Potential therapeutic strategies for non-muscle invasive bladder cancer based on association of intravesical immunotherapy with P-MAPA and systemic administration of cisplatin and doxorubicin

The present study describes the histopathological and molecular effects of P-MAPA (Protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride) intravesical immunotherapy combined with systemic doxorubicin or cisplatin for treatment of non-muscle invasive bladder cancer (NMIBC) in an appropriate animal model. Our results showed an undifferentiated tumor, characterizing a tumor invading mucosa or submucosa of the bladder wall (pT1) and papillary carcinoma in situ (pTa) in the Cancer group. The histopathological changes were similar between the combined treatment with intravesical P-MAPA plus systemic Cisplatin and P-MAPA immunotherapy alone, showing decrease of urothelial neoplastic lesions progression and histopathological recovery in 80% of the animals. The animals treated systemically with cisplatin or doxorubicin singly, showed 100% of malignant lesions in the urinary bladder. Furthemore, the combined treatment with P-MAPA and Doxorubicin showed no decrease of urothelial neoplastic lesions progression and histopathological recovery. Furthermore, Akt, PI3K, NF-kappa B and VEGF protein levels were significantly lower in intravesical P-MAPA plus systemic cisplatin and in intravesical P-MAPA alone treatments than other groups. In contrast, PTEN protein levels were significantly higher in intravesical P-MAPA plus systemic cisplatin and in intravesical P-MAPA alone treatments. Thus, it could be concluded that combination of intravesical P-MAPA immunotherapy and systemic cisplatin in the NMIBC animal model was effective, well tolerated and showed no apparent signs of antagonism between the drugs. In addition, intravesical P-MAPA immunotherapy may be considered as a valuable option for treatment of BCG unresponsive patients that unmet the criteria for early cystectomy425942954CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP490519/2011-3não tem2014/12047-

Alterations In Ubiquitin Ligase Siah-2 And Its Corepressor N-cor After P-mapa Immunotherapy And Anti-androgen Therapy: New Therapeutic Opportunities For Non-muscle Invasive Bladder Cancer.

The present study describes the role of the ubiquitin ligase Siah-2 and corepressor N-CoR in controlling androgen receptor (AR) and estrogen receptors (ERα and ERβ) signaling in an appropriate animal model (Fischer 344 female rats) of non-muscle invasive bladder cancer (NMIBC), especially under conditions of anti-androgen therapy with flutamide. Furthermore, this study describes the mechanisms of a promising therapeutic alternative for NMIBC based on Protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) intravesical immunotherapy combined with flutamide, involving the interaction among steroid hormone receptors, their regulators and Toll-like receptors (TLRs). Our results demonstrated that increased Siah-2 and AR protein levels and decreased N-CoR, cytochrome P450 (CYP450) and estrogen receptors levels played a critical role in the urothelial carcinogenesis, probably leading to escape of urothelial cancer cells from immune system attack. P-MAPA immunotherapy led to distinct activation of innate immune system TLRs 2 and 4-mediated, resulting in increase of interferon signaling pathway, which was more effective in recovering the immunosuppressive tumor immune microenvironment and in recovering the bladder histology features than BCG (Bacillus Calmette-Guerin) treatments. The AR blockade therapy was important in the modulating of downstream molecules of TLR2 and TLR4 signaling pathway, decreasing the inflammatory cytokines signaling and enhancing the interferon signaling pathway when associated with P-MAPA. Taken together, the data obtained suggest that interferon signaling pathway activation and targeting AR and Siah-2 signals by P-MAPA intravesical immunotherapy alone and/ or in combination with AR blockade may provide novel therapeutic approaches for NMIBC.84427-444

Efeitos do tratamento crônico com extrato etanólico de pterodon pubescens no reparo de defeito femoral em ratas ovariectomizadas

O objetivo desse estudo foi avaliar os efeitos do tratamento com extrato etanólico de Pterodon pubescens no reparo de defeito femoral em ratas ovariectomizadas. Foram utilizadas ratas Sprague – Dawley de 2 meses ≈ 200 g (protocolo CEUA-UNICAMP nᵒ 4358-1). A osteoporose foi induzida por ovariectomia bilateral (OVX) e sua eficácia foi confirmada por microtomografia (μCT). O tratamento oral com o extrato etanólico de Pterodon pubescens (EEPp) nas doses de 1, 10 e 100 mg/kg ocorreu por 93 dias consecutivos. Para avaliação do reparo por μCT e histologia, um defeito ósseo foi induzido 90 dias pós-OVX por osteotomia (OST) femoral. Foram realizados testes de von Frey eletrônico (hiperalgesia mecânica/dor), Open-field (locomoção espontânea) e Rotarod (locomoção forçada), bem como monitorados peso e ingesta de ração e água. Foram realizadas análises toxicológicas no soro e em órgãos vitais ex vivo. As diferenças foram consideradas significativas quando P≤0,05. O EEPp (1, 10 e 100 mg/kg) inibiu a hiperalgesia mecânica em 98 ± 9%, 81 ± 9% e 87 ± 4%, não alterou a atividade locomotora espontânea e aumentou em 70 ± 5%, 72 ± 7% e 73 ± 7% a latência de queda no Rotarod, respectivamente. Além disso, a histologia revelou que o EEPp (1, 10 e 100 mg/kg) induziu neoformação óssea na extensão do defeito em 89 ± 3%, 79 ± 6% e 74 ± 10%, respectivamente. As análises de μCT revelaram perda de massa óssea no grupo OVX e mostrou que a dose de 1 mg/kg do EEPp consolidou 70% do defeito ósseo em 40 dias pós-OST. O EEPp não causou inconsistências na ingesta de ração e água dos animais. Porém, o EEPp na dose de 100 mg/kg parece ser hepatotóxica, pois, além de alterar cor e textura, aumentou o peso relativo do coração (6 ± 1%), pulmões (7 ± 2%) e fígado (21 ± 1%), bem como elevou os níveis séricos de GGT em 64 ± 19%.Fil: Bighetto-Cain, Bruna. Universidade Estadual de Campinas (Brasil)Fil: Camilli, José Angelo. Universidade Estadual de Campinas (Brasil)Fil: Camilli, Júlia Constantino. Universidade Estadual de Campinas (Brasil)Fil: Fávaro, Wagner José. Universidade Estadual de Campinas (Brasil)Fil: Garcia, Patrick Vianna. Universidade Estadual de Campinas (Brasil)Fil: Nucci-Martins, Catharina. Universidade Estadual de Campinas (Brasil

Clinical implications of T-Cells CX3CR1+, Toll-like Receptor 4 signaling pathway, and immune checkpoints in Non-Muscle Invasive Bladder Cancer

Background: This study characterized and compared the molecular profiles of CX3C chemokine receptor 1 (CX3CR1, a marker of T-cell differentiation), Toll-like receptor 4 (TLR4)-mediated interferon signaling pathway, and immune checkpoints in the different histological stages of non-muscle invasive bladder cancer (NMIBC), aiming the investigation of these biomarkers as a criterion of clinical response to immunotherapy. Methods: Seventy-five formalin-fixed paraffin-embedded samples of bladder were obtained from 34 to 96-year-old patients (mean 65 years) with NMIBC diagnosis in University of Campinas (UNICAMP) and Paulinia Municipal Hospital/ Brazil. Subsequently, the samples were divided into 3 groups (n= 25 samples per group): pTis group, high-grade pTa group, and pT1 group; and submitted to immunohistochemistry analysis: TLR4-mediated IFN-γ production signaling pathway (TRIF, TBK1, IRF-3, IFN-γ), CX3CR1+CD8+ T-cells, immune checkpoints (PD-1/PD-L1 and CTLA-4), and regulatory T (Treg) cells (FOXP3). The retrospective anonymous study was approved by the local ethics committee (Clinical Trial: RBR-6swqd2). Results: pTis group showed the lowest activation of TLR4-mediated IFN-γ signaling pathway when compared (p<0.01) to high-grade pTa and pT1 groups. Both the immunoreaction intensity and positive cells percentage were lower (p<0.01) for TLR4, TRIF, IRF-3, and IFN-γ in the pTis group with respect to other groups. No statistical difference was found between high-grade pTa and pT1 groups for these biomarkers. Likewise, CX3CR1 immunoreactivities were remarkably lower (p<0.01) in the pTis group in comparison with high-grade pTa and pT1 groups, which did not show statistical differences between them. Furthermore, immune checkpoints (PD-1/PD-L1 and CTLA4) and FOXP3+ Treg cells immunoreactivities were significantly higher (p<0.01) in the high-grade pTa and pT1 compared to the pTis group. Conclusions: Our data demonstrated that pTis stage was characterized by an immunosuppressive microenvironment in comparison with pTa and pT1 stages, showing decreased TLR4-mediated interferon signaling pathway and low activation of CX3CR1+CD8+ T-cells; which implies in low sensitivity to immunotherapy. The larger number of FOXP3+ Treg cells in pTa and pT1 was correlated with intensified immune checkpoints immunoreactivities, indicating higher sensitivity to immunotherapy. Finally, these biomarkers may be useful in the clinical management of patients with NMIBC

Características anatômicas da cateterização da uretra e bexiga de camundongos e ratos fêmeas. Instrumento essencial na pesquisa pré clínica

To present fundamental anatomical aspects and technical skills necessary to urethra and urinary bladder catheterization in female mice and rats. Urethral and bladder catheterization has been widely utilized for carcinogenesis and cancer research and still remains very useful in several applications: from toxicological purposes as well as inflammatory and infectious conditions to functional aspects as bladder dynamics and vesicoureteral reflux, among many others. Animal models are in the center of translational research and those involving rodents are the most important nowadays due to several advantages including human reproducibility, easy handling and low cost. Although technical and anatomical pearls for rodent urethral and bladder access are presented as tackles to the advancement of lower urinary tract preclinical investigation in a broaden sight, restriction to female animals hampers the male microenvironment, demanding future advances262Apresentar aspectos anatômicos fundamentais e habilidades técnicas necessárias para cateterismo da uretra e bexiga em ratos e camundongos fêmeas. Cateterismo vesical tem sido amplamente utilizado na pesquisa do câncer e carcinogênese, além de várias outras aplicações, desde fins toxicológicos, condições inflamatórias e infecciosas até aspectos funcionais como a dinâmica vesical e refluxo vesico-ureteral, entre muitos outros. Os modelos animais estão no centro da investigação de translação e os roedores são os mais importantes devido a várias vantagens, incluindo reprodutibilidade humana, o fácil manuseio e baixo custo. Apesar de permitir o desenvolvimento da investigação pré-clínica do trato urinário inferior, o modelo se restringe aos animais do sexo feminino, de modo que avanços futuros são necessário

Anatomical Features Of The Urethra And Urinary Bladder Catheterization In Female Mice And Rats. An Essential Translational Tool.

To present fundamental anatomical aspects and technical skills necessary to urethra and urinary bladder catheterization in female mice and rats. Urethral and bladder catheterization has been widely utilized for carcinogenesis and cancer research and still remains very useful in several applications: from toxicological purposes as well as inflammatory and infectious conditions to functional aspects as bladder dynamics and vesicoureteral reflux, among many others. Animal models are in the center of translational research and those involving rodents are the most important nowadays due to several advantages including human reproducibility, easy handling and low cost. Although technical and anatomical pearls for rodent urethral and bladder access are presented as tackles to the advancement of lower urinary tract preclinical investigation in a broaden sight, restriction to female animals hampers the male microenvironment, demanding future advances.26 Suppl 2106-1

P-MAPA and interleukin-12 reduce cell migration/invasion and attenuate the toll-like receptor-mediated inflammatory response in ovarian cancer SKOV-3 cells : a preliminary study

Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium–ammonium phospholinoleate–palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment2515CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP401040/2016-00708/20182019/00906-6; 2016/03993-9We would like to give a special thanks to Farmabrasilis-Brazil, FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, grant numbers: 2019/00906-6 and 2016/03993-9), CAPES (grant number: 0708/2018), and CNPq (grant number: 401040/2016-0) by providing financial suppor

Hormonal and ultrastructural reactivity of the ventral lobe of the prostate of rats (Rattus norvegicus) submitted to simultaneous long-term alcohol-nicotine treatment

Orientador: Valeria Helena Alves Cagno QuiteteDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: o álcool e a nicotina têm ação prejudicial sobre o funcionamento da glândula prostática. Contudo, os mecanismos moleculares e celulares envolvidos nas alterações prostáticas, bem como suas correlações com a patogênese glandular permanecem desconhecidos. Assim, os objetivos do presente estudo foram caracterizar as alterações estruturais das células epiteliais e do estroma e, a expressão dos receptores androgênicos e estrogênicos a. na próstata ventral de ratos sob o efeito da ação concomitante de nicotina e etanol, bem como cada uma dessas drogas isoladamente, além dos níveis plasmáticos dos hormônios testosterona e estrógenos. Também, foi objetivo estabelecer associações das interações epitélio-estromal úente a possível pato gênese glandular. Um total de 40 ratos macho (Rattus norvegicus), com 75 dias de idade, foi dividido em quatro grupos: Grupo controle (10 animais) recebeu água ad libitum; Grupo alcoolista (10 animais) recebeu etanol diluído a 10% Gay Lussac (10% v/v) ad libitum; Grupo nicotina (10 animais) recebeu um total de 0,125mg/l00g de peso corpóreo/diariamente de nicotina (Sigma, St. Louis, USA) por via subcutânea; Grupo nicotina-álcool (10 animais) recebeu tratamento simultâneo com etanol e nicotina, nas mesmas concentrações que nos grupos alcoolista e nicotina. Após 90 dias de tratamento, todos os animais foram sacrificados e amostras do lobo ventral da próstata foram coletadas para análises macroscópicas, microscopias de luz e eletrônica de transmissão e imunomarcações dos receptores androgênicos e estrogênicos a., além das dosagens hormonais. Os resultados revelaram: atrofia dos órgãos genitais masculinos como testículos e vesículas seminais; atrofia epitelial; neoplasia intra-epitelial prostática; desorganização das organelas envolvidas no processo secretor; hipertrofia estromal; presença de microácinos e células inflamatórias e célula muscular lisa com fenótipo secretor. Também, houve diminuição significativa dos rúveis séricos de testosterona e aumento dos níveis séricos de estrógenos nos animais dos grupos nicotina álcool, alcoolista e nicotina em relação aos animais controles, bem como a expressão dos seus respectivos receptores. Assim, concluiu-se que fatores extrínsecos como etanol e nicotina são componentes de risco à homeo~tase da glândula prostática, levando a ocorrência da pato gênese do órgão, a qual poderá ser associada à processos tardios de malignescência glandularAbstract: Alcohol and nicotme act negatively on the prostatic gland function. However, molecular and cellular mechanisms mvolved m the prostatic alteration, as well as their correlations with glandular pathogenesis remam unclear. Then, the main objective of this study was to identifY the features of the epithelial and stromal prostatic structure and to characterize the expression of the androgenic and a estrogenic receptors in rats submitted to chronic nicotme and alcohol use simultaneously, besides each one of these drugs alone. Moreover, it was mtended to establish the stromal-epithelial mteraction in a possible glandul~ pathogenesis. A total of 40 male rats, aged 75 days, were divided mto 4 groups: Control group (10 animaIs) received tap water ad libitum; Alcoholic group (10 animaIs) received diluted 10% Gay Lussac ethanol ad libitum; Nicotme group (10 animaIs) received a 0.125 mg/100 g body weight dose ofnicotine daily; Nicotine-Alcohol group (10 animaIs) received simultaneous alcohol and nicotme treatment, m the same concentration as the alcoholic and nicotine groups. Afier 90 days of treatment, the animaIs were sacrificed and samples ITom the ventrallobe ofthe prostate were collected and processed for transmission electron and light microscopies, immunological techniques to androgenic and a estrogenic receptor expression and radioimmunoassay techniques to measure testosterone and estrogen levels. The results showed atrophied testes and seminal vesicle, atrophied prostatic epithelial cells, prostatic mtraepithelial neoplasm, disorganization of cellular organelles mvolved m the glandular process, inflammatory cells, diminished testosterone levels and their receptors besides mcreased estrogen levels. All alterations were verified m the different experimental groups, bemg more mtense m the alcohol-nicotme, alcoholic and nicotme groups, respectively. Then, it can be concluded that extrmsic factors like ethanol and nicotme are risk compounds to prostatic homeostasis, leadmg to occurrence of glandular pathogenesis which could be related to glandular malignancy lateMestradoAnatomiaMestre em Biologia Celular e Estrutura