Virological efficacy of combination therapy with corticosteroid and nucleoside analogue for severe acute exacerbation of chronic hepatitis B.

The short-term prognosis of patients with severe acute exacerbation of chronic hepatitis B (CHB) leading to acute liver failure is extremely poor. We have reported the efficacy of corticosteroid in combination with nucleoside analogue in the early stages, but virological efficacy has not been documented. Our aim was to elucidate the virological efficacy of this approach. Thirteen patients defined as severe acute exacerbation of CHB by our uniform criteria were prospectively examined for virological responses to treatment. Nucleoside analogue and sufficient dose of corticosteroids were introduced as soon as possible after the diagnosis of severe disease. Of the 13 patients, 7 (54%) survived, 5 (38%) died and 1 (8%) received liver transplantation. The decline of HBV DNA was significant between the first 2 weeks (P = 0.02) and 4 weeks (P < 0.01). Mean reduction in HBV DNA during the first 2 weeks was 1.7 ± 0.9 log copies per mL in overall patients, 2.1 ± 0.8 in survived patients and 1.2 ± 0.9 in dead/transplanted patients. The decline of HBV DNA was significant between the first 2 weeks (P = 0.03) and 4 weeks (P = 0.02) in survived patients, but not in dead/transplanted patients. Our study shows that corticosteroid treatment in combination with nucleotide analogue has sufficient virological effect against severe acute exacerbation of CHB, and a rapid decline of HBV DNA is conspicuous in survived patients.

Quantification of hepatitis C virus in patients treated with peginterferon-alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test.

Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG-IFN-alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non-SVR patients with greater accuracy.

Semi-allogeneic dendritic cells injected via the intratumoural injection route show efficient antitumour effects in cooperation with host-derived professional antigen-presenting cells.

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8(+) T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC.

Complete elimination of established neuroblastoma by synergistic action of gamma-irradiation and DCs treated with rSeV expressing interferon-beta gene.

Dendritic cell (DC)-based immunotherapy has been investigated as a new therapeutic approach to intractable neuroblastomas; however, only limited clinical effect has been reported. To overcome the relatively low sensitivity of neuroblastomas against immunotherapy, we undertook a preclinical efficacy study to examine murine models to assess the combined effects of gamma-irradiation pretreatment and recombinant Sendai virus (ts-rSeV/dF)-mediated murine interferon-beta (mIFN-beta) gene transfer to DCs using established c1300 neuroblastomas. Similar to intractable neuroblastomas in the clinic, established c1300 tumors were highly resistant to monotherapy with either gamma-irradiation or DCs activated by ts-rSeV/dF without transgene (ts-rSeV/dF-null) that has been shown to be effective against other murine tumors, including B16F10 melanoma. In contrast, immunotherapy using DCs expressing mIFN-beta through ts-rSeV/dF (ts-rSeV/dF-mIFNbeta-DCs) effectively reduced tumor size, and its combination with gamma-irradiation pretreatment dramatically enhanced its antitumor effect, resulting frequently in the complete elimination of established c1300 tumors 7-9 mm in diameter, in a high survival rate among mice, and in the development of protective immunity in the mice against rechallenge by the tumor cells. These results indicate that the combination of ts-rSeV/dF-mIFNbeta-DCs with gamma-irradiation is a hopeful strategy for the treatment of intractable neuroblastomas, warranting further investigation in the clinical setting.

Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies.

We previously reported the development of a prototype 'oncolytic Sendai virus (SeV) vector' formed by introducing two major genomic modifications to the original SeV, namely deletion of the matrix (M) gene to avoid budding of secondary viral particles and manipulation of the trypsin-dependent cleavage site of the fusion (F) gene to generate protease-specific sequences. As a result, the 'oncolytic SeV' that was susceptible to matrix metalloproteinases (MMPs) was shown to selectively kill MMP-expressing tumors through syncytium formation in vitro and in vivo. However, its efficacy has been relatively limited because of the requirement of higher expression of MMPs and smaller populations of MMP-expressing tumors. To overcome these limitations, we have designed an optimized and dramatically powerful oncolytic SeV vector. Truncation of 14-amino acid residues of the cytoplasmic domain of F protein resulted in dramatic enhancement of cell-killing activities of oncolytic SeV, and the combination with replacement of the trypsin cleavage site with the new urokinase type plasminogen activator (uPA)-sensitive sequence (SGRS) led a variety of human tumors, including prostate (PC-3), renal (CAKI-I), pancreatic (BxPC3) and lung (PC14) cancers, to extensive death through massive cell-to-cell spreading without significant dissemination to the surrounding noncancerous tissue in vivo. These results indicate a dramatic improvement of antitumor activity; therefore, extensive utility of the newly designed uPA-targeted oncolytic SeV has significant potential for treating patients bearing urokinase-expressing cancers in clinical settings.

Impact of deletion of envelope-related genes of recombinant Sendai viruses on immune responses following pulmonary gene transfer of neonatal mice.

We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.

Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes.

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.

Simian lentiviral vector-mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats.

Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal diseases resulting in adult blindness caused by mutations of various genes. Although it is difficult to cure the blindness that results from these diseases, delaying the disease progression may be of great benefit, since the majority of RP diseases are seen in middle age or later. To test a gene therapy strategy for RP using a neurotrophic factor gene, we assessed the effect of simian lentivirus (SIV)-mediated subretinal gene transfer of pigment epithelium-derived factor (PEDF), a potent neurotrophic factor, during the disease progression in Royal College of Surgeons (RCS) rats, a well-accepted animal model of RP. Regional gene transfer via SIV into the peripheral subretinal space at the nasal hemisphere was performed in all animals to monitor site-specific transgene expression as well as the therapeutic effect in each retina. Gene transfer of lacZ and PEDF was observed in the regional pigment epithelium corresponding to the regional gene transfer. Histologically, PEDF gene transfer significantly protected the loss of photoreceptor cells (PCs) corresponding to the regions of the gene transfer, compared to those of control groups during the course of the experiment. The antiapoptotic effect of PEDF on PCs is likely to be a related mechanism, because a significant reduction of terminal dUTP-nicked end labeling-positive PC numbers was found in PEDF-treated eyes compared to those of the control group (P<0.05). PEDF-treated eyes also retained a significant sensitivity to light flash during the experimental course. These findings clearly show that neuroprotective gene therapy using PEDF can protect retinal degeneration and functional defects in individuals with RP.

Recombinant Sendai virus vectors for activated T lymphocytes.

T-lymphocyte-directed gene therapy has potential as a treatment of subjects with immunological disorders. One current limitation of this therapeutic strategy is low gene transfer efficiency, even when complex procedures are used. We report herein that a recombinant Sendai virus vector (SeV) was able to overcome this issue. Using jellyfish enhanced green fluorescent protein gene (EGFP), we found that SeV was able to transduce and express a foreign gene specifically and efficiently in activated murine and human T cells, but not in naive T cells, without centrifugation or reagents including polybrene and protamine sulfate; the present findings were in clear contrast to those demonstrated with the use of retroviruses. The transduction was selective in antigen-activated T cells, while antigen-irrelevant T cells were not transduced, even under bystander activation from specific T-cell responses by antigens ex vivo. Receptor saturation studies suggested a possible mechanism of activated T-cell-specific gene transfer, ie, SeV might attach to naive T cells but might be unable to enter their cytoplasm. We therefore propose that the SeV vector system may prove to be a potentially important alternative in the area of T-cell-directed gene therapy used in the clinical setting.

Simian immunodeficiency virus-based lentivirus vector for retinal gene transfer: a preclinical safety study in adult rats.

Although lentivirus vectors hold promise for ocular gene therapy, they also have potential safety issues, particularly in the case of the current human immunodeficiency virus-based vectors. We recently developed a novel lentivirus vector derived from the nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to minimize these potentials. In this preclinical study, we evaluated whether SIV vector could be efficiently and safely applicable to retinal gene transfer by assessing the transgene expression, retinal function and histology over a 1-year period following subretinal injection in adult rats. The functional assessment via electroretinogram after both titers of SIV-lacZ (2.5 x 10(7) or 2.5 x 10(8) transducing units/ml) injection revealed both the dark and light adaptations to soon be impaired, in a dose-dependent manner, after a buffer injection as well, and all of them recovered to the control range by day 30. In both titers tested, the retinas demonstrated a frequent transgene expression mainly in the retinal pigment epithelium; however, the other retinal cells rarely expressed the transgene. Retinas exposed to a low titer virus showed no significant inflammatory reaction throughout the observation period, and also maintained the transgene expression over a 1-year period. In the retinas exposed to a high titer virus, however, mononuclear cell infiltration persisted in the subretinal area, and the retina that corresponded to the injected area finally underwent degeneration by around day 90. No retinal neoplastic lesions could be found in any animals over the 1-year period. We thus propose that SIV-mediated stable gene transfer might be useful for ocular gene transfer; however, more attention should be paid to avoiding complications when administering high titer lentivirus to the retina.

Airway-directed gene transfer of interleukin-10 using recombinant Sendai virus effectively prevents post-transplant fibrous airway obliteration in mice.

Bronchiolitis obliterans (BO) after lung transplantation prevents a satisfactory prognosis, and recent studies suggested that interleukin-10 (IL-10) gene transfer to distant organs could inhibit BO in rodent models. Although delivery of the therapeutic gene to a local airway would be favored to minimize systemic effects, current limitations include lower gene transfer efficiency to airway epithelium. As recombinant Sendai virus (SeV) can produce dramatically efficient gene transfer to airway epithelium, we determined if SeV-mediated IL-10 gene transfer to the local airway would inhibit bronchial fibrous obliteration in murine tracheal allografts. Administration of cyclosporine A (CsA) significantly promoted not only recovery of the injured airway epithelium but also SeV-mediated IL-10 expression (CsA- versus CsA+ =228+/-78 versus 3627+/-1372 pg/graft with 5 x 10(7) pfu), thereby suggesting the requirement of epithelia for efficient gene transfer. Even at the highest expression, no significant leakage of IL-10 was evident in the systemic circulation, and the induction of interferon-gamma was completely diminished on day 7 by IL-10 gene transfer. As a result, luminal loss was significantly prevented in allografts treated with SeV-IL-10 (luminal opening, all control groups: 0% respectively, and SeV-IL-10 5 x 10(7) pfu: 25.7+/-10.5%), an effect that was enhanced by short-term CsA treatment (SeV-IL-10 5 x 10(7) pfu with CsA: 63.7+/-12.7%). We propose that SeV is a useful vector that can target airway epithelium to prevent BO avoiding putative systemic effect.

Recombinant Sendai virus provides a highly efficient gene transfer into human cord blood-derived hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are a promising target for gene therapy, however, the low efficiencies of gene transfer using currently available vectors face practical limitations. We have recently developed a novel and efficient gene transfer agent, namely recombinant Sendai virus (SeV), and we have here characterized SeV-mediated gene transfer to human cord blood (CB) HSCs and primitive progenitor cells (PPC) using the jelly fish green fluorescent protein (GFP) gene. Even at a relatively low titer (10 multiplicity of infections), SeV achieved highly efficient GFP expression in CB CD34(+) cells (85.5+/-5.8%), as well as more immature CB progenitor cells, CD34(+)AC133(+) (88.2+/-3.7%) and CD34(+)CD38(-) (84.6+/-5.7%) cells, without cytokines prestimulation, that was a clear contrast to the features of gene transfer using retroviruses. SeV-mediated gene transfer was not seriously affected by the cell cycle status. In vitro cell differentiation studies revealed that gene transfer occurred in progenitor cells of all lineages (GM-CFU, 73.0+/-11.1%; BFU-E, 24.7+/-4.0%; Mix-CFU, 59+/-4.0%; and total, 50.0+/-7.0%). These findings show that SeV could prove to be a promising vector for efficient gene transfer to CB HSCs, while preserving their ability to reconstitute the entire hematopoietic series.

Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse.

Several gene transfer methods, including viral or nonviral vehicles have been developed, however, efficacy, safety or handling continue to present problems. We developed a nonviral and plasmid-based method for arterial gene transfer by in vivo electronic pulse, using a newly designed T-shaped electrode. Using rabbit carotid arteries, we first optimized gene transfer efficiency, and firefly luciferase gene transfer via electronic pulse under 20 voltage (the pulse length: P(on)time 20 ms, the pulse interval: P(off) time 80 ms, number of pulse: 10 times) showed the highest gene expression. Exogenous gene expression was detectable for at least up to 14 days. Electroporation-mediated gene transfer of E. coli lacZ with nuclear localizing signal revealed successful gene transfer to luminal endothelial cells and to medial cells. Histological damage was recognized as the voltage was increased but neointima formation 4 weeks after gene transfer was not induced. In vivo electroporation-mediated arterial gene transfer is readily facilitated, is safe and may prove to be an alternative form of gene transfer to the vasculature.

Preclinical and therapeutic utility of HVJ liposomes as a gene transfer vector for hepatocellular carcinoma using herpes simplex virus thymidine kinase.

Although gene therapy has been suggested to be a novel strategy to treat hepatocellular carcinoma (HCC), no study showing the clinical feasibility of vectors to treat HCC has been reported. In this preclinical study, we show evidence indicating that hemagglutinating virus of Japan (HVJ) liposomes are a feasible vector to treat HCC in a clinical setting using ganciclovir (GCV) and herpes simplex virus thymidine kinase (HSV-tk), which is driven by the cytomegalovirus immediate early enhancer/promoter (plasmid pcDNA3/HSV-tk). In in vitro experiments, almost complete tumor cell regression was achieved with the optimal GCV concentration (100 microg/mL) and more than 1/3 regression was seen even with a 20% transduction ratio using HuH7 HCC cells stably transformed by HSV-tk. HVJ liposomes showed a 19.7% (mean) transduction rate of the lacZ gene in a relatively large mass of more than 300 mm3 in vivo, which is a clinically detectable size, implanted into SCID mice. Moreover, a single HSV-tk injection of HVJ liposomes followed by GCV treatment inhibited tumor growth at least within a week, and repeat administration was more effective. Furthermore, subcutaneous injection of an HVJ liposomes vehicle induced no apparent inflammatory response in C3H/HeN mice, whereas lacZ gene transfection resulted in inflammatory pathology, suggesting a lower immunogenicity of the HVJ envelope protein than those of bacteria-derived plasmid DNA or the beta-galactosidase gene product. From these findings, we conclude that HVJ liposomes are a clinically safe and effective gene transfer vector to treat HCC.

Heterogeneous distribution of P53 immunoreactivity in human lung adenocarcinoma correlates with MDM2 protein expression, rather than with P53 gene mutation.

Although the tumor suppressor p53 protein (P53) immunoreactivity and its gene (p53) mutation were reported to be significant prognostic indicators for human lung adenocarcinomas, little is known regarding the relationship between the heterogeneous distribution of P53 and its genetic status in each tumor focus and the clinicopathological significance. To determine how P53 is heterogeneously stabilized in patients, we compared P53 expression to both the p53 allelic mutation in exon 2 approximately 9 by polymerase chain reaction-single strand conformation polymorphism using microdissected DNA fractions, and the immunohistochemical MDM2 expression. Of the 48 positive to P53 in 118 lung adenocarcinomas examined, 10 with heterogeneous P53 expression were closely examined. The higher P53 expression foci in 7 of 10 cases were less differentiated, histologically in respective cases, and were frequently associated with fibrous stroma. Two had genetic mutations in exon 7 of the p53 gene in both the high and low P53 expression foci of cancer tissue indicating no apparent correlation between heterogeneous P53 expression and the occurrence of gene mutation. Immunohistochemical expression of MDM2 was significantly lower in high P53 expression areas (p < 0.05, the mean labeling indices of high and low P53 expression areas being 4.2 +/- 5.4% and 13.6 +/- 12.2%, respectively). In addition, among all the 118 cases examined, MDM2 expression was significantly suppressed in cases of p53 gene mutation, simultaneously with P53 overexpression, as compared with cases without both the p53 mutation and expression (p < 0.001). These findings suggest that the heterogeneous stabilization of P53 in human lung adenocarcinomas could be partly due to suppressed MDM2 expression. The overexpression of non-mutated P53 may afford a protective mechanism in human lung adenocarcinomas.

Clinicopathological and molecular evidence indicating the independence of bronchioloalveolar components from other subtypes of human peripheral lung adenocarcinoma.

Although human lung adenocarcinoma has diverse histological subtypes, the correlation between histological subtypes and occurrence of the p53 gene mutation has been given less attention. We investigated 145 surgically resected lung adenocarcinomas to search for the incidence of p53 mutations and for record data on survival in each histological subtype, according to the new WHO criteria (1999). The frequency of p53 mutation in bronchioloalveolar carcinoma (BAC; 0% in 17 cases) and BAC with invasive growth component (BAC-invasive; 11% in 27 cases), which is conventionally categorized as the mixed subtype in WHO typing, were apparently significantly lower than in other types (non-BAC including acinar, papillary, solid, or mixed histology with these subtypes; 48% in 101 cases; P < 0.01). Multivariate analysis revealed that the histological subtype including BAC-invasive was a strong, independent, and significant prognostic factor (P < 0.03), as were tumor size and pathological stage (P < 0.001 and 0.002, respectively) for overall survival. However, the occurrence of p53 mutation itself was seen to be significant only in case of the univariate analysis. Therefore, histological subtyping may be a better prognostic indicator than is p53 mutation. These findings suggest that the WHO classification with the BAC and BAC-invasive from other histological subtypes may prove useful to predict the outcome for surgically treated patients with lung adenocarcinoma.

Non-penetrating vascular clips anastomosis inhibited intimal thickening under poor runoff conditions in canine autogenous vein grafts.

OBJECTIVE: Late graft failure is still a significant problem, particularly in cases with poor runoff vessels. The main cause of late graft failure is intimal thickening of the anastomotic region. Vascular closure system (VCS) clips may provide ideal anastomosis, since they do not penetrate the wall. Therefore, we examined whether the VCS clips affect intimal thickening under poor runoff conditions in the canine autogenous vein grafts. METHODS: A canine poor runoff model was prepared at both femoral veins. Four weeks after the first surgical procedure, two groups were established according to the two different methods of anastomosis employed. The right femoral vein graft was performed using polypropylene sutures, conventional surgical anastomosis (control group), while the left femoral vein graft was performed using VCS clips anastomosis (VCS group). Four weeks after grafting, the vein grafts were removed and the intimal thickening of proximal, distal anastomosis and midportion of the vein grafts were examined histologically. RESULTS: In the control group, flow rate and variation were 26+/-8 ml/min and 51+/-10 dynes/cm(2), respectively. In the VCS group, the flow rate and variation were 23+/-11 ml/min and 44+/-14 dynes/cm(2), respectively. There were no significant differences between the two groups. The average value of intimal thickening of both the anastomotic region and the midportion of the vein graft in the VCS group was significantly inhibited compared to that of the control group. The number of positive cells of masson trichrome stain in the VCS group was significantly less than that of the control group. CONCLUSIONS: These experiments indicate that VCS clips significantly inhibit intimal thickening under poor runoff conditions in canine autogenous vein grafts to a greater extent compared to suture-constructed anastomosis. One mechanism that may account for the decreased intimal thickening is the inhibition of the expression of transforming growth factor-beta (TGF-beta), because the number of positive cells of masson trichrome stain in the VCS group was significantly less than that of the control group.

Dual action of ZD6169, a novel K(+) channel opener, on ATP-sensitive K(+) channels in pig urethral myocytes.

1. The effects of ZD6169, a novel K(+) channel opener, on both membrane and unitary currents in pig urethra were investigated using patch-clamp techniques. Its effect was also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (Kir6.2C36) which form ATP-sensitive K(+) channels (K(ATP) channels). 2. In current-clamp mode, ZD6169 (< or = 10 microM) induced a concentration-dependent membrane hyperpolarization. Higher concentrations (> or = 30 microM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed. 3. In conventional voltage-clamp configuration, at -50 mV in symmetrical 140 mM K(+) conditions, ZD6169 (100 microM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K(+) current with a similar time course. 4. ZD6169 produced an inward glibenclamide-sensitive K(+) current, demonstrating a bell-shaped concentration-response relationship. 5. In cell-attached configuration in symmetrical 140 mM K(+) conditions, ZD6169 (< or = 30 microM) activated an K(ATP) channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 microM) inhibited the activity of the levcromakalim-induced K(ATP) channels. 6. ZD6169 (100 microM) had no significant effect on the channel activity of Kir6.2C36 in inside-out configuration, although cibenzoline greatly suppressed the channel activity. 7. These results demonstrate that ZD6169 possesses a dual effect on the activity of the K(ATP) channel; activating at low concentration and inhibiting at higher concentration.