Progress towards rabies control and elimination in Vietnam.

Rabies is a fatal viral disease that causes an estimated 59,000 human deaths each year. The majority of these deaths occur in developing countries in Asia. Canine rabies is endemic to Vietnam, which is, however, moving towards the disease's elimination. Many countries, such as Vietnam, have invested tremendous resources in controlling rabies, highlighting the goal of regional and global elimination of this neglected disease. In Vietnam, rabies is recognised as one of five high-priority, zoonotic diseases by the Ministry of Health and the Ministry of Agriculture and Rural Development. Investment by the government and by international partners for rabies prevention and control has played a substantial role in reducing human rabies deaths from 404 cases in 1992 to 74 cases in 2017. The catalyst for this effort was the Prime Minister's creation of the National Rabies Program in 1996, which led to increased support and resources for rabies prevention and control. Interventions carried out since then include the expansion of post-exposure prophylaxis centres throughout the country, the introduction or revision of key legislation and guidelines, and improved multisectoral One Health collaboration. In addition, support from international partners, such as the World Organisation for Animal Health (OIE), the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), and the Centers for Disease Control and Prevention (CDC), has helped to increase awareness, manage dog populations more effectively, and improve Vietnam's surveillance and diagnostic capabilities. To pursue the goal of eliminating dog-mediated rabies in Vietnam, political commitment is crucial. Resources must be made available to enforce the regulations and guidelines that will enable Vietnam to achieve greater canine rabies vaccination coverage. In this paper, the authors provide an overview of the animal and human health systems in Vietnam, as well as past, current and future directions of rabies prevention and control.

La rage est une maladie virale à l'issue mortelle faisant chaque année un nombre estimé de 59 000 victimes humaines. La plupart de ces décès surviennent dans les pays en développement d'Asie. Au Vietnam, la rage canine est endémique mais le pays poursuit activement l'objectif d'éliminer la rage de son territoire. À l'instar du Vietnam, plusieurs pays ont investi des ressources colossales pour contrôler la rage, renforçant ainsi la dimension régionale et mondiale de l'objectif d'élimination de cette maladie négligée. Au Vietnam, la rage figure parmi les cinq zoonoses hautement prioritaires prises en compte par le ministère de la Santé et le ministère de l'Agriculture et du développement rural. Les investissements consacrés à la prévention et au contrôle de la rage par le gouvernement et ses partenaires internationaux ont joué un rôle déterminant dans la réduction du nombre de décès humains dus à la rage, qui est passé de 404 cas en 1992 à 74 cas en 2017. L'élément catalyseur de cet effort a été la création en 1996 du Programme national de lutte contre la rage par le premier ministre de l'époque, ce qui a permis de renforcer les ressources et le soutien dédiés à la prévention et à la lutte contre la rage. Depuis lors, les interventions ont porté sur la création de centres de prophylaxie post-exposition sur tout le territoire, l'introduction ou la révision de la législation et des lignes directrices applicables et l'amélioration de la collaboration Une seule santé. En outre, le soutien de partenaires internationaux tels que l'Organisation mondiale de la santé animale (OIE), l'Organisation mondiale de la santé (OMS), l'Organisation des Nations Unies pour l'alimentation et l'agriculture (FAO) et les Centres pour le contrôle et la prévention des maladies (CDC, États-Unis d'Amérique) a abouti à une meilleure sensibilisation, à une gestion plus efficace des populations de chiens et à un renforcement des capacités de surveillance et de diagnostic au Vietnam. Un engagement politique fort est indispensable pour réussir à éliminer totalement la rage transmise par les chiens au Vietnam. Des ressources doivent être rendues disponibles afin de mettre en oeuvre la réglementation et les lignes directrices pertinentes et d'augmenter ainsi la couverture vaccinale de la population canine du pays. Les auteurs décrivent les systèmes de santé animale et publique du Vietnam ainsi que les orientations passées, actuelles et futures de la prévention et du contrôle de la rage dans le pays.

La rabia es una enfermedad vírica fatal, que según las estimaciones mata a 59 000 personas al año, mayoritariamente en países en desarrollo asiáticos. La rabia canina es endémica en el Vietnam, país que no obstante avanza ahora hacia la eliminación de la enfermedad. Como el Vietnam, muchos países han invertido cantidades colosales de recursos en la lucha antirrábica, subrayando con ello su compromiso con el objetivo de eliminar esta enfermedad desatendida a escala regional y mundial. El Ministerio de Salud y el Ministerio de Agricultura y Desarrollo Rural del Vietnam tienen catalogada la rabia como una de las cinco enfermedades zoonóticas que revisten máxima prioridad. Las inversiones en prevención y control de la rabia realizadas por el gobierno y por asociados internacionales han ayudado sensiblemente a reducir el número de personas muertas por la rabia, que ha pasado de 404 casos en 1992 a 74 en 2017. El catalizador de este esfuerzo fue la creación en 1996, por iniciativa del Primer Ministro, del Programa Nacional contra la Rabia, que se tradujo en un aumento del apoyo y los recursos destinados a prevenir y combatir la enfermedad. Entre otras intervenciones, desde entonces se ha multiplicado en todo el país el número de centros donde se dispensa profilaxis tras la exposición, se han promulgado o revisado leyes, decretos y directrices fundamentales y se ha mejorado la colaboración multisectorial en clave de Una sola salud. Además, el respaldo de asociados internacionales como la Organización Mundial de Sanidad Animal (OIE), la Organización Mundial de la Salud (OMS), la Organización de las Naciones Unidas para la Alimentación y la Agricultura (FAO) o los Centros para el Control y la Prevención de Enfermedades (CDC) de los Estados Unidos ha ayudado a generar una mayor conciencia del problema, a gestionar más eficazmente las poblaciones de perros y a dotar al país de mejores medios de vigilancia y diagnóstico. Para hacer realidad el objetivo de eliminar del Vietnam la rabia transmitida por perros, la voluntad política es un factor clave, pues hay que poner sobre la mesa los recursos necesarios para aplicar los reglamentos y normas que permitirán al país ampliar la cobertura de vacunación canina antirrábica. Tras trazar una panorámica de los sistemas sanitario y zoosanitario del Vietnam, los autores describen el rumbo pasado, presente y futuro de las labores de prevención y control de la rabia en el país.

Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken ß-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Interleukin-10 expression induced by adeno-associated virus vector suppresses proteinuria in Zucker obese rats.

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1±11.6 mg per mg·creatinine versus 88.8±30.0 mg per mg·creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r=-0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity.

Redescription and supplementary molecular characteristics of Aspidogaster ijimai Kawamura, 1915 (Trematoda, Aspidogastrea, Aspidogastridae), a parasite of Cyprinus carpio Linnaeus, 1758 s. lato (Actinopterygii) and freshwater bivalves in East Asia.

A detailed morphological description and ribosomal DNA (rDNA) sequence molecular data for Aspidogaster ijimai from Japan are provided. Morphological analysis, including a description of the cirrus-sac, indicate the conspecificity of Japanese and continental East Asian A. ijimai specimens. Analysis of ITS1-5.8S-ITS2 rDNA sequences of Japanese, Chinese and Russian specimens confirmed the morphological results. Phylogenetic analysis using ITS rDNA sequences confirmed that A. ijimai is a sister species for Aspidogaster chongqingensis. Median-joining network analysis showed an initial molecular differentiation step of Russian specimens from group of Japanese-Chinese samples. Our 28S rDNA results based on maximum likelihood (ML) and Bayesian (BI) phylogenetic analyses indicated well-supported monophyly of the Aspidogaster conchicola + A. ijimai group, a finding that indicates that these species are congeneric. At the same time, our data demonstrated that the genus Lobatostoma is paraphyletic and the family Aspidogastridae is polyphyletic, results that confirm previous studies.

Production and characterization of monoclonal antibodies directed against bovine milk fat globule membrane (MFGM).

Nine hybridomas secreting monoclonal antibodies (mAbs) to bovine milk fat globule membrane (MFGM) were produced from spleen cells of three immunized BALB/c mice. Several MFGM antigens recognized by some mAbs were identified as a 120 kDa protein and 67 kDa (butyrophilin), 57 kDa (PAS-6), 53 kDa (PAS-7), 33 kDa glycoproteins. The other mAbs secreted by four independent hybridoma clones recognized many broad bands ranging from 20 to 200 kDa. The 120 kDa protein and 67 kDa, 57 kDa, 53 kDa glycoproteins were detected by each mAb in the plasma membrane fraction prepared from a lactating bovine mammary gland. Moreover, mammary gland epithelium of a thin section was specifically stained with these mAbs, indicating that these mAbs directed against MFGM recognized membrane proteins and glycoproteins of lactating mammary epithelial cells. Upon heating of the MFGM in phosphate buffer, pH 7.4 at 100 degrees C for 10 min, the antigens still retained most of its reactivity to these mAbs, whereas, proteolytic cleavage by trypsin and chymotrypsin strongly reduced its reactivity to these mAbs by 60% or more except for two mAbs which recognized the 57 and 53 kDa glycoproteins, respectively.

Immunologically cross-reactive 57 kDa and 53 kDa glycoprotein antigens of bovine milk fat globule membrane: isoforms with different N-linked sugar chains and differential glycosylation at early stages of lactation.

Two glycoprotein antigens with molecular masses of 57 kDa (MGP57) and 53 kDa (MGP53) were co-purified from bovine milk fat globule membrane (MFGM) by immunoaffinity chromatography using a monoclonal antibody raised against the MFGM. Their N-terminal sequences of 22 amino acids determined were identical, and the sequence was homologous (about 60% identical) to the deduced amino acid sequence of mouse milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) (Ref. [12], Stubbs, J.D. et al., Proc. Natl. Acad. Sci. USA, 87, 8417-8421, 1990). This suggests that MGP57/53 are bovine MFGM components 15/16 (PAS-6 and PAS-7), which have recently been reported to be bovine homologs of MFG-E8. N-Glycanase treatment of these glycoproteins reduced their molecular masses, and consequently the enzymatically deglycosylated MGP57 and MGP53 converged on a single band of 50 kDa as measured by SDS-PAGE, indicating that the polypeptide portions of these two distinct glycoprotein antigens are very similar or identical and that their N-linked sugar chains contributed to minor difference in their molecular masses. Western blot analyses using lectins also revealed that they were differentially glycosylated; MGP57 was stained with concanavalin A (Con A) more strongly than MGP53, whereas MGP 53 was stained well with soybean agglutinin (SBA). Reactivity with SBA remarkably increased during early stages of lactation. Two-dimensional gel electrophoresis showed that MGP57 and MGP53 were electrically heterogeneous; from day 9 after parturition, both glycoproteins fell in almost the same range of isoelectric points between 6.4 and 7.6, also, such glycoproteins from day 1 after parturition were more acidic, probably due to terminal sialylation of their sugar chains.

Epidemiological and outcome characteristics of major burns in Tokyo.

The Tokyo Burn Unit Association (TBUA) was established in 1983 funded by the Tokyo Metropolitan Government, and is organized by 13 burn units in Tokyo. TBUA covers more than 90% of severe burn patients occurring in Tokyo, and all of the cases are registered according to the burn injury registration format. The purpose of this study is to analyze the registered data and to elucidate epidemiological and outcome characteristics of major burn injuries in Tokyo. The total of 6988 hospitalized patients had data for epidemiological analysis, and 6401 patients had complete data for outcome analysis as well, and were included in this study. The characteristic profiles for the analysis included age, sex, cause of burns, inhalation injury, %BSA, burn index (BI), length of burn unit stay, and outcome, and were analyzed by age groups. The mean age of the patients was 40.4 years, and 63% of them were male. It was noteworthy that 25% of the total patients were elderly patients over 60 years of age. Flame was the most common cause making up 45.6% followed by scalding (32.0%). The overall mortality rate was 15.4%. Inhalation injury was accompanied in 27.3% of burn patients. The mortality rate was 34.6% with inhalation injury, and 8.2% without inhalation injury. Causes of death showed that multiple organ failure made up 36.9% of total mortality, followed by sepsis 25.2 and shock 19.0%. The burn size (%BSA and BI) and inhalation injury were the factors for high mortality rate in all age groups whereas age was a predictor for high mortality in the patients older than 16 years of age. Gender was not a factor for high mortality in any age group. The mortality rate showed mildly decreasing tendency since 1995 for which implementation of skin bank was thought to be responsible.

Gene transfer into vascular cells using adeno-associated virus (AAV) vectors.

OBJECTIVES: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. METHODS: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining. RESULTS: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. CONCLUSIONS: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

Effect of lithium on function and growth of thyroid cells in vitro.

Lithium has been reported to alter thyroid function and cause goiter in some patients. To explain the mechanism of lithium action in the thyroid gland, we studied the effect of lithium on thyroid function and cell growth in FRTL-5 rat thyroid cells and on de novo thyroid hormone formation in primary cultures of porcine thyroid follicles. TSH-induced iodide uptake was suppressed at 2 mM lithium in both FRTL-5 cells and porcine follicles. In porcine thyroid follicles, iodide uptake stimulated by 8-bromo-cAMP, iodine organification, and de novo thyroid hormone formation were also reduced by lithium; however, 2 mM lithium did not inhibit TSH-induced cAMP production. In FRTL-5 cells, lithium also inhibited forskolin-stimulated iodide uptake. These results suggested that lithium exerts its effect at a step involving cAMP signal transduction rather than inhibiting cAMP production. In both FRTL-5 thyroid cells and porcine follicles, lithium enhanced cell growth in basal states (lacking TSH) and with TSH treatment. In porcine thyroid cells, the protein kinase C activator, tetradecanoyl phorbol-13-acetate, increased cell growth, and lithium had an additive effect with tetradecanoyl phorbol-13-acetate on cell growth. To examine the possibility that the action of lithium was mediated by the protein kinase C pathway, porcine cells were incubated with lithium and H7, a selective protein kinase C inhibitor. Lithium-induced cell growth was suppressed to the basal level by H7. These results suggest that lithium exerts its growth-promoting effect through the protein kinase C system.

Enhanced 5-hydroxytryptamine release from vascular adrenergic nerves in spontaneously hypertensive rats.

The release of 5-hydroxytryptamine from the vascular adrenergic nerve by periarterial nerve stimulation in spontaneously hypertensive rats (SHR) was compared with that in normotensive Wistar-Kyoto rats (WKY). The isolated mesenteric vascular bed was perfused at a constant flow rate of 5 ml/min. Vasoconstrictor responses to periarterial nerve stimulation (4, 8, 12, and 16 Hz for 30 seconds) and 5-hydroxytryptamine (1 microM), but not norepinephrine (1 nmol), were significantly greater in SHR than in WKY. After treatment with 5-hydroxytryptamine (1 microM) for 15 minutes, vasoconstrictor responses to periarterial nerve stimulation previously reduced by prazosin (50 nM) were restored and a frequency-dependent pressor response reappeared. However, 5-HT treatment did not significantly affect the pressor response to exogenously administered norepinephrine (1 nmol), which was previously inhibited by prazosin. The degree of the restoration in SHR was significantly greater than that in WKY at all frequencies used. The restoration of the pressor response to periarterial nerve stimulation after 5-hydroxytryptamine treatment did not occur in the presence of the selective 5-hydroxytryptamine2 receptor antagonists ketanserin (10 nM) or LY53857 (10 nM). In the perfused mesenteric vascular bed of both WKY and SHR prelabeled with [3H]5-hydroxytryptamine, periarterial nerve stimulation (4-16 Hz) evoked a frequency-dependent increase in tritium efflux that was abolished by Ca2+-free Krebs-Ringer solution or tetrodotoxin (100 nM) and treatment with 6-hydroxydopamine. The tritium efflux evoked by periarterial nerve stimulation was significantly greater in SHR than in WKY at all frequencies used. These results suggest that the release of 5-hydroxytryptamine from adrenergic nerve endings by periarterial nerve stimulation is enhanced in the mesenteric vascular bed of the SHR.

A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.

Gamma-rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells.

Adeno-associated virus (AAV) vector has several unique properties suited for gene therapy applications. However, relatively low efficiency of transgene expression, which is mainly due to a limited second-strand synthesis from the single-stranded AAV genome, can be a problem in some applications that require potent gene expression such as antitumor applications. Recently, gamma-ray irradiation has been reported to enhance the second-strand synthesis of the AAV genome, and consequently transgene expression. We demonstrate here that an AAV vector harboring the herpes simplex virus type-1 thymidine kinase (HSVtk) is able to kill cancer cells more efficiently when used in combination with gamma-ray irradiation. A human maxillary sinus cancer cell line, NKO-1, was efficiently killed in combination with HSVtk transduction and ganciclovir (GCV), as expected. More importantly, gamma-ray irradiation of practical dosages augmented the cytocidal effect of the HSVtk/GCV system. Southern analysis indicated that gamma-rays enhanced the double-strand synthesis of the rAAV genome in NKO-1 cells. These findings suggest that the combination of rAAVtk/GCV suicide gene therapy with radiotherapy has synergistic effects in the treatment of cancers and may lead to a reduction of the potential toxicity of both rAAVtk/GCV and gamma-ray irradiation.

Early development of Epstein-Barr virus-associated T-cell lymphoma after a living-related renal transplantation.

We herein report a case of Epstein-Barr virus (EBV)-associated T-cell lymphoma that developed within a month after a kidney transplantation. The recipient was a 37-year-old man who had evidence of a previous EBV infection. Cyclosporine, methylprednisolone, and azathioprine were used for immunosuppression, and acute rejection was treated with high-dose methylprednisolone. The lactate dehydrogenase level started to increase on day 24 and thereafter peaked on day 37 while also demonstrating progressive jaundice and a bleeding tendency. A transplant nephrectomy was done on day 37; however, the patient could not recover and eventually died of respiratory failure as a result of diffuse pulmonary edema. A pathological examination of the resected kidney revealed a diffuse proliferation of large atypical lymphoid cells in the parenchyma. Immunohistochemically, the tumor cells were positive for CD45 and T-cell marker, CD45RO, but negative for B-cell markers. EBV-encoded RNA was demonstrated within the neoplastic cells by in situ hybridization.

Effect of endothelium removal on the vasoconstrictor response to neuronally released 5-hydroxytryptamine and noradrenaline in the rat isolated mesenteric and femoral arteries.

1. The role of the vascular endothelium in the vasoconstrictor response to transmural nerve stimulation (TNS) was studied in isolated ring segments of rat mesenteric and femoral arteries. 2. In both types of artery, TNS (1 to 16 Hz) produced frequency-dependent vasoconstriction, which was abolished by 100 nM tetrodotoxin, 10 microM guanethidine or 10 nM prazosin, indicating that the response was mediated by endogenous noradrenaline (NA) released from noradrenergic nerves. NA-mediated vasoconstriction in response to TNS was significantly potentiated by removal of the endothelium. 3. In the presence of 10 nM prazosin, the reduced vasoconstriction in response to TNS was restored by incubation with 10 microM 5-hydroxytryptamine (5-HT) for 20 min. Restoration of the response to TNS was markedly attenuated by treatment with 10 nM ketanserin, 100 nM tetrodotoxin, or 10 microM guanethidine, indicating that the restored response was mediated by 5-HT released from noradrenergic nerves. Vasoconstriction mediated by 5-HT in response to TNS was not modified by removal of the endothelium. 4. In both types of artery with intact endothelium, treatment with 3 microM methylene blue potentiated the NA-mediated contractile response to TNS, but did not potentiate the 5-HT-mediated response to TNS. 5. In both types of artery, the contractile responses to exogenous NA and 5-HT were potentiated by removal of the endothelium. 6. These results suggest that endothelial cells regulate neurogenic vasoconstriction by releasing endothelium-derived relaxing factor. Furthermore, it appears likely that the response to neuronally released 5-HT is not affected by the endothelium.

Atheroprotective effect of estriol and estrone sulfate on human vascular smooth muscle cells.

In patients with atherosclerosis, fibrosclerotic focuses are induced by multiplication of vascular smooth muscle cells (VSMC), and they are regulated by cytokines and regulators. There have been few reports about the atheroprotective effect of estriol (E(3)). Estrone sulfate (E(1)-S) is the predominant estrogen of conjugated equiline estrogens, which is commonly used in hormone replacement therapy, but it should be hydrolyzed by steroid sulfatase (STS) to enter the cells of target tissues. The purpose of this study was to detect STS in VSMC and to investigate whether E(3) and E(1)-S have atheroprotective effects like E(2). First, we detected the presence of STS mRNA in VSMC by in situ hybridization. We then examined the changes in the expression of mRNAs of cytokines, namely, PDGF-A chain, IL-1, IL-6 and TGF-beta, in VSMC, in the presence and absence of E(3) and estrogens. As a result, the expression of PDGF-A chain, IL-1 and IL-6 mRNAs was suppressed by E(3) (P<0.05 vs control) significantly like E(1)-S and E(2), but that of TGF-beta mRNA was not significantly affected by any estrogen. These results indicate that E(1)-S can be hydrolyzed by STS in VSMC, and that E(3) may regulate the cytokines by suppressing the production of mRNAs. It is suggested that there is a possibility of E(1)-S and E(3) having a direct effect on vessels in atherogenesis.

Osteoporosis influences the late period of fracture healing in a rat model prepared by ovariectomy and low calcium diet.

To elucidate the influence of osteoporosis on the fracture healing, we produced a rat osteoporosis model by ovariectomy and by maintaining a low calcium diet; and monitored the healing process radiographically, histologically, and biomechanically for 12 weeks. Radiologic, histologic and biomechanical findings of the fracture areas 6 weeks after making the fractures were almost identical in both the osteoporosis group and the control group. However, 12 weeks after making the fractures, newly generated bones in the osteoporosis group showed histological osteoporotic changes and their bone mineral density on the fracture site decreased. These findings show that estrogen-deficient and low calcium conditions greatly affect the bone in the later period of the healing process, but do not affect remarkably the early healing period. This is clinically important when we consider fracture treatments for patients with osteoporosis due to menopause.