Amado A. Quintar

Expression of Toll-Like Receptor 4 in the Prostate Gland and Its Association With the Severity of Prostate Cancer

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.

Toll-Like Receptor 4 in Rat Prostate: Modulation by Testosterone and Acute Bacterial Infection in Epithelial and Stromal Cells

Abstract The prostate gland is the most inflammation-prone organ in the male reproductive tract. However, little information is available regarding the immunobiology of this gland. Toll-like receptor 4 (TLR4) is considered to be a major sensor of danger signals and a key trigger of the innate immune responses. TLRs have also been implicated in the development of different inflammatory diseases in organs in which epithelial-stromal interactions are critical for homeostasis. The purpose of this work was to evaluate the presence and regulation of TLR4 in the rat prostate. Western blot and immunocytochemical studies revealed that constitutive expression of TLR4 in the rat ventral prostate was localized in the epithelial cells, mainly associated with the rough endoplasmic reticulum, as well as in smooth muscle cells in the stroma. In addition, increased concentrations of TLR4 were found in castrated rats, predominantly in hypertrophied smooth muscle cells. On the other hand, using a bacterial prostatitis model, we observed an increment in the TLR4 cytoplasmic content and migration of this receptor to the apical plasmatic membranes of epithelial cells at 24 h and 48 h post-infection. These findings suggest that the prostate gland is able to recognize pathogens and to initiate immune responses. In addition, TLR4 appears to be implicated in the vital stromal-epithelial interactions that maintain prostate homeostasis during prostatitis, as well as following androgen deprivation.

Acute Inflammation Promotes Early Cellular Stimulation of the Epithelial and Stromal Compartments of the Rat Prostate

It has been proposed that prostatic inflammation plays a pivotal role in the pathophysiology of benign hyperplasia and prostate cancer. However, little information is available about the prostatic reaction to bacterial compounds in vivo. Our aim was therefore to evaluate the early effects of bacterial infection on rat ventral prostate compartments.

Dedifferentiation of prostate smooth muscle cells in response to bacterial LPS

Prostate smooth muscle cells (SMCs) are strongly involved in the development and progression of benign prostatic hyperplasia and prostate cancer. However, their participation in prostatitis has not been completely elucidated. Thus, we aimed to characterize the response of normal SMC to bacterial lipopolysaccharide (LPS).

Budesonide effects on Clara cell under normal and allergic inflammatory condition

Clara cells are nonciliated secretory cells implicated in lung homeostasis by the synthesis of immunomodulatory and host defense products, being one of the most important the CC16 protein. In this study, we compared the effects of budesonide (BUD), an inhaled corticoid, on Clara cell biology and its ability to reverse morphofunctional changes induced in an allergic airway hyper-responsiveness mouse model. In normal mice, exposure to BUD induced morphological changes compatible with a state of maximal differentiation on CC16 positive cells which developed a prominent cupola filled up with numerous mitochondria rich in CYP2E1, a member of the cytochrome P450 family. Consequently, CYP2E1 expression raised significantly. Exposure to OVA provoked hypertrophy of Clara cells and an increment in their number per millimeter of basal membrane. These cells acquired a mucous cell phenotype characterized by a notorious expansion of the secretory granular content. Synthesis of CC16 was greatly up-regulated concurrent to the finding of MUC5AC expression and the increment of epidermal growth factor receptor (EGFR). Mitochondrial content decreased significantly with a consequent reduction in CYP2E1 expression. After BUD treatment of OVA-challenged animals, the majority of Clara cells regained their normal morphology and functional characteristics; CYP2E1 levels raised when compared to the OVA exposed group. The BUD potential to differentiate Clara cells appeared to be important for the regression of the profound changes generated by the allergic injury. These results demonstrated the wide range of stimuli that can modify different aspects of Clara cell biology, and highlighted the effects of budesonide as a modulator of P450 enzymes, which probably contributes to a complementary antiinflamatory activity.

Regulation of surfactant protein D in the rodent prostate

BackgroundSurfactant protein D (SP-D) is an innate immune protein that is present in mucosal lined surfaces throughout the human body, including the male reproductive tract. In the present study, we characterized the regulation of SP-D expression in the mouse and rat prostate.MethodsReal time reverse transcriptase polymerase chain reaction (RT-PCR) and immunostaining were used to characterize SP-D mRNA and protein in the mouse male reproductive tract. In order to evaluate the effects of testosterone on SP-D gene expression, we measured SP-D mRNA levels via real time RT-PCR in prostates from sham-castrated mice and castrated mice. In addition, we used a rat prostatitis model in which Escherichia coli was injected into the prostate in vivo to determine if infection influences SP-D protein levels in the prostate.ResultsWe found that SP-D mRNA and protein are present throughout the mouse male reproductive tract, including in the prostate. We determined that castration increases prostate SP-D mRNA levels (~7 fold) when compared to levels in sham-castrated animals. Finally, we demonstrated that infection in the prostate results in a significant increase in SP-D content 24 and 48 hours post-infection.ConclusionOur results suggest that infection and androgens regulate SP-D in the prostate.

Immunoelectron Microscopy: A Reliable Tool for the Analysis of Cellular Processes

Electron Microscopy is an indispensable tool to investigate the intricate structures of the cell and organelles, and also to study the cellular biological processes implicated in the responses to changes in the microenvironment. However, several cellular events may be missed if conventional ultrastructural studies are not complemented with details concerning the subcellular localization of a wide range of specific proteins which can become rearranged as part of their own dynamic processes. Thus, immunoelectron microscopy emerges as a technique that links the information gap between biochemistry, molecular biology, and ultrastructural studies, by placing macromolecular functions within a cellular context.

Testosterone abrogates TLR4 activation in prostate smooth muscle cells contributing to the preservation of a differentiated phenotype.

Prostate smooth muscle cells (pSMCs) are capable of responding to inflammatory stimuli by secreting proinflammatory products, which causes pSMCs to undergo dedifferentiation. Although it has been proposed that androgens decrease proinflammatory molecules in many cells and under various conditions, the role of testosterone in the prostate inflammatory microenvironment is still unclear. Therefore, our aim was to evaluate if testosterone was able to modulate the pSMCs response to bacterial LPS by stimulating primary pSMC cultures, containing testosterone or vehicle, with LPS (1 or 10 µg/ml) for 24–48 h. The LPS challenge induced pSMCs dedifferentiation as evidenced by a decrease of calponin and alpha smooth muscle actin along with an increase of vimentin in a dose‐dependent manner, whereas testosterone abrogated these alterations. Additionally, an ultrastructural analysis showed that pSMCs acquired a secretory profile after LPS and developed proteinopoietic organelles, while pSMCs preincubated with testosterone maintained a more differentiated phenotype. Testosterone downregulated the expression of surface TLR4 in control cells and inhibited any increase after LPS treatment. Moreover, testosterone prevented IκB‐α degradation and the LPS‐induced NF‐κB nuclear translocation. Testosterone also decreased TNF‐α and IL6 production by pSMCs after LPS as quantified by ELISA. Finally, we observed that testosterone inhibited the induction of pSMCs proliferation incited by LPS. Taken together, these results indicate that testosterone reduced the proinflammatory pSMCs response to LPS, with these cells being less reactive in the presence of androgens. In this context, testosterone might have a homeostatic role by contributing to preserve a contractile phenotype on pSMCs under inflammatory conditions. J. Cell. Physiol. 228: 1551–1560, 2013.

The Initial Segment of the Rat Epididymis Is Able to Uptake Immature Germ Cells Shed by Testicular Damage

The initial segment of the caput epididymidis, the most proximal part of the rat epididymis, has specific functional characteristics. In the present study, the behavior of the epididymal epithelium from this region was evaluated after the exposure to a massive number of immature germ cells in the luminal fluid. The experimental release of immature germ cells from the seminiferous tubules was performed by injecting anti-microtubule compounds into the rete testis and the lumen of seminiferous tubules. Twenty-four hours after nocodazole or colchicine administration, a massive phagocytosis of immature spermatogenic cells, recognized as acrosin-positive structures, was easily observed in the epithelium of the initial segment of the epididymis assessed by light and electron microscopy. Immature germ cells were engulfed by epithelial cells, where most of them were found as cell debris at different stages of degradation. No signs of inflammation were observed either in the lumen or in the interstitium. The phagocytosis of immature germ cells was restricted to the epithelium of the initial segment of the epididymis, suggesting a role for this segment as the first selective barrier for the exclusion of abnormal gametes along the male genital tract.

Restoration of the normal Clara cell phenotype after chronic allergic inflammation

Bronchiolar Clara cells play a critical role in lung homoeostasis. The main goal of this study was to evaluate the effects of chronic allergy on these cells and the efficacy of budesonide (BUD) and montelukast (MK) in restoring their typical phenotypes after ovalbumin‐induced chronic allergy in mice. Chronic allergy induced extensive bronchiolar Alcian blue‐periodic acid‐Schiff (AB/PAS)‐positive metaplasia. In addition, cells accumulated numerous big electron‐lucent granules negative for Clara cell main secretory protein (CC16), and consequently, CC16 was significantly reduced in bronchoalveolar lavage. A concomitant reduction in SP‐D and CYP2E1 content was observed. The phenotypic changes induced by allergy were pharmacologically reversed by both treatments; MK was more efficient than BUD in doing so. MK decreased AB/PAS reactivity to control levels whereas they remained persistently elevated after BUD. Moreover, most non‐ciliated cells recovered their normal morphology after MK, whereas for BUD normal cells coexisted with ‘transitional’ cells that contained remnant mucous granules and stained strongly for CC16 and SP‐D. Glucocorticoids were also less able to reduce inflammatory infiltration and maintained higher percentage of neutrophils, which may have contributed to prolonged mucin expression. These results show that chronic allergy‐induced mucous metaplasia of Clara cells affects their defensive mechanisms. However, anti‐inflammatory treatments were able to re‐establish the normal phenotype of Clara cell, with MK being more efficient at restoring a normal profile than BUD. This study highlights the role of epithelial cells in lung injuries and their contribution to anti‐inflammatory therapies.