Outcomes In Preclinical Models Research Articles

The Effect of PAOPA, a Novel Allosteric Modulator of Dopamine D2 Receptors, on Signaling Proteins Following Sub-Chronic Administration in Rats.

Allosteric modulators of G-protein coupled receptors regulate receptor activity by binding to sites other than the active site and have emerged as a new and highly desirable class of drugs. PAOPA (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide), a peptidomimetic analog of L-prolyl-L-leucyl-glycinamide, is a potent dopamine D2 receptor allosteric modulator. PAOPA has shown therapeutic effects in pre-clinical models of schizophrenia and extrapyramidal dysfunction. In this study, we sought to examine the biomolecular underpinnings of PAOPA's therapeutic outcomes in pre-clinical models of schizophrenia. Following sub-chronic (daily for 7 days) administration of PAOPA, we assessed levels of dopamine D2 receptors, receptor kinases (GRK2 (G protein-coupled receptor kinase 2) and Arrestin- 3), and phosphorylated mitogen-activated protein kinase (MAPKs), namely, extracellular signal- regulated kinases (ERK1/2) in the hippocampus, medial pre-frontal cortex, nucleus accumbens, pre-frontal cortex, and dorsal striatum via protein quantification. Following 7 days of daily PAOPA treatment, we observed decreased GRK2 and increased dopamine D2 receptor expression in the dorsal striatum. These findings potentially underscore the therapeutic mechanism of action of PAOPA for the positive-like symptoms of schizophrenia in pre-clinical animal models. Additionally, we observed a decline in GRK2 in the hippocampus and an increase in phosphorylated ERK1 in the pre-frontal cortex, suggesting a role of PAOPA in treating cognitive and/or affective dysfunction in pre-clinical models. While further studies are required to elucidate the mechanism of action of PAOPA, this study discusses prior investigations and develops an early framework to describe the therapeutic mechanism of action of PAOPA.

Human cord blood-derived regulatory T-cell therapy modulates the central and peripheral immune response after traumatic brain injury.

Traumatic brain injury (TBI) causes a profound inflammatory response within the central nervous system and peripheral immune system, which contributes to secondary brain injury and further morbidity and mortality. Preclinical investigations have demonstrated that treatments that downregulate microglia activation and polarize them toward a reparative/anti‐inflammatory phenotype have improved outcomes in preclinical models. However, no therapy to date has translated into proven benefits in human patients. Regulatory T cells (Treg) have been shown to downregulate pathologic immune responses of the innate and adaptive immune system across a variety of pathologies. Furthermore, cellular therapy has been shown to augment host Treg responses in preclinical models; yet, studies investigating the use of Treg as a therapeutic for TBI are lacking. In a rodent TBI model, we demonstrate that human umbilical cord blood Treg modulate the central and peripheral immune response after injury in vitro and in vivo.

Modulation of autophagy for neuroprotection and functional recovery in traumatic spinal cord injury.

Spinal cord injury (SCI) is a serious central nervous system trauma that leads to loss of motor and sensory functions in the SCI patients. One of the cell death mechanisms is autophagy, which is ‘self-eating’ of the damaged and misfolded proteins and nucleic acids, damaged mitochondria, and other impaired organelles for recycling of cellular building blocks. Autophagy is different from all other cell death mechanisms in one important aspect that it gives the cells an opportunity to survive or demise depending on the circumstances. Autophagy is a therapeutic target for alleviation of pathogenesis in traumatic SCI. However, functions of autophagy in traumatic SCI remain controversial. Spatial and temporal patterns of activation of autophagy after traumatic SCI have been reported to be contradictory. Formation of autophagosomes following therapeutic activation or inhibition of autophagy flux is ambiguous in traumatic SCI studies. Both beneficial and harmful outcomes due to enhancement autophagy have been reported in traumatic SCI studies in preclinical models. Only further studies will make it clear whether therapeutic activation or inhibition of autophagy is beneficial in overall outcomes in preclinical models of traumatic SCI. Therapeutic enhancement of autophagy flux may digest the damaged components of the central nervous system cells for recycling and thereby facilitating functional recovery. Many studies demonstrated activation of autophagy flux and inhibition of apoptosis for neuroprotective effects in traumatic SCI. Therapeutic induction of autophagy in traumatic SCI promotes axonal regeneration, supporting another beneficial role of autophagy in traumatic SCI. In contrast, some other studies demonstrated that disruption of autophagy flux in traumatic SCI strongly correlated with neuronal death at remote location and impaired functional recovery. This article describes our current understanding of roles of autophagy in acute and chronic traumatic SCI, cross-talk between autophagy and apoptosis, therapeutic activation or inhibition of autophagy for promoting functional recovery, and future of autophagy in traumatic SCI.

3174 – TANDEM MYELOID GROWTH FACTOR TREATMENT ENHANCED THE TOTAL HAEMATOPOIETIC STEM CELL (HSC) POOL AND MAGNITUDE OF HSC MOBILISATION

Autologous HSC transplant is a common therapeutic strategy for some haematological malignancies but is unachievable in up to 40% of cases due to insufficient mobilisation of HSCs for successful transplantation. The monocyte-macrophage colony stimulating factor-1 (CSF1) improves HSC transplantation outcomes in preclinical models and clinical trials, but the underlying mechanism of CSF1 induced benefit is unclear. BM macrophages are a key component of the HSC niches and a primary target of granulocyte colony-stimulating factor (G-CSF) induced HSC mobilisation. We examined CSF1 impacts on HSC frequency using a modified CSF1-Fc molecule that has improved drug qualities. Haematopoietic progenitor cells (HPC), but not HSCs or multipotent progenitors (MPP), expressed the CSF1 receptor. Acute CSF1-Fc treatment (4 x daily injection) induced a robust myeloproliferative response that was evident 1 week post the initial injection. At this time, BM HSCs (17 fold) and MPP (19 fold), but not HPC, were significantly reduced. However, at 2 weeks post the first CSF1-Fc injection, after the myeloproliferative event had resolved, the overall pool of HSCs in BM (1.6 fold), and unexpectedly spleen (9.2 fold), was significantly elevated compared to controls. A mobilising regimen of G-CSF started at 2 weeks post CSF1-Fc treatment enhanced HSC mobilisation (2.5 fold) into blood when compared to mice treated with G-CSF only. This increase in mobilised HSCs was verified via competitive secondary transplantation. The data suggest that CSF1 has complex indirect effects on HSCs to increase HSC frequency and promote formation of extramedullary HSC niches. CSF1-induced increased in the total HSC pool may contribute to the reported improvement in transplantation outcomes associated with CSF1 therapy and reveals a novel strategy to increase HSCs prior to mobilisation.

The Impact of Riluzole on Neurobehavioral Outcomes in Preclinical Models of Traumatic and Nontraumatic Spinal Cord Injury: Results From a Systematic Review of the Literature.

Study Design:Systematic review.Objective:To evaluate the impact of riluzole on neurobehavioral outcomes in preclinical models of nontraumatic and traumatic spinal cord injury (SCI).Methods:An extensive search of the literature was conducted in Medline, EMBASE, and Medline in Process. Studies were included if they evaluated the impact of riluzole on neurobehavioral outcomes in preclinical models of nontraumatic and traumatic SCI. Extensive data were extracted from relevant studies, including sample characteristics, injury model, outcomes assessed, timing of evaluation, and main results. The SYRCLE checklist was used to assess various sources of bias.Results:The search yielded a total of 3180 unique citations. A total of 16 studies were deemed relevant and were summarized in this review. Sample sizes ranged from 14 to 90, and injury models included traumatic SCI (n = 9), degenerative cervical myelopathy (n = 2), and spinal cord-ischemia (n = 5). The most commonly assessed outcome measures were BBB (Basso, Beattie, Besnahan) locomotor score and von Frey filament testing. In general, rats treated with riluzole exhibited significantly higher BBB locomotor scores than controls. Furthermore, riluzole significantly increased withdrawal thresholds to innocuous stimuli and tail flick latency following application of radiant heat stimuli. Finally, rats treated with riluzole achieved superior results on many components of gait assessment.Conclusion:In preclinical models of traumatic and nontraumatic SCI, riluzole significantly improves locomotor scores, gait function, and neuropathic pain. This review provides the background information necessary to interpret the results of clinical trials on the impact of riluzole in traumatic and nontraumatic SCI.

Th17 Cells Changing Colors

The transcription factor c-MAF can synergize with IL-27 to transform inflammatory Th17 cells into regulatory IL-10–producing cells that may be beneficial in settings of chronic stimulation. The transcription factor c-MAF can synergize with IL-27 to transform inflammatory Th17 cells into regulatory IL-10–producing cells that may be beneficial in settings of chronic stimulation. CITATION Aschenbrenner D, Foglierini M, Jarrossay D, et al. An immunoregulatory and tissue-residency program modulated by c-MAF in human Th17 cells. Nat Immunol. 2018;19(10):1126-1136. Interleukin (IL)-17A–producing CD4 T (Th17) cells protect hosts against extracellular pathogens and have been linked to transplant rejection and autoimmunity. Some Th17 cells co-express the regulatory cytokine IL-10, which limits their response and may help avoid collateral tissue injury. However, the transcriptional programs underlying IL-10–producing Th17 cells have not been fully explored. The study by Aschenbrenner et al. demonstrates that a subset of human memory Th17 cells acquired anti-inflammatory and immunoregulatory programs upon restimulation, dependent on the transcription factor musculoaponeurotic fibrosarcoma (c-MAF) that largely directed expression of IL-10 and co-inhibitory markers. Aschenbrenner and colleagues isolated CCR6+CCR4+CXCR3– human memory Th17 clones and showed that approximately 25% of these cells upregulated IL-10 and downregulated IL-17A upon restimulation with anti-CD3/CD28. IL-10+ Th17 cells acquired a memory of IL-10 expression associated with permissive H3K4me3 chromatin modifications at the transcriptional start site of the IL-10 locus, and IL-10 production was maintained over several rounds of restimulation. IL-10+ Th17 cells selectively increased expression and binding of the transcriptional co-activator c-MAF to the c-MAF response element (MARE-2) in the IL-10 promoter. This event was not observed in IL-10– Th17 cells, although they expressed basal c-MAF. Collectively, these data suggest a unique epigenetic landscape accompanied by upregulated c-MAF, which results in IL-10 production by Th17 cells. To investigate the global transcriptional profile of Th17 cells, RNA sequencing (RNAseq) was conducted in IL-10+ and IL-10– Th17 subsets. As expected, IL-10+ Th17 cells activated immunoregulatory/tissue homing transcriptional pathways and were enriched in genes encoding regulatory molecules such c-MAF, IL-10, CTLA-4, PD-1, CD69, CCR9 and CXCR6. In contrast, IL-10– Th17 cells exhibited a pro-inflammatory transcriptional profile expressing (IFN)γ, IL-22, IL-1R1, IL-2, IL-23R, and CCR7. Depleting c-MAF using lentivirus-encoding short hairpin RNA reduced expression of IL-10 and of most of the other IL-10+ genes associated with Th17, while a number of pro-inflammatory genes were upregulated. Conversely, ectopic overexpression of c-MAF in IL-10– Th17 cells modestly upregulated IL-10, which was further induced in the presence of IL-27, suggesting synergy between these two pathways. Overall, these data suggest that IL-10+ Th17 cells acquire immunoregulatory and tissue-residency programs, which are in large part defined by c-MAF expression. The IL-10+ and IL-10– Th17 subsets shared considerable plasticity. IL-10– Th17 cells acquired a regulatory phenotype in the presence of IL-27, a cytokine known to induce IL-10, whereas IL-10+ Th17 cells acquired a pro-inflammatory phenotype when restimulated in the presence of IL-1β. Interestingly, IL-27 and IL-1β did not change c-MAF expression levels, such that their effects on IL-10 transcription may depend on altered binding of c-MAF to the IL-10 locus. Functionally, IL-10+ and IL-10– Th17 subsets were able to polarize CD14+ monocytes into anti-inflammatory M2 and pro-inflammatory M1-like macrophages, respectively, in co-culture experiments. Th17 cells are thought to participate in chronic allograft rejection, especially in the setting of lung transplantation. IL-17A is a potent chemoattractant for neutrophils and can induce profibrotic cytokines from allograft epithelial cells and fibroblasts. Therapies targeting IL-17A in select settings of autoimmunity have shown promising results in the clinic. However, IL-17A–targeting in transplant animal models has not resulted in complete attenuation of chronic rejection. The current study suggests that overexpressing c-MAF in T cells or inhibiting its antagonizing pathways, along with IL-27 administration, may help promote regulatory over inflammatory Th17 cells and may improve long-term transplant outcomes. Recent reports demonstrating that c-MAF drives T cell exhaustion and regulates IL-10 in macrophages/Th1 subsets further highlight c-MAF as an attractive therapeutic target. If the proregulatory effects of c-MAF in Th17 cells are conserved in other mammals, it will be exciting to see how targeting T cell–c-MAF influences allograft survival outcomes in preclinical models.

Preoperative Protein or Methionine Restriction Preserves Wound Healing and Reduces Hyperglycemia

BackgroundDietary restriction (DR), defined as reduced nutrient intake without malnutrition, is associated with longevity extension, improved glucose metabolism, and increased stress resistance, but also poor wound healing. Short-term preoperative DR followed by a return to normal feeding after surgery results in improved surgical outcomes in preclinical models. However, the effect of preoperative DR on wound healing and perioperative glucose homeostasis is currently unknown. Here, we tested the effects of two different preoperative DR regimens—protein restriction (PR) and methionine restriction (MR)—on wound healing and perioperative glucose homeostasis using an established murine model of wound healing in both nondiabetic and diabetic mice. Materials and methodsSurgical outcomes were tested using the McFarlane flap in nondiabetic and streptozotocin-induced diabetic mice. Short-term dietary preconditioning included 1 wk of PR or MR diet (1-2 wk) versus an isocaloric complete diet before surgery; all mice were returned to a complete diet postoperatively. Outcome measures of flap wound recovery included skin viability and laser Doppler imaging of flap perfusion and assessment of CD45+ cell infiltration. Glucose homeostasis was assessed by glucose tolerance testing and by perioperative glucose levels in the diabetic cohort. ResultsNo significant differences were observed in percentage of viable skin, perfusion, or immune cell infiltration at 7-10 d after surgery in PR or MR mice compared with controls in healthy or diabetic mice. Preoperative glucose tolerance and postoperative glucose levels were however significantly improved by both PR and MR in diabetic mice. ConclusionsShort-term dietary preconditioning with PR or MR did not impair wound healing in nondiabetic or diabetic mice. However, both regimens reduced preoperative hyperglycemia in diabetic mice. Thus, brief preoperative dietary manipulations stand as strategies to potentially improve perioperative hyperglycemia with no deleterious effects on wound healing in mice.

Combination of 177 Lu-lilotomab with rituximab significantly improves the therapeutic outcome in preclinical models of non-Hodgkin's lymphoma.

To investigate the therapeutic potential of the next-generation anti-CD37 radioimmunoconjugate 177 Lu-lilotomab satetraxetan (177 Lu-lilotomab) in combination with the anti-CD20 antibody rituximab for treatment of mice with non-Hodgkin's lymphoma (NHL) xenografts. Nude mice with subcutaneous (s.c.) Burkitt's lymphoma Daudi xenografts and SCID mice intravenously (i.v.) injected with Mantle cell lymphoma Rec-1 cells were treated with either 177 Lu-lilotomab or rituximab alone or with the combination of both treatments. Tumour volume, body weight, blood counts and clinical status were monitored. CD20 expression was measured using flow cytometry with fluorescence-labelled rituximab. The combination of 177 Lu-lilotomab and rituximab was synergistic for treatment of nude mice with s.c. Daudi xenografts while it was additive for treatment of SCID mice with i.v. injected Rec-1 cells. Binding of rituximab to NHL cells in-vitro was increased by pretreatment with 177 Lu-lilotomab. Treatment of mice with NHL xenografts with 177 Lu-lilotomab synergistically increased tumour suppression of subsequent anti-CD20 immunotherapy and improved survival. If the same effect is confirmed in a recently started clinical study, it could change the way radioimmunotherapy and CD20 immunotherapy would be used in the future.

Approaches to Improve Chemically Defined Synthetic Peptide Vaccines.

Progress made in peptide-based vaccinations to induce T-cell-dependent immune responses against cancer has invigorated the search for optimal vaccine modalities. Design of new vaccine strategies intrinsically depends on the knowledge of antigen handling and optimal epitope presentation in both major histocompatibility complex class I and -II molecules by professional antigen-presenting cells to induce robust CD8 and CD4 T-cell responses. Although there is a steady increase in the understanding of the underlying mechanisms that bridges innate and adaptive immunology, many questions remain to be answered. Moreover, we are in the early stage of exploiting this knowledge to clinical advantage. Several adaptations of peptide-based vaccines like peptide-adjuvant conjugates have been explored and showed beneficial outcomes in preclinical models; but in the clinical trials conducted so far, mixed results were obtained. A major limiting factor to unravel antigen handling mechanistically is the lack of tools to efficiently track peptide vaccines at the molecular and (sub)cellular level. In this mini-review, we will discuss options to develop molecular tools for improving, as well as studying, peptide-based vaccines.

Targeting Coagulase Activity in Staphylococcus aureus Bacteraemia: A Randomized Controlled Single-Centre Trial of Staphylothrombin Inhibition.

Staphylococcus aureus (S. aureus) bacteraemia is frequent and carries a high morbidity and mortality. Coagulases secreted by S. aureus initiate blood coagulation by directly activating prothrombin. This pathogen-activated coagulation is insensitive to most antithrombotic drugs, with the exception of small molecule direct thrombin inhibitors (DTIs). DTIs inhibit the coagulase-prothrombin complex, or staphylothrombin, and improve outcome in preclinical models of S. aureus infection. A single-centre, randomized, controlled feasibility and safety trial of staphylothrombin inhibition with DTIs in patients with S. aureus bacteraemia. Consecutive eligible adult patients with S. aureus positive blood cultures in the University Hospitals Leuven (Belgium) were randomized 1:1 to DTI (oral dabigatran 110 mg twice daily or intravenous argatroban according to activated partial thromboplastin time [aPTT]) for 7 to 10 days, or subcutaneous enoxaparin 40 mg once daily. Primary outcomes were feasibility and safety of DTI in patients with S. aureus bacteraemia. Secondary outcomes include D-dimer evolution (day 0-4) as marker of coagulation activation; inflammatory and microbiological parameters; and clinical outcomes including metastatic infections. Thirty-one percent (94/303) of screened patients were enrolled. Dabigatran plasma levels inhibited staphylothrombin. Clinically relevant bleeding (5/47 vs. 5/47) and thrombotic (7/47 vs. 7/47) complications were similar in both groups. Coagulase inhibition with DTIs was associated with a trend towards faster D-dimer decrease at day 4 (-662 ± 249 ng/mL vs. -40 ± 213 ng/mL for DTI-treated patients vs. control; p = 0.06) and a numerically lower number of persistently positive blood cultures. No differences in inflammatory parameters or other clinical outcomes were observed. Targeting staphylothrombin with DTIs is feasible in a subset of S. aureus bacteraemic patients, with comparable safety to standard thromboprophylaxis. In future studies of staphylothrombin inhibition, feasibility can be further improved by rapid diagnostics and by strategies without concomitant anticoagulant effect.

Immunotherapy: Viral reprogramming.

Two studies have shown that oncolytic virus treatment prior to surgery can prime the tumour immune microenvironment for subsequent immune checkpoint inhibition and lead to better outcomes in preclinical models of breast and brain cancers.

Long-term results of a sirolimus-eluting stent with biodegradable polymer (RAPSTROM™) in de novo coronary stenoses.

To report long-term results of a novel sirolimus-eluting stent with biodegradable polymer BACKGROUND: Newer generation drug-eluting stents are characterized by thin struts, improved platform design and highly biocompatible polymer carrying the antiproliferative drug. The RapstromTM stent, sharing these features, showed promising outcomes in preclinical models and in a first-in-man trial. The present study is a multicenter, non-randomized post-market registry, including patients with de novo coronary artery disease treated with implantation of one or more Rapstrom stents. Primary endpoint of the study was the rate of major adverse cardiac events (MACE) at three-year follow-up. 1073 patients were enrolled, with a high prevalence of diabetes (35%) and acute coronary syndrome at presentation (82%); at three-year follow up, MACE rate was 14.8%, with a low incidence of definite or probable stent thrombosis (0.75%). These data confirm the good clinical performance of the Rapstrom stent, supporting the concept that the combination of thin struts and biodegradable polymer is associated with positive clinical outcomes.

Abstract 12475: Mechanisms of Akt1-mediated Hypothermia Protection After Cardiac Arrest: Metabolism, Inflammation and Contractile Function

Introduction: Induction of intra-arrest cooling (IC) improves cardiac, neurological and survival outcomes in preclinical models of cardiac arrest. Previous studies demonstrate that IC protection is mediated by Akt1 activation through initiating survival signaling. Hypothesis: We hypothesize that IC-induced Akt1 activation modulates protective calcium cycling proteins, metabolic, and inflammatory pathways. Methods: Adult female Akt1 +/- underwent 8 min of KCl-induced asystolic arrest and were randomized to receive IC (30 ± 0.5°C) or normothermia (NT) before CPR (n = 10 per group). 72 h survival and mean blood pressure (MAP) were measured. Indicators of glucose utilization, inflammatory responses and contractile function in heart and brain of Akt1 +/+ and Akt1 +/- mice were assessed (n=5 per group). Results: No difference on survival was seen for IC and NT treated Akt1 +/- mice. IC induced transient improvement in MAP at 30 min following resuscitation (53.6 ± 2.2 vs 71.2 ± 2.6 mmHg p<0.05) and increased phospholamban phosphorylation in the heart of Akt1 +/+ mice which were not seen in Akt1 +/- mice. Compared to IC-treated Akt1 +/+ , pyruvate dehydrogenase phosphorylation and sorbitol accumulation were significantly increased, and NAD and ATP contents were markedly reduced in the hearts and brains of Akt1 +/- mice. IC significantly inhibited IκBα degradation and NF-κB nuclear translocation, and reduced concentrations of blood lactate and Pre-B cell Colony Enhancing Factor in Akt1 +/+ mice, but not Akt1 +/- mice. Conclusion: Akt1 is a critical mediator for TH protection during early post resuscitation in heart and brain through regulating calcium regulatory proteins, enhancing glucose utilization and downregulating inflammatory responses.

Infliximab enhances the therapeutic effects of 5-fluorouracil resulting in tumor regression in colon cancer.

Colon cancer (CC) is among the most common malignant diseases with a dismal survival. Tumor necrosis factor-alpha (TNF-α) has been identified as a therapeutic target in various cancers, and anti-TNF-α treatment has shown promising effects in different cancer models. However, if TNF-α can be targeted in CC, the therapeutic values of anti-TNF-α treatment in CC remain unknown. Our study indicated that TNF-α is highly expressed in CC cell lines and patient tumor samples. High expression of TNF-α is an independent adverse prognosticator of CC. Targeting the TNF-α by its antibody infliximab induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and enhanced apoptosis leading to cell death. The combination of infliximab with 5-fluorouracil showed better responses in vitro and in vivo than 5-fluorouracil alone. In conclusion, this study identified TNF-α as a target of CC and anti-TNF-α treatment synergized with chemotherapy leading to a better outcome in preclinical models.

Pharmacological Inhibition of NAD(P)H Oxidase with the Peptide gp91ds‐tat During Ischemic Stroke Reduces Infarct Volume and Improves Vascular Reactivity in Obese Zucker Rats

Production of reactive oxygen species (ROS) during ischemic stroke and subsequent reperfusion has been linked to increased infarct size and poor outcomes in multiple rodent models. NAD(P)H) oxidase (NOX) has been identified as a principal source of ROS generation during ischemia/reperfusion, and inhibition of NOX during ischemic stroke results in decreased infarct size and improved outcomes in preclinical models, although underlying mechanisms are unclear. Elevated blood glucose increases ROS production by NOX during ischemia/reperfusion, and vascular dysfunction can impair collateral flow; a stroke model that incorporates hyperglycemia and vascular dysfunction may therefore amplify ROS production and lead to larger infarcts and worsened outcomes. As such, the aim of this study was to determine the effects of gp91ds‐tat, a peptide inhibitor of NOX‐2, on infarct size and vascular reactivity in both the ipsilateral (IL) and contralateral (CL) middle cerebral arteries (MCAs) of lean and obese Zucker rats (LZR, OZR) two weeks after ischemic stroke. Strokes were induced in 17 week old rats for 60 min by transient MCA occlusion (tMCAO); 30 minutes into tMCAO, OZR received either gp91ds‐tat (10mg/kg body weight) or vehicle. Changes in cerebral blood flow were monitored by laser Doppler. 15 days after tMCAO, animals were sacrificed and the IL and CL MCAs were isolated and cannulated in an ex vivo microvessel preparation; MCA reactivity was assessed in response to acetylcholine (ACh). Infarct size was measured by TTC stain. Infarct size and mortality rates were increased at 15 days post‐tMCAO in control treated OZR vs. LZR. Vascular dysfunction was worsened in both CL and IL MCA; ACh‐induced vasodilation was reduced 35% in CL‐MCA compared to non‐stroke OZR MCA, while in IL‐MCA, dilation was completely ablated, likely due to reperfusion injury. In contrast, OZR treated with gp91ds‐tat demonstrated reduced infarct size and mortality, as well as improved recovery of cerebral blood flow after reperfusion vs. control OZR. Vascular reactivity improved significantly, as ACh‐induced dilation in CL‐MCA and IL‐MCA increased 12% and 67% respectively vs. control treated OZR, indicating gp91ds‐tat acutely reduced damage from reperfusion injury. Our findings suggest that preexisting metabolic syndrome can worsen neurological and cerebrovascular outcomes following ischemic stroke though a mechanism at least partially mediated by NOX. Targeted inhibition of NOX during ischemia/reperfusion may improve stroke outcomes, especially in patients with history of metabolic syndrome, and warrants further consideration as a treatment avenue for ischemic stroke.Support or Funding InformationNIH COBRE GrantAHA Predoctoral Fellowship

In VivoFluorescence Lifetime Imaging for Monitoring the Efficacy of the Cancer Treatment

Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients.

Abstract T MP91: Association of Gender-Age Interaction With Outcome After Intracerebral Hemorrhage in Multiple Ethnicities

Introduction: Female sex is associated with better functional outcome in preclinical models of intracerebral hemorrhage (ICH). However, recovery is impaired after ischemic stroke as women age, relative to men, independent of stroke severity. We sought to define gender-age interactions for long-term neurological outcome after ICH across ethnicities. Methods: Clinical and radiographic data from subjects in the Ethnic/Racial Variations of Intracerebral Hemorrhage (ERICH; NS069763) and Genetic and Environmental Risk Factors for Hemorrhagic Stroke (GERFHS; NCT00930280) studies prior to January 2013 were used. Relationships between sex, age, and outcome in 473 black, 437 Hispanic, and 801 white subjects were evaluated after ICH using multivariable logistic regression analysis. Poor outcome was defined as modified Rankin ≥3 at 3 months after ICH. Age was modeled as a quadratic; gender-age interaction was modeled as centered cross-products to reduce collinearity. Results: Evidence of an interaction effect for gender and age on outcome at 3 months was found after ICH (p=0.054). The figure shows overlayed plots of the interaction between gender and age with regard to poor outcome. Women had lower risk of poor outcome at younger ages but higher risk at older ages. When testing this effect in different ethnicities, there was significant evidence of a gender-by-age interaction (p=0.005) within black patients. Conclusion: We found evidence of better outcomes in younger aged women than men, independent of comorbidities and severity of stroke. This trend reversed in elderly women. The gender-age interaction in outcome after ICH, seen especially in black subjects, may result from hormonal changes due to aging. Future studies to evaluate the impact of estrogen and progesterone in age/ethnic groups as a potential mediator of outcome are currently under design.

Mesenchymal stem cells in joint disease and repair

Osteoarthritis (OA), a prevalent chronic condition with a striking impact on quality of life, represents an enormous societal burden that increases greatly as populations age. Yet no approved pharmacological intervention, biologic therapy or procedure prevents the progressive destruction of the OA joint. Mesenchymal stem cells (MSCs)-multipotent precursors of connective tissue cells that can be isolated from many adult tissues, including those of the diarthrodial joint-have emerged as a potential therapy. Endogenous MSCs contribute to maintenance of healthy tissues by acting as reservoirs of repair cells or as immunomodulatory sentinels to reduce inflammation. The onset of degenerative changes in the joint is associated with aberrant activity or depletion of these cell reservoirs, leading to loss of chondrogenic potential and preponderance of a fibrogenic phenotype. Local delivery of ex vivo cultures of MSCs has produced promising outcomes in preclinical models of joint disease. Mechanistically, paracrine signalling by MSCs might be more important than differentiation in stimulating repair responses; thus, paracrine factors must be assessed as measures of MSC therapeutic potency, to replace traditional assays based on cell-surface markers and differentiation. Several early-stage clinical trials, initiated or underway in 2013, are testing the delivery of MSCs as an intra-articular injection into the knee, but optimal dose and vehicle are yet to be established.

Gene therapy for misfolding protein diseases of the central nervous system.

Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. Accordingly, most recent studies aim to prevent protein misfolding and/or aggregation as a strategy to treat these pathologies. For instance, state-of-the-art approaches, such as silencing protein overexpression by means of RNA interference, are being tested with positive outcomes in preclinical models of animals overexpressing the corresponding protein. Therapies designed to treat central nervous system diseases should provide accurate delivery of the therapeutic agent and long-term or chronic expression by means of a nontoxic delivery vehicle. After several years of technical advances and optimization, gene therapy emerges as a promising approach able to fulfill those requirements. In this review we will summarize the latest improvements achieved in gene therapy for central nervous system diseases associated with protein misfolding (e.g., amyotrophic lateral sclerosis, Alzheimer's, Parkinson's, Huntington's, and prion diseases), as well as the most recent approaches in this field to treat these pathologies.

Abstract 1136: MK-1775, a selective wee1 kinase inhibitor, overcomes cisplatin resistance associated with high risk TP53 mutations in squamous cell carcinoma of the head and neck.

Abstract Head and neck squamous cell carcinoma (HNSCC) affects over 500,000 patients worldwide annually and over half this number of patients dies from the disease each year. Platinum-based chemotherapy is currently employed in the treatment of advanced HNSCC to decrease distant metastasis and to improve local-regional control through sensitization of external beam radiation. Platinum based agents have been shown to be less effective in the context of specific genomic events such as mutation of TP53 tumor suppressor gene. Recent data from whole-exome sequencing of HNSCC reveals that this gene is mutated in 60-80% of HNSCC. It is unclear whether all mutations in TP53 equally trigger an aggressive HNSCC phenotype which is resistant to platinum based agents. Therefore, we have developed an algorithm termed Evolutionary Gradient and Structure (EGS) which stratifies HNSCC patients based on the type of TP53 mutation and correlates the score (high risk vs. low risk) with clinical outcomes. Based on this system, twelve isogenic cell lines harboring different p53 mutants were created in a p53 null HNSCC cell line background. Cells expressing high risk TP53 mutations were highly resistant to cisplatin relative to wildtype TP53. Decreased cisplatin sensitivity associated with these mutations was found to be driven by their inability to undergo cellular senescence, the primary mechanism of death for cells with wildtype TP53. It has been previously shown that tumors expressing mutant TP53 rely more heavily on activation of the S- and G2-phase checkpoints for mediating the growth arrest needed to repair DNA damage and survive genotoxic stress, making these cells potentially sensitive to G2 checkpoint inhibitors. Wee1 is a tyrosine kinase that has been linked to DNA damage induced G2 arrest, owing to its ability to inactivate cyclin dependent kinase 1 (CDK1)) through phosphorylation of the Tyr15 residue. Recent studies have shown that inhibitors of Wee1 sensitize TP53 mutant colon cancer tumors to cisplatin in preclinical models by abrogating a G2 arrest. Therefore, we assessed the ability of the wee1 kinase inhibitor, MK-1775, to sensitize the HNSCC cells to cisplatin. Treatment of HNSCC cell lines expressing high risk TP53 mutations with MK-1775 and cisplatin resulted in strong synergism with a combination index of 0.07. Furthermore, the addition of MK-1775 to cisplatin did not restore induction of senescence, but rather resulted in increased cell death through mitotic catastrophe. This study demonstrates the potential for a therapeutic strategy aimed at tumors expressing high risk TP53 mutations. Through the use of synthetic lethality it appears possible to restore cell death pathways and improve treatment outcomes in pre-clinical models of aggressive HNSCC. Citation Format: Abdullah A. Osman. MK-1775, a selective wee1 kinase inhibitor, overcomes cisplatin resistance associated with high risk TP53 mutations in squamous cell carcinoma of the head and neck. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1136. doi:10.1158/1538-7445.AM2013-1136