Urinary microbiota is associated to clinicopathological features in benign prostatic hyperplasia.

BACKGROUND: The urinary microbiota of patients with benign prostatic hyperplasia (BPH) has been associated with lower urinary tract symptoms (LUTS), however, little is known about urinary microbiota correlations with clinicopathological parameters associated with BPH. Here, we investigate associations between the urinary microbiota and clinical parameters of patients with BPH undergoing surgery. METHODS: Forty-one patients with BPH undergoing surgery were recruited from two medical centers. Catheterized urine specimens were collected and the microbiota was characterized by 16S rRNA gene sequencing. Patients were segregated into two groups according to each clinical parameter and differences in urinary microbiota diversity and composition were evaluated. RESULTS: Higher prostate weight and prostate-specific antigen (PSA) levels were associated with higher alpha diversity in the urinary microbiota of BPH patients. At the specific microbe level, we found that the greater the prostatic weight, the lower the relative abundance of Streptococcus, while the greater the PSA levels, the higher the abundance of Lactobacillus. Treatment with 5-α-reductase inhibitor was associated with overall urinary microbiota composition, in part due to a higher abundance of Corynebacterium and Anaerococcus in this group. CONCLUSIONS: We demonstrated that the urinary microbiota of BPH patients is associated with clinicopathological features, paving the way for larger studies in which causality between urinary microbiota and BPH can be appropriately explored.

The bladder microbiota is not significantly altered by intravesical BCG therapy.

INTRODUCTION: Bladder microbiota dysbiosis has been associated with several urological disorders. However, dysbiosis markers in bladder cancer have not been identified and little is known about the effect of Bacillus Calmette-Guérin (BCG) intravesical therapy on the bladder microbiota. In this study, we compared the bladder microbiota of patients with non-muscle-invasive bladder cancer (NMIBC) undergoing BCG therapy to nononcological controls. We also longitudinally analyzed the impact of BCG therapy on the bladder microbiota of NMIBC patients and addressed whether bladder microbiota is associated with BCG efficacy. METHODS: We collected catheterized urine samples from males with intermediate/high-risk NMIBC (cancer group, n = 32) or benign prostatic hyperplasia (control group, n = 41). The cancer group also provided urine samples during and after BCG induction. We used 16S rRNA gene sequencing to characterize the bladder microbiota. Bladder microbiota parameters, such as diversity and taxonomic composition, were compared between groups and associated with clinicopathological data and BCG efficacy. RESULTS: We observed no significant differences between the bladder microbiota of NMIBC patients and controls. BCG intravesical instillations did not significantly alter the bladder microbiota of NMIBC patients, and BCG was rarely detected in the bladder during and after BCG therapy. Microbiota diversity and overall composition before BCG induction did not influence disease persistence at 3 months. However, higher abundance of Lactobacillus, Streptococcus, and Cutibacterium in the pre-BCG bladder microbiota was associated with BCG effectiveness. CONCLUSION: We were unable to identify markers of bladder microbiota dysbiosis among male NMIBC patients. Moreover, we demonstrated for the first time using longitudinally collected samples that BCG cannot persist in the bladder microbiota nor significantly alter its diversity and composition. The associations found between bladder microbes and BCG efficacy highlight the potential of microbial-based therapeutic and risk-stratification strategies in the intermediate/high-risk NMIBC setting.

A novel program of infiltrative control in astrocytomas: ADAM23 depletion promotes cell invasion by activating γ-secretase complex.

Background: Infiltration is a life-threatening growth pattern in malignant astrocytomas and a significant cause of therapy resistance. It results in the tumor cell spreading deeply into the surrounding brain tissue, fostering tumor recurrence and making complete surgical resection impossible. We need to thoroughly understand the mechanisms underlying diffuse infiltration to develop effective therapies. Methods: We integrated in vitro and in vivo functional assays, RNA sequencing, clinical, and expression information from public data sets to investigate the role of ADAM23 expression coupling astrocytoma's growth and motility. Results: ADAM23 downregulation resulted in increased infiltration, reduced tumor growth, and improved overall survival in astrocytomas. Additionally, we show that ADAM23 deficiency induces γ-secretase (GS) complex activity, contributing to the production and deposition of the Amyloid-ß and release of NICD. Finally, GS ablation in ADAM23-low astrocytomas induced a significant inhibitory effect on the invasive programs. Conclusions: Our findings reveal a role for ADAM23 in regulating the balance between cell proliferation and invasiveness in astrocytoma cells, proposing GS inhibition as a therapeutic option in ADAM23 low-expressing astrocytomas.

Choice of 16S Ribosomal RNA Primers Impacts Male Urinary Microbiota Profiling.

Accessibility to next-generation sequencing (NGS) technologies has enabled the profiling of microbial communities living in distinct habitats. 16S ribosomal RNA (rRNA) gene sequencing is widely used for microbiota profiling with NGS technologies. Since most used NGS platforms generate short reads, sequencing the full-length 16S rRNA gene is impractical. Therefore, choosing which 16S rRNA hypervariable region to sequence is critical in microbiota profiling studies. All nine 16S rRNA hypervariable regions are taxonomically informative, but due to variability in profiling performance for specific clades, choosing the ideal 16S rRNA hypervariable region will depend on the bacterial composition of the habitat under study. Recently, NGS allowed the identification of microbes in the urinary tract, and urinary microbiota has become an active research area. However, there is no current study evaluating the performance of different 16S rRNA hypervariable regions for male urinary microbiota profiling. We collected urine samples from male volunteers and profiled their urinary microbiota by sequencing a panel of six amplicons encompassing all nine 16S rRNA hypervariable regions. Systematic comparisons of their performance indicate V1V2 hypervariable regions better assess the taxa commonly present in male urine samples, suggesting V1V2 amplicon sequencing is more suitable for male urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future male urinary microbiota studies.

A Key Pathway to Cancer Resilience: The Role of Autophagy in Glioblastomas.

There are no effective strategies for the successful treatment of glioblastomas (GBM). Current therapeutic modalities effectively target bulk tumor cells but leave behind marginal GBM cells that escape from the surgical margins and radiotherapy field, exhibiting high migratory phenotype and resistance to all available anti-glioma therapies. Drug resistance is mostly driven by tumor cell plasticity: a concept associated with reactivating transcriptional programs in response to adverse and dynamic conditions from the tumor microenvironment. Autophagy, or "self-eating", pathway is an emerging target for cancer therapy and has been regarded as one of the key drivers of cell plasticity in response to energy demanding stress conditions. Many studies shed light on the importance of autophagy as an adaptive mechanism, protecting GBM cells from unfavorable conditions, while others recognize that autophagy can kill those cells by triggering a non-apoptotic cell death program, called 'autophagy cell death' (ACD). In this review, we carefully analyzed literature data and conclude that there is no clear evidence indicating the presence of ACD under pathophysiological settings in GBM disease. It seems to be exclusively induced by excessive (supra-physiological) stress signals, mostly from in vitro cell culture studies. Instead, pre-clinical and clinical data indicate that autophagy is an emblematic example of the 'dark-side' of a rescue pathway that contributes profoundly to a pro-tumoral adaptive response. From a standpoint of treating the real human disease, only combinatorial therapy targeting autophagy with cytotoxic drugs in the adjuvant setting for GBM patients, associated with the development of less toxic and more specific autophagy inhibitors, may inhibit adaptive response and enhance the sensibility of glioma cells to conventional therapies.

NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients.

The risk of developing metastatic disease in breast cancer patients is traditionally predictable based on the number of positive axillary lymph nodes, complemented with additional clinicopathological factors. However, since lymph node-negative patients have a 20-30% probability of developing metastatic disease, lymph node information alone is insufficient to accurately assess individual risk. Molecular approaches, such as multigene expression panels, analyze a set of cancer-related genes that more accurately predict the early risk of metastasis and the treatment response. Here, we present N-Myc downstream-regulated gene 4 (NDRG4) epigenetic silencing as a mechanistic biomarker of metastasis in ductal invasive breast tumors. While aberrant NDRG4 DNA hypermethylation is significantly associated with the development of metastatic disease, downregulation of NDRG4 transcription and protein expression is functionally associated with enhanced lymph node adhesion and cell mobility. Here, we show that epigenetic silencing of NDRG4 modulates integrin signaling by assembling ß1-integrins into large punctate clusters at the leading edge of tumor cells to promote an "adhesive switch," decreasing cell adhesion to fibronectin and increasing cell adhesion and migration towards vitronectin, an important component of human lymph nodes. Taken together, our functional and clinical observations suggest that NDRG4 is a potential mechanistic biomarker in breast cancer that is functionally associated with metastatic disease.

High IL-1R8 expression in breast tumors promotes tumor growth and contributes to impaired antitumor immunity.

Tumors develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. The identification of mechanisms leading to local immune dysregulation is critical to improve cancer therapy. We here demonstrate that Interleukin-1 receptor 8 (IL-1R8 - previously known as SIGIRR/TIR8), a negative regulator of Toll-Like and Interleukin-1 Receptor family signaling, is up-regulated during breast epithelial cell transformation and in primary breast tumors. IL-1R8 expression in transformed breast epithelial cells reduced IL-1-dependent NF-κB activation and production of pro-inflammatory cytokines, inhibited NK cell activation and favored M2-like macrophage polarization. In a murine breast cancer model (MMTV-neu), IL-1R8-deficiency reduced tumor growth and metastasis and was associated with increased mobilization and activation of immune cells, such as NK cells and CD8+ T cells. Finally, immune-gene signature analysis in clinical specimens revealed that high IL-1R8 expression is associated with impaired innate immune sensing and T-cell exclusion from the tumor microenvironment. Our results indicate that high IL-1R8 expression acts as a novel immunomodulatory mechanism leading to dysregulated immunity with important implications for breast cancer immunotherapy.

Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose

Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis

Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose.

Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.

Problem-solving capacity for vitreoretinal diseases in a university health center.

PURPOSE: To evaluate the effectiveness of the tertiary care delivered to patients with vitreoretinal diseases in a defined urban population; to substantiate the planning and allocation of resources in order to improve the tertiary eye care delivery system in a specific area. METHODS: Data were collected from consecutive first-time patients between June 1, 2003 and July 31, 2004 in the Department of Ophthalmology, State University of Campinas, São Paulo, Brazil. Problem-solving capacity values were calculated for vitreoretinal surgery and photocoagulation. Data were entered into the Statistical Package for the Social Sciences (version 10.0). RESULTS: Of the 7500 patients referred to the Department, 641 were deemed suitable for analysis. The diagnoses analyzed were retinal detachment (26.0%), diabetic retinopathy (21.0%), and vitreous hemorrhage (7.7%). The median ages were 52, 59 and 57.5, respectively. Of all patients referred for retinal detachment, 26.5% were inoperable. The values obtained for the problem-solving capacity (PSC) showed that 38.1%, 33.0% and 93.5% of those eligible for an ophthalmic intervention (for retinal detachment, vitreous hemorrhage and diabetic retinopathy) had obtained treatment. The main reason for not giving treatment was the unavailability of operating room time and photocoagulation time (87.1%). CONCLUSIONS: This was the first study of tertiary eye care service performance in Latin America. Sight-threatening conditions such as retinal detachment and diabetic retinopathy are not thoroughly covered by the health system in this area. Various ways to reduce the problem are considered. The study has provided valuable information on planning high-complexity eye services in the population in question.

Characterization of C21ORF100 as a Novel Prostate Differentiation Antigen

Differentiation antigens are immunogenic proteins expressed in specific cell lineages or at specific stages of differentiation ina particular tissue. Generally, their expression in normal cells is preserved after neoplasic transformation and this feature hasmade such molecules potential candidates for cancer immunotherapy. Using alignments between expressed sequence tags(ESTs) and the human chromosome 21 sequence, we have identified a novel gene, named C21orf100, which is exclusivelyexpressed in normal prostate and codes for a putative protein of 55 amino acids. Objective: To characterize C21orf100 as anovel prostate differentiation antigen. Material and Methods: C21orf100 mRNA expression was determined by RT-PCR in 22normal tissues and in 65 samples from melanomas and prostate, thyroid, stomach, uterus and breast tumors. The existenceof a humoral immune response against C21orf100 protein in prostate cancer patients was evaluated by immunoblotting usinga His-tagged recombinant protein. Results: As expected for a differentiation antigen, C21orf100 mRNA expression waspredominantly detected in both normal and tumor prostate samples. Antibodies against C21orf100 recombinant protein weredetected in 1 out of 50 (2%) plasma samples from prostate cancer patients and were not detected in the plasma from healthyblood donors. Conclusion: The restricted expression pattern and the detection of antibodies in prostate cancer patients suggestthat C21orf100 is a novel prostate differentiation antigen. However, due to the low frequency of antibody response againstC21orf100 detected among prostate cancer patients, further analysis is necessary to evaluate its potential for cancerimmunotherapy.