Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3ß-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery.

Outcome of acute renal injury in diabetic mice with experimental endotoxemia: role of hypoxia-inducible factor-1 α.

The role of diabetic nephropathy in the outcome of acute renal injury (AKI) is not well defined. Herein we evaluate the outcome of lipopolysaccharide- (LPS-) induced AKI in streptozotocin-induced diabetes, as well as the potential role of Hypoxia Inducible Factor (HIF-1 α ) in this condition. Although 6 h after LPS injection all mice developed a decrease in renal function, proteinuric diabetic mice showed a better recovery of this parameter throughout the study (72 h). Both HIF-1 α and vascular endothelium growth factor (VEGF) were found to be upregulated in diabetic mice. After LPS injection, all animals showed an upregulation of these factors, although it was higher in the diabetic group. Glycated albumin (GA) was found to upregulate HIF-1 α in HK-2 cells, which resulted in increased production of VEGF. Interestingly, LPS cooperated with GA to induce HIF-1 α upregulation. In conclusion, diabetic mice display a better recovery of AKI after experimental endotoxemia. Moreover, these animals showed an increased expression of both HIF-1 α and VEGF that was reproduced by incubating renal cells with GA. Since VEGF is considered a survival factor for tubular cells, our findings suggest that diabetes displays HIF-1 α upregulation that might function as a "precondition state" offering protection from endotoxic AKI.

Influence of thyroid hormone receptors on breast cancer cell proliferation.

BACKGROUND: The involvement of thyroid hormones in the development and differentiation of normal breast tissue has been established. However, the association between breast cancer and these hormones is controversial. Therefore, the objective of the present study was to determine the protein expression pattern of thyroid hormone receptors in different human breast pathologies and to evaluate their possible relationship with cellular proliferation. PATIENTS AND METHODS: The presence of thyroid hormone receptors was evaluated by immunohistochemistry and western blot analysis in 84 breast samples that included 12 cases of benign proliferative diseases, 20 carcinomas in situ and 52 infiltrative carcinomas. RESULTS: TR-alpha was detected in the nuclei of epithelial cells from normal breast ducts and acini, while in any pathological type this receptor was located in the cytoplasm. However, TR-beta presented a nuclear location in benign proliferative diseases and carcinomas in situ and a cytoplasmatic location in normal breast and infiltrative carcinomas. The highest proliferation index was observed in carcinomas in situ, although in infiltrative carcinomas an inverse correlation between this index and the TR-alpha expression was encountered. CONCLUSIONS: The results of this study reveal substantial changes in the expression profile of thyroid hormone receptors suggesting a possible deregulation that could trigger breast cancer development.

Expression of vitamin D3 receptor and retinoid receptors in human breast cancer: identification of potential heterodimeric receptors.

Vitamin D3 (VD) and all-trans-retinoic acid (ATRA) have been postulated as a novel treatment option for breast carcinoma. Since the combined effects of retinoids and VD derivatives are attributed to heterodimeric interactions between members of the nuclear receptor family, the expression patterns of the heterodimers formed by vitamin D3 receptor (VDR) and the retinoid receptors RARs (RAR-alpha, RAR-beta and RAR-gamma) and RXRs (RXR-alpha, RXR-beta and RXR-gamma) have been studied by immunohistochemistry in benign and malignant breast tissues. Present results revealed that immunoexpressions to all receptor types studied were higher in both in situ and infiltrative carcinomas than in benign breast diseases. In a variable number of cases of infiltrative carcinoma, immunostaining appeared in the nucleus, whereas in the other two disorders immunostaining was only cytoplasmic. The correlation established between VDR and the different isoforms of retinoid receptors revealed that VDR seems to select mainly RAR-alpha to form heterodimers and to exert their properties as transcription factor. The results of this study suggest that this heterodimer plays a critical role in cancer malignancy, and its presence indicates those patient groups presenting a better response to adjuvant therapies based on the combination of vitamin D and ATRA.

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle.

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.

Androgen receptor (AR), estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) expression in the testis of the newt, Triturus marmoratus marmoratus during the annual cycle.

Expression of androgen receptor (AR), estrogen receptor alpha (ER-alpha) and estrogen receptor beta (ER-beta) in the testis of the marbled newt (Triturus marmoratus marmoratus) was investigated, with special attention to changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. Primordial germ cells, primary and secondary spermatogonia and spermatocytes showed a positive reaction to the 3 receptor antibodies during the annual reproductive cycle. Follicular cells were positive to AR, ER-alpha and ER-beta during the spermiogenesis and quiescence periods in the glandular tissue. Interstitial cells showed reactivity to AR, ER-alpha and ER-beta in the spermiogenesis and the quiescence periods, and presented no labelling to these receptors in the proliferative period. These findings suggest that, as in mammals, there is an androgen-estrogen regulation of the function and development of the newt testis.

Estrogen receptors alpha and beta in the normal, hyperplastic and carcinomatous human prostate.

Two different estrogen receptors (ER-alpha and ER-beta) have been described, which are differentially involved in regulating the normal function of reproductive tissues. ER-alpha was considered for a long time to be the only estrogen receptor, and it has been detected in the stromal cells of the human prostate but not in the epithelium. To obtain new information about the differential effects of both receptor types, we have investigated their localization in normal prostates, benign prostatic hyperplasia (BPH), and prostatic cancer (PC) by immunohistochemistry, ELISA and Western blot. Epithelial immunostaining was absent in normal prostates and was present in BPH (10% of cells) and PC (80% of cells), whereas about 15% of stromal cells were positively immunostained for ER-alpha in the three types of prostatic specimens studied. Epithelial immunostaining for ER-beta was detected in normal prostates (13% of cells), BPH (30% of cells) and PC (79% of cells), whereas stromal immunostaining for ER-beta was absent in normal and hyperplastic prostates and was present in PC (12% of cells). The complementary presence of both receptor types in the normal prostate (ER-beta in the epithelium and ER-alpha in the stroma) might explain the mechanism of estrogen action in the development of BPH. The increased epithelial immunostaining for both ER-alpha and ER-beta in BPH and PC suggests that the involvement of estrogen receptors in hyperplasia and cancer concerns mainly the epithelium.

Morphometric evaluation of the human prostate.

In order to clarify the ageing-related histological changes in the human prostate, a quantitative morphometric analysis was performed. Complete prostates were obtained at autopsy from 281 men (aged 20-84 years) who died in traffic accidents and presented no clinical symptoms of prostatic disease. The prostates were classified as: histologically normal (n=182), with nodular hyperplasia (n=42), with intraepithelial neoplasia (n=40) and carcinomatous with low Gleason grade (n=20). Each prostate was divided into three regions (periurethral, central and peripheral) and the volume of each region, as well as the average volume occupied by stroma and epithelium in each region were quantified. For each parameter, the average values for each age group were compared. In the histologically normal prostates, an increase with ageing in the total volume and the volume occupied by the central region were observed; these increases were mainly caused by an increase in the stromal volume of the central region in men after 30 years of age. No histologically normal prostates were found in men older than 70 years of age. Nodular prostatic hyperplasia was found in men over 30 years of age and a fluctuation in the total volume throughout ageing was observed. Prostates with intraepithelial neoplasia (PIN) and carcinoma were observed in men aged >20 years and the total volume and those of each prostatic region showed multiple variations, except for the eighth decade where a marked increase with regard to that of the previous decades was observed.

Identification of N- and O-linked oligosaccharides in human seminal vesicles.

The main oligosaccharide residues and the saccharide linkage in infantile and adult human seminal vesicles were studied by means of lectin histochemistry at light and electron microscopy levels. In adult glands, the epithelial cell cytoplasm and luminal content reacted positively to the following residues: (GlcNAc)n (WGA), Galbeta1,3GalNAc (PNA), GalNAcalpha1,3Gal (SBA), GalNAcalpha1,3GalNAc (HPA), Fucalpha1,2Galbeta1,4GlcNAc (UEA-I), and alphaL-Fuc1,6DGlcNAc-O-Melibiosc (AAA). The presence of intense staining in the luminal content suggest that glycoproteins containing these oligosaccharide moieties are secreted by epithelial cells. Adult epithelial cells also reacted to Neu5Acalpha2,6Gal (SNA), Neu5Acalphaa2,3Galbeta1,4GlcNAc (MAA), Galbeta1,4GlcNAc (DSA), branched mannose chains (ConA), Man1,3Man (GNA), and Fucalpha1,2Galbeta1,4GlcNAcFucalpha1,3GlcNAc (LTA) but reaction to these residues was weak (MAA, DSA, ConA, and LTA) or absent (SNA and GNA) in the gland lumen, which suggests that they belong to intracytoplasmic proteins. The chemical and enzymatic treatments used suggest that the residues recognized by SNA, MAA, PNA, DSA, HPA, and SBA belong to O-linked oligosaccharides; those residues localized by ConA and GNA have an N-glycosidic linkage, and those bound by WGA, LTA, UEA-I, and AAA are linked to both N- and O-oligosaccharides. In prepubertal seminal vesicles, reaction in the epithelial cell cytoplasm was similar to that observed in adults, except for GNA and HPA, which showed a weaker reaction. However, the lumen of prepubertal seminal vesicles showed intense reaction to WGA and SBA only. The chemical and enzymatic treatments suggest that the scanty glycoproteins secreted by the prepubertal glands belong to the mucin-type.

Immunoexpressions of p21, Rb, mcl-1 and bad gene products in normal, hyperplastic and carcinomatous human prostates.

A comparative study of the expression of p21, Rb, mcl-1, and bad gene products, which are involved in the control of the cell cycle, was performed in normal, hyperplastic, and carcinomatous human prostates by means of a semiquantitative immunochemical study. This included Western blot, ELISA, and immunohistochemistry procedures. In normal prostates, immunoexpression of the four gene products was scanty or absent. In men with benign prostatic hyperplasia, immunoreactions to the four proteins studied were found in many epithelial cells and some stromal cells. In prostatic carcinoma, the immunostaining pattern was as in hyperplastic prostates but the numbers of both epithelial and stromal cells were higher. Present results indicate that immunoexpression of p21, Rb (both the phosphorylated and dephosphorylated forms), mcl-1, and bad gene products are markedly increased in prostates with proliferative alterations but that these proteins do not discriminate between benignant (hyperplasia) and malignant (adenocarcinoma) prostatic tumours, although immunoexpression is higher in prostatic carcinoma.

IL-2, its receptors, and bcl-2 and bax genes in normal, hyperplastic and carcinomatous human prostates: immunohistochemical comparative analysis.

The presence of interleukin-2 (IL-2) and its receptors (Ralpha, Rbeta, Rgamma), and their relationship with the products of bcl-2 and bax genes was studied in normal prostates, benign prostatic hyperplasia (BPH), and prostatic cancer (PC) by ELISA, Western blot, and immunohistochemistry. A comparative semiquantitative immunohistochemical study was also performed. For all the antibodies assayed, immunoreactions were found in the epithelium and some stromal cells in the three types of specimens studied. These immunoreactions were much more higher in PC samples than in normal prostates. In BPH, immunoreactions were similar to that of normal prostates (bax), similar to that of PC (IL-2 and its three receptors), or intermediate between that of normal prostates and that of PC (bcl-2). Immunoexpressions of IL-2 and its receptors were found in the epithelial basal cells and some stromal cell of normal prostates and might be related to the control of the proliferation-apoptosis equilibrium. The increased expressions of IL-2 and its receptors in the epithelium of prostates in BPH, associated with increased bcl-2 expression which would account for the decrease in the apoptosis index that has been reported in this disorder. The increased expression of both bcl-2 and bax in PC might be involved in the higher apoptosis index reported in PC specimens. Since IL-2 administration seems to have an anti-tumour effect, the increased expression of this interleukin in BPH and PC could be interpreted as an attempt to hinder cell proliferation which would only be efficient at high doses.

Immunohistochemical localization of protein gene product 9.5, ubiquitin, and neuropeptide Y immunoreactivities in epithelial and neuroendocrine cells from normal and hyperplastic human prostate.

This study was designed to investigate (a) the presence of protein gene product 9.5 (PGP 9.5), ubiquitin, and neuropeptide Y (NPY) in the neuroendocrine and secretory epithelium of the human normal prostate and its secretions, and (b) the changes in immunoreactivity to these proteins in men with benign prostatic hyperplasia. Western blotting and light microscopic immunohistochemistry techniques were used and the numerical density of immunoreactive neuroendocrine cells, and the volume fractions of immunostained secretory epithelium were evaluated. Western blotting revealed the presence of the three antigens in both tissue homogenates and prostate secretion. Some neuroendocrine cells immunoreacted to PGP 9.5 and NPY in all the prostate regions of control specimens. Ubiquitin immunoreactivity was detected in the nuclei from both basal cells and secretory epithelial cells. The cytoplasm of the secretory cells and the glandular lumen also showed immunostaining for the three proteins. The numerical densities of both PGP 9.5 and NPY neuroendocrine cells were lower in hyperplasia than in controls. No differences in the volume fraction occupied by epithelial immunostaining to both proteins was found between hyperplastic and control prostates. We concluded that (a) PGP 9.5 and NPY, but not ubiquitin, are common antigens in both neuroendocrine and secretory prostate cells, (b) the three immunoreactive proteins contribute to the prostate secretions, and (c) the secretion of ubiquitin is markedly diminished in the hyperplastic epithelium.(J Histochem Cytochem 48:1121-1130, 2000)

Interferon-gamma and its functional receptors overexpression in benign prostatic hyperplasia and prostatic carcinoma: parallelism with c-myc and p53 expression.

The therapeutic potential of IFN-gamma in prostatic cancer has been documented in several reports, although no immunohistochemical studies of this factor and its receptors in the prostate have been reported. The aim of the present study was to investigate the expression of IFN-gamma and its receptor components (IFN-gamma-Ralpha and IFN-gamma-Rbeta) in normal prostate, benign prostatic hyperplasia (BPH) and prostatic cancer (PC), as well as the possible relationship between this factor and the products of the p53 gene (the wild and mutant forms) and the oncogene c-myc, by means of immunochemical techniques (Western blot, ELISA, and quantification of immunostaining in histological sections). In normal prostate, IFN-gamma and its two receptors were expressed in the basal cells of the epithelium and some stromal cells. In BPH specimens, immunostaining of basal epithelial cells was significantly increased for IFN-gamma and its a receptor, whereas stromal cell immunostaining was significantly increased for IFN-gamma and its b receptor. In addition, columnar epithelial cells immunostained for IFNbeta-Rbeta. PC specimens differed from BPH specimens in the significantly increased immunostaining of epithelial cells for IFN-gamma and its two receptors, and the immunostaining of columnar epithelial cells for IFN-gamma-Ralpha. Immunodetection of wild-p53 was weak and limited to some stromal cells in the three types of specimens. Immunostainings for both mutant-p53 and c-myc were negative in normal prostate, and positive in the epithelium and stromal cells of both BPH and PC specimens. Immunostaining intensity in PC was significantly higher than in BPH. These observations suggest that the expression of both mutant-p53 and c-myc, together with other factors, might be involved in the development of prostatic hyperplasia and neoplasia, while the increased expression of IFN-gamma and its receptors could be regarded as an attempt, although insufficient, to inhibit the uncontrolled cell proliferation.

Characterization of several invertebrate muscle cell types: a comparison with vertebrate muscles.

Ultrastructural classification of invertebrate muscles is complex and not always clear. The aim of the present paper was to establish some criteria that might be useful for classification of invertebrate muscles and for a better understanding of the differences between them. The procedures used were: (1) immunochemical evaluation of those proteins that differentiated striated from smooth muscle (troponin, caldesmon, and calponin), and (2) calculations of several myofilament parameters to establish differences among muscles. The muscles studied were: striated muscles from the rat, Drosophila, the crab Callinectes, and the snail Helix (heart); obliquely striated muscles from the earthworm Eisenia foetida and Helix (mouth); and smooth muscles from the rat, and Helix (retractor, body wall, and intestinal wall). Immunochemical studies revealed that troponin was only present in the striated muscles and the obliquely striated muscle from Eisenia, whereas caldesmon and calponin were only present in the smooth muscles and the obliquely striated muscle from Helix. The highest thick filament/thin filament volume ratio was found in the striated muscles, followed by the obliquely striated muscles, and the smooth muscles. This suggests the order in which the contraction strength decreases. The myofilament length is inversely related to the contraction speed, which was higher in the striated muscles than in the obliquely striated muscles. In vertebrates, the smooth muscle seems to be less rapid than the striated muscle because their myofilaments are longer. This assertion cannot be generalized for invertebrate smooth muscle, because myofilament lengths vary widely in both striated and smooth muscles. In smooth muscles, the presence of apparently unordered electron-dense bodies instead of ordered Z lines and the absence of true sarcomeres permit a certain overlapping of thin filaments increasing the range of shortening.

E-, N- and P-cadherin, and alpha-, beta- and gamma-catenin protein expression in normal, hyperplastic and carcinomatous human prostate.

The expression of E-, N- and P-cadherin, alpha-, beta- and gamma-catenin, and actin was studied by immunohistochemistry, ELISA, and Western blot analysis in normal prostates, and in the prostates of men with benign prostatic hyperplasia and men with prostatic carcinoma, in order to evaluate their possible role in the pathogenesis of these diseases. Present results reveal that the immunophenotype of hyperplastic prostates differs from those of both normal and carcinomatous prostates in the intracellular distribution (observed by immunohistochemistry) and the intensity (measured by ELISA) of immunoreactions to cadherins, catenins, and actin. Hyperplastic prostates differ form normal prostates in the weaker immunoreaction to the three cadherin types, the two catenins, and actin, as well as in the intracellular distribution of P-cadherin, beta- and gamma-catenin, and actin. Differences between benign prostatic hyperplasia and prostatic carcinoma are less marked because hyperplastic prostates differ from carcinomatous prostates only in the weaker immunoreactions to P-cadherin, and alpha-catenin. The most remarkable findings in this study were: (1) alpha-catenin production was elevated in prostatic carcinoma in comparison with benign prostatic hyperplasia and normal prostate; and (2) P-cadherin expression in benign prostatic hyperplasia is reduced with regard to those of normal and carcinomatous prostates. It may be concluded that a decreased immunoreaction to cadherins, catenins, and actin, as well as changes in the intracellular distribution of actin in prostatic cells are not necessarily suggestive of malignancy, because these alterations are also present in BPH, and thus, the loss of cadherin-catenin-mediated adhesion alone is not sufficient to establish an invasive phenotype.