Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer.

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

Responsabilidad profesional derivada del ejercicio de la odontología en Costa Rica: I Parte / Professional responsibility derived from the practice of dentistry in Costa Rica: Part I

El aumento a nivel mundial de las denuncias por responsabilidad profesional en Ciencias de la Salud hace imprescindible que quienes ejerzan esta profesión deban conocer las implicaciones de su labor, así como cumplir con los principios bioéticos y jurídicos para garantizar una sana relación odontólogo-paciente. Es importante comprender que el ejercicio de la odontología en Costa Rica está regulado por normas, códigos y leyes como cualquier otra actividad humana en un Estado de Derecho.

The number of complaints related to professional liability in health sciences has increased worldwide, it is essential for dental professionals to be aware of the implications of their labor, as well as to comply with bioethical and legal principles to ensure a healthy dentist-patient relationship. It is important to understand that the practice of dentistry in Costa Rica is regulated by norms, codes, and laws like any other human activity in a State governed by the rule of law.

Contribution of Host miRNA-223-3p to SARS-CoV-Induced Lung Inflammatory Pathology.

Severe acute respiratory syndrome coronavirus (SARS-CoV) and the closely related SARS-CoV-2 are emergent highly pathogenic human respiratory viruses causing acute lethal disease associated with lung damage and dysregulated inflammatory responses. SARS-CoV envelope protein (E) is a virulence factor involved in the activation of various inflammatory pathways. Here, we study the contribution of host miRNAs to the virulence mediated by E protein. Small RNAseq analysis of infected mouse lungs identified miRNA-223 as a potential regulator of pulmonary inflammation, since it was significantly increased in SARS-CoV-WT virulent infection compared to the attenuated SARS-CoV-ΔE infection. In vivo inhibition of miRNA-223-3p increased mRNA levels of pro-inflammatory cytokines and NLRP3 inflammasome, suggesting that during lung infection, miRNA-223 might contribute to restrict an excessive inflammatory response. Interestingly, miRNA-223-3p inhibition also increased the levels of the CFTR transporter, which is involved in edema resolution and was significantly downregulated in the lungs of mice infected with the virulent SARS-CoV-WT virus. At the histopathological level, a decrease in the pulmonary edema was observed when miR-223-3p was inhibited, suggesting that miRNA-223-3p was involved in the regulation of the SARS-CoV-induced inflammatory pathology. These results indicate that miRNA-223 participates in the regulation of E protein-mediated inflammatory response during SARS-CoV infection by targeting different host mRNAs involved in the pulmonary inflammation, and identify miRNA-223 as a potential therapeutic target in SARS-CoV infection. IMPORTANCE The SARS-CoV-2 pandemic has emphasized the need to understand the mechanisms of severe lung inflammatory pathology caused by human deadly coronaviruses in order to design new antiviral therapies. Here, we identify miRNA-223-3p as a host miRNA involved in the regulation of lung inflammatory response mediated by envelope (E) protein during SARS-CoV infection. miRNAs downregulate the expression of cellular mRNAs and participate in complex networks of mRNA-miRNA interactions that regulate cellular processes. The inhibition of miRNA-223 in infected mice by intranasal administration of antisense RNAs led to changes in the expression of host factors involved in inflammation (cytokines, chemokines, and NLRP3 inflammasome) and in the resolution of lung edema ion transporter CFTR. These results confirmed the contribution of miRNA-223 to the regulation of SARS-CoV-induced pathogenic processes and support the therapeutic potential of inhibiting miRNAs during coronavirus infection using RNA interference approaches.

Toward Tumor Fight and Tumor Microenvironment Remodeling: PBA Induces Cell Cycle Arrest and Reduces Tumor Hybrid Cells' Pluripotency in Bladder Cancer.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1ß. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b+EPCAM+) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers (SOX2, NANOG, miR-302) as compared with non-fused (CD11b-EPCAM+) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10. Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

Genomic Landscape of Vinflunine Response in Metastatic Urothelial Cancer.

BACKGROUND AND AIMS: Metastatic urothelial carcinoma (mUC) remains an incurable disease with limited treatment options after platinum-based chemotherapy and immune checkpoint blockade (ICB). Vinflunine has shown a modest increase in overall survival and remains a therapeutic option for chemo- and immunotherapy refractory tumours. However, biomarkers that could identify responding patients to vinflunine and possible alternative therapies after failure to treatment are still missing. In this study, we aimed to identify potential genomic biomarkers of vinflunine response in mUC patient samples and potential management alternatives. METHODS: Formalin-fixed paraffin-embedded samples of mUC patients (n = 23) from three university hospitals in Spain were used for genomic targeted-sequencing and transcriptome (using the Immune Profile panel by NanoString) analyses. Patients who received vinflunine after platinum-based chemotherapy failure were classified in non-responders (NR: progressive disease ≤ 3 months; n= 11) or responders (R: response ≥ 6 months; n = 12). RESULTS: Genomic characterization revealed that the most common alteration, TP53 mutations, had comparable frequency in R (6/12; 50%) and NR (4/11; 36%). Non-synonymous mutations in KTM2C (4/12; 33.3%), PIK3CA (3/12; 25%) and ARID2 (3/12; 25%) were predominantly associated with response. No significant difference was observed in tumour mutational burden (TMB) between R and NR patients. The NR tumours showed increased expression of diverse immune-related genes and pathways, including various interferon gamma-related genes. We also identified increased MAGEA4 expression as a potential biomarker of non-responding tumours to vinflunine treatment. CONCLUSIONS: Our data may help to identify potential genomic biomarkers of response to vinflunine. Moreover, tumours refractory to vinflunine showed immune signatures potentially associated with response to ICB. Extensive validation studies, including longitudinal series, are needed to corroborate these findings.

Cell Therapies in Bladder Cancer Management.

Currently, bladder cancer (BC) represents a challenging problem in the field of Oncology. The high incidence, prevalence, and progression of BC have led to the exploration of new avenues in its management, in particular in advanced metastatic stages. The recent inclusion of immune checkpoint blockade inhibitors as a therapeutic option for BC represents an unprecedented advance in BC management. However, although some patients show durable responses, the fraction of patients showing benefit is still limited. Notwithstanding, cell-based therapies, initially developed for the management of hematological cancers by infusing immune or trained immune cells or after the engineering of chimeric antigen receptor (CAR) expressing cells, are promising tools to control, or even cure, solid tumors. In this review, we summarize recent cell-based immunotherapy studies, with a special focus on BC.

Análisis de huellas labiales en una muestra de estudiantes de odontología de la Universidad de Costa Rica mediante la clasificación de Renaud: estudio piloto / Lip prints analysis in a sample of dentistry students of the University of Costa Rica based in Renaud's classification: pilot study

Resumen El presente estudio, tuvo como objetivo, analizar los distintos patrones morfológicos presentes en las huellas labiales de una determinada población costarricense y su correlación con las variables, sexo, edad y afinidad biológica. Se analizaron 105 huellas labiales pertenecientes a estudiantes de quinto y sexto año de la carrera de odontología de la Universidad de Costa Rica durante el II semestre del 2016. De las 105 huellas labiales analizadas mediante el método de Renaud, el patrón más prevalente fue el tipo C de manera general, mientras que, de manera individual, en labio superior predomina el patrón tipo A y en labio inferior predomina el patrón tipo C. De la totalidad de muestras analizadas, se destaca que todas revelaron patrones distintos y la correlación estadística entre las variables, sexo, edad y afinidad biológica, permitió conocer la existencia de elementos que podrían ser útiles para identificación de individuos en el ámbito forense. Los resultados demuestran que, con la aplicación de las técnicas adecuadas en la toma de muestras y un análisis estandarizado, las huellas labiales pueden ser utilizadas en el ámbito forense como método de identificación de individuos. Este estudio, además, pone en evidencia la necesidad de realizar más investigación sobre huellas labiales en Costa Rica, no sólo por la necesidad de un aumento en el número de muestras y así obtener mayores resultados estadísticos, si no también, para desarrollar las herramientas necesarias en la toma, revelado y análisis de huellas labiales, así como su aplicabilidad al servicio de la justicia costarricense.

Abstract The objective of this study was to analyze the different morphological patterns present in the lip prints of a certain Costa Rican population and its correlation with the variables, sex, age and biological affinity. 105 lip prints were analyzed belonging to fifth and sixth year students of the dental career of the University of Costa Rica during the second semester of 2016. Of the 105 labials analyzed by the Renaud method, the most prevalent pattern was type C in general, while, individually, in the upper lip, predominantly, the type of pattern, A and the lower lip, predominantly the type C pattern. Of the total of the analyzed samples, the different patterns and the statistical correlation between the variables, sex, age and biological affinity, remarks information that can be useful to identify individuals in the forensic field. The results show that, with the application of the appropriate techniques in the taking of samples and a standardized analysis, the lip prints can be used in the field of forensic science as a method of identification of individuals. This study also highlights the need to conduct more research on lip prints in Costa Rica, not only to increase the number of samples, but also to obtain more extended statistical results. Besides, this research can be used to develop the necessary tools in the taking, development and analysis of lip print. Moreover, the results obtained may be taken into consideration in order to improve forensic research in the Costa Rican justice system.

Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain.

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n=70), ferrets (Mustela putorius furo, n=2), genets (Genetta genetta, n=6), Iberian lynxes (Lynx pardinus, n=6), beech martens (Martes foina, n=8), mongooses (Herpestes ichneumon, n=2), otters (Lutra lutra, n=2), polecats (Mustela putorius, n=2), red foxes (Vulpes vulpes, n=87), wildcats (Felis silvestris, n=2), and wolves (Canis lupus, n=6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003-April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n=1), Asturias (n=69), Basque Country (n=49), Castile-La Mancha (n=38), and Extremadura (n=36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin.

Neuroprotective effects of progesterone and allopregnanolone on long-term cognitive outcome after global cerebral ischemia.

PURPOSE: To assess the longterm neuroprotective effects of progesterone (P4) and allopregnanolone (ALLO) on functional and morphological parameters of the integrity of the hippocampus, after global cerebral ischemia. METHODS: Adult male Sprague-Dawley rats were subjected to a transient severe (20 min) forebrain ischemia (Isch) episode and treated with P4 or ALLO (8 mg/kg i.v.) or its vehicle, at 20 min, 2, 6, 24, 48 and 72 h after ischemia. Rats subjected to Sham procedures, and intact rats were included as nonischemic controls. Three months after ischemia, both the functional (spatial learning and memory, and reference and working memory), and the morphological integrity (dimensions of the hippocampal formation, thickness of the CA1 subfield, and pyramidal neuron population) of the hippocampus and the medial prefrontal cortex(mPFC) were determined. RESULTS: Treatment with P4 or ALLO significantly reduced the impairment in spatial learning and memory, as well as in reference and working memory, and prevented the narrowing of the hippocampus, otherwise induced by ischemia. This better performance of P4 and ALLO treated rats than vehicle (Veh) treated rats, occurred in spite of a loss of pyramidal neurons in the CA1, CA2,CA3 and hilus subfields of the Ammon's horn (remaining neurons: Isch+Veh: 21.0, 35.6, 44.1, and 40.3%; Isch+P4: 19.9, 32.2,41.1, and 32.5%; Isch+ALLO: 25.5, 62.0, 73.7, and 56.7%), and nonsignificant changes in the mPFC, as compared to the Intact group (100%). CONCLUSIONS: Performance of P4 or ALLO treated rats in learning and memory tests suggests that these steroids promoted neural conditions accounting for adequate functioning long after ischemia, in spite of the loss of hippocampal pyramidal neurons.

Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells.

Members of the SR protein family of RNA-binding proteins have numerous roles in mRNA metabolism, from transcription to translation. To understand how SR proteins coordinate gene regulation, comprehensive knowledge of endogenous mRNA targets is needed. Here we establish physiological expression of GFP-tagged SR proteins from stable transgenes. Using the GFP tag for immunopurification of mRNPs, mRNA targets of SRp20 and SRp75 were identified in cycling and neurally induced P19 cells. Genome-wide analysis showed that SRp20 and SRp75 associate with hundreds of distinct, functionally related groups of transcripts that change in response to neural differentiation. Knockdown of either SRp20 or SRp75 led to up- or downregulation of specific transcripts, including identified targets, and rescue by the GFP-tagged SR proteins proved their functionality. Thus, SR proteins contribute to the execution of gene-expression programs through their association with distinct endogenous mRNAs.

Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus.

Progesterone is neuroprotective in models of focal or global ischemia when treatment starts either before the insult or at the onset of reperfusion. In these cases the steroid may act during the occurrence of the early pathophysiological events triggered by ischemia or reperfusion. As opposed to this condition, the aim of the present study was to assess the effect of delayed, post-injury administration of progesterone on the preservation of pyramidal neurons of the hippocampus of rats 21 days after been exposed to global ischemia by the four vessel occlusion model. Progesterone (8 mg/kg, i.v.) or its vehicle, were administered at 20 min, 2, 6, and 24h after the end of ischemia. At histological examination, brains of the ischemic vehicle-treated rats showed a severe reduction of the population of pyramidal neurons in the CA1 and CA2 subfields (12% and 29% remaining neurons, respectively), and a less severe neuronal loss in the CA3 and CA4 subfields of the hippocampus (68% and 63% remaining neurons, respectively), as compared to rats exposed to sham procedures. They also showed a two-fold enlargement of the lateral ventricles and 33% shrinkage of the cerebral cortex as compared to the sham group. Progesterone treatment resulted in a significant preservation of pyramidal neurons in CA1 and CA2 (40% and 62% remaining neurons), with no ventricular dilation and only a mild (12%) cortical shrinkage. Results suggest that progesterone is able to interfere with some late pathophysiological mechanisms leading both to selective neuronal damage in the hippocampal CA1 and CA2 subfields, and to shrinkage of the cerebral cortex.