Reprogramming of prostate cancer-associated stromal cells to embryonic stem-like

BACKGROUND CD90+ prostate cancer-associated (CP) stromal cells represent a diseased cell type found only in tumor tissue. They differ from their normal counterpart in gene expression and inductive signaling. Genetic reprogramming by induced pluripotent stem (iPS) cell technology can effectively change adult cells into stem-like cells through wholesale alteration of the gene expression program. This technology might be used to erase the abnormal gene expression of diseased cells. The resultant iPS cells would no longer express the disease phenotype, and behave like stem cells. METHODS CP stromal cells, isolated from tumor tissue of a surgically resected prostate by anti-CD90-mediated sorting and cultured in vitro, were transfected with in vitro packaged lentiviral expression vectors containing stem cell transcription factor genes POU5F1, LIN28, NANOG, and SOX2. RESULTS Alkaline phosphatase-positive iPS cells were obtained in about 3 weeks post-transfection at a frequency of 10-4. Their colony morphology was indistinguishable from that of human embryonic stem (ES) cells. Transcriptome analysis showed a virtually complete match in gene expression between the iPS and ES cells. CONCLUSIONS Genes of CP stromal cells could be fully inactivated by genetic reprogramming. As a consequence, the disease phenotype was cured. Prostate 72:14531463, 2012. (c) 2012 Wiley Periodicals, Inc.

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

Abstract Background Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. Methods SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. Results Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. Conclusions The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

Quantification of fractal dimension and Shannon’s entropy in histological diagnosis of prostate cancer

Background: Prostate cancer is a serious public health problem that affects quality of life and has a significant mortality rate. The aim of the present study was to quantify the fractal dimension and Shannon’s entropy in the histological diagnosis of prostate cancer. Methods: Thirty-four patients with prostate cancer aged 50 to 75 years having been submitted to radical prostatectomy participated in the study. Histological slides of normal (N), hyperplastic (H) and tumor (T) areas of the prostate were digitally photographed with three different magnifications (40x, 100x and 400x) and analyzed. The fractal dimension (FD), Shannon’s entropy (SE) and number of cell nuclei (NCN) in these areas were compared. Results: FD analysis demonstrated the following significant differences between groups: T vs. N and H vs. N groups (p < 0.05) at a magnification of 40x; T vs. N (p < 0.01) at 100x and H vs. N (p < 0.01) at 400x. SE analysis revealed the following significant differences groups: T vs. H and T vs. N (p < 0.05) at 100x; and T vs. H and T vs. N (p < 0.001) at 400x. NCN analysis demonstrated the following significant differences between groups: T vs. H and T vs. N (p < 0.05) at 40x; T vs. H and T vs. N (p < 0.0001) at 100x; and T vs. H and T vs. N (p < 0.01) at 400x. Conclusions: The quantification of the FD and SE, together with the number of cell nuclei, has potential clinical applications in the histological diagnosis of prostate cancer.

Incidence of Leptospirosis infection in the East Zone of Sao Paulo City, Brazil

Abstract Background Leptospirosis is a zoonosis which is spread through contamined running water. This contaminations is seriously affected by the flooding which occurs in the area surrounding the Aricanduva river. The transmission of the disease results mainly from the contact of water with soil contaminated by the urine of infected animals. We aimed to conduct an epidemiological survey on Leptospirosis cases in Sao Paulo East Zone area. Method The analysis conducted in this study was based on data collected from the health authorities of that region close the Aricanduva river between 2007 and 2008 years, which give the rates of confirmed cases, mortality and death from human Leptospirosis. Other information concerned with the relationships among rainfall index, points of flooding and incidence of Leptospirosis. Results We observed a direct and important water contamination. Records of flooding points and dates of the reported cases in the region showed a direct relationship from which the period of higher rainfall also recorded an increase in cases. The annual record of the city and the region and rainfall regions also presented correlation. Conclusion The association between the indices of flooding and Leptospirosis cases indicates that preventive measures are necessary to avoid exposing the community.

Reorganization of secondary and tertiary health care levels: impact on the outcomes of oral cancer screening in the São Paulo State, Brazil

This study presents the strategies for prevention and early detection of oral cancer by means of screening in the elderly population of São Paulo, the richest and the most populous state of Brazil. This research was a retrospective longitudinal study based on the analysis of secondary data. The variables - number of participating cities, coverage of screening, and number of suspicious and confirmed cases of oral cancer - were divided into two periods: 2001-2004 and 2005-2008. Data were analyzed statistically by the chi-square test at 5% significance level. The implementation of a nationwide public oral health policy in 2004 and the reorganization of the secondary and tertiary health care were evaluated as mediator factors able to interfere in the achieved outcomes. From 2001 to 2008, 2,229,273 oral examinations were performed. There was an addition of 205 participating cities by the end of the studied period (p<0.0001). The coverage of oral cancer screening increased from 4.1% to 16% (p<0.0001). There was a decrease in the number of suspicious lesions (from 9% in 2005 to 5% in 2008) (p<0.0001) and in the rate of confirmed oral cancer cases per 100,000 examinations (from 20.89 in 2001 to 10.40 in 2008) (p<0.0001). After 8 years of screening, there was a decrease in the number of suspicious lesions and confirmed cases of oral cancer in the population. The reorganization of secondary and tertiary health care levels of oral care seems to have contributed to modify these numbers, having a positive impact on the outcomes of oral cancer screening in the São Paulo State.

Influence of cervical preflaring on determination of apical file size in the palatal roots of maxillary molars

PURPOSE: The aim of this study was to investigate the influence of cervical preflaring in determining the initial apical file (IAF) in the palatal roots of maxillary molars, and to determine the morphologic shape of the canal 1 mm short of the apex. METHODS: After preparing standard access cavities the group 1 received the IAF without cervical preflaring (WCP). In groups 2 to 5, preflaring was performed with Gates-Glidden (GG), Anatomic Endodontics Technology (AET), GT Rotary Files (GT) and LA Axxes (LA), respectively. Each canal was sized using manual K-files, starting with size 08 files, and making passive movements until the WL was reached. File sizes were increased until a binding sensation was felt at the WL. The IAF area and the area of the root canal were measured to verify the percentage occupied by the IAF inside the canal in each sample by SEM. The morphologic shape of the root canal was classified as circular, oval or flattened. Statistical analysis was performed by ANOVA/Tukey test (P < 0.05). RESULTS: The decreasing percentages occupied by the IAF inside the canal were: LA>GT=AET>GG>WCP. The morphologic shape was predominantly oval. CONCLUSION: The type of cervical preflaring used interferes in the determination of IAF.

Incidence of lower limb bone fractures in female individuals of Aracaju (Sergipe), Brazil

Studies indicate that the number of hip bone fractures caused by osteoporosis may rise from 1.66 to 6.26 million until 2050, worldwide. For this reason, implementation of preventive measures becomes a necessity. Female individuals are usually more affected due to a variety of factors including old age, early menopause, chronicle disease in the family history, calcium deficit, as well as the lack of physical exercise (sedentary individual). The aim of this study was to estimate the incidence of hip and lower limb fracture in female individuals’ resident in Aracaju city. From the period of January 2008-2009, around of 300 fracture cases were of lower limb analyzed from females. The incidence of femur fractures in women increased according to age group, 66.17 individuals per 10,000 inhabitants (over 60 years-old). These findings allow us to conclude that the incidence of hip and lower limb bone fractures among women over 60 years were more significant in the femur.

Relationship between body image disturbance and incidence of depression: the SUN prospective cohort

[EN] Background: Body image disturbance is an increasing problem in Western societies and is associated with a number of mental health outcomes including anorexia, bulimia, body dysmorphia, and depression. The aim of this study was to assess the association between body image disturbance and the incidence of depression. Methods: This study included 10,286 participants from a dynamic prospective cohort of Spanish university graduates, who were followed-up for a median period of 4.2 years (Seguimiento Universidad de Navarra – the SUN study). The key characteristic of the study is the permanently open recruitment that started in 1999. The baseline questionnaire included information about body mass index (BMI) and the nine figure schemes that were used to assess body size perception. These variables were grouped according to recommended classifications and the difference between BMI and body size perception was considered as a proxy of body image disturbance. A subject was classified as an incident case of depression if he/she was initially free of depression and reported a physician-made diagnosis of depression and/or the use of antidepressant medication in at least one of the follow-up questionnaires. The association between body image disturbance and the incidence of depression was estimated by calculating the multivariable adjusted Odds Ratio (OR) and its 95% Confidence Interval (95% CI), using logistic regression models. Results: The cumulative incidence of depression during follow-up in the cohort was 4.8%. Men who underestimated their body size had a high percentage of overweight and obesity (50.1% and 12.6%, respectively), whereas women who overestimated their body size had a high percentage of underweight (87.6%). The underestimation exhibited a negative association with the incidence of depression among women (OR: 0.72, 95% CI: 0.54 – 0.95), but this effect disappeared after adjusting for possible confounding variables. The proportion of participants who correctly perceived their body size was high (53.3%) and gross misperception was seldom found, with most cases selecting only one silhouette below (42.7%) or above (2.6%) their actual BMI. Conclusion: We found no association between body image disturbance and subsequent depression in a cohort of university graduates in Spain.

Cellular and Molecular Biomarkers for Risk Assessment of Colorectal cancer

The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.

Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth.

Role of new molecular approaches for the early diagnosis of bladder cancer in clinical practice

There is an urgent need to improve the performance of urine cytology for the diagnosis of bladder cancer. In preliminary studies, telomerase activity evaluated by telomeric repeat amplification protocol (TRAP) assay and chromosomal aneuploidy detected by fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer have produced important results. Urine cell-free (UCF) DNA has also been proposed as a potential marker for early bladder cancer diagnosis. In the first study the diagnostic performance of TRAP assay and FISH analysis was assessed, while the second study evaluated the potential role of UCF DNA integrity in early bladder cancer diagnosis. In the first cross-sectional study, 289 consecutive patients who presented with urinary symptoms underwent cystoscopy and cytology evaluation. In the second study, UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. c-Myc, BCAS1 and HER2 gene sequences longer than 250 bp were quantified by real time PCR to verify UCF DNA integrity. In the first study, sensitivity and specificity were 0.39 and 0.83, respectively, for cytology; 0.66 and 0.72 for TRAP; 0.78 and 0.60 for the cytology and TRAP combination; 0.78 and 0.78 for the cytology, TRAP and FISH combination; and 0.65 and 0.93 for the TRAP and FISH combination. In the second study, at the best cutoff of 0.1 ng/µl, UCF DNA integrity analysis showed a sensitivity of 0.73 and a specificity of 0.84 in healthy individuals and 0.83 in symptomatic patients. The preliminary results suggest that these biomarkers could potentially be used for the early diagnosis of bladder cancer, especially in high-risk populations (e.g, symptomatic individuals exposed to occupational risk) who may benefit from the use of noninvasive diagnostic tests in terms of cost-benefit.

Preclinical development of anti-cancer strategies against human HER-2

HER-2 is a 185 kDa transmembrane receptor tyrosine kinase that belongs to the EGFR family. HER-2 is overexpressed in nearly 25% of human breast cancers and women with this subtype of breast cancer have a worse prognosis and frequently develop metastases. The progressive high number of HER-2-positive breast cancer patients with metastatic spread in the brain (up to half of women) has been attributed to the reduction in mortality, the effectiveness of Trastuzumab in killing metastatic cells in other organs and to its incapability to cross the blood-brain barrier. Apart from full-length-HER-2, a splice variant of HER-2 lacking exon 16 (here referred to as D16) was identified in human HER-2-positive breast cancers. Here, the contribution of HER-2 and D16 to mammary carcinogenesis was investigated in a model transgenic for both genes (F1 model). A dominant role of D16, especially in early stages of tumorigenesis, was suggested and the coexistence of heterogeneous levels of HER-2 and D16 in F1 tumors revealed the undeniable value of F1 strain as preclinical model of HER-2-positive breast cancer, closer resembling the human situation in respect to previous models. The therapeutical efficacy of anti-HER-2 agents, targeting HER-2 receptor (Trastuzumab, Lapatinib, R-LM249) or signaling effectors (Dasatinib, UO126, NVP-BKM120), was investigated in models of local or advanced HER-2-positive breast cancer. In contrast with early studies, data herein collected suggested that the presence of D16 can predict a better response to Trastuzumab and other agents targeting HER-2 receptor or Src activity. Using a multiorgan HER-2-positive metastatic model, the efficacy of NVP-BKM120 (PI3K inhibitor) in blocking the growth of brain metastases and the oncolytic ability of R-LM249 (HER-2-retargeted HSV) to reach and destroy metastatic HER-2-positive cancer cells were shown. Finally, exploiting the definition of “oncoantigen” given to HER-2, the immunopreventive activity of two vaccines on HER-2-positive mammary tumorigenesis was demonstrated.

Proteomics-driven approach for the detection of breast cancer biomarkers

Breast cancer (BC) is the most often diagnosed cancer entity of women worldwide. No molecular biomarkers are usable in the clinical routine for the early detection of BC. Proteomics is one of the dynamic tools for the successful examination of changes on the protein level. In this thesis different proteomics-based investigations were performed for the detection of protein and autoantibody biomarkers in serum samples of BC and healthy (CTRL) subjects. First, protein levels of candidates from previous profiling studies were investigated via antibody-microarray platform. Three proteins were found in distinct levels in both groups: secretoglobin family 1D member 1, alpha-2 macroglobulin and inter-alpha-trypsin inhibitor heavy chain family member 4. The second part was dedicated to the de novo exploration of potentially immunogenic tumor antigens (TA’s) with immunoprecipitation and Western immunoblotting followed by identification over mass spectrometry. Autoantibody levels were verified in individual serum profiling via the protein microarray platform. Two autoantibody’ cohorts (anti-Histone 2B and anti-Recoverin) were found in different levels in both groups. The findings of this PhD thesis underline deregulated serum protein and autoantibody levels in the presence of BC. Further investigations are needed to confirm the results in an independent study population.

Development and optimisation of novel 68 Ga-folates - towards early diagnosis of ovarian cancer

Der Folsäure-basierte Radiotracer Etarfolatide (99mTc-EC 20) hat in der Vergangenheit sehr vielversprechende Ergebnisse im Bereich der frühzeitigen Diagnostik von Ovarialkarzinomen gezeigt. Einzelphotonen-Emissionscomputertomographie (SPECT) erlaubt dabei eine Visualisierung der Krankheit in einem sehr frühen Stadium – ermöglicht wird dies durch Folsäure, welche als Target Vektor dient. Um das erfolgreiche Prinzip der Radiofolate auf die Positronen-Emissionstomographie (PET) zu übertragen, welche eine noch höhere räumliche Auflösung ermöglicht, wurden in den letzten fünf Jahren bereits 18F-folate entwickelt. Deren hepatobiliären Exkretionsmuster, verursacht durch die relativ hohe Lipophilie der Strukturen, entsprachen jedoch nicht den Anforderungen. Eine optimierte Bioverteilung der Tracer in vivo kann durch eine generelle Erhöhung der Polarität erfolgen. Die Kombination aus einem polaren 68Ga-Komplex mit Folsäure als Target Vektor stellte den Fokus dieses Projektes dar. Ziel war die Entwicklung eines Radiofolates mit der Tendenz einer raschen renalen Ausscheidung und verringerter hepatobiliärer Anreicherung. Dazu wurde Folsäure regiospezifisch über ihre y-Säure an verschiedene bifunktionelle Chelatoren (BFCs) gekoppelt. Vier verschiedene Reaktionstypen wurden gewählt und durchgeführt: Cu-katalysierte sowie Cu-freie Click Reaktion, Amindbindung und Thioharnstoff Bildung. Es wurden sechs verschiedene Derivate erhalten und mit 68Ga radiomarkiert.