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Impaired insulin receptor (IR) activity has been found in various models of insulin resistance, including models of injury or critical illness and Type 2 diabetes. However, mechanisms that modulate IR function remain unclear. With an animal model of critical-illness diabetes, we found insulin-induced IR tyrosine phosphorylation in the liver was impaired as early as 15 min following trauma and hemorrhage. Possible mechanisms for this defect were examined, including IR protein levels and IR posttranslational modifications. The total amounts of hepatic IRα and IRβ subunits and the membrane localization of the IR were not altered by trauma and hemorrhage, and, likewise, no change in IR tyrosine nitration was found in the liver. However, there was a decrease in the level of protein O-linked β-N-acetlyglucosamine (O-GlcNac) modification on Ser/Thr in the liver following trauma and hemorrhage. Inhibition of JNK increased IR O-GlcNac modification, implicating an involvement of JNK. These findings suggest that a balance between O-GlcNac modification and JNK-induced phosphorylation may exist, with decreased Ser/Thr O-GlcNac modification following trauma and hemorrhage, allowing JNK to phosphorylate the IR on neighboring Ser/Thr residues, which subsequently inhibits IR activity. The present studies suggest potential mechanisms of hemorrhage-induced defects in IR activity and a potential role for acutely decreased O-GlcNac and increased serine phosphorylation of the IR.

Insulin resistance and metabolic dysfunction are common following injury. Polytrauma is defined as combined injuries to more than one body part or organ system, and is common in modern warfare, as well as automobile and industrial accidents. Polytrauma can include any combination of burn injury, fracture, hemorrhage, trauma to the extremities, and blunt or penetrating trauma. Multiple minor injuries are often more deleterious than a more severe single injury. To investigate the mechanisms of development of insulin resistance following injury, we have developed a rat model of polytrauma which combined soft tissue trauma with burn injury and penetrating gastrointestinal (GI) trauma. Male Sprague-Dawley rats were subjected to a laparotomy plus either a 15-18% total body surface area scald burn or a single puncture of the cecum (CLP) with a G30 needle, or the combination of both burn and CLP injuries (polytrauma). We examined the effects of polytrauma which increased markers of hepatic endoplasmic reticulum (ER) stress, and increased hepatic Trib3 mRNA levels coincident with reduced insulin-inducible insulin signaling. Phosphorylation/activation of the insulin receptor (IR) and AKT were decreased at 24, but not 6h following polytrauma. These results demonstrate a complex, time-dependent development of hepatic ER-stress and a diminished response to insulin, which were among the pathological sequelae following polytrauma.

Background Oncolytic adenoviruses provide a promising alternative for cancer treatment. Recently, adjuvant interferon (IFN)-alfa has shown significant survival benefits for pancreatic cancer, yet was impeded by systemic toxicity. To circumvent these problems adenovirus with high-level targeted IFN-alfa expression can be generated. Methods Conditionally replicative adenoviruses (CRAds) with improved virulence and selectivity for pancreatic cancer were generated. The vectors were tested in vitro, in vivo, and in human pancreatic cancer and normal tissue specimens. Results Adenoviral death protein and fiber modifications significantly improved oncolysis. CRAds selectively replicated in vitro, in vivo and showed persistent spread in cancer xenografts. They showed high-level replication in human pancreatic cancer specimens, but not in normal tissues. Improved IFN-CRAd oncolytic efficiency was shown. Conclusions Optimized cyclooxygenase-2 CRAds show highly favorable effects in vitro and in vivo. We report a pancreatic cancer–specific, highly virulent, IFN-expressing CRAd, and we believe that adenovirus-based IFN therapy offers a new treatment opportunity for pancreatic cancer patients.

Polytrauma is a combination of injuries to more than one body part or organ system. Polytrauma is common in warfare, and in automobile and industrial accidents. The combination of injuries can include burn, fracture, hemorrhage, and trauma to the extremities or specific organ systems. Resistance to anabolic hormones, loss of muscle mass, and metabolic dysfunction can occur following injury. To investigate the effects of combined injuries, we have developed a highly reproducible rodent model of polytrauma. This model combines burn injury, soft tissue trauma, and penetrating injury to the gastrointestinal (GI) tract. Adult, male Sprague–Dawley rats were anesthetized with pentobarbital and subjected to a 15–20% total body surface area scald burn, or laparotomy and a single puncture of the cecum with a G30 needle, or the combination of both injuries (polytrauma). In the current studies, the inflammatory response to polytrauma was examined in skeletal muscle. Changes in skeletal muscle mRNA levels of the proinflammatory cytokines TNF‐α, IL‐1β, and IL‐6 were observed following single injuries and polytrauma. Increased expression of the E3 ubiquitin ligases Atrogin‐1/FBX032 and TRIM63/MuRF‐1 were measured following injury, as was skeletal muscle insulin resistance, as evidenced by decreased insulin‐inducible insulin receptor (IR) and AKT/PKB (Protein Kinase B) phosphorylation. Changes in the abundance of IR and insulin receptor substrate‐1 (IRS‐1) were observed at the protein and mRNA levels. Additionally, increased TRIB3 mRNA levels were observed 24 h following polytrauma, the same time when insulin resistance was observed. This may suggest a role for TRIB3 in the development of acute insulin resistance following injury.

Recombinant adenovirus (Ad) vectors can initiate an inflammatory response, limiting its use in gene therapy and basic research. Despite increased efforts to better understand Ad infection, little is known about how it affects cellular metabolic responses. In the current studies, we explored the effects of Ad vectors on insulin signaling molecules and glucose homeostasis. Nonreplicative Ad vectors were injected into rats through the tail vein, and at 4–13 days postinjection insulin signaling and glucose tolerance were examined. Ad vector infection significantly reduced total levels of the insulin receptor (IR), and insulin receptor substrates 1 and 2 (IRS-1, IRS-2) in the liver of rats, resulting in decreased insulin-induced tyrosine phosphorylation of IR, IRS-1, and IRS-2, and decreased interaction of IRS-1 and IRS-2 with phosphoinositide 3-kinase (PI3K). In addition, Ad infection resulted in impaired systemic glucose homeostasis, which recovered by 13 days, after the protein levels of IR, IRS-1, and IRS-2 had started to normalize. Expression of a TNF inhibitor or Kupffer cell depletion attenuated the Ad vector-induced decreases of insulin signaling molecules, indicating a potential role of Kupffer cell activation in this process. These studies provide evidence that systemic administration of Ad vectors can impair insulin signaling in liver, resulting in altered systemic glucose metabolism. Thus, effects of Ad vector infection on insulin action and glucose metabolism need to be considered when Ad vectors are used in research or gene therapy and may be more broadly applicable to other viral agents.

In vivo monitoring of conditionally replicative adenovirus (CRAd) replication and assessing its correlation to CRAd biological effects are necessary for the clinical development of gene therapy. Noninvasive bioimaging is one current approach which can monitor in vivo CRAd replication and functional effect. Here we describe a novel cyclooxygenase-2 (Cox2) promoter-controlled CRAd that was modified to contain firefly luciferase in its E3 region; this modification permitted serial bioluminescence imaging of viral replication in vitro and in vivo. In vitro luciferase expression correlated with viral replication and cytolytic effect. In vivo bioluminescence imaging showed dynamic representation of the viral replication level in athymic nude mice bearing subcutaneous tumor xenografts. Importantly, in vivo luciferase bioluminescence measured 6 days after viral administration significantly correlated with CRAd antitumor effect at day 36. Thus, our system could detect viral replication and predict in vivo therapeutic outcome based on early imaging. Further development of this approach may improve patient safety, enhance clinical trial conduct, and provide mechanistic insight into CRAd function in vivo. (Cancer Sci 2009; 00: 000–000)

Background The addition of interferon (IFN) alpha to adjuvant chemoradiotherapy regimens resulted in remarkable improvements in survival for pancreatic cancer patients. However, systemic toxicities and insufficient levels of IFN at the tumor sites have limited its widespread adoption in treatment schemes. We have previously developed an IFN-expressing conditionally replicative oncolytic adenovirus and demonstrated its therapeutic effects both in vitro and in vivo. Here, the same vectors were tested in a syngeneic and immunocompetent Syrian hamster model to better understand the roles of adenoviral replication and of the pleiotropic effects of IFN on pancreatic tumor growth suppression. Methods Oncolytic adenoviruses expressing human or hamster IFN were designed and generated. Viral vectors were tested in vitro to determine qualitative and quantitative cell viability, cyclooxygenase 2 (Cox2) promoter activity, and IFN production. For the in vivo studies, subcutaneous hamster pancreatic cancer tumors were treated with 1 intratumoral dose of virus. Similarly, 1 intraperitoneal dose of virus was used to prolong survival in a carcinomatosis model. Results All cell lines tested demonstrated Cox2 promoter activity. The oncolytic potential of a replication competent adenovirus expressing the IFN cytokine was clearly demonstrated. These viruses resulted in significant tumor growth suppression and survival increases compared with controls in a hamster model. Conclusion The profound therapeutic potential of an IFN-expressing oncolytic adenovirus for the treatment of pancreatic cancer was demonstrated in a syngeneic Syrian hamster model. These results strongly suggest the potential application of our viruses as part of combination regimens with other therapeutics.

Background: Conditionally replicative adenoviruses represent a promising strategy to address the limited effi cacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host – vector interactions in vivo have not been completely evaluated. Methods: We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fl uorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IXtdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fl uorescence microscopy. Athymic mice ( n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fl uorescence noninvasive imaging system. Correlations between fl uorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson’s correlation coeffi cient ( r ). Results: The red fl uorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding . The fl uorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication effi ciency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations. Conclusions: Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo. [J Natl Cancer Inst 2006;98:203 – 14]

Macrophage-derived factors, including TNF-α, are known as important inducers of insulin resistance. However, the role of macrophages in insulin resistance in the liver is unclear. Hyperglycemia and insulin resistance commonly occur following acute injuries or critical illness, referred to as "critical illness diabetes." In the present studies, the roles of macrophages in hepatic insulin resistance following surgical trauma and hemorrhage were investigated. Intravenous administration of gadolinium chloride or clodronate-liposome resulted in depletion of macrophages in both liver and spleen of rats. Macrophage depletion by either gadolinium chloride or clodronate-liposome did not prevent the development of trauma and hemorrhage-induced insulin resistance in the liver of rats, as indicated by impaired hepatic insulin signaling following a 90-minute hemorrhage period. Similarly, hepatic insulin resistance still developed in rats after removal of the spleen (splenectomy). In contrast, macrophage depletion significantly reversed the hepatic insulin resistance several hours later, following resuscitation. As a comparison, splenectomy resulted in improvement in hepatic insulin signaling following resuscitation, but to a lesser extent, suggesting that both liver and spleen resident macrophages have a role in the continuation of hepatic insulin resistance following resuscitation. These studies demonstrated that the initial development of insulin resistance in liver is macrophage-independent in a rodent model of critical illness diabetes, whereas both liver and spleen macrophages have a role in the later maintenance of the insulin-resistant state, following resuscitation.

To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

The employment of conditionally replicative adenoviruses (CRAd) constitutes a promising alternative for cancer treatment; however, in the case of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor-specific promoter and relative resistance to adenovirus infection have hampered the construction of CRAds with clinically applicable specificity and efficacy. By combining transcriptional targeting with infectivity enhancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) promoter-controlled replicative viral agents for the treatment of EAC. We used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral (Ad) fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob. The Cox-2 promoter was highly active in EAC, whereas showing no significant activity in Cox-2-negative cell lines and primary cells isolated from normal mouse esophagus and stomach. Evaluation of infectivity-enhanced vectors revealed that the transduction and virus-cell binding ability of Ad5/Ad3-chimera were significantly more efficient than that of unmodified and Arg-Gly-Asp (RGD)-modified vectors. All of the Cox-2 CRAds demonstrated replication and subsequent oncolysis in EAC cells but not in Cox-2-negative cells in vitro, thus confirming the dependence of their replication on the Cox-2 promoter activity. Ad5/Ad3 CRAds exhibited significantly improved oncolysis and progeny production compared with unmodified and RGD-modified vectors without sacrificing tumor selectivity. Whereas unmodified and RGD-modified CRAds showed insignificant therapeutic effect in vivo, Ad5/Ad3 CRAds remarkably suppressed tumor growth of established xenografts in mice. Thus, our studies have demonstrated that Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds are selective and potent agents for the treatment of EAC.

Top of pageAbstract Although anti-androgen therapy is initially effective for advanced prostate cancer, all patients eventually progress to hormone-refractory (HR) prostate cancer (PC) due to biological selection. In this stage, a median survival is only 6-12 months. Therefore, new therapeutic approaches for hormone-refractory prostate carcinoma (HRPC) are needed. Conditionally replicative adenoviruses (CRAds) are attractive anti-cancer agents. These agents are designed to replicate specifically in tumor cells followed by spread of the viral progeny to neighboring cancer cells. We used the cyclooxygenase-2 (COX-2) promoter for transcriptional targeting of tumor, which is active in many cancers but minimally active in normal tissues, including the liver. The COX-2 promoter (-1432/+59: COX-2L) was tested with luciferase (Luc) expression vectors in PC-3, Du-145, and LnCap prostate cancer cell lines. Very strong COX-2 promoter activity comparable to the CMV promoter was observed in PC-3 and Du-145 HRPC cell lines. We next examined infectivity enhancement in the PC cells by using fiber modification of Ad5 vectors, including incorporation of an RGD4C motif into the HI loop (RGD) and replacement of the Ad5 knob region with the Ad3 knob (Ad5/3 chimera),. Three CMV promoter driven Luciferase expression vectors with the wild type fiber (Ad5Luc1), RGD insertion (AdRGDLuc1), and Ad5/3 chimera fiber (Ad5/3Luc1) were used. The RGD-modified vector did not show enhanced infectivity in either of these PC cell lines, while the Ad5/3 chimera demonstrated significantly higher levels of infection in PC-3 and Du-145 HRPC cells. To analyze the effect of fiber modification on the cytocidal effect of CRAds we constructed COX-2 CRAds with wild type, RGD-modified, and Ad5/3 chimeric fibers, respectively. All COX-2 CRAds showed replication and subsequent oncolytic killing in both PC-3 and Du-145 cell lines while the COX-2 negative LnCap cell line was not affected. Ad5/Ad3 chimera CRAds demonstrated the strongest cytocidal effect among COX-2 CRAds in PC3 and Du-145 cell lines. Analysis of in vivo anti-tumor efficacy was performed using Du-145 prostate carcinoma subcutaneous xenografts. COX-2 CRAd agents with unmodified, RGD-modified, or Ad5/3 chimera fibers as well as wild type Ad5 and a non-replicative E1 deleted vector were used for intratumoral administration (5×109 vp/tumor). Our data indicate that the anti-tumor effect of COX-2 CRAd with Ad5/3 chimera fiber was better than unmodified or RGD-modified CRAds. The present results demonstrate that selective replication of CRAd under the control of the COX-2 promoter and infectivity enhancement with Ad5/3 chimeric fiber may be combined to generate promising viral therapeutic agents for the treatment of hormone refractory prostate carcinomas.

The employment of conditionally replicative adenoviruses (CRAds) provides a novel way to treat lung cancer. To maintain safety and efficacy in clinical application, a noninvasive detection system of viral replication is required. However, there are currently no available methods to dynamically monitor adenovirus replication and spreading ability in clinical settings. Recently we have shown that the EGFP reporter gene placed in the deleted E3 region under the adenoviral major late promoter could be exploited to noninvasively monitor adenovirus replication. In this study, we developed a system which allows monitoring of CRAd replication via the detection of bioluminescence reporter and applied it to derive novel Cox-2 promoter-based CRAds as therapeutic agents for lung cancer. In our reporter vectors most E3 genes except adenoviral death protein (ADP) were deleted or mutated and replaced with the firefly luciferase gene. We combined the Cox-2 promoter-controlled E1 expression cassette with the E3 vector to generate imaging capable Cox-2 CRAds. We designed six different structures to optimize monitoring including configurations with and without ADP and vectors equipped with different fibers accommodating the coxsackie-adenovirus receptor deficiency of cancer cells. The presence or absence of a poly-adenylation signal following the reporter gene has been examined for effect on stability of transgene expression. In vitro, all viruses showed transgene expression augmentation and killing effect over time and in a dose-dependent manner during infection of A549 human lung adenocarcinoma cells. The detected gene expression closely correlated with the viral DNA quantity resulting from viral replication. No expression or cytocidal effect was observed in mouse hepatoma BNL-1NG-A.2 cells in which human adenoviruses do not productively replicate, indicating the dependence of reporter expression on productive replication. All vectors with ADP showed a stronger killing ability without hampering luciferase expression compared to ADP lacking viruses. In vivo optical studies performed in nude mice with A549 subcutaneous xenografts visualized viral replication after both intratumoral and intravenous administration of Cox2-based CRAds. The bioluminescent signal was detected as early as the next day after adenoviral injection, increased with time, persisted over four weeks, and was comparable to that of a matched wild type E1 vector. Surprisingly, in one mouse, bioluminescence signal from a Cox2Crad was detected not only in the A549 subcutaneous nodule but also in spontaneously developed tumor metastases. Of note, in both in vitro and in vivo studies, Cox2-based CRAds demonstrated efficient replication and subsequent oncolysis in Cox2-positive A549 cells but not in Cox-2-negative A431 cells, thus confirming the dependence of their replication on the Cox-2 promoter activity. These data validate the applicability of our promoter-controlled E3 reporter system for noninvasive monitoring of CRAd replication and further development of oncolytic viruses for lung cancer.

The employment of conditionally replicative adenoviruses (CRAds) offers a promising alternative for cancer treatment. To maintain safety in clinical trials, a noninvasive detection system of viral replication is required. Our previous studies revealed the effective and transcriptionally-restricted cytocidal effect of infectivity-enhanced cyclooxygenase-2 (Cox-2) promoter-driven CRAds. However, there are no currently available methods to noninvasively monitor dynamic adenovirus replication, spreading ability, and extent of persistence in vivo. In this study, we have developed a system which allows monitoring of CRAds replication via the detection of fluorescence or luminescence reporters expressed from the E3 region. We deleted or mutated most E3 genes except adenovirus death protein (ADP), and incorporated the enhanced green fluorescent protein (EGFP) or the firefly luciferase (Luc) into the E3 region under the control of the adenovirus major late promoter (MLP). Since MLP turns on only when the virus is replicating, we hypothesized that the transgene expression would closely represent the viral replication in this configuration. Finally, we combined E1 shuttle vector with E1 expression cassette under control of Cox-2 promoter with E3ADPLuc cassette and generated imaging capable Cox-2 CRAds with different fiber modifications in order to overcome coxsackie-adenovirus receptor deficiency of cancer cells. In addition, we considered the structure of the E3 region with and without ADP since a strong cytocidal effect of ADP may not be optimal for imaging. To assess the replication dependent expression of the reporter gene from the E3 region, a replication permissive human lung adenocarcinoma A549 cells and mouse hepatoma BNL-1NG-A.2 cells, in which human adenoviruses do not productively replicate, were applied in our experiments. In vitro, all replication-competent reporter viruses showed transgene expression augmentation over time and in a dose-dependent manner during infection of A549 cells. The detected signal closely correlated with the viral DNA quantity resulting from viral replication. On the other hand, no EGFP/Luc expression was observed in BNL-1NG-A.2 cells, thus indicating the dependence of fluorescence/luminescent signal on productive replication. In vivo optical studies performed with a custom made in vivo imaging system revealed that after intratumoral administration of adenoviruses with the E3 imaging cassette the EGFP expression could be detected two days post-injection. Cox-2 CRAds with the luciferase expression showed the signal on day one and maintained it over four weeks. Importantly, in vivo reporter detection strongly correlated with the underlying level of replication. These data validate the applicability of our E3 reporter system for noninvasive in vivo monitoring of CRAd replication.