A congenital CMV infection model for follow-up studies of neurodevelopmental disorders, neuroimaging abnormalities, and treatment.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.

Significant antitumor activity of oncolytic adenovirus expressing human interferon-beta for hepatocellular carcinoma.

BACKGROUND: Human interferon-beta (IFN-beta) has been widely used in gene therapy for its antitumor activity but its therapeutic effect is limited. The conditionally replicative adenovirus ONYX-015 (a E1B-55-kDa-deleted adenovirus) targets well to tumor cells, but is not potent enough to cause significant tumor regression. To solve these problems, a tumor-selective replicating adenovirus expressing IFN-beta was constructed in this study. METHODS: The oncolytic adenoviruses were generated by homologous recombination in packaging cells. The expression of the IFN-beta protein was detected by enzyme-linked immunosorbent assay (ELISA). The antitumor efficacy of ZD55-IFN-beta was evaluated in cell lines and human hepatocellular carcinoma xenografts in nude mice. RESULTS: ZD55-IFN-beta can express much more IFN-beta than Ad-IFN-beta because of the replication of the ZD55 vector. Our data showed that ZD55-IFN-beta could exert a strong cytopathic effect on tumor cells (about 100-fold higher than Ad-IFN-beta or ONYX-015). Moreover, no obvious cytotoxic or apoptotic effects were detected in normal cells infected with ZD55-IFN-beta. CONCLUSIONS: The antitumor efficacy of IFN-beta could be significantly improved due to the increased gene expression level from the tumor-selective replicating vector. The oncolytic adenovirus expressing IFN-beta may provide a novel approach for cancer gene therapy.

An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy.

ONYX-015 is an attractive therapeutic adenovirus for cancer because it can selectively replicate in tumor cells and kill them. To date, clinical trials of this adenovirus have demonstrated marked safety but not potent enough when it was used alone. In this paper, we put forward a novel concept of Gene-ViroTherapy strategy and in this way, we constructed an armed therapeutic oncolytic adenovirus system, ZD55-gene, which is not only deleted of E1B 55-kD gene similar to ONYX-015, but also armed with foreign antitumor gene. ZD55-gene exhibited similar cytopathic effects and replication kinetics to that of ONYX-015 in vitro. Importantly, the carried gene is expressed and the expression level can increase with the replication of virus. Consequently, a significant antitumoral efficacy was observed when ZD55-CD/5-FU was used as an example in nude mice with subcutaneous human SW620 colon cancer. Our data demonstrated that ZD55-gene, which utilizing the Gene-ViroTherapy strategy, is more efficacious than each individual component in vivo.

Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells. It may be used as a tool for driving a gene to kill tumors specifically. To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells. A tumor-specific expression plasmid phTERT-CD was constructed, in which the E. coli cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold. Mixed cell experiments showed a strong "bystander effect" on CD-negative cells. Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c. inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days. These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer.

Adenoviral transfer of human interleukin-10 gene in lethal pancreatitis.

AIM: To evaluate the therapeutic effect of adenoviral-vector-delivered human interleukin-10 (hIL-10) gene on severe acute pancreatitis (SAP) rats. METHODS: Healthy Sprague-Dawley (SD) rats were intraperitoneally injected with adenoviral IL-10 gene (AdvhIL-10), empty vector (Adv0) or PBS solution. Blood, liver, pancreas and lung were harvested on the second day to examine hIL-10 level by ELISA and serum amylase by enzymatic assay. A SAP model was induced by retrograde injection of sodium taurocholate through pancreatic duct. SAP rats were then administered with AdvhIL-10, Adv0 and PBS solution by a single intraperitoneal injection 20 min after SAP induction. In addition to serum amylase assay, levels of hIL-10 and tumor necrosis factor-alpha (TNF-alpha) were detected by RT-PCR, ELISA and histological study. The mortality rate was studied and analyzed by Kaplan-Meier and log rank analysis. RESULTS: The levels of hIL-10 in the pancreas, liver and lung of healthy rats increased significantly after AdvhIL-10 injection (1.42 ng/g in liver, 0.91 ng/g in pancreas); while there was no significant change of hIL-10 in the other two control groups. The concentration of hIL-10 was increased significantly in the SAP rats after AdvhIL-10 injection (1.68 ng/g in liver, 1.12 ng/g in pancreas) compared to the other two SAP groups with blank vector or PBS treatment (P<0.05). The serum amylase levels remained normal in the AdvhIL-10 transfected healthy rats. However, the serum amylase level was significantly elevated in the other two control SAP rats. In contrast, serum amylase was down-regulated in the AdvhIL-10 treated SAP groups. The TNF-alpha expression in the AdvhIL-10 treated SAP rats was significantly lower compared to the other two control SAP groups. The pathohistological changes in the AdvhIL-10 treated group were better than those in the other two control groups. Furthermore, the mortality of the AdvhIL-10 treated group was significantly reduced compared to the other two control groups (P<0.05). CONCLUSION: Adenoviral hIL-10 gene can significantly attenuate the severity of SAP rats, and can be used in the treatment of acute inflammation process.

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain.

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

[Killing effect of Ad.TERT-TRAIL on tumor cell lines and its mechanism].

BACKGROUND & OBJECTIVE: Oncolytic adenovirus Ad.TERT, a novel tumor-specific proliferating virus, has been constructed by replacing normal promoter of mild-type adenovirus E1A with promoter of human telomerase reverse transcriptase (hTERT). In vitro and in vivo experiments confirmed that Ad.TERT has antitumor effect. This study was to construct Ad.TERT-TRAIL through inserting apoptosis gene trail into Ad.TERT, and explore its antitumor effect and mechanism. METHODS: Plasmid pZhTERT-trail and adenovirus packaging plasmid pBHGE3 were homologously recombined in HEK293 cells to construct Ad.TERT-TRAIL. Ad.TERT-TRAIL was identified by polymerase chain reaction (PCR), and confirmed by Western blot. Killing effects of Ad.TERT-TRAIL and Ad.TERT on 3 tumor cell lines, SW620, BEL-7404 and Bcap-37, and a normal cell line NHLF were detected by crystal violet dye method or MTT assay. Expression of Caspase-3 in Ad.TERT-TRAIL-, and Ad.TERT-transfected SW620 cells was detected by Western blotu cell apoptosis was detected by flow cytometry (FCM). RESULTS: The 860 bp-length trail gene has been amplified by PCR. Western blot showed trail and E1A only expressed in tumor cells, which confirmed the successful construction of Ad.TERT-TRAIL. Killing effects of Ad.TERT-TRAIL on tumor cells were 10-100 times as strong as that of Ad.TERTu while both of them had little effects on normal cells. After 3 days infection (100 multiple of infection, MOI), survival rate of Ad.TERT-TRAIL-infected SW620 cells was 4%, but that of Ad.TERT-infected SW620 cells was 56%u both viruses had little effects on NHLF cells. Expression of Caspase-3 was higher in Ad.TERT-TRAIL-infected SW620 cells than in Ad.TERT-infected SW620 cells. Apoptosis rate of Ad.TERT-TRAIL-infected SW620 cells was 4 times as high as that of Ad.TERT-infected SW620 cells. CONCLUSIONS: Ad.TERT-TRAIL has much stronger antitumor effect than Ad.TERT. Its effect might relate with inducement effects of trail gene on expression of Caspase-3, and apoptosis of tumor cells.

Human interleukin 10 gene therapy decreases the severity and mortality of lethal pancreatitis in rats.

BACKGROUND: Studies have proven the validity of interleukin-10 (IL-10) in the treatment of experimental pancreatitis. Prophylactic human IL-10 (hIL-10) gene treatment attenuated the severity in cerulein models. Our research aims to study whether the therapeutic hIL-10 gene could decrease both severity and mortality in a lethal pancreatic model. METHODS: Severe acute pancreatitis (SAP) was induced by sodium taurocholate. A plasmid-hIL-10 construct (pcDNA3-hIL-10) complexed with cationic liposomes was administered to SAP rats by a single intraperitoneal injection. Levels of hIL-10 in the pancreas, liver, and lungs were determined by ELISA kits. The severity of pancreatitis was assessed in terms of serum amylase, histology, and tissue tumor necrosis factor alpha (TNF-alpha). Mortality, observed for 7 days, was evaluated for gene therapy or control groups. RESULTS: After hIL-10 gene therapy, hIL-10 levels in the pancreas, liver, and lungs increased significantly and the serum amylase, tissue TNF-alpha, and histological changes in pancreas, liver, and lungs decreased markedly. Therefore, mortality was significantly reduced in the hIL-10 gene therapy group, in which 70% of rats survived in the 7-day observation, while only 10% survived in untreated groups (P < 0.05). CONCLUSION: We found that liposome/hIL-10 gene therapy decreased severity and mortality in SAP, even carried out after SAP establishment, predicting a more convenient shift to clinical applications.