Increased aquaporin 1 expression in the tunica albuginea of Peyronie's disease patients: an in vivo pilot study.

Peyronie's disease (PD) is a localized disorder of the connective tissue of the tunica albuginea (TA) whose etiology has not been elucidated. Although several studies have implicated genetic susceptibility and/or mechanical trauma as triggering events for PD, the underlying molecular mechanisms remain largely unknown. Aquaporin 1 (AQP1) is a water channel protein potentially implicated in connective tissue resistance to mechanical stress, acting primarily by increasing tension within the collagen network. Although it represents a potentially attractive molecular target in PD, to date no studies had ever addressed whether AQP1 is detectable and/or differentially expressed in the TA of these patients. Herein the present study, through immunohistochemical and biochemical approaches, we were able to detect AQP1 expression in the TA of control and PD affected patients. We demonstrated that AQP1-like immunoreactivity and expression are significantly increased in plaques of PD patients Vs controls, implying that AQP1 overexpression might be the consequence of a localized maladaptive response of the connective tissue to repeated mechanical trauma. In summary, these data support the idea that AQP1 might represent a potentially useful biomarker of mechanical injury in the TA and a promising target for the treatment of PD.

Genetic blockade of the dopamine D3 receptor enhances hippocampal expression of PACAP and receptors and alters their cortical distribution.

Dopamine D3 receptors (D3Rs) are implicated in several aspects of cognition, but their role in aversive conditioning has only been marginally uncovered. Investigations have reported that blockade of D3Rs enhances the acquisition of fear memories, a phenomenon tightly linked to the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP). However, the impact of D3R ablation on the PACAPergic system in regions critical for the formation of new memories remains unexplored. To address this issue, levels of PACAP and its receptors were compared in the hippocampus and cerebral cortex (CX) of mice devoid of functional D3Rs (D3R(-/-)) and wild-types (WTs) using a series of comparative immunohistochemical and biochemical analyses. Morphometric and stereological data revealed increased hippocampal area and volume in D3R(-/-) mice, and augmented neuronal density in CA1 and CA2/3 subfields. PACAP levels were increased in the hippocampus of D3R(-/-) mice. Expression of PACAP receptors was also heightened in mutant mice. In the CX, PACAP immunoreactivity (IR), was restricted to cortical layer V in WTs, but was distributed throughout layers IV-VI in D3R(-/-) mice, along with increased mRNAs, protein concentration and staining scores. Consistently, PAC1, VPAC1 and VPAC2 IRs were variably redistributed in CX, with a general upregulation in cortical layers II-IV in knockout animals. Our interpretation of these findings is that disturbed dopamine neurotransmission due to genetic D3R blockade may enhance the PACAP/PAC1-VPAC axis, a key endogenous system for the processing of fear memories. This could explain, at least in part, the facilitated acquisition and consolidation of aversive memories in D3R(-/-) mice.

In vitro antiproliferative effect of trastuzumab (Herceptin(®)) combined with cetuximab (Erbitux(®)) in a model of human non-small cell lung cancer expressing EGFR and HER2.

Lung cancer is the leading cause of cancer death. For this reason, new therapies are needed for the treatment of this devastating disease. In this study, we investigated the effects of combining cetuximab and the trastuzumab on the growth of a model of human non-small cell lung carcinoma cell line (A549). The results were compared with those obtained from a human lung squamous carcinoma cell line (NCI-H226). Both cell lines were treated with cetuximab and trastuzumab, alone or in combination, at various concentrations, for 24, 48 and 72 h. Cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. EGFR and HER-2 mRNA expression was detected by reverse transcription polymerase chain reaction, and the gene amplification status of receptors was evaluated by fluorescence in situ hybridisation. The colorimetric proliferation assay showed that trastuzumab combined with cetuximab significantly inhibited A549 cells at a dose of 40 µg/ml after 72 h of treatment (p < 0.05), while no time-dose dependent inhibition was observed in NCI-H226 cells. The combined treatment influenced both levels of EGFR and HER-2 mRNA in A549 cells and only EGFR mRNA levels in NCI-H226 cells. Fluorescence in situ hybridisation showed that both cell lines were aneuploid for the two genes with equally increased EGFR and CEN7 signals, as well as HER-2 and CEN17 signals, indicating a condition of polysomy without amplification. The preliminary results of this study encourage further investigations to elucidate the downstream events involved and to understand how these mechanisms influence non-small cell lung cancers growth.

Hypothesis on etiopathogenesis, congenital or acquired, of an imperforate distal ureter: a case report.

INTRODUCTION: Ureteral atresia is a rare disease usually associated with a non-functioning kidney. Its association with other urinary anomalies is rare. CASE PRESENTATION: In this study we discuss the possibility of congenital or acquired etiology of a right imperforate distal ureter. Here we report the case of 11-month-old white boy with a right ureteropelvic junction obstruction. He underwent a right pyeloplasty when he was 11-months old, and 3 weeks after surgery a cystoscopy was performed. Two months after the first operation, he underwent a right ureteral meatoplasty and a new pyeloplasty. CONCLUSIONS: To the best of our knowledge, few cases of imperforate distal ureter have been described in the literature. The suspicion of a non-patent terminal ureter, occurring during upper urinary tract surgery, must be intraoperatively clarified to preserve the renal function and to avoid more complex surgical approaches.

In vitro combined treatment with cetuximab and trastuzumab inhibits growth of colon cancer cells.

OBJECTIVES: Overexpression or constitutive activation of epidermal growth factor receptors (EGFR) is involved in growth of human cancers. We investigated effects of EGFR and HER-2 blockade in colon cancer cell lines using cetuximab and trastuzumab, with the aim of developing novel approaches to cancer therapy. MATERIALS AND METHODS: We studied effects of treatment on cell growth, cell cycle distribution, induction of apoptosis, changes in EGFR and HER-2 mRNA-protein expression and EGFR and HER-2 gene copy number in Caco-2, HT-29 and HCT-116 cells. RESULTS: Treatment of cells resulted in no effect in one of the three cell lines and in inhibition of cell proliferation in a time- and dose-dependent manner in the other two, with modulation of EGFR and HER-2 mRNA and protein levels. Differences in sensitivity to cetuximab and trastuzumab were observed. Treatment induced specific changes in cell cycle distribution in both cell lines affected, while apoptosis was not increased. Fluorescence in situ hybridization analysis revealed abnormal copy number of two genes resulting from aneuploidy; this was not responsible for different sensitivity to combination between the two cell lines. CONCLUSIONS: Targeting EGFR and HER-2 simultaneously could have useful applications in colorectal cancer treatment. To improve pharmacological efficacy of cetuximab and trastuzumab combination, molecular mechanisms involved in their activity need to be elucidated.

Effects of hypothyroidism and endocrine disruptor-dependent non-thyroidal illness syndrome on the GnRH-gonadotroph axis of the adult male rat.

Effects of primary hypothyroidism (HYPO) on the male gonadal axis are controversial, with only scanty data on the gonadotroph cell response and no information on GnRH tuberoinfundibular neurons, even in animal models. HYPO has been reported to variably induce hypogonadotropic hypogonadism, a hypergonadotropic state, or to have no effects on basal levels of pituitary gonadotropins, both in adult male rats and humans. Similarly, the exogenous administration of GnRH to HYPO rats and humans may increase or decrease gonadotropin secretion. Since inhibitory effects of HYPO on the GnRH-gonadotropin axis are reversed by replacement with L-T4, it has been suggested that thyroid hormone (TH) may regulate tuberoinfundibular GnRH and pituitary gonadotropin biosynthesis and/or secretion. To shed light on this hypothesis, we conducted immunocytochemical studies on the distribution and immunostaining characteristics of hypophysiotropic GnRH neurons, LH, PRL and vasoactive intestinal polypeptide (VIP) immunoreactive (IR) cells in the pituitary of adult, male rats. We show that HYPO reduces IR-GnRH in a restricted population of tuberoinfundibular perikarya and their proximal axons compared to euthyroid controls, but increases IR-VIP both in pituitary cells in direct association with LH-gonadotrophs and within IR-LH cells, itself. We propose that VIP may serve as a juxtacrine/paracrine/autocrine regulator of LH secretion and that, when GnRH biosynthesis is reduced by HYPO, gonadotropin secretion may be rescued by local activating effects of VIP. Polychlorinated biphenyls (PCB), industry toxicants found in food and water, also have inhibitory effects on the gonadal axis, decreasing fertility and suppressing basal and GnRHinduced LH release in male rats. Since PCB may also exert endocrine disruptor-dependent (EDD) effects on the thyroid axis producing a non-thyroidal illness syndrome (NTIS) (coined EDD-NTIS), we developed a rat model of EDD-NTIS to determine whether central hypothyroidism may contribute to the pathophysiology of PCB-induced hypogonadism. On the basis of preliminary animal data, we speculate that one of the mechanisms for Partial Androgen Deficiency of the Aging Male may involve central hypothyroidism and EDD-NTIS, resulting in inhibition of the GnRH-gonadotroph axis.

Molecular mechanisms for pituitary thyrotroph cell growth.

Neuroendocrine, endocrine and autocrine/paracrine signals contribute to the regulation of basal thyrotroph growth. Thyrotropin-releasing hormone (TRH), somatostatin, thyroid hormone (TH), estrogens (Es) and epidermal growth factor, all may play a role both in normal and tumoral thyrotroph proliferation, acting via either plasma membrane receptors and non-genomic steps or nuclear receptors and gene transcription. Signaling features common to all these ligands are involvement of G protein-coupled receptors, mitogen-activated protein kinase cascade and nuclear polyphosphoinositide cycle. In addition, each growth information, independently from the eliciting factor, may be routed intracellularly following a branched pathway, that often links different transduction systems at common check-points, as the Shc-Grb2-SOS complex. Finally, some ligands (e.g. TRH, TH, Es) may display opposite effects on thyrotroph growth, depending on environmental conditions and state of cell differentiation. These ambiguities of response can be interpreted using a "fuzzy" logic-based model of intracellular signaling. Accordingly, check-points common to different transduction cascades may be envisaged as targets for antitumoral therapy selective to the neoplastic thyrotroph cell.

Correlations between protein kinase C zeta signaling and morphological modifications during rat heart development and aging.

From birth to aging the heart undergoes functional changes reflecting biochemical and ultrastructural modifications which imply apoptosis. This is a physiological process resulting from genetic programs closely associated with development and aging. During development apoptosis eliminates redundant cells leading to heart remodeling, while during aging it eliminates damaged or exhausted cells. In the present paper we analyze some molecular mechanisms involved with heart morphological modifications, especially in the neonatal heart which displays different features in the subendocardial and myocardial area. The high number of subendocardial apoptotic cells and the inverted ratio of Bcl-2/Bax molecule expression in the two heart compartments led us to hypothesize a different metabolism in the myocardium as compared with subendocardium. Moreover, we propose that PKC zeta may mediate this different response by activating Nf-kB pathway and by maintaining the balance between hypertrophic growth and apoptosis involved with remodeling of neonatal heart. Further, we underline that in the aged heart, where this pathway is not activated, such balance is not maintained.

Phospholipase C delta2 expression characterizes the neoplastic transformation of the human gastric mucosa.

The expression, cellular distribution, and activity of PIP(2)-specific phospholipase C (PLC) in healthy human gastric-mucosa cells have been recently studied in our laboratories and a direct evidence for an almost exclusive expression of PLC beta isoforms, with the exception of PLC beta4, has been provided. These results addressed our attention to possible modification of PLC expression and activity during neoplastic transformation of the human gastric mucosa. In the present article we present results indicating that PLC delta2 is markedly expressed in type II intestinal metaplasia and in the adenocarcinoma whereas traces of other PLC isoforms were sometime detected. Interestingly, we found that type I intestinal metaplasia was in the majority of the cases PLC delta2-negative, but when expressed, this type of metaplasia generally considered as benignant, always evolved toward neoplastic transformation. These results therefore readdress the question of surveillance of the patients with type I intestinal metaplasia and suggest that PLC delta2 expression might be a possible marker of gastric malignant transformation.

Involvement of the pathway phosphatidylinositol-3-kinase/AKT-1 in the establishment of the survival response to ionizing radiation.

Ionizing radiation is one of the agents inducing activation of DNA repair, cell cycle arrest, apoptosis and cell death. Here we report evidence for an enhanced activity of DNA polymerase beta, one of the repair enzymes, concomitant to the activation of the pathway phosphatidylinositol-3-kinase/AKT-1 (PI-3-kinase/AKT-1), which delivers a survival signal in Friend erythroleukemia cells exposed to 15 Gy. Significantly, the preincubation of the cellls with PI-3-kinase inhibitors wortmannin and LY 294002, disactivating this pathway, sensitizes the cells to ionizing radiation by further reducing the rate of proliferation without substantial variations of the number of dead cells. Thus, we suggest a role for these enzymes in maintaining survival programs upon exposure to ionizing radiation and in giving to these cells a chance to recover from this stress.

A role for nuclear phospholipase Cbeta 1 in cell cycle control.

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4.

[Male breast cancer. Histochemical and flow cytometry analysis of 6 cases]. / Carcinoma mammario maschile. Analisi immunoistochimica e citofluorimetrica di 6 casi.

The authors studied immunohistochemical and flow cytometric parameters of 6 male breast carcinomas. All patients were ER positive and frequency of aneuplody was 66%. Diploid tumors showed low proliferative activity and S-phase fraction < 3%.

Intermediate filaments of human Sertoli cells in germinal alterations.

Immunohistochemical techniques were used to examine testis biopsy specimens in subjects of different ages, in whom histological examination of the seminiferous tubules revealed a reduction in number, immaturity or absence of germinal elements. Our aim was to detect changes in the expression of the vimentin and cytokeratin (8-18-19) intermediate filaments in Sertoli cells. The use of anti-vimentin antibodies demonstrated intense Sertoli cell positivity in all the cases studied, confirming that germinal alterations do not interfere with the expression of these filaments. The Sertoli cells of the subjects affected by pathological conditions also reacted positively to anti-cytokeratin antibodies. This finding was constant in all the testes, showing evident signs of germinal immaturity. In contrast, anti-cytokeratin positivity was not observed in the control specimens after the pre-pubertal stage. Our findings show that persisting co-expression of vimentin and cytokeratin filaments in the Sertoli cells of productive subjects may be a marker of germinal cell degeneration.

Health care delivery systems: effects on surgical education in Italy.

Since 1989 a national health system (NHS) has been in effect in Italy based on the constitutional principle that health care is a right of all citizens. Until 1992 the NHS had a secondary role in medical education, as the 33 medical schools for postgraduate training are under the control of the Ministry of University. The European Community legislation allowing free movement of M.D.s and specialists between member countries has resulted in standardization of teaching programs and formative curricula in the European Community. Therefore beginning in 1992, every 3 years the Ministry of Health and University establishes the number of specialists that can meet the needs of the NHS and allocates funds to each school for the salary obligations. The actual number of paid residents for surgical specialties is 941 per year (192 for general surgery). Until recently surgical training in Italy was mainly theoretic, as no legislation guaranteed that physicians in training would perform surgery. New legislation, increased exchanges with European hospitals, and improved and loyal cooperation between universities and the NHS will certainly improve the situation in coming years.

[Morphologic and endocrine changes in the male reproductive system after porta-caval anastomosis in rats. Experimental research]. / Alterazioni morfologiche ed endocrine dell'apparato riproduttivo maschile dopo anastomosi porta-cava nel ratto. Ricerche sperimentali.

Quantitative alterations of the sexual hormones are present in cirrhotic patients whose testicular volumes are decreased with tubular atrophy in more than 50% of cases. The authors performed an experimental study utilizing end-to-side portacaval anastomosis in the rat in order to evaluate the consequences of the complete interruption of the portal blood to the liver in the male genital system, i.e. testicular alterations, due to the missing hepatic inactivation. Forty male Sprague-Dawley rats were used for this study. Twenty rats were subjected to end-to-side porta-caval anastomosis according to Lee, 10 rats underwent sham-operation while the remaining 10 rats were utilized as negative controls. The rats were weighed and necropsied three and six months after surgery and the liver and genital organs were weighed. In rats subjected to porta-caval anastomosis loss of weight was shown, about 20 g three months after surgery and 30 g six months after surgery, while other rats (sham-operated and negative controls) showed an increase of its weight, about 60 g after three months and 80 g after six months. In rats subjected to porta-caval anastomosis the liver was hypotrophic and its weight was decreased in comparison with the control group. In the same rats testicular volumes were decreased and hypotrophic in comparison with the control groups with average length 0.5 to 1 cm, while control groups showed values of 1.7 to 2.5 cm. Testosterone levels were 0.50 ng/ml in rats subjected to porta-caval anastomosis while in control groups the levels were 2.20 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological aspects of the epithelium of the trigone of the human urinary bladder: immunohistochemical findings and histogenetic features.

An immunohistochemical method was used to study the structural characteristics of the epithelium of the bladder trigone in 20 premenopausal women. Fragments of mucosa were obtained by cystoscopy from the trigones of the 20 subjects. Monoclonal antibodies (34 beta E12 and 34 beta B4) were used to demonstrate the presence of stratified squamous epithelium-specific cytokeratins in these samples. Based on our results, we believe that the frequent finding of stratified squamous epithelium in the trigone of the female bladder is a consequence of the stimulation of estrogen, which is believed to act on the trigone, but not on the rest of the bladder. This may be due to the complex histogenesis of the bladder. Embryologically, the trigone derives from the mesoderm, while the rest of the bladder derives from the endoderm.

[Morpho-functional considerations on the annulate lamellae of the human oocyte]. / Considerazioni morfo-funzionali sulle lamelle anulate dell'ovocita umano.

Ovarian biopsy specimens from four girls in complete remission state of acute lymphoblastic leukemia, previously treated with antiblastic chemotherapy for about three years, were examined by means of electron microscopy. The normal morphology of residual follicles and, in particular, the observation that annulate lamellae were always present in oocytes of primordial and primary follicles, gave the chance to consider a possible functional role and to suggest a prognosis for future fertility. The similarities in structure to the nuclear envelope and the relation between annulate lamellae and other cellular organelles (especially endoplasmic reticulum and ribosomes) suggest that lamellae may be involved in the release, assembly or activation of stored development information. The annulate lamellae could prove to be an important organelle that participates in the regulation of gene expression. Therefore, the presence of annulate lamellae in oocytes of primordial and primary follicles may present the possibility of a normal development for these cells in which long-lived gene products are synthesized and transported to the cytoplasm for storage and use later in development. The above hypothesis, while leads to consider these patients at risk for low fertility and early menopause, does not consider them as definitely infertile.

[Intramural ganglia of the human gallbladder: morphological and functional observations]. / Gangli intramurali della colecisti umana: rilievi morfologici e funzionali.

The purpose of this study was the reinvestigation of the intrinsic innervation of human gall bladder with an immunohistochemical technique named peroxidase anti-peroxidase (PAP). The antigen demonstrated was the S100 protein normally present in the surface of glial cells, Schwann cells and satellite cells in ganglia. The tissues used were taken from 20 human gall bladders, fixed after surgery. This technique is not specific to demonstrate adrenergic or cholinergic innervation but it reveals just myelinated fibers. The current study was undertaken in order to study the organization and the function of plexus of nerves and ganglia present in the wall of the gall bladder. The neck of the gall bladder was the region in which the higher number of nerve cells and nervous fibers was present. The technique used has demonstrated ganglionated plexus and nerves in submucosal layer, fibromuscular and adventitial layer according to the enteric nervous system. All ganglia are postganglionic stations related with preganglionic cholinergic fibers. These results confirm that the intramural ganglia of the gall bladder are analogous to those of the enteric nervous system according to their common origin.

[Probable correlations ultrastructural anomalies of the central nervous system and hyperammonemia following portacaval anastomosis in rats]. / Corrélations probables entre les anomalies ultrastructurelles du système nerveux central et l'hyperammoniémie après anastomose porto-cave chez le rat.

The correlation between hyperammonemia, during porto-systemic encephalopathy, and brain's lesions in patients died for porto-systemic encephalopathy is not demonstrated. The aim of this study has been to try a demonstration. A histological study of the brain, cerebellum, brainstem, and spinal cord was made in 60 rats: in 40 rats 1, 3 and 6 months after portocaval shunt, and in 20 rats sham operated. The brain, cerebellum, brainsteam and spinal cord have been fixed with paraformaldehyde (4%) and then sectioned for optical and electronic microscopic study. Ammonemia was measured regularly in the 40 rats with portocaval shunt, all the rats have been hyperammonemia since 10 weeks, after this period in 4 rats ammonemia was normal. In 20 rats sham-operated ammonemia was always normal. One month after surgery electronic microscopy revealed changes in the astrocytes characterized by nuclear swelling and lobulation. Three months after surgery this lesion was increased. After six months most lesions were noted in the hyperammoniemic rats. No similar lesions were observed in control rats. These results suggest that hyperammonemia is responsible of nuclear changes in the astrocytes of the patients died from hepatic encephalopathy.