Expression of HPV-16 E6 and E7 oncoproteins alters Chlamydia trachomatis developmental cycle and induces increased levels of immune regulatory molecules.

Introduction: Infection with Human Papillomavirus (HPV) is a recognized risk factor for Chlamydia trachomatis (CT) infection and vice versa. Coinfection of HPV and CT in women is a very common and usually asymptomatic finding that has been linked to increased risk of cervical cancer. It has been demonstrated that CT facilitates the entry of multiple high risk HPV genotypes, leading to damage of the mucosal barrier and interfering with immune responses and viral clearance, which ultimately favours viral persistence and malignant transformation. Although the facilitating effects elicited by CT infection on viral persistence have been reported, little is known about the consequences of HPV infection on CT development. Methods: Herein, we took advantage of a genetically modified human cervical cell line co-expressing HPV-16 major oncogenic proteins E6 and E7, as an experimental model allowing to investigate the possible effects that HPV infection would have on CT development. Results and discussion: Our results show that CT infection of HPV-16 E6E7 expressing cells induced an upregulation of the expression of E6E7 oncoproteins and host cell inhibitory molecules PD-L1, HVEM and CD160. Additionally, smaller chlamydial inclusions and reduced infectious progeny generation was observed in E6E7 cells. Ultrastructural analysis showed that expression of E6 and E7 did not alter total bacterial counts within inclusions but resulted in increased numbers of reticulate bodies (RB) and decreased production of infectious elementary bodies (EB). Our results indicate that during CT and HPV coinfection, E6 and E7 oncoproteins impair RB to EB transition and infectious progeny generation. On the other hand, higher expression of immune inhibitory molecules and HPV-16 E6E7 are cooperatively enhanced in CT-infected cells, which would favour both oncogenesis and immunosuppression. Our findings pose important implications for clinical management of patients with HPV and CT coinfection, suggesting that screening for the mutual infection could represent an opportunity to intervene and prevent severe reproductive health outcomes, such as cervical cancer and infertility.

Comparación de las tinciones con hematoxilina y eosina versus azul de toluidina en la evaluación de los carcinomas cutáneos durante la cirugía micrográfica de Mohs / Comparative study of toluidine blue and hematoxylin and eosin staining in evaluating skin-carcinoma during Mohs micrographic surgery / Comparação da coloração de hematoxilina e eosina versus azul de toluidina na avaliação de carcinomas cutâneos durante a cirurgia micrográfica de Mohs

Resumen: Introducción: la cirugía micrográfica de Mohs es una técnica quirúrgica especializada para el tratamiento del cáncer de piel no melanoma. La histopatología cumple un rol fundamental, y la elección de la tinción es un punto de controversia. Objetivos: comparar el rendimiento de las tinciones de hematoxilina y eosina (HyE) versus azul de toluidina (AT) durante la cirugía. Método: estudio observacional, descriptivo y transversal a partir de noviembre de 2017 hasta mayo de 2018. Se incluyeron las láminas empleadas durante la cirugía en el período mencionado. Estas fueron analizadas por el cirujano de Mohs, tres residentes y una dermopatóloga. Se valoró el rendimiento de ambas tinciones, teniendo en cuenta las características celulares y los elementos del estroma. Resultados: se estudiaron 23 tumores (16 carcinomas basocelulares y 7 carcinomas espinocelulares). Al observarse al microscopio óptico tanto con la tinción de AT como con HyE no se encontraron diferencias significativas entre ambos grupos en lo global, sólo en algunas características, especialmente con la HyE. Conclusiones: es el primer trabajo en Uruguay que compara la eficacia de las dos tinciones durante la cirugía micrográfica de Mohs. Como conclusión tanto la tinción de HyE como el AT son muy buenas técnicas para el diagnóstico de carcinomas cutáneos.

Abstract: Introduction: Mohs micrographic surgery is a specialized surgical technique used to treat nonmelanoma carcinoma. Histopathology plays a vital role in the diagnosis of this condition, and the choice staining method is controversial. Objective: to compare results in the use of hematoxylin and eosin (H&E) versus Toluidine blue (TB) staining during surgery. Method: observational, descriptive and transversal study conducted from November, 2017 until May, 2018 of the slides used during surgeries in the selected period. Slides were analysed by the Mohs surgeon, 3 residents and a dermopathologist to evaluate the results of both staining methods, in consideration of cell features and stromal elements. Results: 23 tumors were analysed (16 Basal Cell carcinomas and 7 Squamous Cell Carcinoma). Microscopic observation of slides prepared with Toluidine blue and hematoxylin and eosin stains did not show significant global differences between both groups, except in terms of a few characteristics, in particular with hematoxylin and eosin stains. Conclusions: this was the first study in Uruguay to evaluate the effectiveness of both staining methods during Mohs micrographic surgery, and it concluded that both Toluidine blue and hematoxylin and eosin stains are very good techniques in evaluating skin-cancer.

Resumo: Introdução: a cirurgia micrográfica de Mohs é uma técnica cirúrgica especializada para o tratamento do câncer de pele não melanoma. A histopatologia desempenha um papel fundamental, onde a escolha da coloração é um ponto de controvérsia. Objetivos: comparar o desempenho das colorações de hematoxilina e eosina versus azul de toluidina durante a cirurgia. Método: estudo observacional, descritivo e transversal de novembro de 2017 a maio de 2018. Foram incluídas as lâminas utilizadas durante as cirurgias no referido período. Estas foram analisadas pelo cirurgião especializado na técnica de Mohs, 3 residentes e um dermatopatologista onde foi avaliado o desempenho de ambas as colorações, levando em consideração as características celulares e os elementos do estroma. Resultados: foram estudados 23 tumores (16 carcinomas basocelulares e 7 carcinomas espinocelulares). Quando observados ao microscópio de luz para coloração AT e H&E, não foram encontradas diferenças significativas entre os dois grupos em geral, apenas em algumas características, especialmente com o H&E. Conclusões: é o primeiro estudo no Uruguai que compara a eficácia dos 2 corantes durante a cirurgia micrográfica de Mohs. Em conclusão, tanto a coloração com hematoxilina e eosina quanto com azul de toluidina são técnicas muito boas para o diagnóstico de carcinomas de pele.

Association between Human Papillomavirus and Chlamydia trachomatis genital infections in male partners of infertile couples.

The prevalence of HPV infection and its relationship with other sexually transmitted infections was analyzed in a cohort of 117 male partners of infertile couples from Cordoba, Argentina. Semen samples and urethral swabs were obtained and the infection with HPV, Chlamydia trachomatis, HSV1, HSV2, Mycoplasma hominis and Ureaplasma urealyticum was analyzed. A prevalence of HPV infection of 27.4% was found. Interestingly, infections by exclusively low risk HPV genotypes or high/intermediate risk HPV genotypes were present in 64.5% and 22.6% of cases, respectively. Low risk-HPV6 was the most frequently detected genotype. Remarkably, HPV and C. trachomatis infections were significantly associated to each other (OR: 11.55, 95% CI 1.14-117.06). No significant differences in sperm quality were found between HPV-positive and HPV-negative patients indicating that HPV male urogenital infection does not impair sperm quality. Our results show a high prevalence of HPV urogenital infection among male partners of infertile couples, and that HPV and C. trachomatis infections are reciprocal risk factors of their co-infection. Moreover, our results suggest that men constitute a reservoir for continued transmission of C. trachomatis and HPV to women highlighting the need for routine screening for these two pathogens in male partners of infertile couples.

Patients with chronic prostatitis/chronic pelvic pain syndrome show T helper type 1 (Th1) and Th17 self-reactive immune responses specific to prostate and seminal antigens and diminished semen quality.

OBJECTIVES: To assess the presence of self-reactive immune responses to seminal and prostate antigens (PAg), biomarkers of inflammation of the male genital tract, and semen quality parameters in patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). PATIENTS, SUBJECTS AND METHODS: Peripheral blood and semen samples were collected from patients with CP/CPPS and age-matched healthy control volunteers. We analysed the lymphoproliferative responses of peripheral blood mononuclear cells (PBMC) to different seminal plasma (SP)-derived and purified PAg, serum autoantibodies specific to PAg, leucocyte subpopulations, and inflammatory cytokines in semen, sperm apoptosis/necrosis, and semen quality parameters. RESULTS: Significantly greater PBMC proliferative responses specific to PAg, with elevated secretion of interferon (IFN)γ and interleukin (IL)-17, were detected in the patients with CP/CPPS vs the controls. Moreover, the patients with CP/CPPS had significantly greater serum immunoglobulin G immune reactivity to SP proteins, such as prostate-specific antigen and prostatic acid phosphatase, than the controls. Inflammation of the male genital tract was exemplified by high levels of IFNγ, IL-17, IL-1ß and IL-8, as well as higher counts of leukocytes, mainly CD4 T lymphocytes and macrophages, in the semen. In addition, this local inflammation was associated with an overall diminished semen quality, i.e., reduced sperm concentration, motility and viability; and higher levels of sperm apoptosis/necrosis in patients with CP/CPPS vs controls. CONCLUSION: Patients with CP/CPPS show T helper type 1 (Th1) and Th17 immune responses specific to PAg associated with chronic inflammation of the male genital tract and reduced semen quality. These immune responses may underlie the induction and development of chronic pelvic pain and inflammation of the male genital tract, which in turn could alter normal prostate functioning and impair semen quality.

Implications of prostate inflammation on male fertility.

The prostate is the seat of three major causes of morbidity: benign prostatic hyperplasia, prostate cancer and prostatitis, three conditions in which inflammation has been implicated. A state of inflammation of the prostate gland, originally incited by an infection, an autoimmune response, a neurogenic stimulus or another trigger may have consequences on prostate functionality. In fact, male fertility depends intrinsically on the content of prostatic fluid factors secreted by the prostatic epithelium. Taking into account that the prostate gland is the major male accessory gland that exerts essential functions for male fertility, a state of local inflammation can alter male fertility by either directly impairing sperm quality or, indirectly, by causing prostate dysfunction. In the present review, we summarise the current knowledge regarding prostatitis due to well-known infections such as Escherichia coli, Chlamydia trachomatis and other commonly identified microorganisms focusing on inflammatory markers detected during these infections and seminal quality and male fertility alterations reported. We also focused on type III prostatitis or chronic nonbacterial prostatitis/chronic pelvic pain syndrome, of unknown aetiology, in which inflammation of an autoimmune origin, neurogenic stimuli or another trigger have been proposed and fertility alterations reported.

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset.

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.

IL-17 is not essential for inflammation and chronic pelvic pain development in an experimental model of chronic prostatitis/chronic pelvic pain syndrome.

Pain and inflammation in the absence of infection are hallmarks in chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) patients. The etiology of CP/CPPS is unclear, and autoimmunity has been proposed as a cause. Experimental autoimmune prostatitis (EAP) models have long been used for studying CP/CPPS. Herein, we studied prostate inflammation induction and chronic pelvic pain development in EAP using IL-12p40-KO, IL-4-KO, IL-17-KO, and wild-type (C57BL/6) mice. Prostate antigen (PAg) immunization in C57BL/6 mice induced specific Th1 and Th17 immune responses and severe prostate inflammation and cell infiltration, mainly composed of CD4 T cells and macrophages. Moreover, chronic pelvic pain was evidenced by increased allodynia responses. In immunized IL-17-KO mice, the presence of a prominent PAg-specific Th1 immune response caused similar prostate inflammation and chronic pelvic pain. Furthermore, markedly high PAg-specific Th1 immune responses, exacerbated prostate inflammation, and chronic pelvic pain were detected in immunized IL-4-KO mice. Conversely, immunized IL-12p40-KO mice developed PAg-specific Th2 immune responses, characterized by high IL-4 secretion and neither infiltration nor damage in the prostate. As observed in wild-type control animals, IL12p40-KO mice did not evidence tactile allodynia responses. Our results suggest that, as in patients, chronic pelvic pain is a consequence of prostate inflammation. After PAg immunization, a Th1-associated immune response develops and induces prostate inflammation and chronic pelvic pain. The absence of Th1 or Th2 cytokines, respectively, diminishes or enhances EAP susceptibility. In addition, IL-17 showed not to be essential for pathology induction and chronic pelvic pain development.

Transglutaminase 4 as a prostate autoantigen in male subfertility.

Autoimmune polyendocrine syndrome type 1 (APS1), a monogenic disorder caused by AIRE gene mutations, features multiple autoimmune disease components. Infertility is common in both males and females with APS1. Although female infertility can be explained by autoimmune ovarian failure, the mechanisms underlying male infertility have remained poorly understood. We performed a proteome-wide autoantibody screen in APS1 patient sera to assess the autoimmune response against the male reproductive organs. By screening human protein arrays with male and female patient sera and by selecting for gender-imbalanced autoantibody signals, we identified transglutaminase 4 (TGM4) as a male-specific autoantigen. Notably, TGM4 is a prostatic secretory molecule with critical role in male reproduction. TGM4 autoantibodies were detected in most of the adult male APS1 patients but were absent in all the young males. Consecutive serum samples further revealed that TGM4 autoantibodies first presented during pubertal age and subsequent to prostate maturation. We assessed the animal model for APS1, the Aire-deficient mouse, and found spontaneous development of TGM4 autoantibodies specifically in males. Aire-deficient mice failed to present TGM4 in the thymus, consistent with a defect in central tolerance for TGM4. In the mouse, we further link TGM4 immunity with a destructive prostatitis and compromised secretion of TGM4. Collectively, our findings in APS1 patients and Aire-deficient mice reveal prostate autoimmunity as a major manifestation of APS1 with potential role in male subfertility.

Bio-efficacy of the essential oil of oregano (Origanum vulgare Lamiaceae. Ssp. Hirtum).

The aim of this study was to investigate the bioactivity of the essential oil isolated from Origanum vulgare L. (EOv). We analyzed the in vivo anti-inflammatory properties in a mouse-airway inflammation model and the in vitro antimicrobial activity, genotoxicity over the anaphase-telophase with the Allium cepa strain and its cytotoxicity/viability in A549 culture cells. In vivo, EOv modified the levels of tumor necrosis factor -α and viable activated macrophages and was capable to mitigate the effects of degradation of conjugated dienes. In vitro, EOv reduced the viability of cultured A549 cells as well as the mitotic index and a number of chromosomal aberrations; however, it did not change the number of phases. We found that EOv presents antimicrobial activity against different Gram (-) and (+) strains, measured by disc-diffusion test and confirmed with a more accurate method, the AutoCad software. We postulate that EOv presents antibacterial, antioxidant and chemopreventive properties and could be play an important role as bioprotector agent.

Classifier ensemble based on feature selection and diversity measures for predicting the affinity of A(2B) adenosine receptor antagonists.

A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

Chlamydia trachomatis infection of the male genital tract: an update.

Chlamydia trachomatis (CT) is the most prevalent cause of sexually transmitted diseases. Although the prevalence of chlamydial infection is similar in men and women, current research and screening are still focused on women, who develop the most severe complications, leaving the study of male genital tract (MGT) infection underrated. Herein, we reviewed the literature on genital CT infection with special focus on the MGT. Data indicate that CT certainly infects different parts of the MGT such as the urethra, seminal vesicles, prostate, epididymis and testis. However, whether or not CT infection has detrimental effects on male fertility is still controversial. The most important features of CT infection are its chronic nature and the presence of a mild inflammation that remains subclinical in most individuals. Chlamydia antigens and pathogen recognition receptors (PRR), expressed on epithelial cells and immune cells from the MGT, have been studied in the last years. Toll-like receptor (TLR) expression has been observed in the testis, epididymis, prostate and vas deferens. It has been demonstrated that recognition of chlamydial antigens is associated with TLR2, TLR4, and possibly, other PRRs. CT recognition by PRRs induces a local production of cytokines/chemokines, which, in turn, provoke chronic inflammation that might evolve in the onset of an autoimmune process in genetically susceptible individuals. Understanding local immune response along the MGT, as well as the crosstalk between resident leukocytes, epithelial, and stromal cells, would be crucial in inducing a protective immunity, thus adding to the design of new therapeutic approaches to a Chlamydia vaccine.

Chemical compositions and properties of Schinus areira L. essential oil on airway inflammation and cardiovascular system of mice and rabbits.

The main purpose was to investigate the effects of essential plant-oil of Schinus areira L. on hemodynamic functions in rabbits, as well as myocardial contractile strength and airways inflammation associated to bacterial endotoxin lipopolysaccharide (LPS) in mice. This study shows the important properties of the essential oil (EO) of S. areira studied and these actions on lung with significant inhibition associated to LPS, all of which was assessed in mice bronchoalveolar lavage fluid and evidenced by stability of the percentage of alveolar macrophages, infiltration of polymorphonuclear leukocytes and tumor necrosis factor-α concentration, and without pathway modifications in conjugated dienes activity. Clinical status (morbidity or mortality), macroscopic morphology and lung/body weight index were unaffected by the administration of the EO S. areira. Furthermore, the ex vivo analysis of isolated hearts demonstrated the negative inotropic action of the EO of S. areira in a mice model, and in rabbits changes in the hemodynamic parameters, such as a reduction of systolic blood pressure. We conclude that EO S. areira could be responsible for modifications on the cardiovascular and/or airway parameters.

Identification of ETP-46321, a potent and orally bioavailable PI3K α, δ inhibitor.

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein, we describe the optimization of imidazo [1,2-a] pyrazines, which allow us to identify compound 14 (ETP-46321), with potent biochemical and cellular activity and good pharmacokinetic properties (PK) after oral dosing. ETP-46321 PK/PD studies showed time dependent downregulation of AKT(Ser473) phosphorylation, which correlates with compound levels in tumor tissue and demonstrating to be efficacious in a GEMM mouse tumor model driven by a K-Ras(G12V) oncogenic mutation. Treatment with ETP-46321 resulted in significant tumor growth inhibition.

Imidazo[1,2-a]pyrazines as novel PI3K inhibitors.

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of its signaling pathway in a wide variety of human cancers. We describe herein a novel series of imidazo[1,2-a]pyrazines as PI3K inhibitors.

Hit to lead evaluation of 1,2,3-triazolo[4,5-b]pyridines as PIM kinase inhibitors.

PIM kinases have become targets of interest due to their association with biochemical mechanisms affecting survival, proliferation and cytokine production. 1,2,3-Triazolo[4,5-b]pyridines were identified as PIM inhibitors applying a scaffold hopping approach. Initial exploration around this scaffold and X-ray crystallographic data are hereby described.

Effects of the organophosphate insecticides phosmet and chlorpyrifos on trophoblast JEG-3 cell death, proliferation and inflammatory molecule production.

Epidemiological data have associated environmental organophosphate insecticide (OP) exposure during pregnancy with fetal growth deficits. To better understand OP injury that may adversely affect pregnancy, we used the JEG-3 choriocarcinoma cell line, which provide a recognized in vitro model to study placental function. The effects of the OP phosmet (Pm) and chlorpyrifos (Cp) on JEG-3 cells viability, proliferation, cell cycle and inflammatory molecule production were evaluated. Both insecticides affected cellular viability in a concentration- and time-dependent manner, inducing apoptosis and decreasing [(3)H]-thymidine incorporation. However, only Pm reduced DNA synthesis independently of cellular death and decreased the cell percentage at the S-phase. Unlike apoptosis, TNFα production varied with the concentration tested, suggesting that other TNFα independent mechanisms might trigger cell death. No induction of the inflammatory molecule nitric oxide was detected. The mRNA levels of pro-inflammatory IL-6, IL-17 and the anti-inflammatory IL-13 cytokines were differentially modulated. These findings show that Pm and Cp generate a specific toxicity signature, altering cell viability and inducing an inflammatory cytokine profile, suggesting that trophoblasts may represent a possible target for OP adverse effects.

IFNß produced by TLR4-activated tumor cells is involved in improving the antitumoral immune response.

Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNß was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNß-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response.

Expression of Toll-like receptor 4 in the prostate gland and its association with the severity of prostate cancer.

BACKGROUND: Chronic inflammation has been postulated to be an important driving force to prostate carcinoma. Toll-like receptors (TLRs) compose a family of receptors mainly expressed on immune cells. Recently, functional TLRs have been shown to be also expressed in numerous cancer cells, but their significance has only recently begun to be explored. The purpose of this study was to investigate the putative role of TLR4 expression in prostate carcinoma. METHODS: To determine if there is an association between TLR4 expression and the malignancy of the tumor, 35 prostate carcinoma samples showing different Gleason grades were analyzed by immunohistochemistry. Also, to explore the functionality of the receptors expressed on the epithelium, we analyzed the type of cytokine response elicited and the signaling pathways involved after TLR4 triggering in the human prostate adenocarcinoma cell line, DU-145. RESULTS: TLR4 is expressed in the normal prostate gland in both stroma and epithelium. TLR4 expression significantly drops to negative values as the Gleason grade augments in both, stroma and epithelium. Moreover, DU-145 cells also exhibit TLR4 expression and respond to TLR4 agonists, activating the transcription factor NF-kappaB and increasing the expression of pro-inflammatory mediators. Inhibition of the molecular adaptors MyD88 and MAL by overexpression of dominant-negative mutants diminished LPS-induced activation of NF-kappaB, showing that DU-145 cells activate the NF-kappaB through MyD88-dependent signaling pathways. CONCLUSIONS: We hypothesize that TLR4 in prostate cells could synergize with innate immune cells contributing to an eventual inflammatory process, which in genetically prone individuals could promote carcinogenesis. Prostate 69: 1387-1397, 2009. (c) 2009 Wiley-Liss, Inc.

Impact of vitamin D receptor activity on experimental autoimmune prostatitis.

Chronic non bacterial prostatitis is a chronic inflammatory syndrome. Its etiology and physiopathology are unclear and treatments are empirical and ineffective in most cases. Autoimmunity has been proposed as an etiology. In the present report, we investigated the impact of vitamin D receptor silencing, by use of VDR-KO NOD mice and the immune-modulating effect of the vitamin D3 analog TX527 on the development of Experimental Autoimmune Prostatitis in NOD mice. VDR-KO NOD mice developed a more aggressive form of autoimmune prostatitis characterized by a greater lymphoproliferative response against prostate antigen in vitro (6.92+/-4.77 vs. 2.47+/-0.41 21 days after disease induction, p<0.05) and higher levels of specific INFgamma secretion (471+/-6 vs. 386+/-5pg/ml, p<0.01). This was accompanied in vivo by more severe lesions and augmented mononuclear cell infiltration in the prostate gland. On the other hand, although analog-treated mice showed a significant reduction in the spleen T-cell specific proliferative response against prostate antigen in vitro, no effect on disease development was observed. We conclude that vitamin D receptor modulation holds the promise of interfering with autoimmune prostatitis. Introduction of more powerful analogs, or combinations with anti-T-cell reagents may represent therapeutic solutions for these group of patients.

Activation of Toll-like receptor 4 on tumor cells in vitro inhibits subsequent tumor growth in vivo.

Although an eruption of information on the role of Toll-like receptor 4 (TLR4), the main receptor for bacterial lipopolysaccharide, in activating macrophages and dendritic cells has emerged, very little is known about the role of TLR4 present on epithelial cells from sterile environments like tumors. The main goal of this work was to investigate the consequences of TLR4 activation present on tumor cells in two different animal models of cancer: the Dunning rat prostate cancer and the B16 murine melanoma models. We show that (a) activating TLR4 signaling in two different tumor cell lines in vitro modifies the tumor outgrowth in vivo; (b) this effect is not due to a direct consequence of TLR4 signaling on the proliferation/apoptosis balance of the tumor cells; (c) the T-cell compartment is somehow involved in the described phenomenon because the inhibitory effect observed is not seen in athymic nude mice; and (d) tumor-infiltrating lymphocytes purified from tumors induced by TLR4-activated cells show strong induction of IFN gamma transcript in detriment of interleukin-10 transcript, suggesting a change in their functionality. We hypothesize that TLR4 signaling in tumor cells in vitro induces the expression of proinflammatory mediators, which could dramatically alter the maturation state of dendritic cells present at the site of inoculation, switching the type of immune response elicited against the tumor. These results open up new avenues for understanding the role of TLR4 in tumor cells and for identifying potential new therapy strategies for cancer.