Metabolic Reprogramming, Autophagy, and Reactive Oxygen Species Are Necessary for Primordial Germ Cell Reprogramming into Pluripotency.

Cellular reprogramming is accompanied by a metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Previous results from our laboratory showed that hypoxia alone is able to reprogram primordial germ cells (PGCs) into pluripotency and that this action is mediated by hypoxia-inducible factor 1 (HIF1). As HIF1 exerts a myriad of actions by upregulating several hundred genes, to ascertain whether the metabolic switch toward glycolysis is solely responsible for reprogramming, PGCs were cultured in the presence of a pyruvate kinase M2 isoform (PKM2) activator, or glycolysis was promoted by manipulating PPARγ. Conversely, OXPHOS was stimulated by inhibiting PDK1 activity in normoxic or in hypoxic conditions. Inhibition or promotion of autophagy and reactive oxygen species (ROS) production was performed to ascertain their role in cell reprogramming. Our results show that a metabolic shift toward glycolysis, autophagy, and mitochondrial inactivation and an early rise in ROS levels are necessary for PGC reprogramming. All of these processes are governed by HIF1/HIF2 balance and strict intermediate Oct4 levels. Histone acetylation plays a role in reprogramming and is observed under all reprogramming conditions. The pluripotent cells thus generated were unable to self-renew, probably due to insufficient Blimp1 downregulation and a lack of Klf4 and cMyc expression.

Cell metabolism under microenvironmental low oxygen tension levels in stemness, proliferation and pluripotency.

Hypoxia is defined as a reduction in oxygen supply to a tissue below physiological levels. However, physiological hypoxic conditions occur during early embryonic development; and in adult organisms, many cells such as bone marrow stem cells are located within hypoxic niches. Thus, certain processes take place in hypoxia, and recent studies highlight the relevance of hypoxia in stem cell cancer physiology. Cellular response to hypoxia depends on hypoxia-inducible factors (HIFs), which are stabilized under low oxygen conditions. In a hypoxic context, various inducible HIF alpha subunits are able to form dimers with constant beta subunits and bind the hypoxia response elements (HRE) in the genome, acting as transcription factors, inducing a wide variety of gene expression. Typically, the HIF pathway has been shown to enhance vascular endothelial growth factor (VEGF) expression, which would be responsible for angiogenesis and, therefore, re-oxygenation of the hypoxic sites. Embryonic stem cells inhibit a severely hypoxic environment, which dictates their glycolytic metabolism, whereas differentiated cells shift toward the more efficient aerobic respiration for their metabolic demands. Accordingly, low oxygen tension levels have been reported to enhance induced pluripotent stem cell (iPS) generation. HIFs have also been shown to enhance pluripotency-related gene expression, including Oct4 (Octamer-binding transcription factor 4), Nanog and Wnt. Therefore, cell metabolism might play a role in stemness maintenance, proliferation and cell reprogramming. Moreover, in the hypoxic microenvironment of cancer cells, metabolism shifts from oxidative phosphorylation to anaerobic glycolysis, a process known as the Warburg effect, which is involved in cancer progression and malignancy.

c-Kit identifies a subpopulation of mesenchymal stem cells in adipose tissue with higher telomerase expression and differentiation potential.

The stromal vascular fraction (SVF) of adipose tissue is an easy to obtain source of adipose tissue-derived stem cells (ADSCs). We and others have achieved significant but suboptimal therapeutic effects with ADSCs in various settings, mainly due to low rates of differentiation into specific cell types and with the downside of undesired side effects as a consequence of the undifferentiated ADSCs. These data prompted us to find new stem cell-specific markers for ADSCs and/or subpopulations with higher differentiation potential to specific lineages. We found a subpopulation of human ADSCs, marked by c-Kit positiveness, resides in a perivascular location, and shows higher proliferative activity and self-renewal capacity, higher telomerase activity and expression, higher in vitro adipogenic efficiency, a higher capacity for the maintenance of cardiac progenitors, and higher pancreatogenic and hepatogenic efficiency independently of CD105 expression. Our data suggests that the isolation of ADSC subpopulations with anti-c-Kit antibodies allows for the selection of a more homogeneous subpopulation with increased cardioprotective properties and increased adipogenic and endodermal differentiation potential, providing a useful tool for specific therapies in regenerative medicine applications.

Double port injector device to reduce endothelial damage in DMEK.

OBJECTIVE: To study endothelial injury from a newly designed asymmetric double port Descemet Membrane Endothelial Keratoplasty (DMEK) injector, both ex-vivo and in clinical practice. DESIGN: Laboratory investigation with an interventional case series study. METHOD: Sixteen rabbit endothelial rolls were tested for injection using a no-touch technique. For each pair of rolls, one endothelial graft underwent injection with a single port Pasteur pipette twice, wheras the other was injected with a novel asymmetric double port injector with a larger diameter entry port than the exit port also twice. Each graft was stained with 4-6-diamidino-2-phenylinidole dihydrochloride and was counted under a fluorescence-inverted microscope before and after injection. The proportion of graft injury was calculated and the differences were analyzed. Subsequently, six patients requiring DMEK underwent surgery using this novel insertion device and endothelial cell loss was calculated 3 months after the surgery. RESULTS: After injection, the mean proportion of endothelial cell survival with the single port pipette was 78.8% (n=8; SD: ±20.9%), whereas the double port injector yielded a survival rate of 96.8% (n=8; SD: ±8.4%). This difference was statistically significant (P=0.008), representing less endothelial injury with the double port device. Early endothelial cell loss after 3 months in the DMEK patients was 26.1% (SD: ±6.1%). CONCLUSION: In our injection model, using a double port injector created significantly less endothelial cell damage than with the single port pipette. Clinically, this device yielded early endothelial cell loss comparable to that of the series performed by experienced DMEK surgeons.

IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

Although many cancers initially respond to cisplatin (CDDP)-based chemotherapy, resistance frequently develops. Insulin-like growth factor-binding protein-3 (IGFBP-3) silencing by promoter methylation is involved in the CDDP-acquired resistance process in non-small cell lung cancer (NSCLC) patients. Our purpose is to design a translational-based profile to predict resistance in NSCLC by studying the role of IGFBP-3 in the phosphatidyl inositol 3-kinase (PI3K) signaling pathway. We have first examined the relationship between IGFBP-3 expression regulated by promoter methylation and activation of the epidermal growth factor receptor (EGFR), insulin-like growth factor-I receptor (IGFIR) and PI3K/AKT pathways in 10 human cancer cell lines and 25 NSCLC patients with known IGFBP-3 methylation status and response to CDDP. Then, to provide a helpful tool that enables clinicians to identify patients with a potential response to CDDP, we have calculated the association between our diagnostic test and the true outcome of analyzed samples in terms of cisplatin IC50; the inhibitory concentration that kills 50% of the cell population. Our results suggest that loss of IGFBP-3 expression by promoter methylation in tumor cells treated with CDDP may activate the PI3K/AKT pathway through the specific derepression of IGFIR signaling, inducing resistance to CDDP. This study also provides a predictive test for clinical practice with an accuracy and precision of 0.84 and 0.9, respectively, (P=0.0062). We present a biomarker test that could provide clinicians with a robust tool with which to decide on the use of CDDP, improving patient clinical outcomes.

Immunosuppressive properties of mesenchymal stem cells: advances and applications.

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-ß, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.

The interstitial lymphatic peritoneal mesothelium axis in portal hypertensive ascites: when in danger, go back to the sea.

Portal hypertension induces a splanchnic and systemic low-grade inflammatory response that could induce the expression of three phenotypes, named ischemia-reperfusion, leukocytic, and angiogenic phenotypes.During the splanchnic expression of these phenotypes, interstitial edema, increased lymph flow, and lymphangiogenesis are produced in the gastrointestinal tract. Associated liver disease increases intestinal bacterial translocation, splanchnic lymph flow, and induces ascites and hepatorenal syndrome. Extrahepatic cholestasis in the rat allows to study the worsening of the portal hypertensive syndrome when associated with chronic liver disease. The splanchnic interstitium, the mesenteric lymphatics, and the peritoneal mesothelium seem to create an inflammatory pathway that could have a key pathophysiological relevance in the production of the portal hypertension syndrome complications. The hypothetical comparison between the ascitic and the amniotic fluids allows for translational investigation. From a phylogenetic point of view, the ancestral mechanisms for amniotic fluid production were essential for animal survival out of the aquatic environment. However, their hypothetical appearance in the cirrhotic patient is considered pathological since ultimately they lead to ascites development. But, the adult human being would take advantage of the potential beneficial effects of this "amniotic-like fluid" to manage the interstitial fluids without adverse effects when chronic liver disease aggravates.

Reacciones adversas psiquiátricas asociadas a nuevos macrólidos. A propósito de tres casos / Psychiatric adverse reactions associated to new macrolides. Three case reports

Los macrólidos representan el 10-15% del mercado mundial de antibióticos orales. Son uno de los grupos de antibióticos más seguros; las reacciones adversas graves son muy raras. Pueden producir reacciones gastrointestinales, hepatotoxicidad y ototoxicidad. Además, las reacciones psiquiátricas se encuentran entre los efectos adversos esporádicamente asociados con su administración, aunque su mecanismo no está bien establecido. Casos descritos en la Food and Drug Administration (FDA) de EE.UU. muestran que la claritromicina y el ciprofloxacino son los antibióticos más frecuentemente asociados con el desarrollo de manía. Este término ha sido denominado antibiomanía. Presentamos tres casos clínicos vistos en una consulta de Atención Primaria en el período de un año con cuadros similares de hiperactividad y agresividad, coincidiendo con la administración de antibióticos de la familia de los macrólidos (AU)

Macrolides represent the 10-15% of the world-wide market of oral antibiotics. They are one of the safer groups of antibiotics, being the severe adverse reactions very rare. They can produce gastrointestinal reactions, hepatotoxicity and ototoxicity. The psychiatric reactions are found sporadically among the adverse effects. Cases reported to the FDA showed that clarithromycin and ciprofloxacin are the most frequent antibiotics associated with the development of mania. The syndrome has been termed antibiomania. We present three clinical cases seen in a Primary Care office in the last year with similar pictures of hyperactivity and aggressiveness coinciding with the administration of antibiotics of the family of the macrolides (AU)

A5 region modulation of the cardiorespiratory responses evoked from parabrachial cell bodies in the anaesthetised rat.

We have examined the importance of the A5 region modulating cardiorespiratory responses evoked from the parabrachial complex (PB) in spontaneously breathing rats. Cardiorespiratory changes were analyzed in response to electrical stimulation and glutamate microinjections into the PB (10-20 nl, 1-2 nmol) before and after ipsilateral microinjection of muscimol (50 nl, 0.25 nmol) or lidocaine (50 nl, 0.5 nmol) within the A5 region. Stimulation of medial parabrachial and Kölliker-Fuse nuclei (mPB-KF) evoked a decrease in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). After muscimol or lidocaine microinjections within the A5 region, the pressor and heart rate responses to mPB-KF stimulation were reduced (P<0.05, both cases). Muscimol within the A5 region altered the respiratory response to glutamate stimulation of mPB-KF, evoking an increase in respiratory rate (P<0.05). Lidocaine abolished the respiratory response to mPB-KF stimulation. Stimulation of the lateral parabrachial nuclei (lPB) caused an increase in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). Muscimol or lidocaine microinjections within A5 region decreased heart rate (P<0.05) and pressor responses (P<0.05) evoked from lPB. The increase of respiratory rate persisted unchanged. To confirm functional interactions between A5 and PB, extracellular recordings of putative A5 neurones were obtained during PB stimulation. Eighty-three A5 cells were recorded, 35 were activated from the mPB-KF (42%). The results indicate that neurones of the A5 region participate in the cardiorespiratory response evoked from the different regions of the PB complex. The possible mechanisms involved in these interactions are discussed.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Germ cell biology--from generation to generation.

Germ cells hold a unique place in the life cycle of animal species in that they are the cells that will carry the genome on to the next generation. In order to do this they must retain their DNA in a state in which it can be used to recapitulate embryonic development. In the normal life cycle, the germ cells are the only cells that retain this ability to recapitulate development, referred to as developmental totipotency. The molecular mechanisms regulating developmental potency are poorly understood. Recently its has been shown that germ cells can be turned into pluripotent stem cells when cultured in specific polypeptide growth factors that affect their survival and proliferation. The ability to manipulate developmental potency in germ cells with growth factors allows the underlying mechanisms to be dissected. Germ cells are also the only cells that undergo the unique reductive division of meiosis. This too is essential for the ability of germ cells to form the gametes that will carry the genome into the next generation. Arguably meiosis is the most important division in the life of a nascent organism. Defects in meiosis can result in embryonic or fetal loss or, if the animal survives, in the birth of an individual with chromosomal abnormalities. Recent advances in our understanding of meiosis have come from knockout mice and studies on genes identified through studies of human infertility. This review will focus on these two key aspects of germ cell biology, developmental potency and meiosis.

Epididymal growth and differentiation are altered in human cryptorchidism.

Despite the knowledge and histological classification of testicular lesions, epididymal lesions associated with cryptorchidism are not well defined and only macroscopic alterations have been reported. We have evaluated the alterations in the growth of both the epithelium and muscular wall of efferent ducts and epididymis in human patients with cryptorchidism from infancy to adulthood. In addition, by cytokeratin immunostaining we have also evaluated the stage of differentiation of each segment along the human postnatal life in these patients. A decrease is shown in the size of efferent and epididymal ducts in cryptorchid children compared with normal, age-matched controls. The height of the epithelium, muscular wall, and lumen of the cryptorchid epididymis were reduced at every age studied. This decrease in all regions was seen even in the testicular quiescent period (1 to 4 years of age). In addition, the cryptorchid epididymis grows more slowly during the transition to the pubertal period. The smaller size of the cryptorchid epididymis in pubertal and adult men compared with that of normal men is due primarily to underdevelopment of the muscular wall and a reduction in epithelial height. The pattern of growth of cryptorchid efferent ducts and ductus epididymides parallels that in normal men, except that development of the lumen and muscular layer in the cauda epididymis region are delayed. Epithelial differentiation, monitored by cytokeratin expression, is minimal in efferent ducts and throughout the epididymis of the cryptorchid male, and this is already seen in children. In conclusion, our immunohistochemical and morphometric results show a reduced development of the human cryptorchid epididymis that is already evident in childhood. They indicate that cryptorchidism is a primary congenital illness of the testis and spermatic ducts, with evident lesions from the first years of life, and suggest that surgical descent would probably not be able to completely reverse these alterations.

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.

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.

Immunoexpression of tumour necrosis factor-alpha and its receptors 1 and 2 correlates with proliferation/apoptosis equilibrium in normal, hyperplasic and carcinomatous human prostate.

Immunohistochemical and semiquantitative study of TNF-alpha, its receptors types 1 (TNFR1) and 2 (TNFR2), cell proliferation (Ki-67 nuclear antigen), and apoptosis (Tunel method) was carried out in human prostates, in normal healthy conditions, as well as in benign prostatic hyperplasia (BPH) and prostatic carcinoma (PC). Cell proliferation was higher in BPH than in normal prostates, and even higher in PC, mainly in neoformations showing a microglandular pattern. The apoptotic index was similar in BPH and normal prostates, and increased significantly in PC with independence of the pattern. In BPH, immunoreaction to TNF-alpha decreased as compared with that of normal prostates, while immunoreactions to both TNF-alpha receptors increased. This suggests a feedback downregulation of the factor, and that the low TNF-alpha activity in BPH are compensated by the increased amount of receptors. In PC, immunoreaction to TNF-alpha and its two receptors increased markedly, suggesting that the TNF-induced effects are also increased. Contrarily to cell proliferation immunoexpression, PC reaction to TNFR2 was stronger in the papillar pattern than in the micrograndular pattern, and this suggests an inverse correlation between TNFR2 expression and cell proliferation.

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.

Ultrastructure and lectin cytochemistry of the cloacal ventral glands in the male newt Triturus marmoratus marmoratus.

Ventral glands are found in the cloacal walls of male urodele amphibians except for sirenids. These glands are mucous, and secrete substances that will form part of the spermatophore used in transfer of sperm during fertilization. Ventral glands are formed by secretory and ductal portions; both possess epithelial and myoepithelial cells with different characteristics. Urodeles have cyclic reproduction, and cloacal ventral glands show seasonal differences with electron microscopy. The glycoproteins secreted by these glands have been studied by means of lectin histochemistry. The labeling was detected mainly in the nuclei, rough endoplasmic reticulum, Golgi complex, and cytosol. Secretory granules in these glands are composed by mucous glycoproteins that bind PNA lectin (which binds galactose) and SBA and HPA lectins (N-acetylgalactosamine), UEA-I (fucose), and LcA (glucose and/or mannose). These findings suggest that the mucins secreted by ventral glands contain both N- and O-linked oligosaccharides. Ventral glands secrete higher quantity and more diverse mucous substances in the reproductive period, as confirmed by lectin-histochemical reactions. Based on these results, the major similarity between ventral cloacal glands and accessory mammalian glands, can be established with bulbourethral glands.

Oncostatin M in the normal human testis and several testicular disorders.

The immunohistochemical reaction to oncostatin M (OSM) was studied in normal human testes at different ages (fetuses, newborns, children, pubertal boys, adults, and elderly men), as well as in several testicular disorders including carcinoma-in-situ cells (CIS), germ cell tumors, benign functioning Leydig cell tumor, androgen insensitivity syndrome, Klinefelter's syndrome, and cryptorchidism. Positive OSM immunostained Sertoli cells were only observed in fetuses. In normal testes, intense OSM immunoreaction was found in the Leydig cells of fetuses, newborns, and adults. Leydig cell immunoreaction was weak in elderly men and absent in children and pubertal boys. In some testicular disorders (Leydig cell tumor, cryptorchidism, and CIS), Leydig cell immunoreaction was as intense as in normal adult testes. This immunoreaction was heterogeneous in androgen insensitivity syndrome and was absent in Klinefelter's syndrome and intratubular seminoma. No recognizable Leydig cells were observed in the other testicular tumors. The findings of our study suggest that, in humans, the down-regulation of OSM immunoexpression in Sertoli cells occurs early, and that OSM immunoreaction in the Leydig cells is associated with functionally active and differentiated Leydig cells.