Ocular administration of acetazolamide microsponges in situ gel formulations.

In the present work, the antiglaucoma drug, acetazolamide, was formulated as microsponges in situ gel for ocular drug delivery aiming an improved therapeutic efficacy and reduction in the systemic side effects of oral acetazolamide. The microsponges were prepared by the quasi emulsion solvent diffusion method and were incorporated into 25% pluronic F-127 in situ gel. Ethyl cellulose polymer in different proportions with drug was used to prepare the microsponges. Different parameters were evaluated to select the best formulation. The formula S2 with drug to polymer ratio (2:1) showed high entrapment efficiency of about 82% and mean particle size of about 10 µm with polydispersity index (PDI) of 0.22, which are suitable characters for ocular delivery. The in situ gels were evaluated for physicochemical properties (pH, gelling capacity, gelation time and rheological properties) and in vivo studies. S2 formulation showed higher therapeutic efficacy compared to free drug in gel. It was non irritant to the rabbit's eye. These results indicated that acetazolamide microsponges in situ gel have potential ability for ophthalmic delivery.

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.

Assessment of natural radioactivity levels in soil samples from some areas in Assiut, Egypt.

The natural radioactivity of soil samples from Assiut city, Egypt, was studied. The activity concentrations of 28 samples were measured with a NaI(Tl) detector. The radioactivity concentrations of (226)Ra, (232)Th, and (40)K showed large variations, so the results were classified into two groups (A and B) to facilitate the interpretation of the results. Group A represents samples collected from different locations in Assiut and characterized by low activity concentrations with average values of 46.15 ± 9.69, 30.57 ± 4.90, and 553.14 ± 23.19 for (226)Ra, (232)Th, and (40)K, respectively. Group B represents samples mainly collected from the area around Assiut Thermal Power Plant and characterized by very high activity concentrations with average values of 3,803 ± 145, 1,782 ± 98, and 1,377 ± 78 for (226)Ra, (232)Th, and (40)K, respectively. In order to evaluate the radiological hazard of the natural radioactivity, the radium equivalent activity (Raeq), the absorbed dose rate (D), the annual effective dose rate (E), the external hazard index (H ex), and the annual gonadal dose equivalent (AGDE) have been calculated and compared with the internationally approved values. For group A, the calculated averages of these parameters are in good agreement with the international recommended values except for the absorbed dose rate and the AGDE values which are slightly higher than the international recommended values. However, for group B, all obtained averages of these parameters are much higher by several orders of magnitude than the international recommended values. The present work provides a background of radioactivity concentrations in the soil of Assiut.

Considerable hazards produced by heavy fuel oil in operating thermal power plant in Assiut, Egypt.

Heavy fuel oil and ash samples were collected from the Assiut thermal power plant in Egypt and subjected to gamma spectrometry analysis for natural radioactivity contents. Considerable results were observed where the ash contains nearly 1,000 times natural radionuclides more than raw oil. The results were confirmed by measuring the samples via using different devices in different institutions. All ash samples had radium equivalent activities and external hazard index values more than 370 Bq/kg and unity respectively. The mean absorbed dose rate was10,650 nGy/h which is nearly 190 times higher than the global average value of 55 nGy/h. The corresponding annual external effective dose is estimated to be 13 mSv/year, which is nearly 30 times higher than that in areas of natural background radiation (0.46 mSv/year).

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.

Immunolocalization of avian beta-defensins in the hen oviduct and their changes in the uterus during eggshell formation.

The aim of this study was to examine whether avian beta-defensin proteins (avbetaDs) exist in the oviduct, and whether those in the uterus are secreted to the eggshell membrane and eggshell. The oviducts of White Leghorn hens at different times of egg formation, eggshell membrane, and eggshell were used. The presence of immunoreactive (ir) avbetaD-3, -11, and -12 was examined by immunohistochemistry and western blot. Two or three types of avbetaDs were identified in the mucosal surface epithelial cells in each oviductal segment. The density of ir-avbetaD-3 and -12 in the uterus was decreased after the egg entered this segment. Western blot analysis confirmed the presence of ir-avbetaD-3, -11, and -12 in the uterus. In the eggshell membrane, only ir-avbetaD-3 was detected on the surface of fibers at the outer layer of the membrane. The ir-avbetaD-3, -11, and -12 were identified in the eggshell matrix by western blot. These results suggest that the surface epithelial cells are the major sites where avbetaDs proteins exist, and the avbetaDs secreted by the uterus cells are likely to be incorporated in the eggshell membrane and eggshell. These avbetaDs may play roles in the innate host defense of the oviduct and egg surface.

Human fascioliasis and anaemia in Dakahlia Governorate, Egypt.

Fasciola infection (fascioliasis) appeared to be endemic in Egypt. Stool samples of fourty eight patients were coprologically diagnosed. According to Fasciola egg counting per gram stool, the severity of infection was divided into light infection in 60.5%, moderate in 27.1% and severe infection in 12.5%. No significant correlation was detected between severity of infection and patients' sex. Complete blood picture, reticylocytic count, serum iron, immunological assays as anti-nuclear, anti-smooth muscle antibody, anti-mitochondrial anti-body, anti-DNA tests and rheumatoid factor and occult blood in stool were investigated. Normocytic normochromic anaemia was detected in 62.5% of the fascioliasis patients, microcytic hypochromic anaemia in 31.3% and macrocytic one in 6.3%. Highly significant negative correlation (R = -0.68) was detected between haemoglobin concentration and egg count per gram faeces. Human fascioliasis was associated with normocytic normochromic anaemia and to a lesser extent microcytic hypochromic anemia.

Cholestasis in human fascioliasis in Dakahlia Governorate, Egypt.

This study was conducted to assess cholestasis in human fascioliasis. Twenty five patients, passing Fasciola eggs, were compared to twenty age- and sex-matched healthy controls. All were subjected to thorough clinical examination, stool analysis, complete blood picture, liver function tests (AST, ALT, SAP, GGT and total serum bilirubin). Autoimmune and viral hepatitis (HCV & HBV) were excluded. All cases were subjected to radiological examinations (chest x-ray and abdominal ultrasonography). The study revealed significant elevation of liver enzymes in the patients compared to the controls (p < 0.001). Calcular and non-calcular cholecystitis were common findings among patients (32% and 24% respectively). Ascites (4%) and dilated intra-hepatic biliary radicals (32%) were encountered; pleural effusion was detected by chest x-ray in 20% of cases. So, fascioliasis should be considered in the diagnosis of cholestasis in Fasciola endemic areas.

Localization of peripheral dopamine D1 and D2 receptors in rat corpus cavernosum.

OBJECTIVE: To detect and locate anatomically peripheral dopamine D1 and D2 receptors in rat cavernosa, as dopamine is important in sexual drive and penile erection through receptors located in the central nervous system. MATERIALS AND METHODS: Corpora cavernosa were obtained from Sprague-Dawley rats; total RNA and membrane proteins were extracted and cryostat sections prepared. The rat brain hypothalamus was used as a control for dopamine D1 and D2 receptors. The presence and expression of peripheral dopamine D1 and D2 receptor mRNAs in rat corpus cavernosa was assessed using reverse transcription and polymerase chain reaction (RT-PCR), and Northern blot hybridization using (32)P-UTP-labelled RNA probes. Concurrently, corresponding proteins from D1 and D2 receptors were assayed and detected by a Western blotting technique. The anatomical location of dopamine D1 and D2 receptor mRNAs in rat penile tissues was identified by in situ hybridization using (35)S-UTP-labelled RNA probes in cryostat sections. Immunohistochemical staining was used to locate peripheral dopamine D1 and D2 receptor proteins in rat corpora cavernosa. RESULTS: Dopamine D1 and D2 receptor gene expression was detected in rat corpora cavernosa. In situ hybridization signals for dopamine D1 and D2 receptor mRNAs were localized to corpus cavernosal tissues and dorsal vessels in the rat penis. Western blot analyses showed peripheral dopamine D1 and D2 receptor proteins in rat corpora cavernosa. Immunohistochemically, peripheral dopamine D1 and D2 receptor proteins were detected in dorsal nerves, dorsal vessels and corpus cavernosal smooth muscle of the rat penile tissues. CONCLUSIONS: Peripheral dopamine D1 and D2 receptors are present in the corpora cavernosa of rats. The functional significance of these receptors and signal transduction pathways in modulating the vascular tone of the penis warrants further investigation.

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.

Comparison of two doses of intravenous immunoglobulin after allogeneic bone marrow transplants.

Intravenous immunoglobulin has been used after bone marrow transplants to prevent infections and acute graft-versus-host disease. However, the minimum dose required for protection is unknown. This may have significant economic implications. A multicenter randomized clinical trial compared the impact of two intravenous immunoglobulin doses on systemic infections and acute graft-versus-host disease in transplant recipients. Either 250 mg/kg or 500 mg/kg was given weekly from day -8 to day +111. Multivariate analysis was used to assess the effect of dose and other risk factors on event-free survival, systemic infection, and acute graft-versus-host disease. The two-dose cohorts had similar event-free survival and infection frequencies. The higher dose was associated with less acute graft-versus-host disease (P = 0.03).

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.

Successful salvage using mismatched umbilical cord blood transplant in an adult with recurrent acute myelogenous leukemia failing autologous peripheral blood progenitor cell transplant: a case history and review.

We report a 26-year-old female with AML, FAB classification M5 who was initially treated with induction therapy consisting of idarubicin and cytarabine followed by high-dose cytarabine and autologous peripheral blood progenitor cell (PBPC) transplant for consolidation. The patient remained in remission for 1 month post-PBPC transplant, when relapse was noted. Reinduction therapy with idarubicin, cytarabine and etoposide was unsuccessful, and the patient underwent an unrelated, two-antigen mismatched umbilical cord blood (UCB) transplant for salvage after melphalan plus total body irradiation. Complications post transplant included veno-occlusive disease, delayed engraftment, and acute grade III graft-versus-host disease (GVHD). The patient remains in remission 1 year post transplant. This study demonstrates the salvage capability of UCB transplantation for refractory leukemia and its potential use in adult patients.

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.