The induction of antigen-specific CTL by in situ Ad-REIC gene therapy.

An adenovirus vector carrying the human Reduced Expression in Immortalized Cell (REIC)/Dkk-3 gene (Ad-REIC) mediates simultaneous induction of cancer-selective apoptosis and augmentation of anticancer immunity. In our preclinical and clinical studies, in situ Ad-REIC gene therapy showed remarkable direct and indirect antitumor effects to realize therapeutic cancer vaccines. We herein aimed to confirm the induction of tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) by Ad-REIC. Using an ovalbumin (OVA), a tumor-associated antigen, expressing E.G7 tumor-bearing mouse model, we investigated the induction and expansion of OVA-specific CTLs responsible for indirect, systemic effects of Ad-REIC. The intratumoral administration of Ad-REIC mediated clear antitumor effects with the accumulation of OVA-specific CTLs in the tumor tissues and spleen. The CD86-positive dendritic cells (DCs) were upregulated in the tumor draining lymph nodes of Ad-REIC-treated mice. In a dual tumor-bearing mouse model in the left and right back, Ad-REIC injection in one side significantly suppressed the tumor growth on both sides and significant infiltration of OVA-specific CTLs into non-injected tumor was also detected. Consequently, in situ Ad-REIC gene therapy is expected to realize a new-generation cancer vaccine via anticancer immune activation with DC and tumor antigen-specific CTL expansion.

Integrin antagonist augments the therapeutic effect of adenovirus-mediated REIC/Dkk-3 gene therapy for malignant glioma.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) was identified as a gene whose expression is reduced in many human cancers. REIC/Dkk-3 expression is also downregulated in malignant glioma and regulates cell growth through caspase-dependent apoptosis. cRGD (EMD121974), an antagonist of integrins, has demonstrated preclinical efficacy against malignant glioma. In this study, we investigated the antiglioma effect of combination therapy using an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) and cRGD. Quantitative real-time reverse-transcription PCR revealed the reduction of REIC/Dkk-3 mRNA levels in malignant glioma cell lines. The reduction of REIC/Dkk-3 protein expression in malignant glioma cell lines was also confirmed with western blot analysis. After treatment with Ad-REIC and cRGD, the proliferative rate of malignant glioma cells was significantly reduced in a time-dependent manner. In vivo, there was a statistically significant increase in the survival of mice treated with Ad-REIC and cRGD combination therapy compared with Ad-REIC monotherapy. We identified an apoptotic effect following monotherapy with Ad-REIC. Moreover, cRGD augmented the antiglioma efficacy of Ad-REIC. These results may lead to a promising new approach for the treatment of malignant glioma.

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a favorable prognosis marker and inhibits cell growth through the apoptotic pathway in cervical cancer.

BACKGROUND: In light of the poor prognosis for cervical cancer, research continues into the development of innovative and efficacious treatment modalities for this disease. We investigated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) and evaluated its clinical importance in cervical cancer. PATIENTS AND METHODS: HAI-2 expression was examined in cervical cancer specimens (n=52) by immunohistochemistry. We further attempted to investigate the biological functions and inhibitory effects of HAI-2 using human papillomavirus (HPV) 16 type SiHa and HPV 18 type HeLa cervical cancer cell lines. RESULTS: There were significant correlations between HAI-2 expression and stage (P=0.017), lymph node metastasis (P=0.005) and ovarian metastasis (P=0.038). Low HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-2 expression (disease-free survival rate, P=0.016; overall survival rate, P=0.021). After transient transfection into the SiHa and HeLa cell lines, HAI-2 showed potential inhibitory effects mediated by reductions in hepsin and matriptase expression, which led to apoptosis by increasing the level of Bak and reducing the level of Bcl-2. CONCLUSIONS: The present findings indicate that low HAI-2 expression in cervical cancer may be associated with a poor prognosis. We propose that HAI-2 may represent a therapeutic target for the treatment of cervical cancer.

Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model.

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

Therapeutic effects of adoptive splenocyte transfer following in situ AdIL-12 gene therapy in a mouse prostate cancer model.

We developed a preclinical prostate cancer model to study the feasibility of adoptive immunotherapy for residual tumor following neo-adjuvant in situ adenoviral-vector-mediated interleukin 12 (AdIL-12) gene therapy. Splenocytes were obtained from mice with orthotopic 178-2 BMA metastatic mouse prostate cancers treated previously with AdIL-12, or a vector with the IL-12 genes plus the costimulatory gene B7-1 (AdIL-12/B7), or a control gene (Adbetagal). The splenocytes were subsequently injected intravenously into syngeneic mice bearing orthotopic 178-2 BMA tumors generated 3 days previously. Significant orthotopic tumor growth suppression was achieved with splenocytes derived from mice whose tumors had been injected with AdIL-12 compared to splenocytes from control Adbetagal mice (P = 0.0005) and splenocytes from AdIL-12/B7-treated mice significantly suppressed spontaneous lung metastases compared to splenocytes from control mice (P = 0.0356). Adoptive transfer of splenocytes from either AdIL-12 (P = 0.004) or AdIL-12/B7 (P = 0.009)-treated mice significantly prolonged survival relative to controls. Transfer of NK and tumor-specific CTL activities was detected and depletion of CD4+ and CD8+ T cells by in vitro antibody-mediated complement lysis of the splenocytes prior to injection abrogated the effects. Systemic IL-12 administration delivered by intramuscular AdIL-12 injection enhanced the antitumor effects of adoptive splenocyte transfer and boosted the CTL response. Our data provide evidence that this form of adoptive immunotherapy can enhance the effectiveness of neo-adjuvant in situ IL-12 gene therapy in cases of persistent malignancy.

Post-operative infection by pathogenic micro-organisms in the oral cavity of patients with prostatic carcinoma.

The aim of this study was to analyse the change in the oral cavity microflora of 14 patients who had undergone a radical prostatectomy for prostatic carcinoma. The detection of micro-organisms in the oral cavity was compared before and after the surgical procedure. Post-operative infection, defined as those patients who had increased Candida species counts and/or pathogenic bacteria only at the post-operative examination, was observed in 10 patients. Six patients showed increased Candida species counts at the post-operative examination compared with the pre-operative examination. In five patients, pathogenic bacterial species were detected at the post-operative examination but not at the pre-operative examination. One patient had detectable pathogenic bacterial species only at the post-operative examination along with increased Candida species counts. Our findings suggest that pre-operative oral hygiene to remove bacterial and Candida species from patients who are scheduled for surgical procedures is important for satisfactory clinical outcomes.

Ny-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade.

NY-ESO-1 mRNA expression in transitional cell carcinoma was investigated by reverse transcription-PCR and immunohistochemistry. NY-ESO-1 mRNA was detected in 20 of 62 (32%) tumor specimens. There was a correlation between NY-ESO-1 expression and tumor grade: 0 of 4 (0%) grade 1 (G1), 6 of 26 (23%) grade 2 (G2), and 14 of 32 (44%) grade 3 (G3) tumors were NY-ESO-1 mRNA positive. Immunohistochemical analysis using NY-ESO-1-specific monoclonal antibody ES121 showed that 2 of 14 NY-ESO-1 mRNA-expressing G3 tumors were positive for NY-ESO-1. No NY-ESO-1 staining was observed in the panel of 30 G1 or G2 tumor specimens, including 6 NY-ESO-1 mRNA-positive cases. Sera from an expanded panel of 124 patients with transitional cell carcinoma were tested for the presence of NY-ESO-1 antibody. Seropositivity was observed in 9 of 72 (12.5%) patients with G3 tumors, whereas none of 52 patients with G1 or G2 tumors produced antibody against NY-ESO-1. In the 9 positive patients with NY-ESO-1 antibody, 4 had muscular invasive tumors, and 5 had carcinoma in situ.

Adenovirus vector carrying REIC/DKK-3 gene: neoadjuvant intraprostatic injection for high-risk localized prostate cancer undergoing radical prostatectomy.

As the First-In-Human study of in situ gene therapy using an adenovirus vector carrying the human REIC (reduced expression in immortalized cell)/Dkk-3 gene (Ad-REIC), we conducted neoadjuvant intraprostatic injections in patients with high-risk localized prostate cancer undergoing radical prostatectomy (RP). Patients with recurrence probability of 35% or more within 5 years following RP, as calculated by Kattan's nomogram, were enrolled. Patients received two ultrasound-guided intratumoral injections at 2-week intervals, followed by RP 6 weeks after the second injection. After confirming the safety of the therapeutic interventions with initially planned three escalating doses of 1.0 × 1010, 1.0 × 1011 and 1.0 × 1012 viral particles (vp) in 1.0-1.2 ml (n=3, 3 and 6), an additional higher dose of 3.0 × 1012 vp in 3.6 ml (n=6) was further studied. All four DLs including the additional dose level-4 (DL-4) were feasible with no adverse events, except for grade 1 or 2 transient fever. Laboratory toxicities were grade 1 or 2 elevated aspartate transaminase/alanine transaminase (n=4). Regarding antitumor activities, cytopathic effects (tumor degeneration with cytolysis and pyknosis) and remarkable tumor-infiltrating lymphocytes in the targeted tumor areas were detected in a clear dose-dependent manner. Consequently, biochemical recurrence-free survival in DL-4 was significantly more favorable than in patient groups DL-1+2+3.

Novel REIC/Dkk-3-encoding adenoviral vector as a promising therapeutic agent for pancreatic cancer.

Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pancreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied. Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.

Vacuolar-type H(+)-ATPase in mouse bladder epithelium is responsible for urinary acidification.

The urine in the mouse bladder was found to be acidic, ranging from pH 5.3 to 5.5 in the daytime and pH 6.0 to 6.3 at night. Administration of bafilomycin A1 or concanamycin A, specific inhibitors of vacuolar-type H(+)-ATPase, into bladder lumen caused neutralization of urinary pH at least for 36 h, whereas inhibitors of mitochondrial ATP synthase (F-type H(+)-ATPase) or P-type H(+)-ATPases did not. Bafilomycin A1-sensitive proton secretion from isolated inside-out bladder was also observed. Immuno-electron microscopy with antibodies against vacuolar H(+)-ATPase revealed that vacuolar-type H(+)-ATPase is rich in luminal plasma membrane and endosomes of superficial cells of the bladder epithelium. These results indicate that vacuolar-type H(+)-ATPases present in luminal plasma membrane of the superficial epithelial cells secrete protons so as to acidify the urine in mouse bladder.

Molecular characterization of two forms of nontoxic-nonhemagglutinin components of Clostridium botulinum type A progenitor toxins.

The entire sequences of the type A nontoxic-nonhemagglutinin gene and an adjacent open reading frame designated as orf 22-a, which are located between the neurotoxin and the HA-35 genes were determined. SDS-PAGE and N-terminal amino acid sequence analyses of the purified type A progenitor toxins (12S, 16S and 19S) indicate that the nontoxic-nonhemagglutinins of 16S and 19S are single peptides of approximately 120k, but that of 12S has a cleavage at the site between Pro-144 and Phe-145 of this protein.

In-vitro activity of arbekacin alone and in combination with vancomycin against gentamicin- and methicillin-resistant Staphylococcus aureus.

In-vitro susceptibility studies were performed on 99 clinical Staphylococcus aureus isolates. A total of 68 of 73 methicillin-resistant S. aureus and 2 of 26 methicillin-susceptible S. aureus were gentamicin-resistant (gentamicin MIC range 16 to 1,024 microg/mL). All 70 gentamicin-resistant isolates contained the aac(6')-Ie-aph(2'')-Ia aminoglycoside resistance gene, and none possessed the aph(2'')-Ic or aph(2'')-Id aminoglycoside resistance genes. The arbekacin MIC for the 70 gentamicin-resistant isolates ranged from 0.25 to 4 microg/mL. The combination of arbekacin plus vancomycin produced synergistic killing against 12 of 13 gentamicin-resistant MRSA isolates. The combination of gentamicin plus vancomycin produced synergistic killing against 7 of the same 13 isolates. Arbekacin may prove useful when used in combination with vancomycin in treating infections caused by gentamicin-resistant MRSA.

In-vitro synergistic activity of the combination of ampicillin and arbekacin against vancomycin-and high-level gentamicin-resistant Enterococcus faecium with the aph(2")-Id gene.

The combination of ampicillin plus arbekacin produced synergistic killing against 8 of 13 vancomycin-, ampicillin-, gentamicin-, and streptomycin-resistant Enterococcus faecium isolates that possess the high-level gentamicin resistance gene, aph(2")-Id. This combination may prove useful in treating infections caused by multiresistant enterococci.

Pre-penile arteries are dominant in the regulation of penile vascular resistance in the rat.

The amount of blood flow into the penis that will produce an erection is dependent on the sum of inflow resistance from the feeder arteries, arterioles and the intra-penile vasculature. In the present study, our objective was to determine quantitatively the contribution to inflow resistance of these different components of the rat penile vasculature. Using methods developed previously, we determined the resistance properties of the isolated perfused whole penis in situ, both in an intact system and after serial transactions of the vessels. These cuts eliminated progressively larger distal segments of the vascular bed. Perfusion pressures were recorded at different flow rates (0.5-3 ml/min/kg body weight) under conditions of maximal dilatation and maximal vasoconstriction induced by methoxamine (MXA, 40 microg/ml). Regardless of the level of vascular tone, the pudendal artery contributes approximately 70% of the total resistance of the penile vasculature. In contrast, the vasculature within the penis (tip, shaft, crus) contributes only about one quarter of the resistance. Penile arterial inflow resistance properties both at maximal vasodilation and maximal alpha1-adrenergic constriction are dominated by the extra-penile vasculature in the rat. The implications of these findings are that alterations in the pudendal-artery (eg vasodilation, vasoconstriction, stenosis) would have primary control of arterial inflow and suggest an important role for pharmacological agents which can promote a more generalized vasodilation (eg phosphodiesterase inhibitors) in contrast to selective corpus cavernosal agents.

Analysis of retrograde ejaculation using color Doppler ultrasonography before and after transurethral collagen injection.

Transurethral bladder neck collagen injection therapy was performed in a patient with retrograde ejaculation. The phenomenon of retrograde ejaculation and its correction after the therapy were clearly demonstrated by color Doppler ultrasonography. To our knowledge this is the first report showing successful observation of retrograde ejaculation using color Doppler ultrasonography.

The penis is not protected--in hypertension there are vascular changes in the penis which are similar to those in other vascular beds.

In hypertension, small arteries in a variety of vascular beds undergo structural changes that increase resistance. To assess whether there are differential structural changes in the penis that accompany hypertension, we began with determining structurally-based vascular resistance properties in penile and hindlimb vascular beds of adult spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats. In anesthetized SHR, the penile and hindlimb vasculature were isolated and perfused, maximum dilation was induced, and a flow-pressure assessment and alpha 1-adrenoceptor agonist concentration-response curves were generated. Both the baseline and maximum constrictor responses were similar in the two beds of each strain, and overall the maximum structurally-based vascular resistance in SHR was higher than in SD rats. Our data suggests that the penile vasculature is not protected from the structural changes that take place in the other vascular beds in hypertension. There does not appear to be an underlying functional control mechanism that protects the penile vasculature from structural changes that may have a negative impact on penile blood flow.

Inhibitory effect of lichen constituents on mutagenicity induced by heterocyclic amines.

Physodic acid, one of the main constituents of Hypogymnia enteromorpha, inhibited the mutagenicity of indirect mutagens, including benzo[a]pyrene and heterocyclic amines in Salmonella typhimurium TA 98. In contrast, it was not effective against direct mutagens such as 6-nitropiperonal and adriamycin. Its antimutagenicity was not associated with free-radical scavenging or antioxidative activities. Physodic acid seemed to inhibit the formation of reactive metabolites, such as N-hydroxy-Trp-P-2, by blocking the hepatic microsomal oxidation systems. Another component of H. enteromorpha, physodalic acid, also inhibited mutagenicity of a heterocyclic amine, Trp-P-2, in S. typhimurium TA 98, even though it was reportedly mutagenic in S. typhimurium TA 100.

Catheter-associated urinary tract infections: impact of catheter materials on their management.

Infection associated with an indwelling catheter is a representative type of biofilm infection occurring in the urinary tract. Since the most effective way to control this intractable infection is the prevention of bacterial attachment and subsequent biofilm formation on the catheter, the importance of catheter materials and anti-bacterial coating cannot be underestimated. The difference in the degree of bacterial attachment among standard catheter materials, the efficacy of silver-coating of catheters in preventing infection and the potency and effectiveness of a new lecithin/silver coating are discussed.

Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters.

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Antioxidative and antimutagenic effects of theaflavins from black tea.

Theaflavins, polyphenolic ingredients of black tea, were observed to inhibit in vitro lipid peroxidation in the erythrocyte membrane ghost and microsomal systems. Theaflavins also showed inhibition of DNA single-strand cleavage and mutagenicity, both induced by hydrogen peroxide. These results suggest that theaflavins scavenge radicals to produce antioxidative and antimutagenic effects. It was also found that the gallic acid moiety of theaflavins is essential for their potent antioxidative activities.