Bladder cancer immunomodulatory effects of intravesical Nitazoxanide, Rapamycin, Thalidomide and Bacillus Calmette-Guérin (BCG).

PURPOSE: To understand the effect of Nitazoxanide (NTZ), Rapamycin, Thalidomide, alone and in combination with BCG on bladder cancer (BC) histopathology and programmed death-ligand 1 (PD-L1) and anti-cytotoxic T lymphocyte antigen 4 (CTLA4) expression. METHODS: Female Fisher-344 rats underwent intravesical N-methyl-N-nitrosourea (MNU) followed by weekly intravesical treatment with saline (controls, n = 10), BCG (n = 10), NTZ (n = 8), BCG plus NTZ (n = 8), Rapamycin (n = 10) BCG plus Rapamycin (n = 10), Thalidomide (n = 10), and BCG plus Thalidomide (n = 10), and euthanized after 8 weeks and their bladders were investigated for BC and PD-L1 and CTLA4 expression. RESULTS: Rapamicyn alone and in combination with BCG had the lowest number of bladder neoplasias in the histopathology exam (1/10). Neoplastic lesions were found in 4/10 BCG recipients, 5/10 Thalidomide recipients, 4/10 Thalidomide plus BCG recipients, 5/8 NTZ and 3/8 NTZ plus BCG recipients. Adding NTZ to BCG increased the expression of PD-L1 and adding Rapamycin or Thalidomide decreased PD-L1 and CTLA4 expression compared to BCG alone. Rapamycin alone significantly increased CTLA4 and slightly increased PD-L1 expression but its combination with BCG significantly decreased both markers. Thalidomide had a similar effect; however, it was only slightly different from the control and BCG alone groups. CONCLUSION: Intravesical BCG combination treatment seems to effectively prevent BC development in an immunecompetent clinically relevant animal model, introducing Thalidomide, Nitazoxanide, and specially Rapamycin as candidates in the intravesical immunotherapy advancement. Our study contributes in understanding the mechanism of cancer immunotherapy.

Castration immunoregulates toll-like receptor-4 in male bladder cancer.

PURPOSE: Among diverse Pattern Recognition Receptors (PRRs), Toll-like receptor-4 (TLR-4) is a key urothelial trigger for innate immune response impacting urothelial bladder carcinoma (BC). Androgen activation promotes immunotolerance, playing an immunoregulatory role by unknown mechanisms. We explored the castration impact on urothelial TLR-4 modulation in carcinogenesis and immunotherapeutic scenario. METHODS: Intact (SHAM) versus castrated male Fisher-344 rats were evaluated in 2 scenarios: (A) Carcinogenesis: After randomization to SHAM (n = 5) and Castration (n = 5), carcinogenesis was induced by four intravesical doses of 1.5 mg/kg n-methyl-n-nitrosourea (MNU) every 15 days. (B) Treatment: After ultrasonographic confirmed MNU-induced papillary BC on week 8, rats were randomized to SHAM (n = 5) and Castration (n = 5) and offered 6 weekly intravesical treatment of 106 CFU of bacillus Calmette Guerin (BCG) in 0.2 ml saline. After 15 weeks the urinary bladders underwent histopathology. Urothelial cell proliferation was measured by Ki-67 immunohistochemistry (IHC), and TLR-4 expression was quantified by IHC and WB. RESULTS: Castration induced higher TLR-4 urothelial expression (p = 0.007) and anticarcinogenic effect with fewer urothelial tumors (60 vs. 80%) and lower urothelial cell proliferation compared to intact animals (p = 0.008). In the intravesical BCG treatment setting, castration has potentialized the BCG activation of TLR-4 (p = 0.007) with no residual in situ carcinoma compared to intact animals, suggesting the potential to amplify the BCG immune response. CONCLUSION: To our knowledge, this is the first description of TLR-4 urothelial expression hormonal modulation. The described castration-mediated immunomodulation will help to improve the knowledge of urothelial cancer gender diversities and PRRs modulations with treatment implications.

Autochthonous male urothelial carcinoma immune competent model: from induction to BCG transurethral treatment.

OBJECTIVE: To describe a new animal model of autochthonous urothelial cancer (UC) accessible by transurethral catheter in males, from induction to treatment. Seven-week-old male Fischer 344 rats were used. The first 10 animals were used to overcome and standardize the technical challenges of safe transurethral catheterization of male rats. The remaining 14 animals underwent intravesical N-Methyl-Nitrosourea (MNU) instillation for UC induction, of which six were randomized to undergo intravesical BCG treatment. The stretched male rat urethra travels 35 mm in a tortuous "S" shaped trajectory with a 180° angle behind the pubic bone, safely traversed by a 20G 36" 0.8 mm epidural catheter in a stretched, straightened urethra inserted after anterior dilation of the penile urethra with a 24G IV catheter. Histopathologic analysis of the urinary bladder demonstrated Stage pT1, pTa, and pTis lesions in the 8 controls, all with increased cell proliferation by Ki-67 expression and no pT1 or pTis in the animals 6 treated with BCG. This pioneering study describes an autochthonous, effective, and accessible transurethral animal model of immune-competent UC in males, and may help with understanding of the biology, immunology, and treatment of UC, which predominates in males.

Boosting bladder cancer treatment by intravesical nitazoxanide and bacillus calmette-guérin association.

PURPOSE: Nitazoxanide (NTZ) has shown a promising antitumoral effect, the current study compared the anti-neoplastic effects of intravesical NTZ and BCG plus NTZ in NMIBC animal model. METHODS: 30 rats, Fisher 344 were instilled with 4 intravesical doses of 1.5 mg/kg of N-methyl-N-nitrosourea (MNU) every 15 days for BC induction. The animals were divided into 3 groups (Group BCG 106 UFC - 1 mg of BCG; Group NTZ - 300 mg/kg of NTZ; Group NTZ + BCG - simultaneous treatment of BCG and NTZ) and received weekly intravesical treatment for 6 consecutive weeks. Animals were submitted to ultrasound imaging and euthanasia, their bladders were collected and histopathological, immunohistochemical tests (ki67 e c-Myc) and Western Blotting (PI3K, mTOR, and p-4E-BP) were performed. RESULTS: Histopathological tests showed 66.67%, 62.5% and 37.5% incidence of BC in animals treated with BCG, NTZ, and NTZ + BCG, respectively. Nuclear positivity for ki-67 in BC animals were 12.4% (IC 10.1-14.6%), 13.2% (IC 10.5-15.9%) and 8.8% (IC 6.0-11.6%) in BCG, NTZ and NTZ + BCG group, respectively (p = 0.063). Between animals with carcinoma, c-Myc strong positive was 40.10% in NTZ, 32.2% in BCG and 19.90% in the NTZ + BCG group (p < 0.001). Blotting has shown mTOR (p = 0.0473) and PI3K inhibition (p = 0.0349) in the presence of BCG, added to 4-EBP inhibition in the presence of NTZ (p = 0.0004). CONCLUSIONS: Results show the possible synergy between the gold standard BC treatment BCG and NTZ, in which multiple targets inhibition such as c-Myc and downstream mTOR, p-4E-BP and PI3K might play a role.

Carcinogenesis and Bacillus Calmette-Guérin (BCG) Intravesical Treatment of Non-Muscle-Invasive Bladder Cancer under Tryptophan and Thymine Supplementation.

PURPOSE: Evaluate tryptophan and thymine (TT) impact on carcinogenesis and intravesical BCG bladder cancer treatment. METHODS: After identification of TT in vitro inhibitory effect in multiple cancer cell cultures, bladder cancer animal model was induced by MNU intravesical instillations and randomized into four groups: Control (n = 9), BCG (n = 9), TT (n = 7), and BCG + TT (n = 8). BCG groups received intravesical 106 CFU BCG in 0.2 ml saline for 6 consecutive weeks and TT groups received 1 g/kg (1:1) of TT via daily gavage. After 15 wk of protocol, animals were euthanized and the urinary bladders submitted to histopathology, immunohistochemistry, and Western blotting. RESULTS: Urothelial cancer was identified in 100%, 85.7%, 44.5%, and 37.5% of Control, TT, BCG, and BCG + TT groups, respectively. Cell proliferation marked by nuclear Ki-67 was higher in the Control compared to animals in the other groups (P = 0.03). BCG, TT, and BCG + TT groups showed proliferative cell decline and TLR4/5 labeling increase in the urothelium. BCG decreased the urothelial VEGF labeling, even in TT association. CONCLUSION: TT inhibit urothelial carcinogenesis and potentiate the intravesical BCG in the treatment of bladder cancer by reducing cell proliferation and activating TLRs.

Fatores prognósticos para o desfecho do tratamento da tuberculose pulmonar em Recife, Pernambuco, Brasil / Prognostic factors for pulmonary tubeculosis outcome in Recife, Pernambuco, Brazil

OBJETIVOS: O objetivo deste estudo caso-controle foi identificar fatores prognósticos para desfecho do tratamento da tuberculose pulmonar em 297 pacientes (Hospital das Clíinicas, Universidade Federal de Pernambuco, Brasil) entre 1994 e 1999. MATERIAS E METODOS: Foram considerados casos indivíduos com alta por óbito, abandono ou falencia do tratamento. Os controles foram indivíduos com alta cura. Foram realizadas análises uni e multivariada com as variáveis independentes sexo; idade; esoclaridade; hábito de fumar; hábito de ingerir álcool; tratamento anterior para tuberculose; resposta ao teste turberculínico; soroligia para HIV; grau de resistencia aos antimicrobianos; resultado da pesquisa direta de bacilos álcool-ácido-resistentes' esquema terapeuticco utilizado. Além disso, repetiramse as análises uni e multivariada considerando como casos apenas os óbitos e os indivíduos com falencia do tratamento. RESULTADOS: A ingestao excessiva de álcool (OR=2,58; P=0,014), a co-infecçao pelo HIV (OR=3,40; P=0,28), o a tratamento anterior para tuberculose (OR=4,89; P0,001) e resistencia a duas ou mais drogas antituberculose (OR=3,49; P=0,017) foram fatores de risco para o insucesso do tratamento. Na segunda análise multivariada, excluindo os casos de abandono, nao houve associaçao entre a o ingestao excessiva de álcool e desfecho do tratamento, mantendo-se as demais associaçoes, o que sugere uma estreita relaçao entre o abandono do tratamento e o estilismo. CONCLUSOES: Os fatores prognósticos para insuccesso do tratamento da tuberculose pulmonar entre os indivíduos estudados estao interrelacionados, sendo de natureza biológica, clínica e social. Devem ser identificados no início do tratamento para que sejam implementados procedimentos diferenciados de acompanhamento, tais como tratamento diretamente surperisionado, de forma a fortalecer o controle da tuberculose em nível local

Objective. The objective of this case-control study was to identify prognostic factors for the outcome of pulmonary tuberculosis among 297 patients who were treated between 1994 and 1999 at the Federal University of Pernambuco Clinics Hospital, in Recife, Pernambuco, Brazil. Methods. The cases were defined as individuals whose treatment ended in death, dropout, or failure. The controls were persons whose treatment ended in cure. The following independent variables were submitted to uni- and multivariate analyses: sex, age, schooling, being a smoker, consuming alcohol, previous treatment for tuberculosis, response to the tuberculin test, HIV serology, resistance to antimicrobial agents, results of direct investigation of alcohol- and acid-resistant bacilli, and treatment approach used. After that, the uni- and multivariate analyses were repeated, including as cases only deaths and individuals with treatment failure. Results. Four risk factors for tuberculosis treatment failure were found: excessive alcohol intake (odds ratio (OR) = 2.58; P = 0.014), co-infection with HIV (OR = 3.40; P = 0.028), previous tuberculosis treatment (OR = 4.89; P < 0.001), and resistance to two or more antituberculosis drugs (OR = 3.49; P = 0.017). In the second multivariate analysis, which excluded dropout cases, no association was found between treatment outcome and excessive alcohol consumption, but the other associations remained. This result suggests a close relationship between alcoholism and treatment dropout. Conclusions. In the group that was studied the prognostic factors for failure of pulmonary tuberculosis treatment were interrelated and were of a biological, clinical, and social character. These factors should be identified at the beginning of treatment in order to allow implementation of specific follow-up procedures such as the strategy of directly observed treatment. This would strengthen tuberculosis control at the local level

Fatores prognósticos para o desfecho do tratamento da tuberculose pulmonar em Recife, Pernambuco, Brasil / Prognostic factors for pulmonary tubeculosis outcome in Recife, Pernambuco, Brazil

OBJETIVOS: O objetivo deste estudo caso-controle foi identificar fatores prognósticos para desfecho do tratamento da tuberculose pulmonar em 297 pacientes (Hospital das Clíinicas, Universidade Federal de Pernambuco, Brasil) entre 1994 e 1999. MATERIAS E METODOS: Foram considerados casos indivíduos com alta por óbito, abandono ou falencia do tratamento. Os controles foram indivíduos com alta cura. Foram realizadas análises uni e multivariada com as variáveis independentes sexo; idade; esoclaridade; hábito de fumar; hábito de ingerir álcool; tratamento anterior para tuberculose; resposta ao teste turberculínico; soroligia para HIV; grau de resistencia aos antimicrobianos; resultado da pesquisa direta de bacilos álcool-ácido-resistentes' esquema terapeuticco utilizado. Além disso, repetiramse as análises uni e multivariada considerando como casos apenas os óbitos e os indivíduos com falencia do tratamento. RESULTADOS: A ingestao excessiva de álcool (OR=2,58; P=0,014), a co-infecçao pelo HIV (OR=3,40; P=0,28), o a tratamento anterior para tuberculose (OR=4,89; P0,001) e resistencia a duas ou mais drogas antituberculose (OR=3,49; P=0,017) foram fatores de risco para o insucesso do tratamento. Na segunda análise multivariada, excluindo os casos de abandono, nao houve associaçao entre a o ingestao excessiva de álcool e desfecho do tratamento, mantendo-se as demais associaçoes, o que sugere uma estreita relaçao entre o abandono do tratamento e o estilismo. CONCLUSOES: Os fatores prognósticos para insuccesso do tratamento da tuberculose pulmonar entre os indivíduos estudados estao interrelacionados, sendo de natureza biológica, clínica e social. Devem ser identificados no início do tratamento para que sejam implementados procedimentos diferenciados de acompanhamento, tais como tratamento diretamente surperisionado, de forma a fortalecer o controle da tuberculose em nível local