In utero tracheal occlusion: surgical approach associated to steroid treatment increase VEGF (Vascular Endothelial Growth Factor) receptors in lungs of fetal rats

Background/Purpose: Congenital diaphragmatic hernia (CDH) presents with hypoplastic lungs and usually leads to pulmonary hypertension and high neonatal mortality. Fetal tracheal occlusion (TO) and prenatal corticotherapy are alternatives to accelerate fetal pulmonary growth and decrease hypoplasia in CDH. VEGF (Vascular Endothelial Growth Factor) production and surfactant production by type II pneumocytes are related with pulmonary maturityand are altered in CDH, but little has been described about VEGF receptors. Our objective was to quantify the receptors of VEGF (VEGFR) and type II pneumocytes, verifying the effects of TO and corticotherapy on normal lungs of fetal rats. Methods: Six groups of 12 Spreague-Dawley rat fetuses (gestation=22 days) were compared:TO, S (sham), C (control), TO+Dex, S+Dex and C+Dex. On the 18.5 gestational day, TO was performed with and without corticotherapy, using dexamethasone. On the 21.5 gestational daybody and lung weights were measured. Immunohistochemistry was carried out for VEGFR-1(Flt-1) and VEGFR-2 (Flk-1), as well as anti-SP-A, microscopic analysis and quantification of immune system marking. Results: Body weight decreased in Sham and lung weight increased in TO and TO+Dex (p<0.05) groups. VEGFR-1 and VEGFR-2 increased in TO and in TO+Dex (p<0.05). Type II pneumocytes (SP-A) decreased both in the TO and in TO+Dex groups in comparison with the Control groupin absolute value (p<0.05), with no differences in relative value to total cell count. Conclusion: TO in association with the use of corticosteroids increased VEGFR-1 and VEGFR-2, while the quantity of type II pneumocytes (SP-A) decreased in relation to the area considered, but not in relation to the total of pulmonary cells.Introdução: A hérnia diafragmática congênita (HDC) causa hipoplasia e hipertensão pulmonar e em geral leva a alta morbidade e mortalidade neonatal. Traqueo-oclusão fetal (TO) e corticoterapia pré-natal são alternativas para acelerar o crescimento pulmonar fetal e diminuir a hipoplasia na HDC. A produção de VEGF (Vascular Endothelial Growth Factor) está relacionada com a maturidade pulmonar e sofre alterações na HDC ainda não elucidadas. Materiais e métodos: Seis grupos de 12 fetos de ratos Spreague-Dawley foram comparados: TO, Sham, Controle, TO+Dex, Sham+Dex e Controle+Dex. No dia 18,5º foi realizada TO com e sem corticoterapia utilizando dexametasona. No 21,5º dia gestacional os pesos corporal e pulmonar foram mensurados. Realizou-se imunohistoquímica para VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), seguida de morfometria. Resultados: O VEGFR-1 estava aumentado no TO (p<0.05)e na TO+Dex (p<0.05). O VEGFR-2 teve aumento significativo quando comparamos TO e TO+Dex com o controle (p<0,05). Conclusão: A TO associada ao uso de corticoesteróide aumentou o número de VEGFR-1 e VEGFR-2 em pulmões de fetos de ratas

The use of microRNA for the treatment of prostate cancer: in vitro and in vivo studies

Introdução: O câncer de próstata (CaP) é o tumor mais comum do homem nos países ocidentais e a segunda causa de óbito por câncer em homens nos EUA, Europa e Brasil. O câncer localizado tem sobrevida câncer especifica elevada quando tratado adequadamente, porém a doença metastática ainda apresenta tratamentos pouco eficientes com sobrevida global de 28%. Os microRNAs (miRNAs) são um grupo de moléculas pequenas de RNA que contém entre 19 a 25 nucleotídeos não codificantes de proteína, com ação fundamental na regulação da expressão gênica. Eles estão envolvidos em processos essenciais nas células normais e neoplásicas como ciclo celular, proliferação, apoptose, metabolismo energético, invasão e metastatização. Objetivos: Realizar estudos in vitro e in vivo usando miRNA em um modelo de câncer de próstata metastático inédito no nosso meio com intuito de analisar o seu potencial como agente terapêutico dessa neoplasia. Métodos: Nos estudos in vitro, três linhagens celulares foram utilizadas (PC3, DU145 e LNCaP). Essas linhagens foram transfectadas com os miRNAs 100, 145 e 373 e seus respectivos antiMiRs utilizando-se lipofectamina. Analisamos a expressão dos genes alvo mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 por PCR quantitativo em tempo real (qRT-PCR). Foram realizados também estudos de apoptose, ciclo celular e ploidia utilizando o citômetro de fluxo. Alterações no potencial de invasão foram avaliadas pela técnica do matrigel. O modelo in vivo pré-clínico foi desenvolvido pela injeção intra-cardíaca da linhagem PC-3M-Luc-C6 em camundongos NUDE com 9 semanas. O crescimento tumoral foi avaliado com o sistema de bioluminescência in vivo. Após o pleno estabelecimento das metástases no dia 21, os animais foram tratados com três injeções na veia da cauda contendo o miRNA conjugado com o atelocolágeno. Os animais foram sacrificados e no dia 48 para análise dos tecidos. Resultados: miR-100 aumenta a apoptose na LNCaP, e reduz a apoptose na DU145. Na linhagem DU145 o miR 100 inibiu a proliferação. Na análise da expressão gênica o miR-100 inibe SMARCA5 na DU145 e PC3 e mTOR na LNCaP, o anti-miR 100 estimula mTOR e SMARCA5 na LNCaP. O miR-145 promoveu aumento da apoptose em 24% na DU145. Na linhagem PC3 o miR-145 age inibindo a proliferação, com uma diferença absoluta de 18% em relação ao seu controle. O miR-145 inibe KRAS e CMYC nas três linhagens e o anti-miR-145 estimula CMYC na DU145 e RAS nas três linhagens O miR-373 reduziu a apoptose em 29% na DU145 e diminui a proliferação com uma diferença absoluta de 13% em relação ao controle. O miR- 373 estimula a MMP9 na DU145 e na LNCaP e inibe o CD44 na PC3. O antimiR- 373 inibe MMP9 na DU145 e LNCaP. Nos estudos in vivo de CaP metastático o miR-100 apresenta tendência a redução no crescimento tumoral (p=0,23) e o miR-145 reduz o crescimento de forma significativa no dia 34 (p=0,02). Após esses dias o tumor volta a crescer de forma agressiva. Os animais tratados com anti-miR-373 não apresentaram alterações em relação aos controles. Conclusões: O miR-100 é um miRNA contexto dependente, com papel supressor tumoral em linhagens de tumor de próstata agressivo e o miR- 373 age in vitro como oncomiR. O miR-145 age como supressor tumoral in vitro e em modelo animal de CaP metastático apresentando resposta terapêutica consistente, podendo ser utilizado no arsenal terapêutico contra essa neoplasia. Estudos futuros devem avaliar o uso dos miRNAs isoladamente ou de forma adjuvante no tratamento do CaP metastáticoIntroduction: Prostate cancer (PCa) is the most common neoplasia of man in Western countries and the second cause of death by cancer in men in the US, Europe and Brazil. The localized cancer has high cancer-specific survival when treated properly, however metastatic disease still presents low effective treatments with 28% of global survival. microRNAs (miRNAs) are a group of small RNA molecules containing from 19 to 25 nucleotides of noncoding protein with fundamental action in the regulation of gene expression. They are involved in key processes in normal and neoplastic cells as cell cycle, proliferation, apoptosis, energy metabolism, invasion and metastasis. Objectives: To carry out studies in vitro and in vivo using miRNA in a novel model of metastatic prostate cancer in our country in order to evaluate its potential as a therapeutic agent of this neoplasia. Methods: In the in vitro studies, three cell lines were used (PC3, DU145 and LNCaP). These cell lines were transfected with miRNAs 100, 145 and 373 and their antiMiRs using lipofectamine. We analyzed the gene expression of mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 by real-time polymerase chain reaction (qRT-PCR). We also performed studies of apoptosis, cell cycle and ploidy using flow cytometer. Changes in the invasion potential were evaluated by the technique of matrigel. The pre-clinical model in vivo was developed by intracardiac injection of PC-3MLuc-C6 cell line in NUDE mice with 9 weeks. Tumor growth was evaluated with an in vivo image system (IVIS). After the full establishment of metastases on day 21, the animals were treated with three injections into the tail vein containing the miRNA plus atelocollagen. The animals were sacrificed on day 48 for tissues analysis. Results: MiR-100 increases apoptosis in LNCaP and reduces apoptosis in DU145. The anti-miR-100 increased apoptosis in 14% in PC3. In cell line DU145, miR-100 inhibited proliferation. In the analysis of gene expression, the miR-100 inhibits SMARCA5 in DU145 and PC3 and mTOR in LNCaP, anti-miR-100 stimulates mTOR and SMARCA5 in LNCaP. The miR-145 promoted an increased in apoptosis by 24% in DU145. In PC3 cell line miR-145 acts by inhibiting the proliferation, with an absolute difference of 18% compared to control. MiR-145 inhibits KRAS and CMYC in the three cell lines and anti-miR-145 stimulates CMYC in DU145 and KRAS in the three cell lines. The miR-373 reduced apoptosis by 29% in DU145 and reduces proliferation with an absolute difference of 13% relative to control. MiR-373 stimulates MMP9 in DU145 and LNCaP cells and inhibits CD44 in PC3. The anti -miR-373 inhibits MMP9 in DU145 and LNCaP. In the in vivo studies of metastatic PCa, miR-100 shows a tendency to decrease tumor growth (p=0.23) and miR-145 reduces tumor growth on day 34 (p=0.02). After those days, the tumor grows back aggressively. Animals treated with anti-miR-373 showed no changes relative to controls. Conclusion: The miR-100 is a context-dependent miRNA, with tumor suppressor role in aggressive tumor cell lines. The miR-373 acts in vitro as oncomiR and miR-145 acts as a tumor suppressor in vitro and in an animal model with consistent therapeutic response and can be used in the therapeutic arsenal against this neoplasia. Future studies should evaluate the use of miRNAs alone or adjuvant in the treatment of metastatic prostate cance

The role of bladder diverticula in the prevalence of acute urinary retention in patients with BPH who are candidates to surgery

ABSTRACT Introduction: The urinary bladder diverticula (BD) secondary to benign prostatic hyperplasia (BPH) is a complication that can lead to urinary stasis, stone, urinary tract infection (UTI) and tumors. It's role in acute urinary retention (AUR) is not totally understood. Objectives: To determine the effect of BD size on AUR rates in patients with BPH candidates to surgery. Subjects and Methods: We performed a retrospective cohort study of 47 patients with BPH and BD who underwent BPH surgery associated to complete bladder diverticulectomy from 2006 to 2016. We analyzed risk factors for AUR in patients with BD using univariate, multivariate and correlation analysis. Results: There was a difference in the size of the diverticula, with 6.8 cm vs. 4.5 cm among patients with and without AUR respectively (p=0.005). The ROC curve showed a correlation between the size of BD and the risk of AUR. The value of 5.15 cm presented a sensitivity of 73% and a specificity of 72%. The area under the curve was 0.75 (p=0.01). Comparing groups with BD >5.0 cm vs. ≤5.0 cm, the AUR incidence was 74% and 27.8% respectively with an OR of 2.65 (1.20-5.85) (p=0.005). In the multivariate analysis, only the size of the diverticula reached statistical significance (p=0.012). Conclusions: The diameter of BD is an independent risk factor for AUR in patients with BPH and BD who are candidates to surgery. A diameter greater than 5.15 cm increases the risk of AUR

Assessment of neurosurgical outcome in children prenatally diagnosed with myelomeningocele and development of a protocol for fetal surgery to prevent hydrocephalus

Prenatal diagnosis of myelomeningocele (MMC) has permitted a better planning for optimum management of the disease. More recently, it has allowed for a possible intrauterine repair of the spinal defect.To describe neurosurgical outcome in children with myelomeningocele and follow-up at a referral center in Fetal Medicine. Patients were characterized for the development of a protocol suitable for fetal surgery, and fetuses who were possible candidates for intrauterine surgery were identified.A retrospective descriptive analysis was performed of 98 cases of fetal myelomeningocele, seen at CAISM-UNICAMP, from January 1994 to December 2002, identifying cases with a possible indication for fetal surgery.Mean gestational age at diagnosis was 29 weeks (17-39); level of lesion was above the sacral region in 92.84%; association with hydrocephalus occurred in 78.57%. During clinical course, 82.5% of patients had neurogenic bladder and 60% had neural and mental deficits. Potential intrauterine repair rate was 11.57%, using criteria from the protocol developed in our service.Myelomeningocele is associated with severe and frequent sequelae. in virtually 12% of our cases, fetal surgery could have been offered as a therapeutic option.Univ Estadual Campinas, Sch Med, Div Pediat Surg, BR-13084970 Campinas, SP, BrazilUniv Estadual Campinas, Sch Med, Div Maternal Fetal Med, BR-13084970 Campinas, SP, BrazilUniv Estadual Campinas, Dept Obstet & Gynecol, BR-13084970 Campinas, SP, BrazilUniv Estadual Campinas, Dept Pediat, Div Neonatol, BR-13084970 Campinas, SP, BrazilUniv Estadual Campinas, Discipline Neurosurg, Fetal Med Grp, BR-13084970 Campinas, SP, BrazilWeb of Scienc

En Bloc Resection of Bladder Tumors (ERBT) using different lasers – Hybrid and Holmium Laser

ABSTRACT Introduction: The En-bloc Resection of Bladder Tumors (ERBT) is a method that offers more benefits compared to the traditional Transurethral Resection of Bladder Tumor (TURBT) (1, 2). Recent studies have shown that ERBT offers better pathological analysis and oncological outcomes (3-6). Thulium and holmium are the most frequently used lasers for this procedure, with the hybrid laser being a new addition that combines thulium and diode to improve hemostatic properties (5, 7-9). Objective: This report aims to discuss the use of two types of lasers, hybrid and holmium, for ERBT. Material and Methods: Two case studies were conducted. The first case featured a 68-year-old male with two tumors measuring 1.5cm and 2cm. The hybrid laser was used for the procedure. The second case involved a 70-year-old female with a 5cm tumor on the posterior bladder wall, and holmium laser was used with morcellation of the tumor. The quality of histopathological analysis was evaluated. The perioperative data and the entire procedure of the two cases were documented in a step-by-step video. Results: Both lasers demonstrated excellent results without technical difficulties. There was no bleeding, and both patients were discharged with one day of hospitalization. The detrusor muscle was present without artifacts, and the morcellation did not affect the analysis. The first case showed a pT1G3, and the second case showed a pT2 urothelial carcinoma. The hybrid laser exhibited superior hemostatic capacity compared to the holmium laser. Conclusion: ERBT can use hybrid or holmium lasers without affecting histopathological analysis, even with morcellation

Comprehensive study of gene and microRNA expression related to epithelial-mesenchymal transition in prostate cancer.

Prostate cancer is the most common cancer in men, and most patients have localized disease at the time of diagnosis. However, 4% already present with metastatic disease. Epithelial-mesenchymal transition is a fundamental process in carcinogenesis that has been shown to be involved in prostate cancer progression. The main event in epithelial-mesenchymal transition is the repression of E-cadherin by transcription factors, but the process is also regulated by microRNAs. The aim of this study was to analyze gene and microRNA expression involved in epithelial-mesenchymal transition in localized prostate cancer and metastatic prostate cancer cell lines and correlate with clinicopathological findings. We studied 51 fresh frozen tissue samples from patients with localized prostate cancer (PCa) treated by radical prostatectomy and three metastatic prostate cancer cell lines (LNCaP, DU145, PC3). The expression of 10 genes and 18 miRNAs were assessed by real-time PCR. The patients were divided into groups according to Gleason score, pathological stage, preoperative PSA, biochemical recurrence, and risk group for correlation with clinicopathological findings. The majority of localized PCa cases showed an epithelial phenotype, with overexpression of E-cadherin and underexpression of the mesenchymal markers. MiRNA-200 family members and miRNAs 203, 205, 183, 373, and 21 were overexpressed, while miRNAs 9, 495, 29b, and 1 were underexpressed. Low-expression levels of miRNAs 200b, 30a, and 1 were significantly associated with pathological stage. Lower expression of miR-200b was also associated with a Gleason score ≥ 8 and shorter biochemical recurrence-free survival. Furthermore, low-expression levels of miR-30a and high-expression levels of Vimentin and Twist1 were observed in the high-risk group. Compared with the primary tumor, the metastatic cell lines showed significantly higher expression levels of miR-183 and Twist1. In summary, miRNAs 200b, 30a, 1, and 183 and the genes Twist1 and Vimentin might play important roles in the progression of prostate cancer and may eventually become important prognostic markers

Loss of TIMP-1 immune expression and tumor recurrence in localized prostate cancer

Introduction and objective: Overexpression of MMPs has been related to biochemical recurrence after radical prostatectomy. TIMP1 and TIMP2 are controllers of MMPs and the aim of this study is to evaluate the expression levels of MMPs and their regulators using immunohistochemistry in tissue microarray of localized prostate cancer (PC). Materials and Methods: Immune-expression of MMP-9, MMP-2, TIMP1, TIMP-2, MMP-14 and IL8, were analyzed by immunohistochemistry in radical prostatectomy specimens of 40 patients with localized PC who underwent surgery between September 1997 and February 2000. Protein expression was considered as categorical variables, negative or positive. The results of the immune-expression were correlated to Gleason score (GS), pathological stage (TNM), pre-operatory PSA serum levels and biochemical recurrence in a mean follow up period of 92.5 months. Results: The loss of TIMP1 immune-expression was related to biochemical recurrence. When TIMP1 was negative, 56.3% patients recurred versus 22.2% of those whose TIMP1 was positive (p=0.042). MMP-9, MMP-2, IL8 and MMP-14 were positive in the majority of PC. TIMP-2 was negative in all cases. Conclusion: Negative immune-expression of TIMP1 is correlated with biochemical recurrence in patients with PC possibly by failing to control MMP-9, an important MMP related to cancer progression

Clinicopathological Features of 51 Patients with Localized Prostate Cancer Treated by Radical Prostatectomy.

<p>Clinicopathological Features of 51 Patients with Localized Prostate Cancer Treated by Radical Prostatectomy.</p

Kaplan-Meier biochemical recurrence-free survival curve based on miR-200b mean expression (P = 0.049, Log rank test).

<p>Patients with miR-200b expression levels ≤14.690 showed significantly shorter biochemical recurrence-free survival.</p