Differential expression of novel biomarkers (TLR-2, TLR-4, COX-2, and Nrf-2) of inflammation and oxidative stress in semen of leukocytospermia patients.

Chronic genitourinary inflammation results in Leukocytospermia (LCS), an elevated number of white blood cells (WBCs) in semen, which, in association with oxidative stress, may suppress sperm function, and manifest as male factor infertility. The current clinical diagnosis of LCS employs manual enumeration of WBCs and requires complex staining and laboratory skills or measurement of inflammatory cytokines and chemokines levels. Many patients with idiopathic infertility are asymptomatic. In search of better inflammatory markers for LCS, we evaluated expression of toll-like receptors 2 and 4 (TLR-2/4), cyclooxygenase-2 (COX-2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) in semen samples of age-matched infertile patients with and without LCS. We employed the usage of specific Western blot evaluation, cytokine array; immunofluorescence microscopy (IFM) followed by computer-based analysis, and other molecular approaches. As compared with non-LCS patients (n = 38), semen samples from LCS patients (n = 47) displayed significantly lower total sperm count (p < 0.01), motility (p < 0.0001), normal head count (p < 0.0001), and a significantly higher white blood cell count (p < 0.0001). Differential cytokine profiling of seminal plasma by antibody array revealed up-regulation of several pro-inflammatory chemokines in LCS samples. Western blot analysis of LCS seminal plasma (n = 15) also showed a significant increase in expression of TLR-2 (p < 0.001) and 4 (p < 0.01), COX-2 (p < 0.001), and Nrf-2 (p < 0.001) as compared with semen samples from non-LCS patients (n = 15). Computer-based objective IFM analysis of spermatozoa from LCS patients showed increased expression of TLR-4 (p < 0.001), Cox-2 (p < 0.01), and (Nrf-2) (p < 0.01). Significant differences in the subcellular localization of these proteins were evident in the sperm head and tail segments of LCS samples. Altogether, these observations suggest that TLR-2/4, COX-2, and Nrf-2 can serve as novel biomarkers of inflammation and oxidative stress. Therefore, developing a rapid assay for these biomarkers may facilitate early diagnosis and management of LCS especially in idiopathic and asymptomatic male infertility patients.

Adipose tissue-derived stem cell therapy for prevention and treatment of erectile dysfunction in a rat model of Peyronie's disease.

Peyronie's disease (PD) is a localized connective tissue disorder that involves the tunica albuginea (TA) of the penis. While surgical correction remains the gold standard, the search for an effective and less invasive therapy continues. The objective of this study was to evaluate the effects of intratunical injection of adipose tissue-derived stem cells (ADSCs) for the prevention and treatment of erectile dysfunction in a rat model of PD. Twenty-four male Sprague-Dawley rats (300-350 g) were randomly divided into four groups: sham, PD, PD + ADSC (prevention) and PD + ADSC (treatment). All rats underwent penile injections into the TA with 50 µL vehicle (sham) or 0.5 µg transforming growth factor (TGF)-ß1 (remaining groups). The ADSC groups received intratunical injections with 0.5 million rat-labelled ADSCs on day 0 (prevention) or day 30 (treatment). Forty-five days following TGF-ß1 injection, rats underwent cavernous nerve stimulation (CNS) with total intracavernous-to-mean arterial pressure ratio (ICP/MAP) and total ICP recorded to measure response to therapy. Tissues were evaluated histologically and for mRNA expression of tissue inhibitors of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs) and zymographic activity of MMPs. Statistical analysis was performed by analysis of variance followed by the Tukey test for post hoc comparisons. In both prevention and treatment groups, intratunical injection of ADSCs resulted in significantly higher ICP/MAP and total ICP in response to CNS compared with the PD group. Local injection of ADSCs prevented and/or reduced Peyronie's-like changes by decreasing the expression of TIMPs, and stimulating expression and activity of MMPs. This study documents the preventive and therapeutic benefits of ADSC on penile fibrosis and erectile function in an animal model of PD.

Novel role for the transient receptor potential channel TRPM2 in prostate cancer cell proliferation.

We have identified a novel function for a member of the transient receptor potential (TRP) protein super-family, TRPM2, in prostate cancer cell proliferation. TRPM2 encodes a non-selective cation-permeable ion channel. We found that selectively knocking down TRPM2 with the small interfering RNA technique inhibited the growth of prostate cancer cells but not of non-cancerous cells. The subcellular localization of this protein is also remarkably different between cancerous and non-cancerous cells. In BPH-1 (benign), TRPM2 protein is homogenously located near the plasma membrane and in the cytoplasm, whereas in the cancerous cells (PC-3 and DU-145), a significant amount of the TRPM2 protein is located in the nuclei in a clustered pattern. Furthermore, we have found that TRPM2 inhibited nuclear ADP-ribosylation in prostate cancer cells. However, TRPM2 knockdown-induced inhibition of proliferation is independent of the activity of poly(ADP-ribose) polymerases. We conclude that TRPM2 is essential for prostate cancer cell proliferation and may be a potential target for the selective treatment of prostate cancer.

Gene transfer of prepro-calcitonin gene-related peptide restores erectile function in the aged rat.

Erectile dysfunction in the aging male is caused, in part, by inadequate relaxation of the corpora cavernosal smooth musculature. Calcitonin gene-related peptide (CGRP), a peptide neurotrasmitter localized in the corpora cavernosa, is down-regulated in the aging rat penis. We examined the hypothesis that this reduction in CGRP may contribute to decreased cavernosal smooth muscle relaxation. Therefore, we sought to determine whether adenoviral-mediated gene transfer of prepro-CGRP (AdRSVCGRP) could enhance erectile responses in aged rats. We found a significant decrease in CGRP concentrations and in cAMP and cGMP levels in aged rat cavernosal tissue compared to younger rats. Aged rats also had significantly lower erectile function as determined by cavernosal nerve stimulation compared to younger rats. Five days after transfection with AdRSVCGRP, these aged rats had an approximately threefold increase in cavernosal CGRP levels compared to animals transfected with adenoviruses encoding nuclear-targeted beta-galactosidase (AdRSV beta gal). The AdRSVCGRP-transfected animals also demonstrated an increase in CGRP mRNA and immunohistochemical localization of CGRP in the smooth muscle of the corpora cavernosa. In addition, cAMP levels in the corpora cavernosa were significantly increased, whereas cGMP levels remained unchanged. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by chemiluminescence and was observed in cavernosal tissue 5 days after transfection with AdRSV beta gal. More importantly, 5 days after administration of AdRSVCGRP, a significant increase was observed in the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of CGRP can physiologically improve erectile function in the aged rat.

Evaluation of nitric oxide synthase and arginase in the induction of a Peyronie's-like condition in the rat.

Peyronie's disease is an idiopathic, localized connective tissue disorder of the penis, involving the tunica albuginea of the corpus cavernosum and adjacent areolar space. Current proposals as to the origin of Peyronie's disease suggest that fibrosis and collagen changes of the tunica are the result of an inflammatory process following vascular trauma. Our laboratory and other investigators have recently proposed an animal model for the study of Peyronie's disease. When transforming growth factor-beta1 (TGF-beta1) was injected into the rat tunica albuginea, tissue fibrosis was observed at 6 weeks. Therefore, our aim was to assess arginase II, endothelial and inducible nitric oxide synthase isoforms, and nitrotyrosine levels--all factors involved in inflammatory reactions--in the cavernosal tissue of saline-injected and TGF-beta1-injected rats after 6 weeks in order to evaluate the roles these enzymes may play in the induction of a Peyronie's-like condition in the rat. To examine the expression of endothelial nitric oxide synthase (eNOS), iNOS, and arginase II protein, and mRNA in the corpus cavernosum, immunoblot analysis, and reverse transcriptase-polymerase chain reaction were performed. We also determined immunohistochemically the expression of nitrotyrosine, a marker of peroxynitrite formation, in the rat penis. After 6 weeks, iNOS protein and gene expression was up-regulated and eNOS protein and gene expression was down-regulated in the corpora cavernosa of the TGF-beta1-injected penises. Furthermore, arginase II protein expression as well as immunohistochemical localization of nitrotyrosine was significantly higher in the TGF-beta1-injected corpora cavernosa. These results suggest that iNOS is the key control element for peroxynitrite formation, arginase II expression, and eNOS down-regulation in the induction of a Peyronie's-like condition in the rat.

In vivo gene expression profile analysis of metallothionein in renal cell carcinoma.

The antiapoptotic and mitogenic responses of metallothionein (MT) have been well documented in vitro. While MT protein overexpression, frequently encountered in a number of human primary tumors, has been shown to be correlated with disease progression, little information is available on the in vivo isoform expression of MT. In this study we have demonstrated the occurrence of MT proteins and further defined their differential expression profile in human primary renal cell carcinoma (RCC). Pooled normal human kidney RNA and paired biopsy specimens (tumor and control) obtained from 11 patients diagnosed with RCC with tumor grade ranging from 1-3 and a pathological staging of T2-T3 (N0M0) were used for the study. Samples were analyzed for the presence of MT protein using immunohistochemical (IHC) analysis and for MT isoform-specific mRNA expression by reverse transcriptase polymerase chain reaction. Metallothionein protein assumed both cytoplasmic and nuclear staining in cancer cells and was detected in eight of 11 samples (72%) with polyclonal antibodies. The immunoreactivity of MT protein, but not its cellular localization, in RCC specimens suggests a relationship between and advanced disease. While alterations in the basal level of expression of MT-1E, MT-1F and MT-1X genes remained unchanged, significant up-regulation of MT-2A and down-regulation of MT-1A and MT-1G transcripts was observed in RCC tissue specimens when compared with controls. Intriguingly, the paired RCC biopsy specimens had lower MT-1H transcripts than pooled normal human controls. We here provide the first report of the differential expression of MT isoforms in human RCC and that this data further support the role of MT-2A in tumorigenesis.

Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat.

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by a variety of nitric oxide synthases (NOS). It has been demonstrated that a decrease in NOS activity, as observed in aging, is associated with a diminished erectile response. The objective of this study was to determine if adenoviral-mediated gene transfer of eNOS could reverse age-related erectile dysfunction in the rat. Two groups of animals were transfected with adenoviruses: (1) aged rats (60 weeks) with AdRSVbetagal; and (2) aged rats (60 weeks) with AdRSVeNOS. Five days after transfection, these study animals underwent cavernosal nerve stimulation (CNS) to assess erectile function and their responses were compared with young (20 weeks) control rats. Cross-sections of the rat penises transfected with AdRSVeNOS were examined after trichrome staining. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by a galacto-light chemiluminescent reporter gene assay in cavernosal tissues of rats administered AdRSVbetagal. The transgene expression of eNOS was examined by RT-PCR in rats transfected with AdRSVbetagal and AdRSVeNOS. eNOS and iNOS protein levels were measured by Western blot analysis, and cGMP levels were assessed in cavernosal tissue by enzyme immunoassay. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue for up to 30 days, with peak expression registered at 5 days after intracavernosal administration of AdRSVbetagal. Cross-sections of the rat penises transfected with the AdRSVeNOS revealed no pathological (morphological or histological) changes. Five days after administration of AdRSVeNOS, eNOS protein, mRNA and cGMP levels in the corpora cavernosa were significantly increased (P<0. 05), while iNOS protein levels remained unchanged (P>0.05). In conclusion, enhanced expression of eNOS employing an adenoviral vector significantly increased the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the aged rat.

Potential role of rel/nuclear factor-kappaB in the pathogenesis of interstitial cystitis.

PURPOSE: Despite assertive investigation in the last 2 decades, interstitial cystitis remains an unresolved problem in clinical urology, and its etiology and the mechanisms involved in its pathogenesis are still a matter of conjecture. Recently nuclear factor (NF)-KB has been implicated in chronic inflammatory diseases, and is thought to be a key regulator of genes involved in response to infection, inflammation and stress. We document the presence, pattern and distribution of NF-kappaB in bladder biopsies from patients with interstitial cystitis. MATERIALS AND METHODS: Bladder biopsies from 7 women clinically diagnosed with interstitial cystitis according to National Institute for Diabetes and Digestive and Kidney Diseases criteria and 5 women diagnosed with urinary incontinence were used for immunohistochemical localization of p65, an NF-kappaB subunit. RESULTS: Our immunohistochemical localization experiments indicate that NF-kappaB was predominantly activated in bladder urothelial cells and cells of the submucosal layer in biopsies from patients with interstitial cystitis compared to controls. While activation was evident by intense nuclear localization of NF-kappaB in all interstitial cystitis specimens, diffuse and faint immunostaining was observed in control samples. The results also indicate that activation of NF-kappaB correlated with disease occurrence. CONCLUSIONS: The fact that NF-kappaB is capable of transactivating pro-inflammatory mediators, which in turn can amplify NF-kappaB activation by a positive regulatory loop, suggests that inflammatory and/or immune responses in interstitial cystitis can be exacerbated possibly by persistent activation of this nuclear factor. We believe that our study provides a novel basis for investigating the role of NF-kappaB activation in the pathophysiology of interstitial cystitis and further opens a frontier for the development of an innovative therapeutic approach to interstitial cystitis.

Activation of nuclear factor kappaB: potential role in metallothionein-mediated mitogenic response.

The antiapoptotic response and enhanced cellular proliferation observed in neoplastic cells on overexpression of metallothionein (MT) have been well documented. We have investigated the mechanisms associated with this phenomenon by using MT inducers that increased MT transcripts and stimulated growth in MCF-7 cells. A MT antisense phosphorothioate oligonucleotide inhibited growth induction by >50%, suggesting a potential role of MT in mediating the mitogenic effects of these agents. Mobility shift assays using oligonucleotides encompassing the consensus nuclear factor kappaB (NFkappaB) binding site and anti-MT antibody revealed activation and a specific interaction of NFkappaB with MT. Cotransfection experiments using expression and reporter constructs demonstrated that MT caused transactivation of NFkappaB. Gel shift assays using purified proteins showed a specific interaction between MT and the p50 subunit of NFkappaB. These data indicate that MT may be involved in the interaction of NFkappaB with the DNA-binding domain and further suggest a potential role for NFkappaB in mediating the antiapoptotic effects of MT.

Perinatal hypocuprosis affects synthesis and composition of neonatal lung collagen, elastin, and surfactant.

To investigate the role of iron, ascorbate, and fructose on copper depletion and the effect of copper depletion on neonatal lung collagen, elastin, and surfactant, female rabbits were fed a control diet [10 parts per million (ppm) copper], a basal marginal copper diet (1.5 ppm), or a basal diet containing a high concentration of iron (1,750 ppm), ascorbic acid (1%, wt/wt), or fructose (20% of carbohydrates, wt/wt) or a combination of iron, ascorbic acid, and fructose throughout gestation. Whereas 10% of neonates in the control group died in the first 24 h, 27-67% of the offspring of rabbits fed the marginal copper diet died. Birth weight was also lower for the pups of the females fed the marginal copper diets. Lungs of neonates born to females fed iron or ascorbate and marginal copper diets had low levels of copper, high proportions of acid-extractable, high-molecular-weight collagen, and low lysyl-oxidase activities, consistent with incomplete maturation of collagen. The bronchoalveolar lavage fluids of newborns whose mothers were fed marginal copper diets alone or in combination with iron and/or ascorbate had lower levels of total surfactant phospholipids than the fluids from lungs of control newborns. The lower surfactant phospholipid content of these groups could be attributed mainly to lower phosphatidylcholine and, in particular, dipalmitoylphosphatidylcholine levels. These results suggest that high maternal intakes of iron, ascorbate, or their combination in pregnancy deplete biologically available copper, which in turn induces neonatal lung abnormalities.

The effect of various dietary zinc concentrations on the biological interactions of zinc, copper, and iron in rats.

Three groups (14 rats each) were fed one of the following diets for 8 wks: a control purified basal diet containing 12 ppm zinc, 5 ppm copper, and 35 ppm iron; the basal diet with less than 2 ppm zinc; or the basal diet supplemented with 1000 ppm zinc. Rats fed the zinc-deficient diet had decreased weight gain, moderate polydipsia, and intermittent mild diarrhea. The zinc-supplemented rats had a cyclical pattern of food intake and weight loss from weeks 5 to 8. Tissue concentrations suggest that zinc and copper were not mutually antagonistic with chronic dietary imbalances. If tissue element concentrations reflected intestinal uptake, then competition and/or inhibition of intestinal uptake occurred between zinc and iron. The fluctuations in tissue element concentrations that occurred with increased duration of the study were at variance with previous studies of shorter time periods. The dietary proportions of zinc, copper, and iron appear to influence zinc, copper, and iron metabolism at the intestinal and cellular transport levels over a given period of time.

A review on biochemical roles, toxicity and interactions of zinc, copper and iron: IV. Interactions.

While the major impetus of most of our present knowledge of these metals emphasizes their deficiencies or toxicities, little has been done on their metabolic interactions. Such interactions acknowledge the importance of nutritional deficiencies or toxicities in the biospheres. The effect of dietary zinc supplementation on the bioavailability of copper and iron is a matter of conjecture. Likewise, further research is needed before a unifying hypothesis can be established on the effect of imbalances or interactions among copper and iron. Such mineral imbalance studies will be of value in determining their dietary requirements and in appraising circumstances in which risk to human and animal health may arise.

A review of the biochemical roles, toxicity and interactions of zinc, copper and iron: III. Iron.

Because of its essentiality, iron occurs in tissues of all living matter especially aerobes. Iron compounds have been utilized in various aspects of argo-industry. Iron-binding proteins and their vital role in intestinal uptake, distribution and storage of iron are concisely examined. Ceruloplasmin (cuproenzyme) is imperative for iron mobilization from storage sites for hemoglobin synthesis. There is evidence of relationship between enhance susceptibility to infection and iron deficiency. The extent to which behavioral abnormalities and iron depletion are related remains to be established. The efficacy of chelating agents such as desferroxamine, bicarbonates and ethylene diamine tetraacetic acid (EDTA) against iron overload has been tested.

A review of the biochemical roles, toxicity and interactions of zinc, copper and iron: II. Copper.

Copper is an essential nutrient for living matter. Through its cuproenzymes, copper displays a variety of metabolic functions. Atomic absorption spectrophotometry, radioisotope studies and establishment of cell and molecular biology have provided the necessary tools to study copper absorption, metabolism, physiology and biochemistry. The vital role of metallothionein in copper homeostasis is examined. Ceruloplasmin represents the molecular link between copper and iron metabolism. The genetic predisposition of copper toxicity has been attributed to the cause of Wilson's disease in humans. The interrelationships between copper and other dietary factors is addressed.

A review of the biochemical roles, toxicity and interactions of zinc, copper and iron: I. Zinc.

Zinc is essential for biological functions of all living matter. Zinc is necessary for growth, appetite, testicular maturation, skin integrity, mental activity, wound healing and immunocompetence. Zinc is required for the metabolic activities of over 70 metalloenzymes. The intestinal competition of zinc with copper, iron, lead, calcium and cadmium may accentuate nutritional deficiencies or toxicities from these environmental metals. A unifying hypothesis is not yet established for the effects or imbalances among these elements. These interactions will be of substantial practical importance in estimating dietary recommendations, in validating prophylactic measures, and in the assessment of situations in which human and animal health may be at risk.