Interaction between Akkermansia muciniphila and Diet Is Associated with Proinflammatory Index in School-Aged Children.

BACKGROUND: Imbalance in the intestinal microbiota can lead to chronic low-grade inflammation. Diet may influence this association. In this study, we aimed to evaluate the interaction between Akkermansia muciniphila (A. muciniphila) and dietary patterns using a proinflammatory index. METHODS: We conducted a cross-sectional study with school-aged children. We quantified the relative abundance (RA) of A. muciniphila in feces using a polymerase chain reaction. We collected dietary information through employing a food frequency questionnaire and generated dietary patterns using principal component analysis. We generated a proinflammatory index from serum levels of interleukin-6, interleukin-10, tumor necrosis factor alpha, and adiponectin validated by receptor operating characteristic curves. We evaluated the association between A. muciniphila and the proinflammatory index using logistic regression, including an interaction term with dietary patterns. RESULTS: We found that children with a low RA of A. muciniphila and a high intake of simple carbohydrates and saturated fats had increased odds of being high on the proinflammatory index. However, when the consumption of this dietary pattern is low, children with a low RA of A. muciniphila had decreased odds of being high on the proinflammatory index. CONCLUSIONS: Our results suggest that the simultaneous presence of A. muciniphila and diet have a more significant impact on the presence of being high on the proinflammatory index compared to both factors separately.

Interferon-tau (IFN-τ) Has Antiproliferative Effects, Induces Apoptosis, and Inhibits Tumor Growth in a Triple-negative Breast Cancer Murine Tumor Model.

BACKGROUND/AIM: Resistant triple-negative breast cancer (TNBC) is a subtype of this disease that is resistant to conventional chemotherapy agents. IFN-τ is a cytokine that has recently been shown to have immunoregulatory and antitumor effects. The present study aimed to examine the antiproliferative and apoptosis effects of IFN-τ in breast cancer cells and the antitumor effect in a murine tumor model of TNBC. MATERIALS AND METHODS: Murine breast cancer 4T1 cells were cultured and treated with ovine IFN-τ and through MTT and Caspase-Glo 3/7 assays, viability and cell death were determined. In addition, the antitumor effect of IFN-τ was determined in a murine tumor model of TNBC. RESULTS: Ovine IFN-τ showed a concentration-dependent antiproliferative effect on 4T1 murine breast cancer cells. Also, treatment of 4T1 cells with IFN-τ induced the activation of caspase 3 and 7, which is indicative of apoptotic cell death. Moreover, we detected an increase in the expression of type I interferon receptor (IFNAR1/2) in cells treated with IFN-. The intratumoral application of IFN-τ in mice inhibited tumor growth compared to the control non-treated group, and the effect was associated with the increased expression of GM-CSF. CONCLUSION: Ovine IFN-τ may be an effective immunotherapeutic cytokine for the treatment of TNBC.

High Relative Abundance of Staphylococcus aureus and Serum Cytokines Are Associated with Cardiometabolic Abnormalities in Children.

Background: The alteration in the composition of the gut microbiota has been associated with an increased risk of developing cardiovascular and metabolic diseases. The present study evaluated the association between the relative abundance (RA) of intestinal Staphylococcus aureus and the inflammatory response with cardiometabolic alterations in children. Methods: This cross-sectional study included 1142 children (age 6-12 years), which were classified by degree of adiposity. Anthropometry, cardiometabolic markers, and RA of intestinal S. aureus were measured. Cytokine concentrations were available in 626 children. Path coefficients (PC) were estimated by path analysis. Results: RA of S. aureus was positively associated with cholesterol PC = 24.98 (95% CI 10.76 to 39.21) and negatively with triglycerides PC = -13.10 (95% CI -22.73 to -3.48). Body mass index (BMI) Z-scores had significant mediation effects on the association between RA of S. aureus with waist circumference PC = 2.87 (95% CI 0.58 to 5.16), triglycerides PC = 6.63 (95% CI 1.29 to 11.98), low-density lipoproteins (LDL) PC = 1.73 (95% CI 0.27 to 3.18), and high-density lipoproteins PC = -1.20 (95% CI -2.19 to -0.22). Interleukin 6 (IL-6) was negatively associated with glucose PC = -3.01 (95% CI -5.85 to -0.17) and LDL PC = -8.65 (95% CI -16.54 to -0.77), and interleukin 10 (IL-10) was positively associated with glucose PC = 3.37 (95% CI 0.47 to 6.26). Conclusions: It is suggested that the RA of S. aureus, IL-6, and IL-10 are associated with cardiometabolic alterations in children, where BMI Z-scores have an important mediating effect for the development of these.

Induction of Therapeutic Protection in an HPV16-Associated Mouse Tumor Model Through Targeting the Human Papillomavirus-16 E5 Protein to Dendritic Cells.

HPV E5 is an oncoprotein mainly expressed in premalignant lesions, which makes it an important target for a vaccine to prevent or cure cervical cancer (CC). In this study, we evaluated whether E5 targeted to DEC-205, present in dendritic cells (DCs), could induce a therapeutic protection against HPV16-induced tumor cells in a mouse model. The HPV-16 E5 (16E5) protein was cross-linked to a monoclonal antibody (mAb) specific to mouse DEC-205 (anti-DEC-205:16E5) or to an isotype control mAb (isotype:16E5). Rotavirus VP6 was cross-linked to the mouse anti-DEC-205 mAb (anti-DEC-205:VP6) as a non-specific antigen control. BALB/c mice were inoculated subcutaneously (s.c.) with the 16E5-expressing BMK-16/myc tumor cells, and 7 and 14 days later the mice were immunized s.c. with the conjugates, free 16E5 or PBS in the presence of adjuvant. Tumor growth was monitored to evaluate protection. A strong protective immune response against the tumor cells was induced when the mice were inoculated with the anti-DEC-205:16E5 conjugate, since 70% of the mice controlled the tumor growth and survived, whereas the remaining 30% developed tumors and died by day 72. In contrast, 100% of the mice in the control groups died by day 30. The anti-DEC-205:16E5 conjugate was found to induce 16E5-specific memory T cells, with a Th1/Th17 profile. Both CD4+ and CD8+ T cells contributed to the observed protection. Finally, treating mice that had developed tumors with an anti-PD-1 mAb, delayed the tumor growth for more than 20 days. These results show that targeting 16E5 to DEC-205, alone or combined with an immune checkpoint blockade, could be a promising protocol for the treatment of the early stages of HPV-associated cancer.

Health status and gerontological evaluation in Mexican older adults in the face of the COVID-19 pandemic.

INTRODUCTION: Older adults constitute the most vulnerable population group to the COVID-19 pandemic. In Mexico, their biopsychosocial conditions might intensify their vulnerability. METHOD: Affiliation to health systems, health conditions and gerontological evaluation of 3,218 older adults were analyzed following the methodology of the PAHO-Mexico Health, Well-being and Aging Survey. RESULTS: 88.6 % of older adults referred being affiliated to health systems; 30.2 %, 52.4 %, 10.3 %, 4.1 % and 5.6 % referred suffering from diabetes mellitus, high blood pressure, chronic obstructive pulmonary disease, heart disease and cerebrovascular disease, respectively; 15.6 % reported urinary incontinence, and 11.3%, fecal incontinence; 12.1 % of the women referred having suffered from breast cancer at some point, and 6.3 %, cervical cancer. The habit of smoking tobacco was observed in 11.1 %, risk of malnutrition in 32.8 %, established malnutrition in 4.1 %, functional dependence for basic and instrumental activities of daily life in 16.3 % and 17.6 %, respectively. CONCLUSION: Comprehensive gerontological evaluation is essential for efficient care of older adults who suffer from COVID-19, and for adequate care of the effects or health conditions at the conclusion of the confinement imposed by the pandemic.

INTRODUCCIÓN: Los adultos mayores constituyen el grupo más vulnerable ante la pandemia por COVID-19; en México, sus condiciones biopsicosociales podrían potenciar su vulnerabilidad. MÉTODO: Se analizó afiliación a sistemas de salud, condiciones de salud y evaluación gerontológica de 3218 adultos mayores conforme a la metodología de la Encuesta Salud, Bienestar y Envejecimiento OPS-México. RESULTADOS: 88.6 % de los adultos mayores refirió afiliación a un sistema de salud; 30.2, 52.4, 10.3, 4.1 y 5.6 % indicaron padecer diabetes mellitus, hipertensión arterial, enfermedad pulmonar obstructiva crónica, enfermedad cardiaca y evento vascular cerebral, respectivamente; 15.6 % reportó incontinencia urinaria y 11.3 %, fecal; 12.1 % de las mujeres indicó haber padecido en algún momento cáncer de mama y 6.3 %, cáncer cervicouterino. Se observó hábito de fumar tabaco en 11.1 %, riesgo de malnutrición en 32.8 %, malnutrición establecida en 4.1 %, dependencia funcional para las actividades básicas en 16.3 % e instrumentales de la vida diaria en 17.6 %. CONCLUSIÓN: La evaluación gerontológica integral es fundamental para la atención eficiente de los adultos mayores que padecen COVID-19 y para la adecuada atención por los efectos o condiciones de salud al terminar el confinamiento por la pandemia.

Frecuencia de sintomatología depresiva y tratamiento en adultos mayores con acceso a servicios de salud / Frequency of depressive symptoms and treatment in older adults with access to health services / Frequência de sintomas depressivos e tratamento em idosos com acesso a serviços de saúde / Fréquence de la symptomatologie dépressive et de son traitement chez les personnes âgées ayant accès aux services de santé

Resumen Objetivo: Determinar la frecuencia de sintomatología depresiva y su tratamiento en personas adultas mayores, afiliadas a las instituciones de seguridad social de salud en México. Material y métodos: Estudio descriptivo transversal con 3,114 participantes de 7 estados del país, los cuales fueron evaluados psicológicamente, empleando la Escala de Depresión Geriátrica (GDS) de Yesavage, también se analizó su afiliación a seguridad social, funcionalidad (índices Barthel y Lawton) y estado cognoscitivo (Minimental de Folstein). El análisis de la información se realizó empleando el paquete estadístico SPSS (Statistical Package for the Social Sciences), así como los datos sociodemográficos de los participantes. Resultados: Se observó cobertura en el 88.5% de la población. La prevalencia de depresión es del 27.4% y el 96.8% de ellos no recibe tratamiento, además el 18.9% presenta dependencia funcional, el 16.2% deterioro cognoscitivo y el 7.4% ha sufrido al menos una caída con lesiones. Únicamente el 11.2% del total de enfermos fue diagnosticado en su clínica. Conclusión: La prevalencia de depresión no tratada en adultos mayores con acceso a servicios de salud es altamente significativa, es urgente la generación de protocolos, programas y capacitación de profesionales de salud que permitan identificar la patología y atender de manera integral a dicho grupo etario.

Abstract Objective: to determine the frequency of depressive symptoms and their treatment in older adults affiliated to the social health security institutions in Mexico. Material and methods: a descriptive cross-sectional study with 3,114 participants from 7 states of the country who were psychologically evaluated using the Geriatric Depression Scale (GDS) by Yesavage, also analyzed their affiliation to social security, functionality (Barthel and Lawton index) and cognitive status (Minimental State Examination of Folstein). The analysis of the information was carried out using the statistical package SPSS (Statistical Package for the Social Sciences) as well as the sociodemographic data of the participants. Results: Coverage was observed in 88.5% of the population. Prevalence of depression was 27.4% and 96.8% of them did not receive treatment. In addition 18.9% had functional dependence, 16.2% had cognitive impairment and 7.4% had suffered at least one fall with injuries. Only 11.2% of all participants were diagnosed in their clinic. Conclusion: The prevalence of untreated depression in older adults with access to health services is highly significant. It is urgent the generation of protocols, programs and training of health professionals that allow to identify the pathology and provide comprehensive care for that age group.

Sumário Objetivo: determinar a frequência dos sintomas depressivos e seu tratamento em idosos afiliados às instituições de saúde previdenciária do México. Material e métodos: estudo descritivo transversal com 3.114 participantes de 7 estados do país, avaliados psicologicamente pela Escala de Depressão Geriátrica Yesavage {GDS}, analisando também sua afiliação à seguridade social, funcionalidade (índices de Barthel e Lawton) e estado cognitivo. (Minimental de Folstein). A análise das informações foi realizada no programa estatístico SPSS (Statistical Package for the Social Sciences), além dos dados sociodemográficos dos participantes. Resultados: A cobertura foi observada em 88,5% da população. A prevalência de depressão é de 27,4% e 96,8% deles não recebem tratamento; além disso, 18,9% possuem dependência funcional, 16,2% comprometimento cognitivo e 7,4% sofreram pelo menos uma queda com lesões. Apenas 11,2% de todos os pacientes foram diagnosticados em sua clínica. Conclusão: A prevalência de depressão não tratada em idosos com acesso a serviços de saúde é altamente significativa, é urgente a geração de protocolos, programas e treinamento de profissionais de saúde para identificar a patologia e prestar assistência integral a essa faixa etária.

Résumé Objectif: Déterminer la fréquence de la symptomatologie dépressive et de son traitement chez les personnes âgées affiliées aux institutions de santé de la sécurité sociale au Mexique. Matériel et méthodes: Étude descriptive transversale avec 3 114 participants de 7 états du pays, évalués psychologiquement avec l'échelle de dépression gériatrique de Yesavage (GDS). Leur affiliation à la sécurité sociale, leur fonctionnalité (indices de Barthel et Lawton) et leur état cognitif (Folstein Minimal) ont également été évalués. L'analyse de ces informations, ainsi que des données sociodémographiques des participants, a été réalisée à l'aide du logiciel statistique SPSS (Statistical Package for the Social Sciences). Résultats: La couverture sociale a été observée chez 88,5 % de la population. La prévalence de la dépression est de 27,4% et 96,8% d'entre eux ne reçoivent pas de traitement. De plus, 18,9% présentent une dépendance fonctionnelle, 16,2% une déficience cognitive et 7,4% ont fait au moins une chute avec blessures. Seulement 11,2 % du nombre total des participants ayant une dépression ont été diagnostiqués dans leur clinique. Conclusion: La prévalence de la dépression non traitée chez les personnes âgées ayant accès aux services de santé est très importante. Il est urgent de générer des protocoles, des programmes et des formations pour les professionnels de santé qui permettent d'identifier la pathologie et de fournir une attention globale pour cette tranche d'âge.

Estado de salud y valoración gerontológica en adultos mayores mexicanos ante la pandemia por COVID-19 / Health status and gerontological evaluation in Mexican older adults in the face of the COVID-19 pandemic

Resumen Introducción: Los adultos mayores constituyen el grupo más vulnerable ante la pandemia por COVID-19; en México, sus condiciones biopsicosociales podrían potenciar su vulnerabilidad. Método: Se analizó afiliación a sistemas de salud, condiciones de salud y evaluación gerontológica de 3218 adultos mayores conforme a la metodología de la Encuesta Salud, Bienestar y Envejecimiento OPS-México. Resultados: 88.6 % de los adultos mayores refirió afiliación a un sistema de salud; 30.2, 52.4, 10.3, 4.1 y 5.6 % indicaron padecer diabetes mellitus, hipertensión arterial, enfermedad pulmonar obstructiva crónica, enfermedad cardiaca y evento vascular cerebral, respectivamente; 15.6 % reportó incontinencia urinaria y 11.3 %, fecal; 12.1 % de las mujeres indicó haber padecido en algún momento cáncer de mama y 6.3 %, cáncer cervicouterino. Se observó hábito de fumar tabaco en 11.1 %, riesgo de malnutrición en 32.8 %, malnutrición establecida en 4.1 %, dependencia funcional para las actividades básicas en 16.3 % e instrumentales de la vida diaria en 17.6 %. Conclusión: La evaluación gerontológica integral es fundamental para la atención eficiente de los adultos mayores que padecen COVID-19 y para la adecuada atención por los efectos o condiciones de salud al terminar el confinamiento por la pandemia.

Abstract Introduction: Older adults constitute the most vulnerable population group to the COVID-19 pandemic. In Mexico, their biopsychosocial conditions might intensify their vulnerability. Method: Affiliation to health systems, health conditions and gerontological evaluation of 3,218 older adults were analyzed following the methodology of the PAHO-Mexico Health, Well-being and Aging Survey. Results: 88.6 % of older adults referred being affiliated to health systems; 30.2 %, 52.4 %, 10.3 %, 4.1 % and 5.6 % referred suffering from diabetes mellitus, high blood pressure, chronic obstructive pulmonary disease, heart disease and cerebrovascular disease, respectively; 15.6 % reported urinary incontinence, and 11.3%, fecal incontinence; 12.1 % of the women referred having suffered from breast cancer at some point, and 6.3 %, cervical cancer. The habit of smoking tobacco was observed in 11.1 %, risk of malnutrition in 32.8 %, established malnutrition in 4.1 %, and functional dependence for basic and instrumental activities of daily life in 16.3 % and 17.6 %, respectively. Conclusion: Comprehensive gerontological evaluation is essential for efficient care of older adults who suffer from COVID-19, and for adequate care of the effects or health conditions at the conclusion of the confinement imposed by the pandemic.

Bovine Interferon-Tau Activates Type I interferon-Associated Janus-signal Transducer in HPV16-positive Tumor Cell.

The mechanisms of signal transduction by interferon-tau (IFN-τ) are widely known during the gestation of ruminants. In trophoblast cells, IFN-τ involves the activation of the JAK-STAT pathway, and it can have effects on other cell types, such as tumor cells. Here we report that the HPV16-positive BMK-16/myc cell treated with ovine IFN-τ, results in the activation of the canonical JAK-STAT and non-canonical JAK-STAT pathway. The MAPK signaling pathway was activated, we detected the proteins MEK1, MEK2, Raf1, STAT3, STA4, STAT5 and STAT6. Moreover, IFN-τ induced the expression of MHC Class I, MX and IP10 in the tumor cells and this response may be associated with the viral replication and with the anti-proliferative and the immunoregulatory effects of IFN-τ.

Transregulation of microRNA miR-21 promoter by AP-1 transcription factor in cervical cancer cells.

BACKGROUND: Gene expression profiles have demonstrated that miR-21 expression is altered in almost all types of cancers and it has been classified as an oncogenic microRNA. Persistent HPV infection is the main etiologic agent in cervical cancer and induces genetic instability, including disruption of microRNA gene expression. In the present study, we analyzed the underlying mechanism of how AP-1 transcription factor can active miR-21 gene expression in cervical cancer cells. METHODS: To identify that c-Fos and c-Jun regulate the expression of miR-21 we performed RT-qPCR and western blot assays. We analyzed the interaction of AP-1 with miR-21 promoter by EMSA and ChIP assays and determined the mechanism of its regulation by reporter construct plasmids. We identified the nuclear translocation of c-Fos and c-Jun by immunofluorescence microscopy assays. RESULTS: We demonstrated that c-Fos and c-Jun proteins are expressed and regulate the expression of miR-21 in cervical cancer cells. DNA sequence analysis revealed the presence of AP-1 DNA-binding sites in the human miR-21 promoter region. EMSA analyses confirmed the interactions of the miR-21 upstream transcription factor AP-1. ChIP assays further showed the binding of c-Fos to AP-1 sequences from the miR-21 core promoter in vivo. Functional analysis of AP-1 sequences of miR-21 in reporter plasmids demonstrated that these sequences increase the miR-21 promoter activation. CONCLUSIONS: Our findings suggest a physical interaction and functional cooperation between AP-1 transcription factor in the miR-21 promoter and may explain the effect of AP-1 on miR-21 gene expression in cervical cancer cells.

Estudio de validación de cuatro diferentes criterios para el diagnóstico de síndrome metabólico en población infantil / Validation study of four different criteria for the diagnosis of metabolic syndrome in children

Resumen Introducción: El síndrome metabólico (SM) es un problema de salud pública, el cual no cuenta con estrategias adecuadas de prevención, diagnóstico y tratamiento para población infantil. Los criterios existentes son controversiales y no son aplicables en los niños. Asimismo, varían según autores y comités de expertos; lo que podría tener importantes consecuencias en el diagnóstico de SM, impactando el tratamiento oportuno y el pronóstico del individuo. Objetivo: Validar criterios (NCEP-ATPIII; Cook, Ford y Duncan, et al; Ferranti, et al; Cruz, et al; e IDF1) para el diagnóstico de SM en niños mexicanos. Metodología: Estudio transversal de 2599 niños entre 6 y 16 años, residentes de la Ciudad de México. Se consideró SM con tres o más de los cinco componentes en los distintos criterios; y dos o más componentes con la presencia de obesidad central para IDF. Se consideró como Gold Standard la combinación de los cinco criterios diagnósticos. Para identificar el mejor valor predictivo se calculó sensibilidad, especificidad, valor predictivo positivo (VPP), valor predictivo negativo (VPN) y razón de verosimilitud. Resultados: Se observó una mayor proporción de individuos diagnosticados con SM con el criterio de Ferranti, et al. en comparación con los demás criterios evaluados. Nuestra propuesta ad hoc presentó una alta sensibilidad (0,89) y especificidad (0,90) frente al Gold Standard aplicado. Conclusión: El criterio propuesto por nosotros contiene una elección de componentes sencillos y de bajo costo, que facilitará su aplicación, permitiendo la unificación en el diagnóstico, tratamiento y pronóstico poblacional, reduciendo los índices de morbimortalidad en mexicanos.

Abstract Introduction: Metabolic syndrome (MS) is a public health problem without appropriate strategies for prevention, diagnosis and treatment in children. Existing criteria are controversial and not applicable for pediatric population, with variations according to different authors and expert committees, which could have important consequences in MS diagnosis, treatment and prognosis. Objective: To validate different definitions (NCEP-ATPIII; Cook, Ford and Duncan, et al; Ferranti, et al; Cruz, et al; and IDF1) for metabolic syndrome diagnosis in Mexican children. Methodology: Cross-sectional study of 2599 children aged between 6 and 16 years, residents of Mexico City. MS was defined as the presence of three or more of the five components in the different criteria; and two or more components with the presence of central obesity for IDF. The Gold Standard was considered as the combination of the five diagnostic criteria. To identify the best predictive value, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratio were calculated. Results: A greater proportion of individuals diagnosed with the Ferranti, et al criterion was observed in comparison with the other criteria evaluated. We proposed an ad hoc criteria which showed a high sensitivity (0,89) and specificity (0,90) compared to the Gold Standard applied. Conclusion: Our diagnostic criteria contains a choice of simple and low-cost components that will facilitate its application in health institutions and will unify-diagnostic criteria, treatment, and prognosis, reducing morbidity and mortality rates in Mexican population.

3D-Hydrogel Based Polymeric Nanoreactors for Silver Nano-Antimicrobial Composites Generation.

This study underscores the development of Ag hydrogel nanocomposites, as smart substrates for antibacterial uses, via innovative in situ reactive and reduction pathways. To this end, two different synthetic strategies were used. Firstly thiol-acrylate (PSA) based hydrogels were attained via thiol-ene and radical polymerization of polyethylene glycol (PEG) and polycaprolactone (PCL). As a second approach, polyurethane (PU) based hydrogels were achieved by condensation polymerization from diisocyanates and PCL and PEG diols. In fact, these syntheses rendered active three-dimensional (3D) hydrogel matrices which were used as nanoreactors for in situ reduction of AgNO3 to silver nanoparticles. A redox chemistry of stannous catalyst in PU hydrogel yielded spherical AgNPs formation, even at 4 °C in the absence of external reductant; and an appropriate thiol-functionalized polymeric network promoted spherical AgNPs well dispersed through PSA hydrogel network, after heating up the swollen hydrogel at 103 °C in the presence of citrate-reductant. Optical and swelling behaviors of both series of hydrogel nanocomposites were investigated as key factors involved in their antimicrobial efficacy over time. Lastly, in vitro antibacterial activity of Ag loaded hydrogels exposed to Pseudomona aeruginosa and Escherichia coli strains indicated a noticeable sustained inhibitory effect, especially for Ag-PU hydrogel nanocomposites with bacterial inhibition growth capabilities up to 120 h cultivation.

Interferon-Tau has Antiproliferative effects, Represses the Expression of E6 and E7 Oncogenes, Induces Apoptosis in Cell Lines Transformed with HPV16 and Inhibits Tumor Growth In Vivo.

Interferon tau (IFN-τ) is a promising alternative antiviral and immunotherapeutic agent in a wide variety of diseases including infectious, neurodegenerative, autoimmune and cancer due to its low toxicity in comparison with other type I interferon´s. The objective of our study was established the effect of the bovine IFN-τ on human (SiHa) and murine (BMK-16/myc) cells transformed with HPV 16 and evaluates the antitumor effect in a murine tumor model HPV 16 positive. We determine that bovine IFN-τ has antiproliferative effects, pro-apoptotic activity and induces repression of viral E6 and E7 oncogenes (time- and dose-dependent) on human and murine cells transformed with HPV 16 similar to the effects of IFN-ß. However, IFN-τ induces greater antiproliferative effect, apoptosis and repression of both oncogenes in BMK-16/myc cells compared to SiHa cells. The differences were explained by the presence and abundance of the type I interferon receptor (IFNAR) in each cell line. On the other hand, we treated groups of tumor-bearing mice (HPV16 positive) with IFN-τ and showed the inhibition tumor growth effect in vivo. Our finding indicates that bovine IFN-τ may be a good candidate for immunotherapy against cervical cancer.

In vivo Antitumor Effect of an HPV-specific Promoter driving IL-12 Expression in an HPV 16-positive Murine Model of Cervical Cancer.

Human papillomavirus (HPV) is a DNA virus that infects epithelial cells and has been implicated in the development of cervical cancer. Few therapeutic strategies have been designed for the treatment of cervical intraepithelial neoplasia, a precursor of cervical cancer. In these early stages, the HPV E2 protein is the most important viral factor involved in viral gene expression and plays crucial roles during the vegetative viral cycle in epithelial cells. Papillomavirus E2 binds specifically to palindromic ACCN6GGT sequences, referred to as the E2 binding sites (E2BS), which are concentrated within the viral long control region, and which are responsible for regulation of the HPV protein's expression. Here, we consider E2BS as a candidate sequence to induce the expression of antiviral therapeutic genes selectively in HPV-infected cells expressing the E2 protein. This study focuses on the use of an HPV-specific promoter comprised of four E2BS to drive the expression of IL-12, leading to an antitumor effect in an HPV-positive murine tumor model. The therapeutic strategy was implemented via viral gene therapy using adenoviral vectors with recombinant E2 and IL-12 genes and E2BS-IL-12. We demonstrate that the HPV-specific promoter E2BS is functional in vitro and in vivo through transactivation of HPV E2 transcription factor.

Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells.

BACKGROUND: Expression of the microRNA miR-21 has been found to be altered in almost all types of cancers and it has been classified as an oncogenic microRNA or oncomir. Due to the critical functions of its target proteins in various signaling pathways, miR-21 is an attractive target for genetic and pharmacological modulation in various cancers. Cervical cancer is the second most common cause of death from cancer in women worldwide and persistent HPV infection is the main etiologic agent. This malignancy merits special attention for the development of new treatment strategies. In the present study we analyze the role of miR-21 in cervical cancer cells. METHODS: To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression in a cervical intraepithelial neoplasia-derived cell lines using siRNAs. The effect of miR-21 on gene expression was assessed in cervical cancer cells transfected with the siRNA expression plasmid pSIMIR21. We identified the tumor suppressor gene PTEN as a target of miR-21 and determined the mechanism of its regulation throughout reporter construct plasmids. Using this model, we analyzed the expression of miR-21 and PTEN as well as functional effects such as autophagy and apoptosis induction. RESULTS: In SiHa cells, there was an inverse correlation between miR-21 expression and PTEN mRNA level as well as PTEN protein expression in cervical cancer cells. Transfection with the pSIMIR21 plasmid increased luciferase reporter activity in construct plasmids containing the PTEN-3'-UTR microRNA response elements MRE21-1 and MRE21-2. The role of miR-21 in cell proliferation was also analyzed in SiHa and HeLa cells transfected with the pSIMIR21 plasmid, and tumor cells exhibited markedly reduced cell proliferation along with autophagy and apoptosis induction. CONCLUSIONS: We conclude that miR-21 post-transcriptionally down-regulates the expression of PTEN to promote cell proliferation and cervical cancer cell survival. Therefore, it may be a potential therapeutic target in gene therapy for cervical cancer.

MicroRNA miR-16-1 regulates CCNE1 (cyclin E1) gene expression in human cervical cancer cells.

MicroRNAs are involved in diverse biological processes through regulation of gene expression. The microRNA profile has been shown to be altered in cervical cancer (CC). MiR-16-1 belongs to the miR-16 cluster and has been implicated in various aspects of carcinogenesis including cell proliferation and regulation of apoptosis; however, its function and molecular mechanism in CC is not clear. Cyclin E1 (CCNE1) is a positive regulator of the cell cycle that controls the transition of cells from G1 to S phase. In CC, CCNE1 expression is frequently upregulated, and is an indicator for poor outcome in squamous cell carcinomas (SCCs). Thus, in the present brief communication, we determine whether the CCNE1 gene is regulated by miR-16-1 in CC cells. To identify the downstream cellular target genes for upstream miR-16-1, we silenced endogenous miR-16-1 expression in cell lines derived from CC (C-33 A HPV-, CaSki HPV16+, SiHa HPV16+, and HeLa HPV18+ cells), using siRNAs expressed in plasmids. Using a combined bioinformatic analysis and RT-qPCR, we determined that the CCNE1 gene is targeted by miR-16-1 in CC cells. SiHa, CaSki, and HeLa cells demonstrated an inverse correlation between miR-16-1 expression and CCNE1 mRNA level. Thus, miR-16-1 post-transcriptionally down-regulates CCNE1 gene expression. These results, suggest that miR-16-1 plays a vital role in modulating cell cycle processes in CC.

Utility of microRNAs and siRNAs in cervical carcinogenesis.

MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3'-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells.

Role of IL-10 and TGF-ß1 in local immunosuppression in HPV-associated cervical neoplasia.

Cervical cancer is a worldwide disease that constitutes a significant public health problem, especially in developing countries, not only due to its high incidence but also because the most affected population comprises women who belong to marginalized socio-economic classes. Clinical and molecular research has identified immunological impairment in squamous intraepithelial cervical lesions and cervical cancer patients. Human Papillomavirus (HPV) has several mechanisms for avoiding the immune system: it down-regulates the expression of interferon and upregulates interleukin (IL)-10 and transforming growth factor (TGF)-ß1 to produce a local immunosuppressive environment, which, along with altered tumor surface antigens, forms an immunosuppressive network that inhibits the antitumor immune response. In this review we analyzed the available data on several deregulated cellular immune functions in patients with NIC I, NIC II and NIC III and cervical cancer. The effects of immunosuppressive cytokines on innate immune response, T-cell activation and cellular factors that promote tumor cell proliferation in cervical cancer patients are summarized. We discuss the functional consequences of HPV E2, E6, and E7 protein interactions with IL-10 and TGF-ß1 promoters in the induction of these cytokines and postulate its effect on the cellular immune response in squamous intraepithelial cervical lesions and cervical cancer patients. This review provides a comprehensive picture of the immunological functions of IL-10 and TGF-ß1 in response to HPV in humans.

Inhibitory activity of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) on transformed cells by human papillomavirus.

In this study, we investigated the effects of the aqueous extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, obtained from three localities (China; and Morelos and Michoacan, Mexico) on cervical cells transformed by human papillomavirus (HeLa and SiHa) and C-33A cancer cells. The cells were plated in DMEM medium supplemented, and were incubated in the presence of different concentrations of G. lucidum for 24 h. Cell proliferation was determined by MTT colorimetric assay and viability by trypan blue assay. Inhibitory dose was determined (IC50) of the three different extracts of G. lucidum in the culture cell lines mentioned above. The apoptosis process was confirmed by nuclear DNA fragmentation and the cell cycle was determined by flow cytometry. The results showed that aqueous extracts G. lucidum obtained from three localities produced inhibition in the proliferation of VPH transformed cells; they also induced apoptosis and cell cycle arrest in HeLa, SiHa, and C-33A cancer cells. Therefore, it was found that aqueous extracts G. lucidum obtained from three different locations produced inhibitory effect on cancer cells and may have a potential therapeutic use for the prevention and treatment of this disease.

The relationship between the antitumor effect of the IL-12 gene therapy and the expression of Th1 cytokines in an HPV16-positive murine tumor model.

OBJECTIVE: The goal of the present study was to investigate the effect of IL-12 expressed in plasmid on the Th1 cytokine profile in an experimental HPV16-positive murine tumor model and the association with the IL-12's antitumor effect. METHODS: Mice were injected with BMK-16/myc cells to establish HPV16-positive tumor and then pNGVL3-mIL-12 plasmid; pcDNA3 plasmid or PBS was injected directly into tumor site. The antitumor effect of the treatment was evaluated and the cytokines expression profile in each tumor tissue was analyzed. RESULTS: Treatment with pNGVL3-mIL-12 plasmid had a significant antitumor effect, and a Th2-Th3-type cytokines prolife was detected in the murine tumor model with expression of the cytokines IL-10, IL-4, and TGF-ß1. However, after the tumor was treated with three intratumoral injections of plasmid containing IL-12 cDNA, it showed a cytokine profile associated with Th1 with expression of IL-2, IL-12, and IFN-γ cytokines and reduced expression of IL-10, IL-4, and TGF-ß1. CONCLUSIONS: The treatment with the IL-12 gene in the experimental HPV16-positive tumor model promoted the activation of the cellular immune response via expression of a Th1-type cytokine profile and was associated with the inhibition of tumor growth. Thus, IL-12 treatment represents a novel approach for gene therapy against cervical cancer.