A simple detection system for adenovirus receptor expression using a telomerase-specific replication-competent adenovirus.

Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunohistochemistry. Here, we show a simple system for detection and assessment of functional CAR expression in human tumor cells, using the green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 h after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy.

Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells.

Heparan sulfate proteoglycans is a major component of the cell surface and extracellular matrix and functions as a barrier against cationic molecules and macromolecules. Heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. To inhibit human heparanase expression in human cancer cells, we constructed an adenoviral vector carrying a full-length human heparanase cDNA in an antisense orientation (Ad-AS/hep). Increased heparanase expression in T.Tn human esophageal cancer cells and A549 human lung cancer cells after infection with an adenovirus vector expressing the human heparanase gene (Ad-S/hep) was specifically inhibited by simultaneous infection with Ad-AS/hep in a dose-dependent manner. A modified Boyden chamber assay demonstrated that infection with Ad-AS/hep significantly inhibited in vitro invasion of A549 cells after Ad-S/hep infection. Moreover, intrathoracic administration of Ad-AS/hep reduced the number and size of heparanase-expressing A549 tumors implanted intrathoracically into BALB/c-nu/nu mice. Our results suggest that heparanase contributes to the invasive phenotype of tumor cells, and that antisense-mediated inhibition of heparanase activity may be efficacious in the prevention of pleural dissemination.

Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells.

Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many different signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no effect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53- and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a specific inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitin-proteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer.

Optical coherence tomography evaluation of idiopathic macular hole treatment by gas-assisted posterior vitreous detachment.

PURPOSE: To report a case of idiopathic macular hole, with vitreoretinal traction confirmed by optical coherence tomography that was successfully treated by a single intravitreous perfluoropropane (C(3)F(8)) gas bubble injection. METHODS: Case report. A 65-year-old patient with idiopathic macular hole (stage 2, one eye) received an intravitreous gas injection and was prospectively followed with optical coherence tomography. RESULTS: A complete posterior vitreous detachment was achieved within 6 weeks after gas injection. Visual acuity improved from 20/80 to 20/25 by 10 months of follow-up. Optical coherence tomography disclosed vitreoretinal traction release and macular hole closure. No complications were related to the procedure. CONCLUSION: This simple procedure can assist a complete posterior vitreous detachment with relief of the hyaloid-foveolar traction, facilitating macular hole closure.

In vivo characteristics of a transplantable Marek's disease lymphoblastoid cell line, MDCC-MSB1-41C.

A Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, was highly transplantable and lethal for chickens. Autopsies showed extensive metastasis in various organs. The transplantabilities of the parent cell line, MDCC-MSB1, and another derivative line, MDCC-MSB1-33C, were transient. MD virus (MDV) could be isolated from the kidneys but not from the peripheral blood leukocytes of chickens inoculated with the MSB1-41C cell line. In addition, anti-MDV antibodies were produced both in chickens inoculated with this cell line and in controls raised with inoculated chickens, but several attempts to isolate MDV from this cell line in vitro failed.

Morphological studies of Gross virus-induced lymphoblasts by scanning electron microscopy.

The surface of Gross virus-induced murine lymphoblasts and C-type virus particles budding from these cells were investigated under the scanning electron microscope (SEM). The cells appeared spindle-shaped or roughly-rounded with extensive surface features consisting of microvilli, blebs and ruffled membranes. C-type virus particles were detected on the cell membrane as small spherical particles, distinguishable from the microvilli. Clustered virions were observed in some cases. However, the distribution of virions appeared to be random. The surface of the virion was smooth and had no globular units at high magnification. These morphological observations were confirmed in ultrathin sections.

Reactivatable latency of three avirulent strains of herpes simplex virus type 1 after intranasal inoculation in mice.

In order to elucidate the mechanism of latent infection of herpes simplex virus (HSV), reactivatable latency of three avirulent strains (SKO-1B, -GCr Miyama, SKa) of HSV type 1 was comparatively examined in a mouse latency model. The SKO-1B strain showed high rate of virus reactivation from explanted trigeminal ganglia without n-butyrate enhancement, while the other two strains showed a very low rate of virus reactivation in the absence of n-butyrate. In the presence of n-butyrate, however, the rate of the -GCr Miyama strain jumped to a comparable level with that of SKO-1B, although the rate of SKa remained at a low level. A more precise follow-up experiment changing the virus dose highlighted the difference of the ability to reactivate from the latent state between SKO-1B and -GCr Miyama. Virus titer in trigeminal ganglia during acute phase, infectivity to cell lines of neural origin, and susceptibility to acyclovir and phosphonoacetate were assayed to know the reasons for the variation in the ability of reactivatable latency among these strains. It was concluded that the reduced infectivity to neural cells, and limited ability of reactivatable latency shown by the SKa strain could mainly be attributed to the deficiency of thymidine kinase activity.

Neurovirulent strains of herpes simplex virus type 1 are not necessarily competent for reactivatable latency.

Ability of two neurovirulent strains (F and +GC (LPV) Miyama) of herpes simplex virus type 1 (HSV-1) to establish and maintain reactivatable latency in trigeminal ganglia (TG) was compared after intranasal inoculation of mice. The +GC (LPV) Miyama strain showed a very low rate of virus reactivation in explant cultures of TG, while the F strain showed a high rate of reactivation. These data indicate that neurovirulent strains of HSV-1 are not always competent for reactivatable latency, although most virulent strains of HSV-1 thus far reported were competent for reactivatable latency.

Transretinal feeder vessel ligature in von Hippel-Lindau disease.

PURPOSE: To present a new technique called transretinal feeder vessel ligature for the treatment of retinal angiomas. METHODS: Case report of a patient with peripheral retinal angiomas previously treated unsuccessfully with photocoagulation who responded to this new, alternative surgical treatment. RESULTS: The retinal angiomas decreased in size although two new feeder vessels appeared and the lesions showed a regression pattern after additional laser therapy over the vascular tumors. CONCLUSIONS: A transretinal feeder vessel ligature in association with vitrectomy and photocoagulation may be useful for some advanced or non-responsive cases of retinal angiomas.

[Structure and assembly of human beta herpesviruses].

This review is concerned with the structure and assembly of HCMV, HHV6 and HHV7. A characteristic ultrastructural feature common to all these viruses is a distinct tegumentary coating of intracytoplasmic capsids. The tegument structure is also distinctly seen in the virions of HHV6 and HHV7. Morphologically, acquisition of the tegument was observed to have taken place in the cytoplasm. Immunoelectron microscopic studies of HCMV infected cells, however, have demonstrated the existence of a tegument protein, pp150, on the surface of intranuclear capsids as well as on capsids in the cytoplasm and in extracellular virions. In addition, another tegument protein, pp65 has been detected within the matrix of cytoplasmic and extracellular dense bodies but not in virions. The molecular mechanism of the assembly of beta herpesviruses was also discussed.

A novel translational approach for human malignant pleural mesothelioma: heparanase-assisted dual virotherapy.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that is related to asbestos exposure. MPM is characterized by rapid and diffuse local growth in the thoracic cavity, and it has a poor prognosis because it is often refractory to conventional therapy. Although MPM is an extraordinarily challenging disease to treat, locoregional virotherapy may be useful against this aggressive disease because of the accessibility by intrapleural virus delivery. In this study, we show that telomerase-specific, replication-selective adenovirus OBP-301 can efficiently infect and kill human mesothelioma cells by viral replication. Intrathoracic administration of virus significantly reduced the number and size of human mesothelioma tumors intrathoracically implanted into nu/nu mice. A high-definition, fluorescence optical imaging system with an ultra-thin, flexible fibered microprobe clearly detected intracellular replication of green fluorescent protein-expressing oncolytic virus in intrathoracically established mesothelioma tumors. As the extracellular matrix (ECM) may contribute to the physiological resistance of a solid tumor by preventing the penetration of therapeutic agents (including oncolytic viruses), we also examined whether the co-expression of heparanase, an endoglucuronidase capable of specifically degrading heparan sulfate, that influences the physiological barrier to macromolecule penetration, can modify the permeability of the ECM, resulting in profound therapeutic efficacy. Co-injection of OBP-301 and a replication-defective adenovirus (Ad-S/hep)-expressing heparanase resulted in more profound antitumor effects without apparent toxicity in an orthotopic pleural dissemination model. Our results suggest that intrathoracic dual virotherapy with telomerase-specific oncolytic adenovirus in combination with heparanase-expressing adenovirus may be efficacious in the prevention and treatment of pleural dissemination of human malignant mesothelioma.

Virus-mediated oncolysis induces danger signal and stimulates cytotoxic T-lymphocyte activity via proteasome activator upregulation.

Dendritic cells (DCs) are the most potent antigen-presenting cells and acquire cellular antigens and danger signals from dying cells to initiate antitumor immune responses via direct cell-to-cell interaction and cytokine production. The optimal forms of tumor cell death for priming DCs for the release of danger signals are not fully understood. OBP-301 (Telomelysin) is a telomerase-specific replication-competent adenovirus that induces selective E1 expression and exclusively kills human cancer cells. Here, we show that OBP-301 replication produced the endogenous danger signaling molecule, uric acid, in infected human tumor cells, which in turn stimulated DCs to produce interferon-gamma (IFN-gamma) and interleukin 12 (IL-12). Subsequently, IFN-gamma release upregulated the endogenous expression of the proteasome activator PA28 in tumor cells and resulted in the induction of cytotoxic T-lymphocytes. Our data suggest that virus-mediated oncolysis might be the effective stimulus for immature DCs to induce specific activity against human cancer cells.

Giant cell-forming variants of the Miyama strain of type 1 herpes simplex virus which differ in fusion activity.

During serial passages of a non-giant cell-forming variant (-GCr) of the Miyama strain of type 1 herpes simplex virus, a new giant cell-forming variant named +GC(81) was isolated. CPE induced by this isolate was compared with that by -GCr and also by +GC(LPV), a derivative strain of the +GC variant of the Miyama strain. Rounding of single cells was observed after infection with -GCr. Remarkable syncytial formation was induced by +GC(LPV), the syncytia containing hundreds of nuclei, while small giant cells were formed by +GC(81). The reason for the appearance of +GC variants that differ in fusion capacity is discussed.

In vitro cytopathology and pathogenicity to inbred mice shown by five variants of a laboratory strain of type 1 herpes simplex virus.

The in vitro cytopathology and the neurovirulence to inbred mice demonstrated by five variants originally derived from one laboratory strain (Miyama) of type 1 herpes simplex virus (HSV-1) were studied comparatively. Three of the variants are syncytial [+GC (LPV), +GC (SPV), +GC (81)] and two are non-syncytial [-GCr and -GCf]. The size of plaques produced by the five variants was found to be in the order of +GC (LPV) greater than +GC (81) greater than +GC (SPV) greater than -GCf greater than -GCr. The pathogenicity of these variants was compared in three kinds of inbred mice (AKR, C 3 H/He and C 57 BL) after intraperitoneal (IP) or intracerebral (IC) inoculation. The +GC (LPV) variant was the most virulent as shown by the highest mortality of mice by either route of inoculation. The other four variants caused death of mice only after IC inoculation, and among these variants, +GC (81) was shown to be the most virulent. These data indicate that so far as these five variants of the Miyama strain of HSV-1 are concerned, neurovirulence is positively correlated with their cell fusion activity or the size of plaques which they produce. Pre-IP-inoculation with any of the less virulent variants [-GCr, +GC (SPV) and +GC (81)] protected mice from subsequent lethal infection with +GC (LPV) by the same route of inoculation.

Ultrahigh-resolution scanning electron microscopy of MDCK cells infected with influenza viruses.

Ultrahigh-resolution scanning electron microscopy of MDCK cells infected with influenza viruses was carried out by the uncoated uranyl acetate staining preparation method. Ridge-like protrusions were detected on virus particles and were considered to be an indication of aggregated glycoprotein spikes. Bundles of filamentous virus particles along with bacillary virus particles were encountered on MDCK cells infected with freshly isolated strains of type A virus. Filamentous virus particles that were twisted like ropes were observed on MDCK cells infected with strains of type B virus.

Mechanism of differences in pathogenicity between two variants of a laboratory strain of herpes simplex virus type 1.

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV-1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the -GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated -GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV-1 was shown to be age-dependent.

Electron microscopic study on vaccinia virus release.

FL cells infected with the IHD-W strain of vaccinia virus were studied by scanning and transmission electron microscopy. A large number of naked virus particles were found to accumulate beneath the host cell plasma membrane and to protrude from the cell surface. It was seen in some cases that naked viral particles were released by budding not only from the cell surface but also from the surface of cytoplasmic packets which were seen along the cell periphery.