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The oncolytic mutant vesicular stomatitis virus VSVΔ51 achieves robust efficacy in multiple extracranial tumor models. Yet for malignancies of the brain, direct intratumoral infusion of VSVΔ51 causes lethal virus-induced neuropathology. Here, we have developed a novel therapeutic regime that uses peripheral immunization with a single sub-lethal dose of VSVΔ51 to establish an acute anti-viral state that enables the safe intracranial (IC) infusion of an otherwise lethal dose of VSVΔ51 within just 6 hr. Although type I interferons alone appeared insufficient to explain this protective phenotype, serum isolated at early time points from primed animals conferred protection against an IC dose of virus. Adaptive immune populations had minimal contributions. Finally, the therapeutic utility of this novel strategy was demonstrated by peripherally priming and intracranially treating mice bearing aggressive CT2A syngeneic astrocytomas with VSVΔ51. Approximately 25% of animals achieved complete regression of established tumors, with no signs of virus-induced neurological impairment. This approach may harness an early warning system in the brain that has evolved to protect the host against otherwise lethal neurotropic viral infections. We have exploited this protective mechanism to safely and efficaciously treat brain tumors with an otherwise neurotoxic virus, potentially widening the available treatment options for oncolytic virotherapy in the brain., Graphical Abstract

Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in phase 1 clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may be efficacious only in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. Therefore, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe "cytokine storm." Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFN-β), tumor necrosis factor alpha (TNF-α) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.

SummaryTo identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.

Sensory changes are thought to be involved in gastro-esophageal reflux disease (GERD). The esophageal multimodal pain model can be used to investigate sensations in response to distension, heat, electric current and acid.The aim of this study was to provide normal values for this model in the normal state and in the acid induced sensitized state.Fifty-three healthy men (20-38 years old) underwent esophageal stimulation with distension, heat and electrical current before and after sensitization with 0.1 N HCl acid. Stimulus intensities at painful and non-painful thresholds and referred pain areas were measured. The percentage of individual participants sensitized to each modality was calculated. In 22 subjects the pre-acid tests were repeated on three subsequent visits.To reach moderate pain, subjects tolerated mean distension of 29.1 ± 11 mL, heat stimulation time of 141 ± 33 s, and mean current of 17.6 ± 6.4 mA. After acid exposure, significantly reduced thresholds were observed for mechanical (24%), heat (11%) and electrical (14%) stimulation (P values 0.05). The percentage of subjects sensitized, defined as reductions in thresholds of ≥10% or ≥20% after acid perfusion, was as follows: for distension 77%/62%, for heat 48%/28%, and for current 58%/44%. The model showed good reliability (intra-class correlations 0.6).Normal values for healthy young men are now provided for the normal and the sensitized state. The percentage of subjects sensitized after acid stimulation are thoroughly documented, and depends on stimulation type and the cut-off value chosen.

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.

The K48R mutant ubiquitin can exert profound in vivo protective effects against a variety of insults, including agents of direct clinical relevance. The manipulation of the ubiquitin/proteasome pathway has enormous potential for clinical benefit, and it is not unreasonable to expect that such benefits will include diseases of aging.

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference.

Background Clinical data suggest gender differences in gastrointestinal pain, but very little experimental data exist. Esophageal painful thresholds to mechanical, thermal, electric, and chemical stimuli can be measured with the esophageal multimodal pain model. The aim was to measure the effect of gender and mild esophagitis on esophageal pain perception. Methods Thirty-five healthy asymptomatic volunteers [19 men, median age 29 (22–56 years)] underwent upper GI endoscopy, 24 h pH/impedance measurement, and multimodal esophageal pain stimulation before and after sensitization with acid. Stimulus intensities at painful thresholds were recorded. Key Results Men had higher pain thresholds (PT) to mechanical stimulation (mean volume: men 20.9 ± 10 mL vs women 15.2 ± 6.8 mL, P = 0.02) and more men tolerated the maximum acid challenge (58% vs 20%, P = 0.03). There were no differences between genders for PT to 1 thermal stimulation [mean stimulation time (men, women): heat; 20 ± 5 s vs 21 ± 6 s or cold; 33.3 ± 20.1 s vs 20.7 ± 21.4 s, P > 0.2], 2 electrical current (mean current: men 17.6 ± 9.2 mA vs women 12.9 ± 3.7 mA, P = 0.11), or 3 acid volume [median volume: men 200 (20;200) mL vs women 133 (40;200) mL, P = 0.2]. Fifteen asymptomatic subjects had mild esophagitis (10 men, all Los Angeles A). There were no differences in esophageal PT between subjects with normal endoscopy or mild esophagitis (all P > 0.3). Conclusions & Inferences The effects of gender and mild esophagitis on esophageal multimodal pain perception have been measured in asymptomatic volunteers. The study suggests that gender, not mild esophagitis, tends to influence mechanical and chemical esophageal pain.

Xeroderma pigmentosum (XP) is an orphan autosomal recessive disorder of DNA repair. When exposed to genotoxic stress, XP patients have reduced capacity to remove bulky adducts by nucleotide excision repair and are thus greatly predisposed to cancer. Unfortunately, given the nature of their underlying genetic defect, tumor-bearing XP patients cannot be treated with conventional DNA damaging therapies. Engineered strains of the poxvirus Vaccinia have been shown to cure cancer in numerous preclinical models, and based on promising Phase I/II clinical trials have recently been approved for late phase evaluation in humans. As poxviruses are nongenotoxic, we investigated whether clinical-candidate strains of Vaccinia can safely and effectively treat cancers arising from XP. In vitro, Vaccinia virus was highly cytotoxic against tumor-derived cells from XP patients, on average 10- to 100-fold more so than on nontumor derived control cells from similar patients. In vivo, local or systemic administration of Vaccinia virus led to durable tumor resolution in both xenograft and genetic models of XP. Importantly, Vaccinia virus was well tolerated in the genetic models, which are each null for a critical component of the DNA repair process. Taken together, our data suggest that oncolytic Vaccinia virus may be a safe and effective therapy for cancers arising from XP, and raise the possibility of similar therapeutic potential against tumors that arise in patients with other DNA repair disorders.

Understanding the mechanism of cisplatin (CDDP) action may improve therapeutic strategy for ovarian cancer. Although p53 and FLICE-like inhibitory protein (FLIP) are determinants of CDDP sensitivity in ovarian cancer, the interaction between p53 and FLIP remains poorly understood. Here, using two chemosensitive ovarian cancer cell lines and various molecular and cellular approaches, we show that CDDP induces p53-dependent FLIP ubiquitination and degradation, and apoptosis in vitro. Moreover, we showed that Itch (an E3 ligase) forms a complex with FLIP and p53 upon CDDP treatment. These results suggest that p53 facilitates FLIP down-regulation by CDDP-induced FLIP ubiquitination and proteasomal degradation. [Cancer Res 2008;68(12):4511–7]

Rheumatoid arthritis (RA) is an erosive joint disease affecting about 1% of the population. The joint destruction is primarily mediated by special cells called fibroblast-like synoviocytes (FLS), which undergo an expansion forming a pannus that destroys the joint. Apoptosis has been rarely detected in the synovial lining. This has lead to the identification of pro-survival factors that are expressed in FLS at the sites of the pannus including mutant p53, hrd1, sentrin, and NF-kappaB. Current anti-inflammatory modalities only bring upon temporary relief and do not treat the pannus. Therefore, the FLS remain intact, joint destruction proceeds, patients relapse and eventually become resistant to all forms of therapy. To date, surgical removal of the pannus remains the only option to help delay further joint destruction. Therefore, we believe the future should hold a less invasive approach using a class of novel drugs called proteasome inhibitors to attenuate the growth of the FLS. We suggest the use of a novel proteasome inhibitor PS-341 to treat RA patients. PS-341 has been shown to induce apoptosis in many cancer cell lines and has lead to successful outcomes in phase II and III clinical trials of multiple myeloma. Moreover, PS-341 has been shown to sensitize a variety of cell lines to chemotherapeutic drugs, some of which are used as conventional therapy in RA. We hypothesize that PS-341 alone and/or in combination with conventional RA therapies could induce apoptosis in FLS in vitro and in vivo thereby treating the pannus. Prior to clinical use extensive research examining the effects of PS-341 in animal models of arthritis would be essential in order to understand the effects proteasome inhibition in disease biology. Overall, the purpose of our hypothesis is to suggest a realistic and alternative treatment for patients with refractory and non-refractory arthritic disease.

Background In yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed. Results In this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics. Conclusion Our results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.

Benzo[a]pyrene exerts its mutagenic effects via induction of benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts. Such helix-distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway. Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. In the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLeta), causing increased BPDE-induced mutagenicity. To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin (K63R-Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin (WT-Ub). We show that PCNA is poly-ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity. In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R-Ub expressing cells, of which the majority (74%) was G-->T transversion. BPDE treatment caused an enhanced recruitment of POLeta to the replication machinery of the K63R-Ub expressing cells, where it co-localized with PCNA. Suppression of POLeta expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability.

Background Abnormal visceral sensory function has been demonstrated in patients with diabetes mellitus and diabetic autonomic neuropathy seems to be involved in the development and progression of gastrointestinal tract dysfunction. The possibility of multimodal (e.g. mechanical, electrical, thermal and chemical) stimulation in, e.g. the esophagus has developed visceral pain research. The major advantage is involvement of distinctive receptors, various sensory nerves and different pain pathways mimicking clinical pain that favours investigation of central pain mechanisms involved in allodynia and hyperalgesia. Hence the rationale of the presents study was to explore the nervous system by assessment of esophageal sensitivity to multimodal stimulations. Methods Throughout an euglycemic clamp, 31 healthy volunteers (age 44.3 ± 10.6 (mean ± SD) years; 11men) and 31 patients (age 46.3 ± 11.7 years; 10 men) with insulin dependent diabetes mellitus (duration 31.3 ± 13.1 years) were included in the study. By use of a multimodal oesophageal probe, sensitivity to heat, mechanical distension and electrical stimulation was assessed in the lower oesophagus. Results For heat stimulation patients had increased sensitivity in the sensory range with shorter stimulus duration until pain tolerance threshold (122 ± 3.8 sec vs. 136 ± 3.7 sec; p = 0.006) and larger area under the temperature curve (2380 ± 1847 vs. 1409 ± 1450; p = 0.03). There were no differences between groups for mechanical stimulation (maximum pressure (39 ± 57mmHg vs. 24 ± 48mmHg; p = 0.3; maximum volume 59 ± 21ml vs. 62 ± 25 ml; p = 0.56). As an overall finding, patients tolerated higher electrical stimulation intensities (p = 0.02), dominated by discrimination in the sensory range: At sensory detection threshold (VAS1) 21.1 ± 12.4 mA vs. 16.3 ± 5.5 mA (p = 0.03); at moderate sensation (VAS3) 27.5 ± 13.3 mA vs. 21.5 ± 5.0 mA (p = 0.03) however at pain detection threshold (VAS5) 31.6 ± 13.1mA vs. 28.8 ± 5.2 mA; the trend was insignificant (p = 0.3). Conclusion Patients with insulin dependent diabetes mellitus had modality-specific alterations of esophageal sensitivity. Heat stimulation activates selectively mucosal receptors whereas on the contrary electrical stimulation depolarizes the free nerve endings non-selectively. Hence, due to the different sensitivity profile, the esophageal neuropathy is most likely a result of both peripheral and central neuropathy caused by longstanding diabetic neuronal damage.

Background Gastrointestinal (GI) symptoms, such as nausea, vomiting, bloating, postprandial fullness, and abdominal pain, are frequent in patients with diabetes mellitus (DM). The pathogenesis is complex and multi-factorial. To determine easy accessible and valid biomarkers for disordered GI function in DM patients, we aimed to study esophageal mechanical parameters and their relation to symptoms typically arising from the digestive tract. Methods Seventeen patients with longstanding DM and GI symptoms and 13 healthy controls were studied using ultrasound monitored esophageal distension. The sensory response was recorded and their symptoms registered. Biomechanical parameters, such as compliance and stiffness were computed from luminal diameters during distension based on the ultrasound images and from pressure data. Biomechanical and sensory parameters were correlated with the clinical data. Key Results Diabetes patients had reduced esophageal sensitivity compared with controls (P = 0.046). The esophageal compliance was reduced (P = 0.004) and the esophageal stiffness was increased (P = 0.004) in the diabetes patients. Among patients, both postprandial fullness/early satiety and bloating correlated negatively to the esophageal compliance parameters (all P

2, HS=1) showed a significant reduction in the total level of VGSC expression. 2 out of 8 samples (NS=2) showed a significant increase in trafficking of VGSC to the membrane. Despite the fact that some samples caused an increase in the VGSC at the membrane and/ or a decrease in the total level of expression of VGSC, this did not relate to patient subtype. These data show that calcium mobilization is a robust marker of HS supernatants. However, only a subset of patients show changes in VGSC trafficking which appears to be independent of their pain status. Supernatants varied considerably in their effect suggesting that the mediator content of this ‘inflammatory soup' varies on a patient-by-patient basis therefore potentially mediating different aspects of hypersensitivity.

Endotoxic shock is a serious systemic inflammatory response to an external biological stressor. The responsiveness of NF-kappaB is built upon rapid protein modification and degradation involving the ubiquitin proteasome pathway. Using transgenic mice, we have obtained in vivo evidence that interference with this pathway can alleviate the symptoms of toxic shock. We posit that administration of proteasome inhibitors may enhance the survival of patients with septic shock.