PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro

<p>Abstract</p> <p>Background</p> <p>High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.</p> <p>Methods</p> <p>Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal <it>in vitro </it>model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.</p> <p>Results</p> <p>Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation <it>in vitro</it>, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.</p> <p>Conclusion</p> <p>The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.</p

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARβ and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas

Nutritional status and functional capacity of hospitalized elderly

Background: The nutritional status of the aging individual results from a complex interaction between personal and environmental factors. A disease influences and is influenced by the nutritional status and the functional capacity of the individual. We asses the relationship between nutritional status and indicators of functional capacity among recently hospitalized elderly in a general hospital.Methods: A cross-sectional study was done with 240 elderly (women, n = 127 and men, n = 113) hospitalized in a hospital that provides care for the public and private healthcare systems. The nutritional status was classified by the MNA (Mini Nutritional Assessment) into: malnourished, risk of malnutrition and without malnutrition (adequate). The functional autonomy indicators were obtained by the self-reported Instrumental Activity of Daily Living (IADL) and Activity of Daily Living (ADL) questionnaire. The chi-square test was used to compare the proportions and the level of significance was 5%.Results: Among the assessed elderly, 33.8% were classified as adequate regarding nutritional status; 37.1% were classified as being at risk of malnutrition and 29.1% were classified as malnourished. All the IADL and ADL variables assessed were significantly more deteriorated among the malnourished individuals. Among the ADL variables, eating partial (42.9%) or complete (12.9%) dependence was found in more than half of the malnourished elderly, in 13.4% of those at risk of malnutrition and in 2.5% of those without malnutrition.Conclusion: There is an interrelationship between the nutritional status of the elderly and reduced functional capacity

An in vitro approach to study effects of prebiotics and probiotics on the faecal microbiota and selected immune parameters relevant to the elderly

The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS) and inulin both stimulated bifidobacteria compared to other treatments (p<0.05). Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05). IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05). To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters

Imaging of Bubonic Plague Dynamics by In Vivo Tracking of Bioluminescent Yersinia pestis

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response

Characteristics of undernourished older medical patients and the identification of predictors for undernutrition status

<p>Abstract</p> <p>Background</p> <p>Undernutrition among older people is a continuing source of concern, particularly among acutely hospitalized patients. The purpose of the current study is to compare malnourished elderly patients with those at nutritional risk and identify factors contributing to the variability between the groups.</p> <p>Methods</p> <p>The study was carried out at the Soroka University Medical Center in the south of Israel. From September 2003 through December 2004, all patients 65 years-of-age or older admitted to any of the internal medicine departments, were screened within 72 hours of admission to determine nutritional status using the short version of the Mini Nutritional Assessment (MNA-SF). Patients at nutritional risk were entered the study and were divided into malnourished or 'at risk' based on the full version of the MNA. Data regarding medical, nutritional, functional, and emotional status were obtained by trained interviewers.</p> <p>Results</p> <p>Two hundred fifty-nine elderly patients, 43.6% men, participated in the study; 18.5% were identified as malnourished and 81.5% were at risk for malnutrition according to the MNA. The malnourished group was less educated, had a higher depression score and lower cognitive and physical functioning. Higher prevalence of chewing problems, nausea, and vomiting was detected among malnourished patients. There was no difference between the groups in health status indicators except for subjective health evaluation which was poorer among the malnourished group. Lower dietary score indicating lower intake of vegetables fruits and fluid, poor appetite and difficulties in eating distinguished between malnourished and at-risk populations with the highest sensitivity and specificity as compare with the anthropometric, global, and self-assessment of nutritional status parts of the MNA. In a multivariate analysis, lower cognitive function, education <12 years and chewing problems were all risk factors for malnutrition.</p> <p>Conclusion</p> <p>Our study indicates that low food consumption as well as poor appetite and chewing problems are associated with the development of malnutrition. Given the critical importance of nutritional status in the hospitalized elderly, further intervention trials are required to determine the best intervention strategies to overcome these problems.</p

Genomic profiling identifies common HPV-associated chromosomal alterations in squamous cell carcinomas of cervix and head and neck

<p>Abstract</p> <p>Background</p> <p>It is well known that a persistent infection with high-risk human papillomavirus (hrHPV) is causally involved in the development of squamous cell carcinomas of the uterine cervix (CxSCCs) and a subset of SCCs of the head and neck (HNSCCs). The latter differ from hrHPV-negative HNSCCs at the clinical and molecular level.</p> <p>Methods</p> <p>To determine whether hrHPV-associated SCCs arising from different organs have specific chromosomal alterations in common, we compared genome-wide chromosomal profiles of 10 CxSCCs (all hrHPV-positive) with 12 hrHPV-positive HNSCCs and 30 hrHPV-negative HNSCCs. Potential organ-specific alterations and alterations shared by SCCs in general were investigated as well.</p> <p>Results</p> <p>Unsupervised hierarchical clustering resulted in one mainly hrHPV-positive and one mainly hrHPV-negative cluster. Interestingly, loss at 13q and gain at 20q were frequent in HPV-positive carcinomas of both origins, but uncommon in hrHPV-negative HNSCCs, indicating that these alterations are associated with hrHPV-mediated carcinogenesis. Within the group of hrHPV-positive carcinomas, HNSCCs more frequently showed gains of multiple regions at 8q whereas CxSCCs more often showed loss at 17p. Finally, gains at 3q24-29 and losses at 11q22.3-25 were frequent (>50%) in all sample groups.</p> <p>Conclusion</p> <p>In this study hrHPV-specific, organ-specific, and pan-SCC chromosomal alterations were identified. The existence of hrHPV-specific alterations in SCCs of different anatomical origin, suggests that these alterations are crucial for hrHPV-mediated carcinogenesis.</p

A novel approach to locate Phytophthora infestans resistance genes on the potato genetic map

Mapping resistance genes is usually accomplished by phenotyping a segregating population for the resistance trait and genotyping it using a large number of markers. Most resistance genes are of the NBS-LRR type, of which an increasing number is sequenced. These genes and their analogs (RGAs) are often organized in clusters. Clusters tend to be rather homogenous, viz. containing genes that show high sequence similarity with each other. From many of these clusters the map position is known. In this study we present and test a novel method to quickly identify to which cluster a new resistance gene belongs and to produce markers that can be used for introgression breeding. We used NBS profiling to identify markers in bulked DNA samples prepared from resistant and susceptible genotypes of small segregating populations. Markers co-segregating with resistance can be tested on individual plants and directly used for breeding. To identify the resistance gene cluster a gene belongs to, the fragments were sequenced and the sequences analyzed using bioinformatics tools. Putative map positions arising from this analysis were validated using markers mapped in the segregating population. The versatility of the approach is demonstrated with a number of populations derived from wild Solanum species segregating for P. infestans resistance. Newly identified P. infestans resistance genes originating from S. verrucosum, S. schenckii, and S. capsicibaccatum could be mapped to potato chromosomes 6, 4, and 11, respectively