses). We did not find statistically significant differences in age (41 vs. 40 years), nor gender (40.9% women vs. 44.7%), nor proportion that caused sick leave (45% and 36.8%), nor size, activity of the company or occupation of the worker. Where differences are found was in the meantime of exposure before the recognition/notification, significantly higher in the OD than in the sentinel (94.1 vs. 22.2 months), 72 months longer. CONCLUSIONS: The cases detected by both systems are similar but, due to the diagnostic complexity, the recognition is delayed as OD, which demonstrates the usefulness of a more agile system for reporting suspicions of this work pathology.

OBJETIVO: El asma ocupacional (AO), aunque causa gran discapacidad laboral, debido a su complejidad diagnóstica es de difícil reconocimiento por los sistemas oficiales de registro. Para mejorar su detección se han desarrollado sistemas de sospecha. Tras años de experiencia en Navarra, nos planteamos caracterizar los casos de sospecha y compararlos con los declarados como enfermedad profesional (EEPP). METODOS: Se identificaron los casos de AO notificados como sucesos centinela en el Programa de Vigilancia Epidemiológica en Salud Laboral en Navarra desde enero de 2010 a diciembre de 2015 y los casos declarados como EEPP (asma). La población a estudio fue la población navarra ocupada (357.200 de media). En los casos con consentimiento se compararon las características clínicas y laborales con las de EEPP. Mediante el programa informático SPSS Statistics® 20v se calcularon diferencias de medias mediante prueba T para muestras independientes y diferencias de porcentajes de distribución mediante Test de Chi cuadrado, test exacto de Fisher y test no paramétricos cuando no se cumplían condiciones de aplicación. Como indicador de notificación se calculó la incidencia acumulada por 100.000 trabajadores para cada año de seguimiento del programa. RESULTADOS: En el período de estudio se notificaron 79 sucesos centinela (en 22 se concluyó la investigación) y 50 casos de EEPP ( 24% recaídas). No encontramos diferencias estadísticamente significativas en la edad (41 vs 40 años), ni el sexo (40,9 % mujeres vs 44,7% hombres), ni la proporción que causó baja laboral (45% y 36,8%), ni el tamaño, la actividad de la empresa o la ocupación del trabajador. Donde sí se encontramos diferencias fue en el tiempo medio de exposición antes de la declaración/notificación, significativamente mayor en las EEPP que en los centinela (94,1 vs 22,2 meses), 72 meses mayor. CONCLUSIONES: Los casos detectados por ambos sistemas son similares pero, debido a la complejidad diagnóstica, se retrasa la declaración como EEPP, lo que evidencia la utilidad de un sistema más ágil de notificación de sospechas para esta patología laboral.

A hispanolone-derived diterpenoid inhibits M2-Macrophage polarization in vitro via JAK/STAT and attenuates chitin induced inflammation in vivo.

Macrophages are highly plastic cells that adopt different functional phenotypes in response to environmental signals. Classically activated macrophages (M1) exhibit a pro-inflammatory role, mediating host defense against microorganisms or tumor cells; whereas alternatively activated macrophages (M2) perform a range of physiological processes, including inflammation, wound repair and tissue remodeling. Interestingly, M2 macrophages have been involved in pathological settings such as tumor progression, parasitic infection and respiratory disorders. Consequently, the search of new agents able to control macrophage polarization is on the basis of new therapeutic strategies. In the present study, we have evaluated the effect of the hispanolone derivative 8,9-dehydrohispanolone-15,16-lactol (DHHL) on M2 macrophage polarization. Our results reveal that DHHL significantly inhibited IL-4- or IL-13-stimulated M2 macrophage activation, as showed by reduced expression of M2 markers. In addition, DHHL suppressed IL-4-induced STAT-6 and JAK-1 tyrosine phosphorylation, suggesting that this compound inhibited M2 polarization by suppressing the JAK-STAT signaling pathway. Finally, DHHL prevented eosinophil recruitment and the presence of F4/80+-CD206+ M2-like macrophages in an in vivo model of M2 polarization via administration of chitin. Collectively, these results confirm DHHL as a novel regulator of macrophage polarization suitable to design future therapies towards M2-macrophages mediated pathologies.

Tumor suppressor ARF regulates tissue microenvironment and tumor growth through modulation of macrophage polarization.

Tumor microenvironment has been described to play a key role in tumor growth, progression, and metastasis. Macrophages are a major cellular constituent of the tumor stroma, and particularly tumor associated macrophages (TAMs or M2-like macrophages) exert important immunosuppressive activity and a pro-tumoral role within the tumor microenvironment. Alternative-reading frame (ARF) gene is widely inactivated in human cancer. We have previously demonstrated that ARF deficiency severely impairs inflammatory response establishing a new role for ARF in the regulation of innate immunity. On the basis of these observations, we hypothesized that ARF may also regulates tumor growth through recruitment and modulation of the macrophage phenotype in the tumor microenvironment. Xenograft assays of B16F10 melanoma cells into ARF-deficient mice resulted in increased tumor growth compared to those implanted in WT control mice. Tumors from ARF-deficient mice exhibited significantly increased number of TAMs as well as microvascular density. Transwell assays showed crosstalk between tumor cells and macrophages. On the one hand, ARF-deficient macrophages modulate migratory ability of the tumor cells. And on the other, tumor cells promote the skewing of ARF-/- macrophages toward a M2-type polarization. In conclusion, these results demonstrate that ARF deficiency facilitates the infiltration of macrophages into the tumor mass and favors their polarization towards a M2 phenotype, thus promoting tumor angiogenesis and tumor growth. This work provides novel information about the critical role of ARF in the modulation of tumor microenvironment.

8,9-Dehydrohispanolone-15,16-lactol diterpene prevents LPS-triggered inflammatory responses by inhibiting endothelial activation.

Endothelial activation contributes to lung inflammatory disorders by inducing leucocyte recruitment to pulmonary parenchyma. Consequently, vascular-targeted therapies constitute promising strategies for the treatment of inflammatory pathologies. In the present study, we evaluated the effect of 8,9-dehydrohispanolone-15,16-lactol diterpene (DT) on lung endothelium during inflammation. Lung endothelial cells pre-treated with DT and activated with lipopolysaccharide (LPS) or tumour necrosis factor-α (TNF-α) exhibited reduced expression of the pro-inflammatory cytokines Cxcl10, Ccl5 and Cxcl1, whereas the anti-inflammatory molecules IL1r2 and IL-10 were induced. Consistent with this result, DT pre-treatment inhibited nuclear factor κB (NF-κB) nuclear translocation, by interfering with IκBα phosphorylation, and consequently NF-κB transcriptional activity in endothelium activated by LPS or TNF-α. Furthermore, DT, probably through p38 signalling, induced transcriptional activation of genes containing activator protein 1 (AP-1)-binding elements. Inhibition of p38 prevented IL1r2 mRNA expression in endothelium incubated with DT alone or in combination with LPS or TNF-α. Accordingly, conditioned medium (CM) from these cells failed to stimulate leucocytes as measured by a reduction in adhesive ability of the leucocyte cell line J774 to fibronectin (FN). Additionally, DT reduced the expression of the endothelial adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) after activation. Similarly, expression of VCAM-1 and ICAM-1 molecules on the lung endothelial layer of C57/BL6 mice pre-treated with DT and challenged with LPS were unchanged. Finally, inhibition of vascular adhesion molecule expression by DT decreased the interaction of J774 cells with lung endothelial cells in an inflammatory environment. Our findings establish DT as a novel endothelial inhibitor for the treatment of inflammatory-related diseases triggered by Gram-negative bacteria or by the associated cytokine TNF-α.

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation.

Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells.

Critical role of p38 MAPK in IL-4-induced alternative activation of peritoneal macrophages.

Alternative activation of macrophages plays an important role in a range of physiological and pathological processes. This alternative phenotype, also known as M2 macrophages, is induced by type 2 cytokines such as IL-4. The binding of IL-4 to its receptor leads to activation of two major signaling pathways: STAT-6 and PI3K. However, recent studies have described that p38 MAPK might play a role in IL-4-dependent signaling in some cells, although its role in macrophages is still controversial. In this study, we investigated whether p38 MAPK plays a role in the polarization of macrophages in mice. Our results reveal that IL-4 induces phosphorylation of p38 MAPK in thioglycollate-elicited murine peritoneal macrophages, in addition to STAT-6 and PI3K activation. Furthermore, p38 MAPK inactivation, by gene silencing or pharmacological inhibition, suppressed IL-4-induced typical M2 markers, indicating the involvement of p38 MAPK in the signaling of IL-4 leading to M2-macrophage polarization. Moreover, p38 MAPK inhibition blocked phosphorylation of STAT-6 and Akt, suggesting that p38 MAPK is upstream of these signaling pathways. Finally, we show that in an in vivo model of chitin-induced M2 polarization, p38 MAPK inhibition also diminished activation of M2 markers. Taken together, our data establish a new role for p38 MAPK during IL-4-induced alternative activation of macrophages.

Demographic, clinical, and genetic characteristics of patients with medullary thyroid cancer in the past 16 years in Castilla-La Mancha.

OBJECTIVE: Medullary thyroid cancer is a rare tumor that is more aggressive and has a worse prognosis than differentiated thyroid cancer. The purpose of this study was to report the demographic, clinical, and genetic characteristics of patients seen in the health care system of the community of Castilla-La Mancha over a 16-year period. PATIENTS AND METHODS: Data were collected through a review of patients' medical records. RESULTS: The medical records of 58 patients (mean age at diagnosis, 51 years; range, 6-82 years; 63.8% women) were reviewed. Prevalence rate was 2.84 cases per 100,000 inhabitants, with a high variability between areas (range, 0-5.4 cases per 100,000 inhabitants). Familial cases accounted for 34.5% of all medullary thyroid cancers, and the most common mutation was C634Y. The condition was most commonly diagnosed following palpation of a cervical lump (70.6%). At diagnosis, 56 of 58 patients underwent ultrasound and 8 of 58 patients were tested for serum calcitonin. Tumor multicentricity was reported in 59 and 50% of patients with multiple endocrine neoplasia syndrome type 2A and 2B, respectively, and in no sporadic cases. Fifty-two percent of patients had an advanced stage (iii or iv) at diagnosis. Median follow-up was 36 months (interquartile range, 14-210); 11 patients were lost to follow-up. CONCLUSIONS: In Castilla-La Mancha, medullary thyroid cancer is diagnosed by cervical ultrasound, rather than calcitonin assay. There is a high prevalence of both familial and sporadic medullary thyroid cancer, and a significant variability in the type of proto-oncogen rearranged during transfection mutation as compared to the rest of the Spanish population.

Role of the tumor suppressor ARF in macrophage polarization: Enhancement of the M2 phenotype in ARF-deficient mice.

The ARF locus is frequently inactivated in human cancer. The oncosuppressor ARF has indeed been described as a general sensor for different situation of cellular stress. We have previously demonstrated that ARF deficiency severely impairs inflammatory responses in vitro and in vivo, establishing a role for ARF in the regulation of innate immunity. The aim of the present work was to get further insights into the immune functions of ARF and to evaluate its possible contribution to the polarization of macrophages toward the M1 or M2 phenotype. Our results demonstrate that resting Arf(-/-) macrophages express high levels of Ym1 and Fizz-1, two typical markers of alternatively-activated macrophages (M2). Additionally, Arf(-/-) peritoneal macrophages showed an impaired response to lipopolysaccharide (a classical inducer of M1 polaryzation) and a reduced production of pro-inflammatory cytokines/chemokines. Moreover, upon stimulation with interleukin-4 (IL-4), an inducer of the M2 phenotype, well established M2 markers such as Fizz-1, Ym1 and arginase-1 were upregulated in Arf(-/-) as compared with wild type macrophages. Accordingly, the cytokine and chemokine profile associated with the M2 phenotype was significantly overexpressed in Arf(-/-) macrophages responding to IL-4. In addition, multiple pro-angiogenic factors such as VEGF and MMP-9 were also increased. In summary, these results indicate that ARF contributes to the polarization and functional plasticity of macrophages.

Metabolic control and treatment patterns in patients with type 1 diabetes in Castilla-La Mancha: the DIAbetes tipo 1 in Castilla La Mancha study.

OBJECTIVE: To assess glycemic control, the degree of control of cardiovascular risk factors, and treatment schemes used in patients with type 1 diabetes mellitus (T1DM) in Castilla-La Mancha (Spain). PATIENTS AND METHODS: A cross-sectional, multicenter study on adult patients with T1DM seen at outpatient endocrinology clinics for 12 months (from September 2009 to August 2010). Diabetes duration was > 5 years in all cases. Sociodemographic, clinical, anthropometric, and laboratory variables were collected, as well as treatment data. A multivariate logistic regression analysis was used to assess variables independently associated to good glycemic control. RESULTS: A total of 1465 patients (48.5% women) with a mean age of 39.4±13.5 years and a mean diabetes duration of 19.4±10.6 years, were enrolled. Mean glycosylated hemoglobin (HbA1c) level was 7.8%, and 26% had HbA1c values ≤7%. Predictors of good glycemic control (HBA1c ≤7%) included intensive insulin treatment [odds ratio (OR): 2.56], non-smoking status (OR: 1.66), and a higher educational level (OR: 1.33). Fifteen percent of patients were obese, 35% had dyslipidemia, 23% were hypertensive, and 26% smoked. Four or more of the recommended control goals were achieved by 68% of patients, but more than 33% required additional drug treatment. CONCLUSIONS: Glycemic control was inadequate in this cohort of T1DM patients. Promotion of healthy attitudes and intensification of insulin treatment may improve glycemic control. Prevalence of cardiovascular risk factors is high, although a great proportion of patients achieve good lipid and blood pressure control.

Labdanolic acid methyl ester (LAME) exerts anti-inflammatory effects through inhibition of TAK-1 activation.

Labdane derivatives obtained from the diterpenoid labdanediol suppressed NO and PGE(2) production in LPS-stimulated RAW 264.7 macrophages. However, mechanisms involved in these inhibitory effects are not elucidated. In this study, we investigated the signaling pathways involved in the anti-inflammatory effects of labdanolic acid methyl ester (LAME) in peritoneal macrophages and examined its therapeutic effect in a mouse endotoxic shock model. LAME reduced the production of NO and PGE(2) in LPS-activated macrophages. This effect involved the inhibition of NOS-2 and COX-2 gene expression, acting at the transcription level. Examination of the effects of the diterpene on NF-κB signaling showed that LAME inhibits the phosphorylation of IκBα and IκBß, preventing their degradation and the nuclear translocation of the NF-κB p65 subunit. Moreover, inhibition of MAPK signaling was also observed. A further experiment revealed that LAME inhibited the phosphorylation of transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1), an upstream signaling molecule required for IKK and mitogen-activated protein kinases (MAPKs) activation. Inflammatory cytokines such as IL-6, TNF-α and IP-10 were downregulated in the presence of this compound after stimulation with LPS. Additionally, LAME also improved survival in a mouse model of endotoxemia and reduced the circulatory levels of cytokines (IL-6, TNF-α). In conclusion, these results indicate that labdane diterpene LAME significantly attenuates the pro-inflammatory response induced by LPS both in vivo and in vitro.