Extended Time Points Research Articles

[89Zr]Zr-DFO-TOC: a novel radiopharmaceutical for PET imaging of somatostatin receptor positive neuroendocrine tumors

BackgroundNeuroendocrine tumors (NETs) are clinically diverse types of tumors that can arise anywhere in the body. Previous studies have shown that somatostatin receptors (SSTRs) are overexpressed on NET cell membranes relative to healthy tissue, allowing for tumor targeting through radiolabeled somatostatin analogs (SSAs). This work aims to develop a novel 89Zr-labeled tracer incorporating the SSA, octreotide (TOC), for positron emission tomography (PET) imaging of SSTR + NETs and predictive dosimetry calculations, leveraging the excellent nuclear (t½ = 3.27 days, β+ = 22.3%, β+avg = 395.5 keV) and chemical characteristics (+ 4 oxidation state, preferential coordination number of 7/8, favorable aqueous chemistry) of 89Zr. In combination with 89Zr, the known radiochemistry with the chelator deferoxamine (DFO) gives reason to believe that this radiopharmaceutical incorporating an octreotide conjugate will be successful in studying the suitability of detecting SSTR + NETs.ResultsRadiochemical tracer assessment indicated that amounts as low as 0.1 nmol DFO-TOC can be effectively radiolabeled with 89Zr, while maintaining ≥ 95% radiochemical yield. The stability of the compound was found to maintain radiochemical yields of 89.6% and 88.7% on the benchtop and in mouse serum, respectively, after 9 days. Receptor binding and competitive receptor blocking assays compared AR42J (high SSTR expression), PC-3 (moderate SSTR expression), and PANC-1 (minimal SSTR expression) cell lines at time points up to 6 days. In vitro studies demonstrated highest uptake in AR42J cells, and statistically significant differences in tracer uptake were seen after 1 h. Internalization assays showed maximum internalization after 3 h for all cell lines.ConclusionsIn this work, [89Zr]Zr-DFO-TOC was synthesized with radiochemical yields ≥ 95% and was found to remain stable in vitro at extended time points. In vitro cell studies demonstrated a statistically significant difference between receptor binding and blocking experiments. The development of this work shows potential to positively impact patient care through the predictive dosimetry calculations for the FDA-approved therapeutic agent [177Lu]Lu-DOTA-TATE, while allowing for imaging at extended timepoints and should be studied further.

Unleashing innovation: 3D-printed biomaterials in bone tissue engineering for repairing femur and tibial defects in animal models - a systematic review and meta-analysis.

3D-printed scaffolds have emerged as an alternative for addressing the current limitations encountered in bone reconstruction. This study aimed to systematically review the feasibility of using 3D bio-printed scaffolds as a material for bone grafting in animal models, focusing on femoral and tibial defects. The primary objective of this study was to evaluate the efficacy, safety, and overall impact of these scaffolds on bone regeneration. Electronic databases were searched using specific search terms from January 2013 to October 2023, and 37 relevant studies were finally included and reviewed. We documented the type of scaffold generated using the 3D printed techniques, detailing its characterization and rheological properties including porosity, compressive strength, shrinkage, elastic modulus, and other relevant factors. Before incorporating them into the meta-analysis, an additional inclusion criterion was applied where the regenerated bone area (BA), bone volume (BV), bone volume per total volume (BV/TV), trabecular thickness (Tb. Th.), trabecular number (Tb. N.), and trabecular separation (Tb. S.) were collected and analyzed statistically. 3D bio-printed ceramic-based composite scaffolds exhibited the highest capacity for bone tissue regeneration (BTR) regarding BV/TV of femoral and tibial defects of animal models. The ideal structure of the printed scaffolds displayed optimal results with a total porosity >50% with a pore size ranging between 300- and 400µM. Moreover, integrating additional features and engineered macro-channels within these scaffolds notably enhanced BTR capacity, especially observed at extended time points. In conclusion, 3D-printed composite scaffolds have shown promise as an alternative for addressing bone defects.

Immunogenicity and Protective Efficacy of a Multi-Antigen Mycobacterium tuberculosis Subunit Vaccine in Mice.

There is an urgent need for an effective TB vaccine capable of controlling both acute and chronic Mycobacterium tuberculosis infection in populations with diverse genetic backgrounds. In this study, we characterised the immunogenicity and protective efficacy of a novel protein-in-adjuvant subunit vaccine. The protein component is a fusion protein of three different M. tuberculosis antigens, which we termed CysVac5: CysD, a major component of the M. tuberculosis sulfate activation pathway that is highly expressed during the chronic stage of M. tuberculosis infection, is fused with two major secreted mycobacterial antigens, Ag85B and MPT83. Vaccination of C57BL/6 mice with CysVac5, formulated in a monophosphoryl lipid A (MPLA) and dimethyldioctadecylammonium (DDA) adjuvant combination, resulted in the potent generation of polyfunctional CD4+ T cells secreting multiple cytokines, including IFN-γ, IL-2, TNF and IL-17, against each of the three components of the fusion protein. Furthermore, vaccination with CysVac5-MPLA/DDA conferred significant protection against infection in mouse lungs, which was greater than that afforded by BCG at extended time points post-challenge. The generation of antigen-specific and protective immunity was also observed in CysVac5 vaccinated BALB/c mice, indicating the vaccine could display efficacy across multiple genetic backgrounds. These results indicate that the CysVac5 vaccine has broad immunogenicity, is effective in controlling both acute and chronic phases of M. tuberculosis infection in mice, and warrants further investigation to assess its potential to control pulmonary TB.

An IL-17-DUOX2 axis controls gastrointestinal colonization by Candida albicans.

Candida albicans is a ubiquitous fungus in the human gut microbiome as well as a prevalent cause of opportunistic mucosal and systemic disease. There is currently little understanding, however, as to how crosstalk between C. albicans and the host regulates colonization of this key niche. Here, we performed expression profiling on ileal and colonic tissues in germ-free mice colonized with C. albicans to define the global response to this fungus. We reveal that Duox2 and Duoxa2 , encoding dual NADPH oxidase activity, are upregulated in both the ileum and colon, and that induction requires the C. albicans yeast-hyphal transition and the hyphal-specific toxin candidalysin. Hosts lacking the IL-17 receptor failed to upregulate Duox2/Duoxa2 in response to C. albicans , while addition of IL-17A to colonoids induced these genes together with the concomitant production of hydrogen peroxide. To directly define the role of Duox2/Duoxa2 in fungal colonization, antibiotic-treated mice lacking intestinal DUOX2 activity were evaluated for C. albicans colonization and host responses. Surprisingly, loss of DUOX2 function reduced fungal colonization at extended time points (>17 days colonization) and increased the proportion of hyphal cells in the gut. IL-17A levels were also elevated in C. albicans -colonized mice lacking functional DUOX2 highlighting cross-regulation between this cytokine and DUOX2. Together, these experiments reveal novel links between fungal cells, candidalysin toxin and the host IL-17-DUOX2 axis, and that a complex interplay between these factors regulates C. albicans filamentation and colonization in the gut.

Abstract 3198: An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer

Abstract Background: Every breast cancer diagnosis is unique in its own way. Triple-negative breast cancer (TNBC), has a highly aggressive phenotype characterized by a rapid growth rate profile, increased risk of metastasis and recurrence. TNBC is devoid of epidermal growth factor receptor 2, estrogen, and progesterone receptors. All three are commonly present in other forms of breast cancer disease. The aim of this study was to develop a cell membrane lipid-extracted nanoliposomes (CLENs) for selective targeting compared to conventional nanoliposomal varieties. An additional objective was to investigate the role of microenvironment on targeting using cell model of TNBC, and representations of non-target tissue environments for comparison. Method: Lipid extracts (LE) obtained from 4T1, a murine mammary carcinoma cell line from a BALB/cfC3H mouse was used to formulate the CLENs (cell membrane lipid-extracted nanoliposomes) with different ratios of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid used in most conventional drug delivery liposomes. Different lipid compositions and ratios were employed to evaluate cellular uptake in target, non-target and off-target cells. The target cell line, 4T1, off-target and non-target cell populations included CRL-2089, normal breast fibroblasts, SKBR3 a (human breast cancer), and A549 a (lung cancer) cell lines. Other studies involved determination of molecular charge and zeta potential of various nanoliposomal preparations and corresponding fluorescence studies in vitro. Results: The physicochemical properties of CLENs such as particle size and surface charge characteristics were vital when assessing benefit of the lipid-based preparations. The size of the CLENs typically ranged between 130 - 165 nm and zeta potential values were negatively-charged. The inclusion of 70 mol% of LE in preparation of 4T1-CLENs demonstrated increased cellular uptake and binding by the 4T1 target cells compared to the control DOPC (100%). The highest degree of selectivity was observed in early cell binding events, and with studies involving more extended incubation time points under different experimental conditions. Off-target cells demonstrated reduced uptake by comparison. Overall, 4T1-CLENs was most efficient when applied against intended target 4T1 cells, when compared to relevant control cell populations. In conclusion, studies performed to date suggest the notion that lipid extracts derived from TNBC improves targeting. Citation Format: Christiana Ayertey, Parmida Amid, Mohammed H. Almozain, Robert B. Campbell. An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3198.

Evaluation of [89Zr]Zr-DFO-2Rs15d Nanobody for Imaging of HER2-Positive Breast Cancer.

One of the most aggressive forms of breast cancer involves the overexpression of human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in ∼25% of all breast cancers and is associated with increased proliferation, increased rates of metastasis, and poor prognosis. Treatment for HER2-positive breast cancer has vastly improved since the development of the monoclonal antibody trastuzumab (Herceptin) as well as other biological constructs. However, patients still commonly develop resistance, illustrating the need for newer therapies. Nanobodies have become an important focus for potential development as HER2-targeting imaging agents and therapeutics. Nanobodies have many favorable characteristics, including high stability in heat and nonphysiological pH, while maintaining their low-nanomolar affinity for their designed targets. Specifically, the 2Rs15d nanobody has been developed for targeting HER2 and has been evaluated as a diagnostic imaging agent for single-photon emission computed tomography (SPECT) and positron emission tomography (PET). While a construct of 2Rs15d with the positron emitter 68Ga is currently in phase I clinical trials, the only PET images acquired in preclinical or clinical research have been within 3 h postinjection. We evaluated our in-house produced 2Rs15d nanobody, conjugated with the chelator deferoxamine (DFO), and radiolabeled with 89Zr for PET imaging up to 72 h postinjection. [89Zr]Zr-DFO-2Rs15d demonstrated high stability in both phosphate-buffered saline (PBS) and human serum. Cell binding studies showed high binding and specificity for HER2, as well as prominent internalization. Our in vivo PET imaging confirmed high-quality visualization of HER2-positive tumors up to 72 h postinjection, whereas HER2-negative tumors were not visualized. Subsequent biodistribution studies quantitatively supported the significant HER2-positive tumor uptake compared to the negative control. Our studies fill an important gap in understanding the imaging and binding properties of the 2Rs15d nanobody at extended time points. As many therapeutic radioisotopes have single or multiday half-lives, this information will directly benefit the potential of the radiotherapy development of 2Rs15d for HER2-positive breast cancer patients.

Evaluation of SARS-CoV-2 antibody point of care devices in the laboratory and clinical setting.

SARS-CoV-2 antibody tests have been marketed to diagnose previous SARS-CoV-2 infection and as a test of immune status. There is a lack of evidence on the performance and clinical utility of these tests. We aimed to carry out an evaluation of 14 point of care (POC) SARS-CoV-2 antibody tests. Serum from participants with previous RT-PCR (real-time polymerase chain reaction) confirmed SARS-CoV-2 infection and pre-pandemic serum controls were used to determine specificity and sensitivity of each POC device. Changes in sensitivity with increasing time from infection were determined on a cohort of study participants. Corresponding neutralising antibody status was measured to establish whether the detection of antibodies by the POC device correlated with immune status. Paired capillary and serum samples were collected to ascertain whether POC devices performed comparably on capillary samples. Sensitivity and specificity varied between the POC devices and in general did not meet the manufacturers’ reported performance characteristics, which signifies the importance of independent evaluation of these tests. The sensitivity peaked at ≥20 days following onset of symptoms, however sensitivity of 3 of the POC devices evaluated at extended time points showed that sensitivity declined with time. This was particularly marked at >140 days post infection. This is relevant if the tests are to be used for sero-prevalence studies. Neutralising antibody data showed that positive antibody results on POC devices did not necessarily confer high neutralising antibody titres, and that these POC devices cannot be used to determine immune status to the SARS-CoV-2 virus. Comparison of paired serum and capillary results showed that there was a decline in sensitivity using capillary blood. This has implications in the utility of the tests as they are designed to be used on capillary blood by the general population.

In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

Vanadium pentoxide nanoparticle mediated perturbations in cellular redox balance and the paradigm of autophagy to apoptosis

The redox-active transition metals such as copper, iron, chromium, vanadium, and silica are known for its ROS generation via mechanisms such as Haber-Weiss and Fenton-type reactions. Nanoparticles of these metals induce oxidative stress due to acellular factors owing to their small size and more reactive surface area, leading to various cellular responses. The intrinsic enzyme-like activity of nano vanadium has fascinated the scientific community. However, information concerning their cellular uptake and time-dependent induced effects on their cellular organelles and biological activity is lacking. This comprehensive study focuses on understanding the precise molecular interactions of vanadium pentoxide nanoparticles (VnNp) and evaluate their specific “nano” induced effects on MDA-MB-231 cancer cells. Understanding the mechanism behind NP-induced ROS generation could help design a model for selective NP induced toxicity, useful for cancer management. The study demonstrated the intracellular persistence of VnNp and insights into its molecular interactions with various organelles and its overall effects at the cellular level. Where triple-negative breast cancer MDA-MB-231 cells resulted in 59.6% cell death towards 48 h of treatment and the normal fibroblast cells showed only 15.4% cell death, indicating an inherent anticancer property of VnNp. It acts as an initial reactive oxygen species quencher, by serving itself as an antioxidant, while; it was also found to alter the cellular antioxidant system with prolonged incubation. The VnNp accumulated explicitly in the lysosomes and mitochondria and modulated various cellular processes including impaired lysosomal function, mitochondrial damage, and autophagy. At more extended time points, VnNp influenced cell cycle arrest, inhibited cell migration, and potentiated the onset of apoptosis.Results are indicative of the fact that VnNp selectively induced breast cancer cell death and hence could be developed as a future drug molecule for breast cancer management. This could override the most crucial challenge of chemo-resistance that still remain as the main hurdle to cancer therapy.

Targeted Ultrasound Contrast Imaging of Tumor Vasculature With Positively Charged Microbubbles.

Molecular ultrasound imaging of tumor vasculature is being actively investigated with microbubble contrast agents targeted to neovasculature biomarkers. Yet, a universal method of targeting tumor vasculature independent of specific biomarkers, or in their absence, would be desirable. We report the use of electrostatic interaction to achieve adherence of microbubbles to tumor vasculature and resulting tumor delineation by ultrasound imaging. Microbubbles were prepared from decafluorobutane gas by amalgamation of aqueous micellar medium. Distearoyl phosphatidylcholine (DSPC) and polyethylene glycol (PEG)-stearate were used as microbubble shell-forming lipids; cationic lipid distearoyl trimethylammoniumpropane (DSTAP) was included to introduce positive electrostatic charge. Microbubbles were subjected to flotation in normal gravity, to remove larger particles. Murine colon adenocarcinoma tumor (MC38, J. Schlom, National Institutes of Health) was inoculated in the hind leg of C57BL/6 mice. Contrast ultrasound imaging was performed under isoflurane anesthesia, using a clinical imaging system in low power mode, with tissue signal suppression (contrast pulse sequencing, 7 MHz, 1 Hz; Mechanical Index, 0.2). The ultrasound probe was positioned to monitor the tumor and contralateral leg muscle; microbubble contrast signal was monitored for 30 minutes or more, after intravenous bolus administration of 2.10 microbubbles. Individual time point frames were extracted from ultrasound video recording and analyzed with ImageJ. Mean bubble diameter was ~1.6 to 2 μm; 99.9% were less than 5 μm, to prevent blocking blood flow in capillaries. For cationic DSTAP-carrying microbubbles, contrast signal was observed in the tumor beyond 30 minutes after injection. As the fraction of positively charged lipid in the bubble shell was increased, adherent contrast signal in the tumor also increased, but accumulation of DSTAP-microbubbles in the normal muscle increased as well. For bubbles with the highest positive charge tested, DSTAP-DSPC molar ratio 1:4, at 10 minutes after intravenous administration of microbubbles, the contrast signal difference between the tumor and normal muscle was 1.5 (P < 0.005). At 30 minutes, tumor/muscle contrast signal ratio improved and reached 2.1. For the DSTAP-DSPC 1:13 preparation, tumor/muscle signal ratio exceeded 3.6 at 10 minutes and reached 5.4 at 30 minutes. Microbubbles with DSTAP-DSPC ratio 1:22 were optimal for tumor targeting: at 10 minutes, tumor/muscle signal ratio was greater than 7 (P < 0.005); at 30 minutes, greater than 16 (P < 0.01), sufficient for tumor delineation. Cationic microbubbles are easy to prepare. They selectively accumulate in the tumor vasculature after intravenous administration. These microbubbles provide target-to-control contrast ratio that can exceed an order of magnitude. Adherent microbubbles delineate the tumor mass at extended time points, at 30 minutes and beyond. This may allow for an extension of the contrast ultrasound examination time. Overall, positively charged microbubbles could become a universal ultrasound contrast agent for cancer imaging.

Long-term neural regeneration following injury to the peroneal branch of the sciatic nerve in sheep.

Peripheral nerves (PNs) are frequently injured as a result of trauma or disease. Development of therapies to regenerate PNs requires the use of animal models, typically beginning in rodents and progressing to larger species. There are several large animal models of PN regeneration that each has their benefits and drawbacks. Sheep have been used in PN studies due to their similarities in body weight to humans and the ease and lesser expense in their care and housing relative to other species. We have investigated the use of sheep for studies of PN regeneration and have developed and tested an injury model in the peroneal branch of the sciatic nerve. Three experimental groups were tested on mature sheep: a bisection; a 5-cm reverse autograft; and sham surgery. Protocols were developed for the post-operative care for animals with this injury, and regeneration was tracked for extended time points via compound muscle action potentials (CMAPs) and endpoint assessments of nerve morphometry, muscle mass and muscle fibrosis. Results indicate the practical viability of this PN injury model and show distinctions in the degree and rate of regeneration between bisection and reverse autograft that persisted 14months. This long-term study shows bisections lead to significantly improved CMAPS and muscle mass and lesser muscle fibrosis as compared to reverse autograft. The persistence of these discernable changes between two relatively similar experimental groups out to extended time points is an indication of the sensitivity of this nerve section and its potential applicability for comparative studies.

Investigating the role of Fzd‐7 in liver donation and regeneration

The Wnt‐Fzd‐LRP5/6‐β‐catenin signaling axis has important roles in liver homeostasis, regeneration and disease. Through use of liver‐ and cell‐type‐specific gene knockout mouse models, we and others have confirmed roles for β‐catenin and LRP5/6 in regulating metabolic zonation of the liver as well as initiation of liver regeneration after partial hepatectomy. Similarly, using cell‐type‐specific Wls gene knockout models we have identified endothelial cells and Kupffer cells as significant sources of Wnt proteins responsible for regulating β‐catenin signaling in liver regeneration. However, little is known about the actual frizzled (Fzd) receptor(s) that are critical to transmitting the Wnt signals that regulate metabolic zonation and liver regeneration. We do know that Fzd‐7 is highly expressed in some liver cancers. Here, we further investigate the role of Fzd‐7 in metabolic zonation and liver regeneration after partial hepatectomy. We used microdissection to isolate cells from 3 major zones of the liver (peri‐portal, mid‐zonal, pericentral) to assess the expression of Fzd receptors in each zone by qPCR. We observed ~3‐fold higher expression of Fzd‐7 in the pericentral zone, indicating a possible role in metabolic zonation. We also examined and found upregulation of Fzd‐7 gene expression at 12–24h after hepatectomy in mice. Therefore, we obtained Fzd‐7‐floxed mice to conditionally delete Fzd‐7 in the liver and investigate its role in metabolic zonation and liver regeneration. We injected Fzd‐7 floxed mice with AAV8‐TBG‐Cre to conditionally knockout Fzd‐7 from hepatocytes. Two weeks after administration of AAV8‐TBG‐Cre, we performed PCR analysis and confirmed successful knockout of the Fzd‐7 mRNA as well as detection of the floxed‐deletion in the liver DNA. However, analysis of GS and CYP2e1 showed no defect in metabolic zonation at 2 weeks post AAV injection. We also performed partial hepatectomy 14 days after AAV‐TBG‐Cre injection and analyzed the regenerating livers. Hepatocyte proliferation by BrdU staining was marginally lower in the AAV8‐Cre injected mice at 40h post hepatectomy indicating a possible delay in liver regeneration. Further characterization has shown compensatory increases in expression of other Fzd receptors in the AAV8‐Cre livers which may explain the lack of overt phenotype in these mice. Since Fzd‐7 protein may have longer half‐life, extended time points after AAV8‐TBG‐Cre injection to Fzd‐7‐floxed mice are currently under investigation. Additional characterization with Alb‐Cre‐Fzd‐7 liver knockouts is also being done. In conclusion, Fzd‐7 expression is highest in zone‐3 and is also upregulated early after hepatectomy. While acute deletion of Fzd‐7 did not reveal changes in zonation, a decrease in hepatocyte proliferation during regeneration was evident. Fzd‐7 may be an important mediator of Wnt‐β‐catenin signaling in the liver.Support or Funding InformationDepartment of Pathology and Pittsburgh Liver Research Center, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, PA

Risk Factors for Postoperative Opioid Use in Arthroscopic Shoulder Labral Surgery

To determine the correlation between preoperative and postoperative opioid use in patients undergoing arthroscopic shoulder labral repair, as well as patient risk factors associated with increased postoperative opioid use after the procedure. A retrospective review of all patients undergoing arthroscopic shoulder labral surgery at a single institution between August 2013 and November 2017 was performed. Patients were stratified as opioid nonusers, acute users, or chronic users based on preoperative consumption. Patient demographic characteristics, injury characteristics, surgical interventions, and postoperative opioid use for the first 12 months after surgery were then analyzed. A total of 340 patients were included in this study. The average age was 26.3 years (range, 13-68 years), and the average body mass index was 27.5 (range, 18.4-45.0). Preoperative opioid users (acute and chronic) were found to continue to receive opioid medications at extended time points beyond 2 months postoperatively compared with nonusers (P < .001). Patients with intraoperatively identified SLAP tears experienced more preoperative pain and required more postoperative opioid prescriptions (P = .018). On stratification for other common shoulder instability injury patterns, no differences were found between the number of postoperative opioid prescriptions filled and the presence of Bankart lesion, Hill-Sachs lesion, reverse Hill-Sachs lesion, anterior labroligamentous periosteal sleeve avulsion, glenolabral articular disruption, or humeral avulsion of the glenohumeral ligament (P > .05). In patients undergoing arthroscopic labral surgery, the chronicity of preoperative opioid use, number of concomitant procedures at the time of initial surgery, and presence of biceps tenodesis were found to significantly increase postoperative opioid demand. Orthopaedic surgeons should recognize risk factors for increased opioid use postoperatively and adapt treatment strategies and patient counseling accordingly. Level III, retrospective cohort study.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Acute Stress Persistently Alters Locus Coeruleus Function and Anxiety-like Behavior in Adolescent Rats

Stress is a physiological state characterized by altered neuroendocrine signaling, behavioral arousal, and anxiety. Chronic or traumatic stress may predispose individuals for multiple somatic and psychiatric illnesses. The locus coeruleus (LC) is a major node in the stress response that integrates input from multiple stress responsive neural circuits and releases norepinephrine (NE) throughout the central nervous system (CNS) to promote vigilance and anxiety. Many mood disorders associated with prior stress are characterized by chronically altered noradrenergic signaling, yet the long-term impact of an acute stressor on LC function is not clear. To determine how acute stress could affect anxiety-like behavior as well as LC function at immediate and extended time points, rats underwent simultaneous exposure to physical restraint and predator odor. Rats underwent behavioral testing immediately or one week after stressor exposure and were then sacrificed for whole-cell patch-clamp recordings of LC neurons. Stress caused an immediate increase in anxiety-like behaviors in the elevated plus maze (EPM), as well decreased excitatory synaptic transmission and increased spontaneous discharge in LC neurons. These effects persisted for seven days after stress. Importantly, the excitability of LC neurons was increased one week post-stress, but not immediately after, suggesting a long-term adaptation by the system. Rats tested in the open field one week after stress also showed increased anxiety-like behaviors. These findings show that a single acute stressor is capable of precipitating long-lasting changes in the LC function that may be related to some of the behavioral effects of stress, potentially contributing to stress-induced disease pathogenesis.

Long-Term Deficits in Behavior Performances Caused by Low- and High-Linear Energy Transfer Radiation

Efforts to protect astronauts from harmful galactic cosmic radiation (GCR) require a better understanding of the effects of GCR on human health. In particular, little is known about the lasting effects of GCR on the central nervous system (CNS), which may lead to behavior performance deficits. Previous studies have shown that high-linear energy transfer (LET) radiation in rodents leads to short-term declines in a variety of behavior tests. However, the lasting impact of low-, medium- and high-LET radiation on behavior are not fully defined. Therefore, in this study C57BL/6 male mice were irradiated with 100 or 250 cGy of γ rays (LET ∼0.3 KeV/μm), 10 or 100 cGy of 1H at 1,000 MeV/n (LET ∼0.2 KeV/μm), 28Si at 300 MeV/n (LET ∼69 KeV/μm) or 56Fe at 600 MeV/n (LET of ∼180 KeV/μm), and behavior metrics were collected at 5 and 9 months postirradiation to analyze differences among radiation qualities and doses. A significant dose effect was observed on recognition memory and activity levels measured 9 months postirradiation, regardless of radiation source. In contrast, we observed that each ion species had a distinct effect on anxiety, motor coordination and spatial memory at extended time points. Although 28Si and 56Fe are both regarded as high-LET particles, they were shown to have different detrimental effects on behavior. In summary, our findings suggest that GCR not only affects the CNS in the short term, but also has lasting damaging effects on the CNS that can cause sustained declines in behavior performance.

Abstract 3678: Imaging AZD1152HQPA Accurin™ nanoparticle accumulation in preclinical tumors

Abstract AZD1152HQPA Accurin is a passively targeted nanoparticle containing the active metabolite of the Aurora B kinase prodrug AZD1152. In preclinical studies, AZD1152HQPA Accurin displays reduced bone marrow toxicity associated with AZD1152 together with increases in anti-tumour activity. Nanoparticles are proposed to enable delivery and retention of drug to the tumour thereby increasing efficacy and modifying therapeutic index. While it is possible to demonstrate increased duration of modulation of pharmacodynamic biomarkers and drug exposure by nanoparticle delivery, directly demonstrating that nanoparticles accumulate in the tumour is challenging. To investigate the intra tumoural distribution of AZD1152HQPA Accurin nanoparticles we have employed mass spectrometry techniques to image samples derived from human colorectal cancer SW620 studies in rats. We have separately identified the nanoparticle itself, drug encapsulated in nanoparticle, and drug released from nanoparticle; the latter by detecting a metabolite that is only generated from released drug. First MALDI (matrix assisted laser desorption ionization) which offers 20 μm spatial resolution, was used to detect AZD1152-hQPA (released and encapsulated). Second LESA (liquid extraction surface analysis), which offers 1000 μm spatial resolution, was used to detect drug and metabolite, enabling co-distribution to be demonstrated. Finally DESI (desorption electrospray ionization) which offers 100 μm spatial resolution was used to detect drug, metabolite and nanoparticle constituent showing the co-distribution in more detail. Using this combination of approaches it is possible to demonstrate the presence of nanoparticle-delivered AZD1152HQPA metabolite in the tumour at extended timepoints; co-located with nanoparticle and unchanged (encapsulated) drug. At these time points no drug remains if delivered as a simple IV infusion. Collectively this imaging mass spectrometry analysis demonstrates AZD1152HQPA Accurin accumulation in pre-clinical tumours and confirms that the Accurin accesses the tumour and achieves sustained drug release within the tumour. Citation Format: Richard Goodwin, John Swales, Anna Nilsson, Per Andren, Nicola Strittmatter, Zoltan Takats, Colin Howes, Paula Taylor, Susan Ashton, Philip Jewsbury, Simon T. Barry. Imaging AZD1152HQPA Accurin™ nanoparticle accumulation in preclinical tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3678. doi:10.1158/1538-7445.AM2015-3678

Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.

The influence of Trp53 in the dose response of radiation-induced apoptosis, DNA repair and genomic stability in murine haematopoietic cells.

Apoptotic and DNA damage endpoints are frequently used as surrogate markers of cancer risk, and have been well-studied in the Trp53+/- mouse model. We report the effect of differing Trp53 gene status on the dose response of ionizing radiation exposures (0.01-2 Gy), with the unique perspective of determining if effects of gene status remain at extended time points. Here we report no difference in the dose response for radiation-induced DNA double-strand breaks in bone marrow and genomic instability (MN-RET levels) in peripheral blood, between wild-type (Trp53+/+) and heterozygous (Trp53+/-) mice. The dose response for Trp53+/+ mice showed higher initial levels of radiation-induced lymphocyte apoptosis relative to Trp53+/- between 0 and 1 Gy. Although this trend was observed up to 12 hours post-irradiation, both genotypes ultimately reached the same level of apoptosis at 14 hours, suggesting the importance of late-onset p53-independent apoptotic responses in this mouse model. Expected radiation-induced G1 cell cycle delay was observed in Trp53+/+ but not Trp53+/-. Although p53 has an important role in cancer risk, we have shown its influence on radiation dose response can be temporally variable. This research highlights the importance of caution when using haematopoietic endpoints as surrogates to extrapolate radiation-induced cancer risk estimation.

Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

The success of stem cell-based cartilage repair requires that the regenerate tissue reach a stable state. To investigate the long-term stability of tissue engineered cartilage constructs, we assessed the development of compressive mechanical properties of chondrocyte and mesenchymal stem cell (MSC)-laden three dimensional agarose constructs cultured in a well defined chondrogenic in vitro environment through 112 days. Consistent with previous reports, in the presence of TGF-β, chondrocytes outperformed MSCs through day 56, under both free swelling and dynamic culture conditions, with MSC-laden constructs reaching a plateau in mechanical properties between days 28 and 56. Extending cultures through day 112 revealed that MSCs did not simply experience a lag in chondrogenesis, but rather that construct mechanical properties never matched those of chondrocyte-laden constructs. After 56 days, MSC-laden constructs underwent a marked reversal in their growth trajectory, with significant declines in glycosaminoglycan content and mechanical properties. Quantification of viability showed marked differences in cell health between chondrocytes and MSCs throughout the culture period, with MSC-laden construct cell viability falling to very low levels at these extended time points. These results were not dependent on the material environment, as similar findings were observed in a photocrosslinkable hyaluronic acid (HA) hydrogel system that is highly supportive of MSC chondrogenesis. These data suggest that, even within a controlled in vitro environment that is conducive to chondrogenesis, there may be an innate instability in the MSC phenotype that is independent of scaffold composition, and may ultimately limit their application in functional cartilage repair.