Epidemiology and Molecular Biology of HPV Variants in Cervical Cancer: The State of the Art in Mexico.

Cervical cancer (CC) continues to be a major public health problem in Mexico, ranking second among cancers in women. A persistent infection with human papillomaviruses (HPV) is the main risk factor for CC development. In addition, a significant fraction of other cancers including those of the anus, oropharynx, and penis are also related to HPV infection. In CC, HPV-16 is the most prevalent high-risk HPV type, followed by HPV-18, both being responsible for 70% of cases. HPV intratype variant lineages differ in nucleotide sequences by 1-10%, while sublineages differ by 0.5-1%. Several studies have postulated that the nucleotide changes that occur between HPV intratype variants are reflected in functional differences and in pathogenicity. Moreover, it has been demonstrated that HPV-16 and -18 intratype variants differentially affect molecular processes in infected cells, changing their biological behavior that finally impacts in the clinical outcome of patients. Mexico has participated in providing knowledge on the geographical distribution of intratype variants of the most prevalent HPVs in premalignant lesions of the cervix and cervical cancer, as well as in other HPV-related tumors. In addition, functional studies have been carried out to assess the cellular effects of intratype variations in HPV proteins. This review addresses the state of the art on the epidemiology of HPV-16 and HPV-18 intratype variants in the Mexican population, as well as their association with persistence, precancer and cervical cancer, and functional aspects related to their biological behavior.

The Polyphenols α-Mangostin and Nordihydroguaiaretic Acid Induce Oxidative Stress, Cell Cycle Arrest, and Apoptosis in a Cellular Model of Medulloblastoma.

Medulloblastoma is a common malignant brain tumor in the pediatric age. The current therapeutics present serious collateral effects. Polyphenols α-mangostin and nordihydroguaiaretic acid (NDGA) exert potent antitumoral activity in different cancer models, although their antitumoral effects have not been described in medulloblastoma cells yet. This study aimed to examine the proapoptotic effects of these polyphenols on human medulloblastoma cells. Medulloblastoma cell line Daoy was incubated with increasing concentrations of α-mangostin or NDGA for 24 h. The cell viability was analyzed using crystal violet and trypan blue dyes. Determination of the glutathione (GSH)/glutathione disulfide (GSSG) ratio and levels of carbonylated proteins was performed to evaluate the oxidative stress. Cell cycle progression and induction of cell death by fluorochrome-couple and TUNEL assays were evaluated using flow cytometry assays. Individual treatments with α-mangostin or NDGA decreased the viability of Daoy cells in a dose-dependent manner, inducing G2/M and S-G2/M cell cycle arrest, respectively. Both polyphenols induced cell death and increased oxidative stress. Very interestingly, α-mangostin showed more potent effects than NDGA. Our results indicate that α-mangostin and NDGA exert important cytostatic and cytotoxic effects in the Daoy cell line. These data highlight the potential usefulness of these compounds as an alternative strategy in medulloblastoma treatment.

Mexican Consensus on Hodgkin's Lymphoma.

Hodgkin's lymphoma is due to the clonal transformation of cells originating from B lymphocytes, generating the pathognomonic binucleate Reed-Sternberg cells. Hodgkin's lymphoma is a B cell disease with a bimodal distribution, with higher incidence in adolescence and the third decade of life, showing a second peak in people over 55 years of age. Classic Hodgkin lymphoma cells routinely undergo gene expression reprogramming, as they lose the expression of most of the typical B-cell genes and acquire the expression of multiple genes that are typical of other types of cells in the immune system. The treatment algorithm will depend on whether it is classic or predominantly lymphocytic HL, if it is early stage with unfavorable prognostic markers or not, the initial management regimen, and whether there is bulky disease, among the most relevant variables.

El linfoma de Hodgkin (LH) se debe a la transformación clonal de células originadas en los linfocitos B, lo que genera las células binucleadas patognomónicas de Reed-Sternberg. El LH es una enfermedad de células B con una distribución bimodal, con mayor incidencia en la adolescencia y la tercera década de la vida y un segundo pico en personas mayores de 55 años. Las células del LH clásico habitualmente sufren una reprogramación de la expresión génica, ya que pierden la expresión de la mayoría de los genes típicos de las células B y han adquirido la expresión de múltiples genes que son típicos de otros tipos de células del sistema inmunitario. El algoritmo de tratamiento dependerá si se trata de LH clásico o de predominio linfocítico, si es un estadio temprano con marcadores de pronóstico desfavorables o no, el esquema inicial de manejo y si existe enfermedad voluminosa, entre las variables más relevantes.

Mexican consensus on Hodgkin's lymphoma.

El linfoma de Hodgkin (LH) se debe a la transformación clonal de células originadas en los linfocitos B, lo que genera las células binucleadas patognomónicas de Reed-Sternberg. El LH es una enfermedad de células B con una distribución bimodal, con mayor incidencia en la adolescencia y la tercera década de la vida y un segundo pico en personas mayores de 55 años. Las células del LH clásico habitualmente sufren una reprogramación de la expresión génica, ya que pierden la expresión de la mayoría de los genes típicos de las células B y han adquirido la expresión de múltiples genes que son típicos de otros tipos de células del sistema inmunitario. El algoritmo de tratamiento dependerá si se trata de LH clásico o de predominio linfocítico, si es un estadio temprano con marcadores de pronóstico desfavorables o no, el esquema inicial de manejo y si existe enfermedad voluminosa, entre las variables más relevantes.Hodgkin's lymphoma (HL) is due to the clonal transformation of cells originating from B lymphocytes, generating the pathognomonic binucleate Reed-Sternberg cells. HL is a B cell disease with a bimodal distribution, with higher incidence in adolescence and the third decade of life, showing a second peak in people over 55 years of age. Classic Hodgkin lymphoma cells routinely undergo gene expression reprogramming, as they lose the expression of most of the typical B-cell genes and acquire the expression of multiple genes that are typical of other types of cells in the immune system. The treatment algorithm will depend on whether it is classic or predominantly lymphocytic HL, if it is early stage with unfavorable prognostic markers or not, the initial management regimen, and whether there is bulky disease, among the most relevant variables.

Consensus on hemophilia in Mexico.

Hemophilia is a hemorrhagic disorder with a sex-linked inherited pattern, characterized by an inability to amplify coagulation due to a deficiency in coagulation factor VIII (hemophilia A or classic) or factor IX (hemophilia B). Sequencing of the genes involved in hemophilia has provided a description and record of the main mutations, as well as a correlation with the various degrees of severity. Hemorrhagic manifestations are related to levels of circulating factor, mainly affecting the musculoskeletal system and specifically the large joints (knees, ankles, and elbows). This document is a review and consensus of the main genetic aspects of hemophilia, from the inheritance pattern to the concept of women carriers, physiopathology and classification of the disorder, the basic and confirmation studies when hemophilia is suspected, the various treatment regimens based on infusion of the deficient coagulation factor as well as innovative factor-free therapies and recommendations for the management of complications associated with treatment (development of inhibitors and/or transfusion-transmitted infections), or secondary to articular hemorrhagic events (hemophilic arthropathy). Finally, relevant reviews of clinical and treatment aspects of hemorrhagic pathology characterized by acquired deficiency of FVIII secondary to neutralized antibodies named acquired hemophilia.

La hemofilia es un trastorno hemorrágico con patrón de herencia ligado al sexo, caracterizado por una incapacidad en la amplificación de la coagulación ocasionada por la deficiencia del factor VIII (hemofilia A o clásica) o del factor IX (hemofilia B). La secuenciación de los genes involucrados en la hemofilia ha permitido la descripción y registro de las principales mutaciones, así como la correlación con los diversos grados de severidad. Las manifestaciones hemorrágicas se relacionan con los niveles de factor deficiente circulante, afectando principalmente al sistema musculoesquelético y en particular a las grandes articulaciones (rodillas, tobillos y codos). El presente documento hace una revisión y consenso de los principales aspectos genéticos de la hemofilia, desde el patrón de herencia y el concepto de mujeres portadoras, la fisiopatología y clasificación de la enfermedad, los estudios básicos y de confirmación ante la sospecha de hemofilia, y de los diversos esquemas de tratamiento basados en la infusión del factor de coagulación deficiente hasta las terapias innovadoras libres de factor, así como de las recomendaciones para el manejo de las complicaciones asociadas al tratamiento (desarrollo de inhibidores y/o infecciones transmitidas por transfusión) o secundarias a los eventos hemorrágicos a nivel articular (artropatía hemofílica). La parte final del documento revisa los aspectos clínicos y de tratamiento relevantes de una patología hemorragica caracterizada por la deficiencia adquirida del FVIII mediada por anticuerpos neutralizantes denominada hemofilia adquirida.

Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for Gastric Cancer with Peritoneal Carcinomatosis: Multicenter Study of Spanish Group of Peritoneal Oncologic Surgery (GECOP).

BACKGROUND: Gastric cancer (GC) with peritoneal carcinomatosis (PC) is traditionally considered a terminal stage of the disease. The use of a multimodal treatment, including cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC), can benefit these patients. Our goal was to evaluate the morbidity and survival outcomes of these patients. METHODS: This is a retrospective, multicenter study from a prospective national database of patients diagnosed with PC secondary to GC treated with CRS and HIPEC from June 2006 to October 2017. RESULTS: Eighty-eight patients from seven specialized Spanish institutions were treated with CRS and HIPEC, with median age of 53 years; 51% were women. Median Peritoneal Cancer Index (PCI) was 6, and complete cytoreduction was achieved in 80 patients (90.9%). HIPEC was administered in 85 cases with 4 different regimens (Cisplatin + Doxorubicin, Mitomycin-C + Cisplatin, Mitomycin-C and Oxaliplatin). Twenty-seven cases (31%) had severe morbidity (grade III-IV) and 3 patients died in the postoperative period (3.4%). Median follow-up was 32 months. Median overall survival (OS) was 21.2 months, with 1-year OS of 79.9% and 3-year OS of 30.9%. Median disease-free survival (DFS) was 11.6 months, with 1-year DFS of 46.1% and 3-year DFS of 21.7%. After multivariate analysis, the extent of peritoneal disease (PCI ≥ 7) was identified as the only independent factor that influenced OS (hazard ratio [HR] 2.37, 95% confidence interval [CI] 1.26-4.46, p = 0.007). CONCLUSIONS: The multimodal treatment, including CRS and HIPEC, for GC with PC can improve the survival results in selected patients (PCI < 7) and in referral centers.

Type-specific persistence and clearance rates of HPV genotypes in the oral and oropharyngeal mucosa in an HIV/AIDS cohort.

BACKGROUND: Oral high-risk human papillomavirus (HR-HPV) infections are frequent and persistent among the HIV-positive population and are associated with an increased risk for head and neck cancer (HNC). In this study, we sought to determine the incidence, persistence and clearance of HPV infections in oral and oropharyngeal samples from HIV/AIDS subjects. METHODS: A longitudinal, observational and analytical study was performed with an ongoing cohort of HIV/AIDS subjects in Mexico City (September 2013-February 2015). The study was approved by institutional committees, and demographic and clinical data were registered. At the baseline and three-month visits, oral examinations and cytobrush samples were obtained. DNA was purified, quantified and used to detect an HPV-L1 gene fragment by nested PCR, using MY09/MY11 and GP5 + /GP6 + primers. HPV DNA products were purified, sequenced and typed according to HPV databases. Risk factors were assessed, and a multivariate modelling approach was used to determine independent effects. RESULTS: This study included 97 HIV/AIDS individuals (91% men [86.4% of which are men who have sex with men], median age: 36 years, 72.2% under HAART). From the baseline visit, HPV was observed in 55.7% (HR-HPV: 26.8%; HPV-18: 24.1%), with a higher HPV-positive samples for smokers (61.1 vs 32.6%, P = .005). The three-month overall HPV incidence was 33.9%; type-specific HPV persistence was 33.3% (HR-HPV: 13.3%); and 13 of the 33 (39.4%) baseline HPV-positive individuals cleared the infection (HR-HPV: 53.8%). CONCLUSIONS: Although HR-HPV persistence was low, and clearance of the infection was observed in most cases, a close follow-up is necessary, given the increase in HNC among HIV-subjects, particularly HPV-related cancer.

Regulation of Cellular Metabolism by High-Risk Human Papillomaviruses.

The alteration of glucose metabolism is one of the first biochemical characteristics associated with cancer cells since most of these cells increase glucose consumption and glycolytic rates even in the presence of oxygen, which has been called “aerobic glycolysis" or the Warburg effect. Human papillomavirus (HPV) is associated with approximately 5% of all human cancers worldwide, principally to cervical cancer. E6 and E7 are the main viral oncoproteins which are required to preserve the malignant phenotype. These viral proteins regulate the cell cycle through their interaction with tumor suppressor proteins p53 and pRB, respectively. Together with the viral proteins E5 and E2, E6 and E7 can favor the Warburg effect and contribute to radio- and chemoresistance through the increase in the activity of glycolytic enzymes, as well as the inhibition of the Krebs cycle and the respiratory chain. These processes lead to a fast production of ATP obtained by Warburg, which could help satisfy the high energy demands of cancer cells during proliferation. In this way HPV proteins could promote cancer hallmarks. However, it is also possible that during an early HPV infection, the Warburg effect could help in the achievement of an efficient viral replication.

Impact of human papillomavirus coinfections on the risk of high-grade squamous intraepithelial lesion and cervical cancer.

OBJECTIVE: The molecular and epidemiologic effect of human papillomavirus (HPV) coinfections in the risk of developing cervical cancer is yet unclear. The aim of this study was to determine the frequency HPV coinfections at different stages of cervical lesions in the development of cervical cancer and the impact of HPV specific type interactions on high-grade squamous intraepithelial lesions (HSIL) and invasive cervical cancer (ICC) risk. METHODS: HPV testing was performed in 931 cervical samples diagnosed as: negative for intraepithelial lesion or malignancy (NILM); low-grade squamous intraepithelial lesion (LSIL); HSIL; and ICC. For HPV detection and typing two sets of primers from the L1 region were used in the polymerase chain reaction method (PCR) (MY09/MY11/HMB01 and L1C1/L1C2.1/L1C2.2) and HPV type was determined by PCR product sequence. To look for multiple HPV infections, the E6 nested multiplex PCR method was performed in all DNA samples. Odds ratios were calculated as indexes of the strength of the association between the sample category (LSIL/NILM or ICC/HSIL) and the presence of a given viral combination. RESULTS: In HPV positive samples, coinfections are as common in ICC/HSIL as in LSIL/NILM (47.12% and 40.17%, respectively). There is an increased risk to ICC/HSIL when multiple high-risk HPV types are present. The coinfection of HPV68 with HPV16 increases the risk of ICC/HSIL (OR=14.54, P=0.012, after multivariate adjustment), related to the presence of HPV16 or HPV68 alone. CONCLUSIONS: These results sustain that specific HPV coinfections confer an increased risk to develop ICC/HSIL.

Promising predictive molecular biomarkers for cervical cancer (Review).

Cervical cancer (CC) constitutes a serious public health problem. Vaccination and screening programs have notably reduced the incidence of CC worldwide by >80%; however, the mortality rate in low­income countries remains high. The staging of CC is a determining factor in therapeutic strategies: The clinical management of early stages of CC includes surgery and/or radiotherapy, whereas radiotherapy and/or concurrent chemotherapy are the recommended therapeutic strategies for locally advanced CC. The histopathological characteristics of tumors can effectively serve as prognostic markers of radiotherapy response; however, the efficacy rate of radiotherapy may significantly differ among cancer patients. Failure of radiotherapy is commonly associated with a higher risk of recurrence, persistence and metastasis; therefore, radioresistance remains the most important and unresolved clinical problem. This condition highlights the importance of precision medicine in searching for possible predictive biomarkers to timely identify patients at risk of treatment response failure and provide tailored therapeutic strategies according to genetic and epigenetic characteristics. The present review aimed to summarize the evidence that supports the role of several proteins, methylation markers and non­coding RNAs as potential predictive biomarkers for CC.

High-risk human papillomavirus (HPV) DNA sequences in metaplastic breast carcinomas of Mexican women.

BACKGROUND: Metaplastic carcinoma, an uncommon subtype of breast cancer, is part of the spectrum of basal-like, triple receptor-negative breast carcinomas. The present study examined 20 surgical specimens of metaplastic breast carcinomas, for the presence of high-risk Human papillomavirus (HPV), which is suspected to be a potential carcinogenic agent for breast carcinoma. METHODS: Mastectomy specimens from patients harboring metaplastic breast carcinoma, as defined by the World Health Organization (WHO), and who attended the Instituto Nacional de Cancerologia in Mexico City, were retrieved from the files of the Department of Pathology accumulated during a 16-year period (1995-2008). Demographic and clinical information was obtained from patients' medical records. DNA was extracted from formalin-fixed, paraffin-embedded tumors and HPV type-specific amplification was performed by means of Polymerase chain reaction (PCR). Quantitative Real-time (RT) PCR was conducted in HPV positive cases. Statistically, the association of continuous or categorical variables with HPV status was tested by the Student t, the Chi square, or Fisher's exact tests, as appropriate. RESULTS: High-risk HPV DNA was detected in eight (40%) of 20 metaplastic breast carcinomas: seven (87.5%) HPV-16 and one (12.5%) HPV-18. Mean age of patients with HPV-positive cases was 49 years (range 24-72 years), the same as for HPV-negative cases (range, 30-73 years). There were not striking differences between HPV + and HPV- metaplastic carcinomas regarding clinical findings. Nearly all cases were negative for estrogen, progesterone and Human epidermal growth factor receptor 2 (HER2), but positive for Epidermal growth factor receptor (EGFR). CONCLUSIONS: High-risk HPV has been strongly associated with conventional breast carcinomas, although the subtle mechanism of neoplastic transformation is poorly understood. In Mexican patients, the prevalence of HPV infection among metaplastic breast carcinomas is higher than in non-metaplastic ones, as so the HPV viral loads; notwithstanding, HPV viral loads show wide variation and remain even lower than cervical and other non-cervical carcinomas, making it difficult to assume that HPV could play a key role in breast carcinogenesis. Further studies are warranted to elucidate the meaning of the presence of high-risk HPVDNA in breast carcinomas.

Hyperthermic Intraperitoneal Chemotherapy and Recirculation with CO2: A Safe Technique.

INTRODUCTION: Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) has evolved as a treatment for peritoneal carcinomatosis in various tumors after a careful and complete cytoreductive surgery, and it demonstrated much better and longer survival than more traditional therapeutic schemas. Our objective has been to examine the safety, efficacy and survival achieved with closed technique with CO2-agitation system Combat PRS® (Peritoneal Recirculation System: PRS). To achieve this, we compared the appearance of adverse events, mortality and survival with the described using classic techniques (open, closed without CO2-agitation) for the treatment of selected patients with peritoneal carcinomatosis; Materials and methods: We studied overall survival, disease-free survival and safety (morbidity and mortality) of the administration of HIPEC through a closed method technique with CO2 recirculation (Combat PRS®) in 482 patients from 11 Spanish hospitals; Results: The mortality of our technique (1.66%) was similar to other published techniques (open, closed). Morbidity exhibited a 9.96% rate of Clavien-Dindo (CD) III/IV complications in 482 patients, which was lower than in other series. Survival (overall survival (OS) and disease-free survival (DFS)) was similar to previously published results: 86% 1y-OS, 54% 3y-OS, 77% 1y-DFS and 31% 3y-DFS; Conclusion: The procedure with closed PRS with CO2 agitation is as safe as standard open and closed procedures for the administration of HIPEC after complete cytoreductive surgery, with similar and very low mortality (1.66%) and lower morbidity (9.96% CD III and IV in our series vs range of 20-40% in the majority of different series); only Kusamura had similar results, with 12% in 205 patients, using the closed technique without CO2 agitation).

HPV Prevalence and Predictive Biomarkers for Oropharyngeal Squamous Cell Carcinoma in Mexican Patients.

BACKGROUND: Worldwide prevalence of Oropharyngeal Squamous Cell Carcinoma (OPSCC) has increased, affecting mostly young males. OPSCC associated with Human Papillomavirus (HPV) infection exhibits particular characteristics in terms of response to treatment, hence HPV has been proposed as a prognostic factor. The impact of HPV positivity and associated biomarkers on OPSCC in the Mexican population has not been addressed. Therefore, the analysis of OPSCC prognostic markers in the Mexican population is necessary. METHODS: Retrolective study in Mexican OPSCC patients, where HPV prevalence, p16 and EGFR levels were assessed using INNO-LiPA and immunohistochemistry. RESULTS: We found an HPV prevalence of 57.6% in OPSCC cases treated at a reference center in Mexico. HPV and p16 positivity, as well as EGFR, associate with better outcomes in OPSCC patients, and they also promote reduced death risk. Notably, HPV presence and p16 positivity showed a significant association with disease-free survival (DFS), with a HR of 0.15 (p = 0.006) and a HR of 0.17 (p = 0.012), respectively, indicating a possible role as predictive biomarkers in Mexican OPSCC patients. CONCLUSIONS: Our results reflect the clinical utility of p16 analysis to improve overall survival (OS) and to predict recurrence in oropharyngeal cancer. These results position p16 and HPV as predictive biomarkers for OPSCC.

HLA-G 3'UTR Polymorphisms Are Linked to Susceptibility and Survival in Spanish Gastric Adenocarcinoma Patients.

HLA-G is a non-classical class I HLA molecule that induces tolerance by acting on receptors of both innate and adaptive immune cells. When overexpressed in tumors, limits surveillance by the immune system. The HLA-G gene shows several polymorphisms involved in mRNA and protein levels. We decided to study the implication of two polymorphisms (rs371194629; 14bp INS/DEL and rs1063320; +3142 C/G) in paired tissue samples (tumoral and non-tumoral) from 107 Spanish patients with gastric adenocarcinoma and 58 healthy control individuals, to assess the possible association of the HLA-G gene with gastric adenocarcinoma susceptibility, disease progression and survival. The presence of somatic mutations involving these polymorphisms was also analyzed. The frequency of the 14bp DEL allele was increased in patients (70.0%) compared to controls (57.0%, p=0.025). In addition, the haplotype formed by the combination of the 14bp DEL/+3142 C variants is also increased in patients (54.1% vs 44.4%, p=0.034, OR=1.74 CI95% 1.05-2.89). Kaplan-Meier analysis revealed that 14bp DEL/DEL patients showed lower 5-year life-expectancy than INS/DEL or INS/INS (p=0.041). Adjusting for TNM staging (Cox regression analysis) disclosed a significant difference in death risk (p=0.03) with an expected hazard 2.6 times higher. Finally, no somatic mutations were found when comparing these polymorphisms in tumoral vs non-tumoral tissues, which indicates that this is a preexisting condition in patients and not a de novo, tumor-restricted, event. In conclusion, the variants predominant in patients were those increasing HLA-G mRNA stability and HLA-G expression, clearly involving this molecule in gastric adenocarcinoma susceptibility, disease progression and survival and making it a potential target for immunotherapeutic approaches.

Raloxifene and n-Acetylcysteine Ameliorate TGF-Signalling in Fibroblasts from Patients with Recessive Dominant Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by mutation of the COL7A1 gene. RDEB is associated with high levels of TGF-ß1, which is likely to be involved in the fibrosis that develops in this disease. Endoglin (CD105) is a type III coreceptor for TGF-ß1 and its overexpression in fibroblasts deregulates physiological Smad/Alk1/Alk5 signalling, repressing the synthesis of TGF-ß1 and extracellular matrix (ECM) proteins. Raloxifene is a specific estrogen receptor modulator designated as an orphan drug for hereditary hemorrhagic telangiectasia, a rare vascular disease. Raloxifene stimulates endoglin synthesis, which could attenuate fibrosis. By contrast, the antioxidant N-acetylcysteine may have therapeutic value to rectify inflammation, fibrosis and endothelial dysfunction. Thus, we present here a repurposing strategy based on the molecular and functional screening of fibroblasts from RDEB patients with these drugs, leading us to propose the repositioning of these two well-known drugs currently in clinical use, raloxifene and N-acetylcysteine, to counteract fibrosis and inflammation in RDEB. Both compounds modulate the profibrotic events that may ultimately be responsible for the clinical manifestations in RDEB, suggesting that these findings may also be relevant for other diseases in which fibrosis is an important pathophysiological event.

Human skin equivalents cultured under hypoxia display enhanced epidermal morphogenesis and lipid barrier formation.

Human skin equivalents (HSEs) are three-dimensional cell models mimicking characteristics of native human skin (NHS) in many aspects. However, a limitation of HSEs is the altered in vitro morphogenesis and barrier formation. Differences between in vitro and in vivo skin could have been induced by suboptimal cell culture conditions, of which the level of oxygen in vitro (20%) is much higher than in vivo (0.5-8%). Our aim is to study how external oxygen levels affect epidermal morphogenesis and barrier formation in HSEs. In the present study, fibroblast and keratinocyte monocultures, and HSEs were generated under 20% (normoxia) and 3% (hypoxia) oxygen level. In all cultures under hypoxia, expression of hypoxia-inducible factor target genes was increased. Characterization of HSEs generated under hypoxia using immunohistochemical analyses of morphogenesis biomarkers revealed a reduction in epidermal thickness, reduced proliferation, similar early differentiation, and an attenuated terminal differentiation program compared to normoxia, better mimicking NHS. The stratum corneum ceramide composition was studied with liquid chromatography coupled to mass spectrometry. Under hypoxia, HSEs exhibited a ceramide composition that more closely resembles that of NHS. Consequently, the lipid organization was improved. In conclusion, epidermal morphogenesis and barrier formation in HSEs reconstructed under hypoxia better mimics that of NHS.

Cutting efficiency evaluation of Quantec Series 2000 orifice openers with computed tomography (CT).

INTRODUCTION: Orifice openers or orifice shapers are used to shape the coronal part of the root canals during endodontic treatment. OBJECTIVES: The aim of this work was to analyse the cutting efficiency of Quantec Series 2000 system orifice shapers by means of computerised tomography (CT). MATERIALS AND METHODS: Mandibular molars were selected, preoperative CT was performed and the cross-sections and surface area of the root canals were measured. Following access opening and preparation of the coronal part of the canals with Quantec Series 2000 orifice shapers, a further CT was obtained of all the teeth, the previous measurements were repeated and the two sets of measurements were compared. RESULTS: Although the canals suffered modifications due to the action of the instruments, they remained centred and retained their original morphology; the changes were greater in the most coronal part of the canals. CONCLUSIONS: the orifice shapers of the system under study kept the canals centred on their original position, so they do not present great aggressiveness during endodontic treatment; the canals that were initially the widest were those that showed the greatest modification; the CT system employed in the study showed itself to be an effective, non-invasive system for studying the changes that had taken place inside the root canals.

Third canal in the mesial root of permanent mandibular first molars: review of the literature and presentation of 3 clinical reports and 2 in vitro studies.

INTRODUCTION: Systematic anatomical studies corroborate the anatomical complexity of the root canal system. Deviations from the norm such as multiple orifices, apical deltas, accessory canals and other variations are frequent. OBJECTIVES: To present clinical reports of mandibular molars with three canals in the mesial root and two in vitro studies of the morphology of these canals, together with a review of the literature. MATERIALS AND METHODS: Three clinical reports are presented of mandibular molars where three canals in the mesial roots were treated endodontically. Two in vitro studies were performed, one using computerized tomography and the other with scanning electron microscopy, to describe the morphology of the mesial root canals of permanent first molars. The scientific literature on this anatomical situation was also reviewed. RESULTS: The tomographic study found that the prevalence of a third mesial canal in the 27 teeth analysed was 14.81%; the microscopic study of 25 teeth found it to be 12%. The review of the literature encountered that the authors who report cases such as these found prevalences ranging from 1% to 15%. CONCLUSIONS: From the teeth analysed in vitro, it is concluded that a third mesial canal may be present in a mandibular first molar in approximately 13% of cases. Clinically, the third canal is difficult to find and exhibits a very variable morphology, which may present anastomosis with the other canals.

SOX2 as a New Regulator of HPV16 Transcription.

Persistent infections with high-risk human papillomavirus (HPV) constitute the main risk factor for cervical cancer development. HPV16 is the most frequent type associated to squamous cell carcinomas (SCC), followed by HPV18. The long control region (LCR) in the HPV genome contains the replication origin and sequences recognized by cellular transcription factors (TFs) controlling viral transcription. Altered expression of E6 and E7 viral oncogenes, modulated by the LCR, causes modifications in cellular pathways such as proliferation, leading to malignant transformation. The aim of this study was to identify specific TFs that could contribute to the modulation of high-risk HPV transcriptional activity, related to the cellular histological origin. We identified sex determining region Y (SRY)-box 2 (SOX2) response elements present in HPV16-LCR. SOX2 binding to the LCR was demonstrated by in vivo and in vitro assays. The overexpression of this TF repressed HPV16-LCR transcriptional activity, as shown through reporter plasmid assays and by the down-regulation of endogenous HPV oncogenes. Site-directed mutagenesis revealed that three putative SOX2 binding sites are involved in the repression of the LCR activity. We propose that SOX2 acts as a transcriptional repressor of HPV16-LCR, decreasing the expression of E6 and E7 oncogenes in a SCC context.