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PubMed1976

[Determination of antibody affinity using threshold values of serum activity in the passive hemagglutination test].

Agglutination of erythrocytes of the antigenic diagnostic agents with the IgG antibodies is attributed to the fact that the reactive centres of the same molecule of the antibody attached to the antigenic determinants located on different erythrocytes. A possibility of agglutination of erythrocytes under conditions of the maximal dilution of the immune serum depended on the number of molecules located on a single erythrocyte. When the erythrocytes are sensitized with the optimal dose of the antigen those serum antibodies are involved in the passive hemagglutination test (PHAT) which are characterized by the mean integrated association constant. When the antibodies participate in the PHAT with several batches of the erythrocytic diagnostic agent characterized by an average number of the antigen molecules on a single erythrocyte there appears a possibility of determining the extent of heterogeneity of the antibodies. Not only the amount of antibodies, but also their affinity can be ascertained by the PHAT, and also the heterogeneity of antibodies can be evaluated.

Antibodies↗
PubMed1979

Serologic response in human hepatitis A: detection of antibody by radioimmunoassay and immune adherence hemagglutination.

An indirect solid-phase radioimmunoassay (RIA) for detection of antibody to the hepatitis A antigen (anti-HAV) was developed using polystyrene pearls as the solid phase and hepatitis A antigen (HAAg) extracted from marmoset livers. This RIA was compared to an immune adherence hemagglutination assay (IAHA) which employed HAAg derived from the stools of chimpanzees collected during acute hepatitis A. Anti-HAV was detected in the sera of 15 humans with naturally acquired hepatitis A infection. Sensitivity and specificity were greater using the RIA, permitting the detection of anti-HAV as early as the time of onset of jaundice. Either seroconversion or a significant increase in the titer of anti-HAV was demonstrated following hepatitis A exposure in paired sera from six patients by both techniques. No significant difference in anti-HAV responses was noted between patients with icteric compared to anicteric hepatitis A or between children and adults with hepatitis A.

Adolescent↗
PubMed1997

Efficient in vivo gene transfer into the heart in the rat myocardial infarction model using the HVJ (Hemagglutinating Virus of Japan)--liposome method.

The lack of efficient treatment for myocardial infarction remains an unresolved problem in the field of cardiovascular disease. Gene therapy may be a potential therapeutic strategy for the treatment of myocardial infarction. However, current methods of in vivo gene transfer into the heart are limited by their low efficiency and/or potential toxicity. In the present study, we developed an efficient technique of gene transfer into the intact heart in vivo using the Sendai virus (HVJ: Hemagglutinating Virus of Japan)--liposome method. We used the beta-galactosidase gene, luciferase gene and human angiotensin converting enzyme (ACE) gene as markers. In vivo gene transfer into the rat heart was performed as follows: (1) direct injection into the rat heart, (2) incubation within the pericardium, and (3) infusion into a coronary artery. Direct injection of the HVJ-liposome complex containing the beta-galactosidase vector into the rat heart resulted in limited staining of beta-galactosidase 3 days after transfection. To compare transfection efficiency between "naked" plasmid DNA transfection and the HVJ-liposome method, we also transfected the luciferase reporter gene into the heart. Luciferase activity was significantly higher in hearts transfected by the HVJ-liposome method than that in hearts transfected by direct "naked" plasmid transfection (P < 0.01). To confirm the successful gene in the protein level, we measured ACE activity in the hearts. Cardiac ACE activity was significantly increased in hearts transfected with human ACE gene as compared to hearts transfected with control vector (P < 0.01). On the other hand, incubation of HVJ-liposome complex, containing beta-galactosidase vector, within the pericardium resulted in widespread staining of cardiac myocytes and fibroblasts, mainly located in several surface layers beneath the pericardium. More importantly, widespread stained areas of beta-galactosidase were also observed in the middle of the myocardium around the vasa vasorum. We also examined the efficiency of gene transfer by the HVJ-liposome method in a rat myocardial infarction model. In the infarction model, using the pericardium incubation approach, staining for beta-galactosidase was observed in the viable cells around the infarction area. Finally, direct infusion of the HVJ complex, containing the beta-galactosidase vector, into coronary artery also resulted in widespread staining of beta-galactosidase in cardiac myocytes around the microvasculature. Using direct injection, we found significant injury to the myocardium and severe fibrosis at the injection site, whereas no apparent injury was observed using pericardium incubation and coronary infusion. There was no evidence of cytotoxicity or inflammation caused by the HVJ-liposome complex itself. Overall, we have established an efficient in vivo gene transfer method into the heart using the HVJ-liposome method. Direct infusion into the coronary artery resulted in widespread transfection without damaging the myocytes; incubation within the pericardium demonstrated the usefulness of the HVJ-liposome method for studying cardiac function and as a means of gene therapy for cardiovascular diseases.

Animals↗
PubMed2002

Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system.

We have developed a simple method for converting the lipid envelope of an inactivated virus to a gene transfer vector. Hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector was constructed by incorporating plasmid DNA into inactivated HVJ particles. This HVJ envelope vector introduced plasmid DNA efficiently and rapidly into various cell lines, including cancer cells and several types of primary cell culture. Efficiency of gene transfer was greatly enhanced by protamine sulfate and centrifugation. Fluorescein isothiocyanate-labeled oligodeoxynucleotides (FITC-ODN) were also delivered to cells at > 95% efficiency. When HVJ envelope vector was injected into organs directly, reporter gene expression was observed in organs including liver, brain, skin, uterus, tumor masses, lung, and eye. When HVJ envelope vector containing luciferase gene was injected into mouse tail vein, luciferase gene expression was detected primarily in spleen. FITC-ODN were also delivered to spleen cells by intravenous injection of HVJ envelope. These results suggest that HVJ envelope vector will be useful for both ex vivo and in vivo gene therapy experiments.

Animals↗
PubMed1998

Spread of swine hemagglutinating encephalomyelitis virus from peripheral nerves to the CNS.

Swine hemagglutinating encephalomyelitis virus (HEV) strain 67N was inoculated into the sciatic nerve or the right leg crural muscle of rats. In both cases, the virus was isolated first from the caudal half of the spinal cord on day 2 after inoculation, and from the rostral half of the spinal cord and the brain on day 3. The virus titers in the brain reached a maximum when the infected rats developed CNS symptoms on day 5. Using confocal laser scanning microscope, fluorescent positive cells were first found in the lumbar dorsal root ganglion (DRG) and spinal cord ipsilateral of the inoculated leg on day 3. Antigen positive neurons were found bilaterally in the lumbar DRG and spinal cord on day 4. On day 5 specific fluorescence was observed in the neurons of the cerebral cortex, hippocampus, brainstem and Purkinje cells in the cerebellum.

Animals↗
PubMed1995

Genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus OC43 as studied by the use of bovine coronavirus S gene-specific probes.

The genomic relationship of porcine hemagglutinating encephalomyelitis virus (HEV) to bovine coronavirus (BCV) and human coronavirus (HCV) strain OC43 was examined by dot blot hybridization assays. Two BCV S gene-specific probes were generated by polymerase chain reaction from the avirulent L9-strain of BCV. Probes were located in the S1 and the S2 region of the peplomeric (S) glycoprotein gene. The S1 probe (726 bp) hybridized with BCV and HCV-OC43, but not with HEV under moderate stringency hybridization conditions (50 degrees C). Only slight signals were present with mouse hepatitis virus (MHV) and no signals were observed with feline infectious peritonitis virus (FIPV) or canine coronavirus (CCV). At high stringency conditions (60 degrees C) the S1 probe hybridized with BCV only. Using the S2 probe (680 bp) under moderate stringency conditions, hybridization signals were obtained with BCV, HCV-OC43 and HEV (strains 67N, NT9, VW572). The signals obtained by the three HEV strains were altogether weaker than with BCV and HCV-OC43. The S2 probe did not react with MHV, FIPV and CCV. At high stringency the S2-specific probe hybridized with BCV and HCV-OC43 but did not hybridize with HEV. Nucleotide sequence analysis of the region covering the S2 probe in HEV revealed 92.6% nucleotide sequence homology to BCV and 91.9% to HCV-OC43. In contrast, the region covering the S1 probe in HEV could not be amplified using the BCV S1-specific primers. The hybridization and sequencing results thus indicate a closer genomic relationship between BCV and HCV-OC43 than there is between HEV and BCV or HCV-OC43 respectively.

Animals↗
PubMed1978

[A new influenza subunit vaccine: hemagglutinating antibodies one year after vaccination (author's transl)].

The antibody response to a new influenza subunit vaccine was compare d one year after vaccination with the responses induced by two other influenza vaccines. The subunit vaccine was given either in a high dose form containing 2100 IU, or in a low dose form containing 700 IU. As comparison a split vaccine was used containing 800 IU and AI(OH)3 as adjuvant and a whole virus vaccine containing 2100 IU. Of the 399 vaccinated subjects which had taken part in this study 151 were available for hemagglutination inhibiting (HAI) antibody determinations one year after vaccination. Protection rates assessed for the respective groups on the assumption that serum HAI titers of 1 : 32 or greater confer protection. With the high dose of subunit vaccine 85% of volunteers were considered still to have protective titers one year after vaccination, compared with 77% of those who received the whole virus vaccine. Although the high dose subunit vaccine and whole virus vaccine induced similarly high protective levels lasting at least one year, the reactions observed on vaccination were significantly less with the subunit preparation. The lower dose of subunit vaccine induced lower levels of protection (60%) after one year, and lower mean HAI titers than the high dose subunit vaccine. Nevertheless protection was superior to that of the split virus adjuvant vaccine. The addition of adjuvant thus does not seem materially to improve the immune response to influenza virus antigens. An increase of antigen content can however be seen as a practical alternative for achieving higher antibody levels. The subunit vaccine would appear to be particularly suitable in this respect as even with a higher dose there is no increase in reactogenicity.

Adjuvants, Immunologic↗
PubMed1977

Special O:K:H serotypes among enterotoxigenic E. coli strains from diarrhea in adults and children. Occurrence of the CF (colonization factor) antigen and of hemagglutinating abilities.

O:H serotypes previously found to be prevalent among a number of toxigenic strains from several geographic areas were examined for polysaccharide K antigen type. Members of each O:H serotype had the same type of K antigen and were found to be characterized by a certain fermentation pattern. Some O:H serotypes had no K antigen. The serofermentative types defined were: 06:K15:H16, 08:K40:H9, 015:H11, 025:K7:H42, 025:K98:H-, 078:H11, 078:H12, and 0149:H10. Some strains of the last-mentioned serotype, which were suspected of having caused a food-borne infection, had K88. This serotype belongs to the group of strains causing diarrhea in swine. The surface antigen (CF) described as a colonization factor [5] was demonstrated in 078:H-, 078:H11, and 078:H12 strains; but not in any strain of the other serotypes nor in any of 248 strains belonging to 078 but not isolated from cases of human diarrhea. Presence of the CF antigen was correlated with presence of a mannose-resistant ability to cause agglutination of human red cells. Behavior of the other serotypes as regards hemagglutinating abilities was examined and 025:K7:H42 strains were found to be very similar to the 078 strains in this respect.

ABO Blood-Group System↗
PubMed1989

Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption.

Human rotaviruses (Wa, KUN, MO) showed hemagglutination (HA) only with fixed 1-day-old chicken erythrocytes, and their HA activities were completely destroyed by trypsin activation of virions. Simian SA-11 and bovine NCDV had HA activities not only against fixed erythrocytes but also against fresh erythrocytes from various species. Their HA activities against fixed erythrocytes were also inhibited by trypsin activation, but those against fresh erythrocytes were not. Neuraminidase treatment of fixed erythrocytes did not inhibit HA by trypsin-untreated rotaviruses. In contrast, HA of fresh human erythrocytes by SA-11 and NCDV was completely inhibited by neuraminidase treatment of erythrocytes or glycophorin A, the major erythrocyte sialoglycoprotein. Adsorption and infection of SA-11 and NCDV to monkey kidney MA104 cells were also inhibited by neuraminidase treatment of cells. Adsorption and infection of human rotaviruses were not, however, affected by treatment of cells with neuraminidase from Vibrio cholerae or Arthrobacter ureafaciens or with potassium periodate. Therefore, HA of fixed chicken erythrocytes by trypsin-untreated human and animal rotaviruses may be independent of sialic acids, whereas that of fresh erythrocytes by SA-11 and NCDV is sialic acid dependent and probably mediated by glycophorin A. Sialic acids also constitute an essential part of the cellular receptors for SA-11 and NCDV, whereas those for human rotaviruses were quite resistant to treatments known to destroy major types of sialic acids.

Animals↗
PubMed1992

Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes.

The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove alpha 2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain alpha 2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.

Animals↗
PubMed1994

Gene transfer and the expression of a foreign gene in vivo in post-mitotic neurons of the adult rat brain using the hemagglutinating virus of the Japan-liposome method.

Neurons in the adult rat brain were transfected in vivo with a simple plasmid that harbored the gene for beta-galactosidase from Escherichia coli under control of a chicken beta-actin promoter by use of the hemagglutinating virus of Japan (HVJ) and liposomes. Cells that expressed beta-galactosidase were detected only in the target area of the central nervous system for 10 days by light microscopic analysis. Since electron microscopic analysis revealed that the products of the histochemical reaction were predominantly associated with the nuclear membrane and the endoplasmic reticulum of positive cells, it appeared that the products were translated endogenously and had not been entrapped by endocytosis. Furthermore, the products were observed in typical neuronal cells with a large, round, and pale nuclei, and with direct axo-somatic and axo-dendritic synaptic contacts. This report suggests the possibility of introducing functionally significant genes into neurons in targeted areas of the adult central nervous system.

Animals↗
PubMed1995

An evaluation of a hemagglutination-inhibition test for the detection of antibodies to herpes simplex virus type 1.

BACKGROUND: We have recently demonstrated the ability of herpes simplex virus type 1 (HSV-1) to agglutinate mouse red blood cells, and identified glycoprotein C (gC-1) as a major virus hemagglutinin. Based on this a classical hemagglutination-inhibition (HI) assay was developed. OBJECTIVES: Regarding significant structural differences between HSV-1 gC-1 and its herpes simplex virus type 2 (HSV-2) counterpart, gC-2, the possibility of application of a classical HI assay for the detection of HSV-1-specific antibodies was explored. STUDY DESIGN: HI antibody titers were compared with those of gC-1-specific enzyme-linked immunoassay (ELISA), and with the results of the standard gG-1- and gG-2-specific immunodot enzymatic assays for the detection of type-specific antibodies to HSV-1 and HSV-2 respectively. RESULTS: The sensitivity of HI test was 89% and 97% of that gC-1-ELISA and gG-1-immunodot respectively. Approximately 21% of serum specimens, defined as containing antibodies specific for only HSV-2, showed low HI titers. Heterotypic reactivity with purified gC-1 antigen was also observed in both ELISA and immunoblot assays. CONCLUSION: Antibodies detectable in HI assay were mainly HSV-1-specific; however, a limited degree of serologic reactivity between HSV-2-specific sera and HSV-1 hemagglutinin also occurred. Thus, our results confirmed prevalent opinion about the presence of a limited number of antigenic determinants shared by HSV-1 gC-1 and HSV-2 gC-2.

Journal Article↗
PubMed2004

Hemagglutinating virus of Japan protein is efficient for induction of CD4+ T-cell response by a hepatitis B core particle-based HIV vaccine.

By using the hepatitis B core (HBc) protein gene as a carrier, HIV-1 env V3 gene was inserted into the carrier gene, and the HIV gene was expressed inside a chimeric HIV-HBc particle (HIV-HBc), which was a unique candidate for induction of HIV-specific CTL activity. This was seen significantly in mice without the need of an adjuvant, because other responses specific for the HIV peptide such as T-cell proliferation and antibody production were not induced. However, when hemagglutinating virus of Japan (HVJ) protein was incorporated into an anionic liposome containing HIV peptide (HIV-HVJ-liposome) and was used as a booster immunization in HIV-HBc primed animals, the HIV-specific T-cell response and enhanced CTL activity were clearly induced in consecutively immunized animals. Furthermore, the HIV-specific humoral immune response was also induced and a neutralization activity was detected in the immune sera. Thus, when an HIV peptide antigen is expressed inside the virus like a particle of HBc, it can induce both cellular and humoral immunities when an HVJ-HIV-liposome, but not an HIV-liposome, is inoculated as the booster antigen. The HVJ-stimulated splenocytes secreted IL-18 and IL-12 to synergistically enhance the secretion of IFN-gamma in vitro. These findings suggest that the HVJ protein is effective at inducing the HIV-specific immunities, if used as part of a booster antigen in the consecutive immunization regimen.

AIDS Vaccines↗
PubMed2005

Magnetic resonance imaging using hemagglutinating virus of Japan-envelope vector successfully detects localization of intra-cardially administered microglia in normal mouse brain.

Although the therapeutic use of microglia has received some attention for the treatment of brain diseases, few non-invasive techniques exist for monitoring the cells after administration. Here, we present a technique using magnetic resonance imaging (MRI) to track microglia injected intra-cardially. We labeled microglia expressing enhanced green fluorescent protein with superparamagnetic iron oxide (Resovist) using the hemagglutinating virus of Japan-envelope vector. We injected labeled microglia into the left ventricle of the heart of mice. After monitoring exogenously administered microglia in the mouse brain in vivo using T(2)*-weighted MRI at a magnetic field of 7T, we compared the MR images with histochemical localization of exogenous microglia in vitro. MRI revealed clear signal changes attributable to Resovist-containing microglia in the mouse brain. Histochemistry demonstrated the presence of exogenous microglia in the brain at the same locations shown by MRI. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for brain disease.

Animals↗
PubMed2005

Nonviral HVJ (hemagglutinating virus of Japan) liposome-mediated retrograde gene transfer of human hepatocyte growth factor into rat nervous system promotes functional and histological recovery of the crushed nerve.

Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve.

Analysis of Variance↗
PubMed2006

Effect of AP-1 decoy using hemagglutinating virus of Japan-liposome on the intimal hyperplasia of the autogenous vein graft in mongrel dogs.

Intimal hyperplasia is the leading cause of late vein graft failure. Smooth muscle cell proliferation and migration is the underlying mechanism. Pharmacological approaches to prolong vein graft patency have produced limited results. AP-1 proteins play a role in the expression of many genes involved in cellular proliferation and cell cycle progression. Previously we reported inhibition of vascular smooth muscle cell migration, proliferation, and intimal hyperplasia in the balloon-injured rat carotid artery using an AP-1 decoy with HVJ-liposomes. In this report, we evaluated the effect of the AP-1 decoy on intimal hyperplasia in a large animal model. The jugular vein was transfected with hemagglutinating virus of Japan-liposomes containing the AP-1 decoy or scrambled oligonucleotides. An interposition graft was performed with the pretreated jugular vein between the transected femoral arteries. The graft was harvested at 16 weeks after the procedure. The intimal area was compared: the intimal area of the AP-1 decoy-treated versus control group was 47.3 +/- 15.2 versus 102.3 +/- 15.9 (P < .05), respectively. In conclusion, AP-1 decoy using HVJ-liposomes effectively prevented intimal hyperplasia of an autogenous vein graft in mongrel dogs.

Animals↗
PubMed2005

DNA vaccine using hemagglutinating virus of Japan-liposome encapsulating combination encoding mycobacterial heat shock protein 65 and interleukin-12 confers protection against Mycobacterium tuberculosis by T cell activation.

We investigated the immunogenicity and protective efficacy of DNA vaccine combinations expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) using gene gun bombardment and the hemagglutinating virus of Japan (HVJ)-liposome method. A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of Hsp65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100-fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated Hsp65 DNA and mIL-12DNA (Hsp65 + mIL-12/HVJ). The HVJ-liposome method improved the protective efficacy of the Hsp65 DNA vaccine compared to gene gun vaccination. Hsp65 + mIL-12/HVJ induced CD8+ cytotoxic T lymphocyte activity against Hsp65 antigen. Most importantly, Hsp65+mIL-12/HVJ vaccination resulted in a greater degree of protection than that evoked by BCG. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells and cytokines (IFN-gamma and IL-2) production upon stimulation with Hsp65 and antigens from M. tuberculosis. These results suggest that Hsp65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to BCG vaccine.

Animals↗
PubMed1998

Hemagglutinating virus of Japan-liposome-mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff.

PURPOSE: Late graft failure is a critical problem, particularly in the presence of poor runoff vessels. Intimal hyperplasia is considered to be the main cause of graft failure. We have already reported that intimal thickening of experimental vein grafts in dogs with poor runoff vessels is more pronounced than that in dogs with normal vessels. We and others also have reported that production of nitric oxide (NO) in the endothelium of canine vein grafts is impaired. In the present study, we asked whether in vivo gene transfer of endothelial cell NO synthase (ecNOS) would inhibit intimal hyperplasia of autogenous vein grafts implanted in limbs with poor distal runoff in dogs. METHODS: After exposing femoral veins, the nuclear-targeted lac Zgene, bovine ecNOS cDNA, or control vector plasmid encapsulated in the hemagglutinating virus of Japan-liposomes was infused intraluminally, followed by incubation for 10 minutes at room temperature under a distending pressure of 100 mm Hg. Twenty reversed vein grafts were implanted under normal runoff conditions, and 4 days later these were used to confirm gene transfer to the vein grafts. Twelve reversed vein grafts were implanted under conditions of poor runoff, and 4 weeks after the operation intimal thickening was evident. RESULTS: In vein grafts under normal runoff conditions, lac Z gene transfer exhibited diffuse and frequent X-Gal-positive signals in both medial and adventitial layers 4 days after implantation (n = 3). In case of the ecNOS gene-transferred vein grafts, bovine ecNOS protein was mainly detected in medial smooth muscle cells and adventitial cells 4 days after implantation, determined using immunohistochemical techniques and bovine ecNOS specific antibody (n = 3). In addition, ecNOS-transferred vessels showed intense purple signals by reduced nicotinamide adenine dinucleotide phosphate diaphorase and nitroblue tetrazolium reaction, in both medial and adventitial layers, whereas weak NOS activity was recognized at the adventitial vasa vasorum of the untreated veins or control vector transferred veins (n = 3, respectively). In vein grafts under poor runoff conditions, the intimal thickness at 4 weeks after implantation was significantly reduced by ecNOS gene transfer (n = 4; 90.0 +/- 7.6 microns and 1.18 +/- 0.07 mm2) in comparison with buffer-treated vessels (n = 4; 195.8 +/- 25.7 microns and 2.62 +/- 0.48 mm2) or vector vehicle-treated vessels (n = 4; 193.0 +/- 15.8 microns and 2.65 +/- 0.22 mm2). CONCLUSIONS: Our findings show that gene transfer of ecNOS inhibited intimal hyperplasia of canine vein grafts caused by poor runoff conditions, as a result of an increased local production of NO. Thus ecNOS gene transfer warrants further study as a possible approach to prevent late graft failure.

Animals↗