2024 Abstracts

Introduction: Mitochondria transplantation has emerged as a promising regenerative therapy for vascular disease due to the significant contribution of mitochondrial dysfunction in inflammation, ischemia-reperfusion injury, and oxidative stress. However, current transplantation strategies suffer from a lack of specificity, limited uptake, uncontrolled biodistribution, and overall subpar efficiency To combat this, we are engineering targeted mitochondrial delivery systems (MDS) using modular polymer coatings that allow easy exchange and combination of tissue-specific and cell-penetrating peptides to improve the efficacy of mitochondrial transplantation. We previously showed that collagen binding peptide (CBP) preferentially binds with the basement membrane protein collagen IV, suggesting it can be exploited to target the vascular endothelium after injury. We pose that CBP can be combined with MDS coating to improve mitochondria transplantation to vascular endothelial cells after injury.

Methods: Mitochondria were isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) and stained with Mitotracker Red. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] (DSPE-PEG) was conjugated with cysteine-modified collagen-binding peptide labeled with FITC (CBP). NMR is being employed to confirm successful covalent modification. Isolated mitochondria were coated with DSPE-PEG-CBP conjugates at varying polymer: mitochondria mass ratios and imaged using fluorescent microscopy to confirm coating. Dynamic light scattering was used to monitor particle size. Mitochondria function was determined using the Seahorse Mito Stress Test assay to measure the oxygen consumption rate.

Results: Under fluorescence microscopy, isolated mitochondria (red) can be visualized with polymer coating (green). The polymer coating did not significantly change the average mitochondria particle size 399.6±149.3nm (uncoated), 426.4±76.2nm (1.5:1 mass ratio), and 411.7±152.9 nm (3:1 mass ratio) with dynamic light scattering analysis. CBP retained its binding affinity to collagen after conjugation with DSPE. The polymer coating also did not impact mitochondrial function, with no significant differences in the basal respiration, maximum respiration, and ATP production among groups.

Conclusion: In summary, DSPE-PEG peptide polymer conjugates are an effective method to coat mitochondria while preserving function. In the future, we plan to demonstrate that coated mitochondria can rescue endothelial cells from oxidative stress before advancing to in vivo studies to show that MDS target damaged endothelium and improve healing in a carotid balloon injury model.

Introduction: Cardiovascular diseases are a leading cause of mortality and morbidity worldwide, often necessitating the replacement of damaged blood vessels with vascular grafts. Conventional vascular grafts suffer from limitations such as poor long-term patency and the inability to provide real-time information about graft performance. This project aims to develop a novel generation of vascular grafts that are not only biocompatible but also electrically conductive. These bioelectronic vascular grafts will pave the way for smart vascular prostheses with built-in bio-sensing capabilities, addressing the shortcomings of current vascular graft technologies.

Methods: Highly soluble, self-doped conductive polymer sulfonated poly(3,4-ethylenedioxythiophene) (S-PEDOT) was synthesized via oxidative polymerization. Rat aortas were decellularized and subsequently incubated in varying concentrations of S-PEDOT or S-EDOT at 37°C. After incubation or polymerization, all aortas underwent a 24-hour leaching step at room temperature to remove unbound material. Conductivity of the modified aortas were measured via 4-point probe. To evaluate in vitro biocompatibility of S-PEDOT incorporation, human umbilical vein endothelial cells (HUVECs) and aortic smooth muscle cells (HAoSMCs) were cultured on collagen hydrogels incubated with S-PEDOT. Morphological assessment with phase contrast microscopy and live/dead stains were performed after 48 hours. Thrombogenicity was assessed with a whole blood coagulation assay. To assess biocompatibility in vivo, S-PEDOT-incorporated aorta sections were implanted subcutaneously in Sprague-Dawley rats for two weeks. Masson’s trichrome and immunofluorescence staining for macrophages (CD68+ cells) were performed to assess inflammation. Additional immunofluorescence staining for vascular and immune cells is currently underway.

Results: Modified grafts demonstrated thorough monomer and polymer penetration as seen by a change in color through and along the length of the vascular wall. Conductivity was increased by an order of magnitude between aortas incubated in 10- and 100-mM S-EDOT before polymerization. Vascular cells demonstrated some rounding, but overall maintained viability on S-PEDOT-modified collagen over 48 hours. Collagen gels with S-PEDOT performed similarly to unmodified collagen gels in response to whole blood incubation, both demonstrating higher thrombogenicity than tissue culture plastic, as expected. No evidence of infection, seroma, or delayed wound healing was observed in subcutaneous implantation studies, though nuclear infiltration was noted to be qualitatively elevated.

Conclusions: Conductive vascular grafts can be generated by coating decellularized aortas with S-PEDOT via in situ polymerization or incubation in previously polymerized S-PEDOT. Aortas incubated in 100 mM S-EDOT and polymerized in situ demonstrate improved conductivity. S-PEDOT-modified extracellular matrix materials demonstrate hemocompatibility and biocompatibility both in vitro and in vivo.

Introduction: A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through serine, one-carbon, glycine and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression.

Methods: MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione and 2-hydroxyglutarate concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA.

Results: Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, glutathione and 2-hydroxyglutarate after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.01), peaking at 15 min. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01) including neural, EMT and ER-BC-related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p < 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased DNA breaks. scRNAseq revealed OA increased the numbers of cells within cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. BSL1, HS1 and LP3 had an increase dependence on serine and antioxidants upon OA.

Conclusions: Metabolism of OA results in a metabolic shift toward serine and methionine increasing the production of SAM, glutathione and 2-hydroxyglutarate which have implications for oncogenesis. The increased SAM and 2-hydroxyglutarate fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress.

Introduction: Aortic remodeling remains an important contributor to the pathogenesis of aortic disease, including occlusive, aneurysmal, and dissection disease states. The paucity of medical therapies has driven considerable interest in elucidating the pathogenesis of these conditions; new therapeutic targets are critically needed. Prior studies have identified sympathetic nervous system signaling (SNS) as a critical regulator of arterial wall homeostasis with potent effects on inflammation and vascular remodeling. This study seeks to evaluate the impact of sustained denervation on morphology and wall homeostasis of the abdominal aorta. We hypothesized that topical phenol denervation of the rat abdominal aorta would induce sustained reduction in nerve fiber density with concomitant pathologic changes to tissue morphology.

Methods: Male and female Sprague-Dawley rats (N=12), aged 3 months, underwent midline laparotomy for infrarenal aorta exposure. Chemical denervation was induced via a one-time topical application of 10% phenol (n=6), while sham controls received phosphate-buffered saline (n=6). Animals were allowed to recover and subsequently sacrificed after 6 months for analysis encompassing morphology, histology, and immunohistochemistry.

Results: At 6 months post-treatment, abdominal aortas subjected to phenol denervation exhibited a significant reduction in nerve fiber density compared to sham controls (92.8 ± 20.3 fibers/mm2 vs 170.4 ± 79.0 fibers/mm2, p=0.042). Denervated aortas demonstrated increased elastin fragmentation breakage scores (2.8 ± 1.1 vs 1.1 ± 0.4, p=0.0056). Phenol denervated rats also had significantly decreased expression of vascular smooth muscle proteins α-SMA (32.6 ± 3.9% vs 24.6 ± 3.3%, p=0.0034) and MYH11(14.2 ± 5.7% vs 21.6 ± 3.5%, p=0.023), as well as elevated adventitial microvascular density (58.9 ± 25.2 vessels/mm2 vs sham control 18.5 ± 8.7 vessels/mm2, p=0.0040).

Conclusion: Single-timepoint phenol-based chemical denervation induces sustained alterations in abdominal aortic morphology and vascular remodeling over a 6-month period. These findings underscore the potential of the SNS as a therapeutic target for aortic pathologies.

Introduction: Patients aged 65 years and older account for an increasing proportion of those who suffer from traumatic brain injury (TBI). Aged TBI patients experience increased morbidity and mortality compared to young TBI patients. Our prior data demonstrated that anti-CD49d antibody (aCD49d Ab), an FDA-approved drug that blocks α4 integrin, abrogates infiltration of T-cells into the injured brain, improves survival, and attenuates neurocognitive deficits. Yet, the molecular mechanisms underlying the therapeutic effects of aCD49d Ab remained unexplored. We hypothesized that aCD49d Ab treatment would mitigate neuroinflammation-associated CD8+ T cell cytotoxicity after TBI in aged mice.

Methods: Young (12 weeks; N=20) and aged (80 weeks; N=20) male C57BL/6 mice underwent TBI via controlled cortical impact vs. sham injury. 300 ug of aCD49d Ab, or isotype control, were administered 2 hours post-TBI via intraperitoneal injection. Dosing was repeated every two weeks. Brains were harvested two months post-TBI, and CD45+ cells were isolated via fluorescence-activated cell sorting. 10x Single-cell RNA coupled with T-cell receptor sequencing was employed to determine the T-cell transcriptome and clonality. Ipsilateral brain tissue homogenates were collected for Luminex-based cytokine analysis and high-parameter flow cytometry to assess T cell subtypes and PD-1, CD69, and Nur77 expression.

Results: Mice incurred age-associated toxic cytokine and chemokine responses long-term post-TBI. aCD49d Ab attenuated this response along with a T helper (Th)1/Th17 immunological shift and produced a neuroprotective Th2 response (p=0.0209). Aged mouse brains had more clonally expanded CD8 T+ cells than young mouse brains after TBI (74% vs. 23%). aCD49d Ab reduced clonal expansion in aged mouse brains compared to isotype treatment after TBI (61% v. 74%). Furthermore, by blocking their infiltration, aCD49d Ab remediated a population of cytotoxic CD8 T+ cells associated with neuroinflammation. Flow cytometry measuring PD-1 expression further validated that aCD49d Ab significantly reduced CD8+ T cell cytotoxicity in aged mouse brains compared to isotype control (p<0.01).

Conclusion: We hypothesized that aCD49d Ab treatment would mitigate neuroinflammation-associated CD8+ T cell cytotoxicity after TBI in aged mice. We found that aCD49d Ab treatment produced a neuroprotective Th2 response along with remediation of the cytotoxicity of CD8+ T cells. Meanwhile, no such effect and phenotypic changes were observed in young mice post-TBI with aCD49d Ab treatment. Our prior work combined with the current findings indicates that infiltrating T cells may be a promising, age-specific, therapeutic target for treating TBI.

Introduction: Traumatic brain injury (TBI) is an underrecognized public health threat. Our prior data demonstrates that TBI leads to a significant infiltration of immune cells into the injured brain over time with the capability of worsening the index injury. We also found that restoring a pre-injury gut microbial community structure via fecal microbiome transplantation (FMT) attenuated neuroinflammation and mitigated secondary injury after TBI. Based on these data, we hypothesized that Short Chain Fatty Acids (SCFA) supplementation, metabolic byproducts of a typical gut microbial community structure, would attenuate neuroinflammation after TBI.

Methods: Male C57BL/6 mice, aged 14 weeks, underwent controlled cortical impact (TBI) or sham injury. Post-TBI, groups received either a mixture of SCFAs or NaCl vehicle in drinking water for four weeks. At 30 days, immune cells from mouse brains were analyzed via Fluorescent Activated Cell Sorting and single-cell RNA sequencing. Data processing included normalization and integration using Seurat, with further transcriptional analysis of microglia using differential gene expression and enrichment analysis.

Results: Post-TBI SCFA supplementation resulted in the downregulation of TBI phenotypes in microglia. According to gene expression, the most significant decrease was in the level of TGF-beta-associated microglia in TBI (1.42) compared to SCFA_TBI (-0.31) and apoptosis-associated microglia ((TBI (1.47) vs. SCFA_TBI (-0.34)). Most interestingly, SCFA treatment stabilized homeostatic microglia levels in SCFA_TBI mice (-0.40) compared to TBI alone (-1.23) (A-C). Consistent with previous findings, immune-related Reactome pathway analyses showed that TBI led to a significant down-regulation of neuroprotective heat shock genes (Hspa1a, Hsp90aa1, and Hspa8) and up-regulation of Uba5a and Apoe (D-I). With SCFA supplementation post-TBI, the aforementioned transcriptional changes were reversed with up-regulation of the neuroprotective heat shock genes (Hspa1a, Hspa1b, Hspb1) and enrichment in GO terms related to neuronal recovery (Astrocyte and Microglial Activation) (J-L).

Conclusions: We hypothesized that SCFA supplementation after TBI would reduce long-term neuroinflammation in mice. SCFA supplementation altered immune cell infiltration and attenuated inflammatory gene expression in TBI mice compared to salt vehicle control. The neuroprotective effects of SCFA supplementation mirror our previously published FMT results, suggesting that taxa-specific metabolites generated within the gut microbiome contribute to the neuroinflammatory response after TBI. This study highlights the role of dietary supplementation as a potential treatment paradigm in TBI.

Introduction: Tissue expansion (TE) is the leading form of post-mastectomy breast reconstruction. However, patients who receive radiation therapy have a high risk of complications. Currently, there is no FDA-approved skin treatment to improve TE in post-mastectomy radiation therapy (PMRT) patients. Using a porcine TE model, we are investigating the therapeutic potential of acellular dermal matrix (ADM) and/or topical deferoxamine (DFO) treatment in limiting the negative effect of radiation.

Methods: Yucatan minipigs underwent subcutaneous TE placement along bilateral flanks. Half of the expanders were wrapped in ADM, and half of the expanders received DFO treatment. All expanders underwent 2 weekly injections of 30 cc of saline with cutometer measurements and 3D images taken before and after every injection. One week after final injection, the minipigs received a single dose of 20 Gy radiation. After 8 weeks of resting period with DFO patch treatment on the assigned grids, the minipigs will be euthanized.

Results: Our novel porcine TE model is representative of prepectoral placement of TE during breast reconstruction. We observed progressive skin growth induced by mechanical forces applied by the tissue expander in all expanded grids. Cutometer analysis showed use of DFO or ADM does not improve elasticity in expanded skin before radiation. The R2 value is calculated based on the cutometer's measurements of total deformation divided by total distensibility where the closer the value is to 1, indicates increased elasticity. The average R2 value for pre-irradiated expanded skin only was 0.496, pre-irradiated expanded skin with DFO was 0.546, pre-irradiated expanded skin with ADM was 0.564, pre-irradiated expanded skin with ADM and DFO was 0.467, pre-irradiated control skin on the left of the pig was 0.805 and pre-irradiated control skin on the right of the pig was 0.653. Lastly, irradiated skin showed clear phenotypic changes compared to non-irradiated controls.

Conclusions: We demonstrate a viable animal model to evaluate tissue expansion and methods to decrease radiation-induced skin injury. The decrease in elasticity in the expanded skin likely reflects the temporary changes in extracellular matrix reorganization and probable capsule formation due to expansion. Further isogeometric analysis will be used to calculate skin growth and deformation and the final radioprotective effect of DFO and ADM will be analyzed when the experiment is complete. This framework has the potential to optimize reconstructive efforts and reduce radiation-induced injury with clear translational potential for human patients.

Introduction: Apolipoprotein E (APOE) alleles impact pathogenesis and risk for multiple human diseases. The APOE 4 allele is the strongest genetic risk factor for sporadic Alzheimer’s disease (AD) and portends a worse outcome after traumatic brain injury (TBI). Meanwhile the APOE 2 allele is the strongest genetic protective factor for AD. Our laboratory recently demonstrated that APOE2 targeted replacement mice have superior neurocognitive outcomes after TBI. It is also known that the gut microbial community structure has marked allelic disparity between the APOE genotypes offering a putative mechanism for the neurologic protection seen in the APOE2 allelic variant via the gut-brain axis. We hypothesized that fecal microbiota transplant (FMT) of donor stool from APOE2 targeted replacement mice would restore the gut microbial community structure of wild type TBI mice to a preinjury state while APOE4 FMT would lead to continued TBI-induced dysbiosis.

Methods: C57Bl/6 male mice (14-weeks; n=20) underwent TBI via a controlled cortical impact or sham-injury. 2 hours post injury, mice underwent FMT with donor stool from either naive C57Bl/6 mice or APOE2 and APOE4 targeted replacement mice via oral gavage. Repeat gavage was performed weekly for 4 weeks. Stool pellets were collected 5 days pre-injury and at 5- and 45-days post TBI for 16s ribosomal RNA sequencing.

Results: Principal coordinate analysis (PCoA ) of stool at 45 days post TBI demonstrated difference in the beta diversity (unique microbial communities) among mice treated with FMT from APOE2 and APOE4 targeted replacement mice. Permutational multivariate analysis of variance (PERMNOVA) of the fecal microbiome revealed these differences to be significant. APOE2 FMT treatment demonstrated no preserved beta diversity in TBI mice as compared to sham injured controls (p=0.13) whereas APOE4 FMT showed a marked difference in beta diversity in TBI as compared to sham injured mice (p=0.04).

Conclusion: We hypothesized that fecal microbiota transplant (FMT) of donor stool from APOE2 targeted replacement mice restore the gut microbial community structure of wild type TBI mice to a preinjury state while APOE4 FMT would lead to continued TBI-induced dysbiosis. Our data shows a significant difference in beta diversity between APOE4 FMT and its sham control, which supports our hypothesis. These studies provide a putative mechanism for the neuroprotection in APOE allelic variants in both human disease and in our laboratory model of TBI. Ongoing studies within these cohorts include neurocognitive testing and neuroimaging which will be correlated to taxa-specific metabolic differences between allelic variants.

Introduction: Breast cancer (BC) risk reducing drugs have minimal impact due to their adverse effects and low acceptance by risk-eligible women. Further, they are ineffective against hormone receptor negative (HR-) BC. Novel agents with reduced toxicity and sufficient efficacy extending beyond HR+ BC are needed. We showed previously that licochalcone A (LicA) from licorice has antioxidant/anti-inflammatory effects, inhibits aromatase, and blocks estrogen genotoxic metabolism. We hypothesize that LicA prevents HR+ and HR- BC through reprogramming metabolism and antioxidant pathways.

Methods: We prepared microstructures from the fresh tissue of contralateral unaffected breast of 6 postmenopausal women with unilateral BC. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing followed by differential gene expression, pathway identification, and metabolism flux analyses. The NanoString metabolism panel was employed in 6 additional subjects. We monitored proliferation of HR- and HR+ pre-malignant and malignant, as well as BRCA-mutated cells using live cell imaging. Xenograft models of HR+ and HR- tumors in nude mice received 28-day treatment with vehicle or LicA (80 mg/kg.day, s.c.) and the tumor growth was monitored. Using LC-MS/MS we measured LicA in the blood and various tissues of BALB/c mice receiving oral LicA (100 mg/kg) and assessed PK.

Results: We observed significant (combined enrichment scores > 4 and FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system. This was accompanied by significant downregulation of NF-kB1-dependent inflammatory pathway. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis, transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose-5-phosphate formation, suggesting an antioxidant and antiproliferative environment. LicA suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cell lines. It reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) xenograft models. Oral bioavailability in serum and breast was in the low micromolar range and was sufficient to show efficacy.

Conclusions: Our data suggest that LicA is an excellent candidate for HR+ and HR- BC prevention by reprogramming metabolism and antioxidant pathways leading to antiproliferation. Next, we will test an optimized oral formulation of LicA in intraductal models of HR+ and HR- precancer lesions in immunocompetent mice to further establish its preventive efficacy.

Introduction: Traumatic brain injury (TBI) affects nearly 3 million people each year in the US. Patients aged 65 years and older account for an increasing proportion TBI victims. We previously published that fecal microbiota transplant (FMT) restores gut microbial community structure, decreases neuroinflammation leading to better neurocognitive outcomes in young TBI mice. However, the potential benefits of FMT in aged TBI subjects has yet to be determined. We hypothesized that restoring a youthful gut microbiome will improve neurocognitive outcomes in aged mice after TBI.

Methods: Aged C57BL/6 mice (84 weeks old, n=4-7 per group) were subjected to severe TBI or sham injury via an open-head controlled cortical impact. Two hours post injury the mice FMT via oral gavage with stool from uninjured young (10-14 weeks old) or aged donors. At two weeks post-injury mice underwent neurocognitive testing in the elevated zero maze— a measure of species-normal anxiety. The time spent in closed and open spaces were measured to calculate percent of time spent in the open region of the maze. Two-way ANOVA with Tukey's multiple comparisons testing was used for statistical analysis and data are reported as the +/- standard error of the mean.

Results: Vehicle-treated sham mice spent 18% of the measured time in the open region of the maze to establish a baseline. TBI decreased the time spent in the open region of the maze for both vehicle- and aged FMT-treated aged mice (means of 4.00±1.06% and 5.24±1.47%; adjusted p-values of 0.032 and 0.042) as compared to vehicle-treated sham. FMT from aged donors resulted in a marked decrease in the time spent in the open region as compared to mice receiving young FMT (means of 9.22±1.53% and 20.16±2.12%; adjusted p-values of 0.05). However, FMT did not affect the time spent in the open region following TBI.

Conclusions: We hypothesized that restoring youthful gut microbiome will improve neurocognitive outcomes in aged TBI. On the contrary, FMT from young donors following TBI did not attenuate alterations in species-normal anxiety in aged mice. This is contrary to our results in young mice where FMT markedly attenuated neurocognitive deficits after TBI. These variable result are age-dependent and suggest a different pathophysiology of injury in aged vs. young subjects. Interestingly, FMT with stool from aged mice significantly altered species normal anxiety in sham injured mice—suggesting a potential detrimental effect of an aged gut microbial community structure on neurocognition.

Introduction: Approximately 3 million Americans sustain a TBI every year. Men have a higher incidence of traumatic injury and are disproportionately represented in pre-clinical and clinical studies of TBI resulting in a critical unmet research need to understand the differences in the pathophysiology of injury between sexes. Recent studies from our lab demonstrated a markedly divergent transcriptional response to TBI within the microglia of male and female mice 8 hours post-injury. We postulated that this divergent transcriptional response would lead to long-term neurocognitive sex differences in TBI subjects. We hypothesized that female mice would have preserved neurocognition after TBI as compared to male mice.

Methods: Young adult male mice (14-15 weeks, n=9) and female mice (14-15 weeks, n= 15) had TBI induced via a controlled cortical impact to induce a severe TBI vs. sham injury. At 60 days post-injury, neurocognitive outcomes were assessed with open field testing (OF) and Fear Conditioning (FC). Data was analyzed using two-way ANOVA and Tukey’s multiple comparison test.

Results: Male mice demonstrated significantly higher rates of velocity in the OF test as compared to female mice after TBI. (12.4 ± 0.9 cm/sec vs. 10.6 ± 2.2 cm/sec, p<0.0001). However, female TBI mice had spent less time in the center of the OF as compared to male TBI indicating increased anxiety (34.5 ± 9.5 % time in center vs. 40.4 ± 11.1 % time in center, p<0.01). Furthermore, female TBI mice had attenuation of associative learning and memory deficits after TBI as compared to sham (31.4 ± 2.9 % time freezing vs. 37.0 ± 4.5 % time freezing, p<0.03), while male TBI mice had a 45% decline decrease in associative learning and memory as compared sham (34.8 ± 7 % time freezing vs. 61.0 ± 6.3 % time freezing, p<0.0007).

Conclusion: We hypothesized that female mice would have preserved neurocognition after TBI as compared to male mice. Female TBI mice had a normal level of locomotion in the OF and preservation of contextual fear as compared to male TBI mice. However, they demonstrated an increase in post-traumatic anxiety-like behavior and increased generalized activity as compared to male mice after TBI. These data suggest marked sex-linked differences in neurocognitive outcomes after TBI, which correlate with our previously presented transcriptional sex differences between the microglia after TBI. This data further supports the need for sex to be evaluated as an independent variable in future clinical trial design.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection/ COVID-19 pandemic, had increased the overall numbers of morbidity and mortality worldwide. To date, worldwide statistics from the World Health Organization show that SARS-CoV-2 has infected more than 676 million people and resulted in more than 6.88 million deaths. Despite the advances in the production of COVID- 19 vaccine peptides, there is still a need for an effective approach for patient response predictions (especially among immunocompromised patients) for better patient management and reduction of mortality.

Methods: Peripheral Blood Mononuclear Cells (PBMCs) from human subjects with and without history of SARS-CoV-2 infection were stimulated with SARS-CoV-2 viral antigenic (S/M/N) peptide pool. The viral-specific responding cell proliferation was measured using 3H-thymidine (3H-TdR) incorporation assay and the specific cell subsets that responded to SARS-CoV-2 antigenic stimulation were defined using the carboxyfluorescein succinimidyl ester (CFSE) dilution assay. Responder CD69+, CD4+ and CD8+ T cells were also isolated from the stimulated PBMCs, stained with fluorochrome-conjugated antibodies, and loaded onto an IsoCode chip for single cell deep functional analysis using the IsoPlexis' IsoLight technology (https://isoplexis.com/solutions/isolight-system/).

Results: As compared to the control, subjects with a history of COVID-19 infection had a very strong proliferative response, particularly to the spike protein, which even surpassed the positive control polyclonal stimulation (Fig la). CFSE dilution assay showed robust proliferation of CD3 cells (comprises of both CD4 and CD8 subsets) and a minor population of non- T cells (Fig 1 b). Single cell analysis indicated the presence of polyfunctional heterogeneity among the responder CD4+ and CD8+ T cells which was driven by the expression of TNF-α, Interleukin-2 and MIP-lb. Additionally, the strong responses induced by SARS-CoV-2 peptides were predominated by effector secretions followed by stimulatory, chemoattractive (cytokines/chemokines) and regulatory secretions.

Conclusions: Single cell deep functional analysis on T cells from COVID-19 responders based on the released secretomes/ proteins as compared to the conventional bulk cell analysis, would pave the path to ensure improved patient response prediction and management. This can be a stepping-stone to provide clear indication if immunosuppressed transplant patients will require modifications to the vaccination scheme.

Introduction: Peripheral artery disease (PAD) is a prevalent disease that causes ischemic injury to lower extremity tissues. Current treatments, such as angioplasty, stenting or bypass surgery aim to improve blood flow, but do not address the associated muscle degeneration. Butyrate has been linked to improved skeletal muscle health via genetic modulation. It is hypothesized that sustained butyrate delivery to ischemic tissues in PAD will protect muscle fibers from degeneration.

Methods: Poly(lactic-co-glycolic acid) (PLGA 50:50, Mw 7,000 – 17,000 Da) with or without sodium butyrate (9 mg/50 mg PLGA) were fabricated into microspheres using a water-in-oil-in-water double emulsion process. Microsphere morphology was characterized with scanning electron microscopy (SEM), size distribution via light microscopy, encapsulation efficiency and release studies with UV-vis spectroscopy. A murine hindlimb ischemia model (male C57BL/6 mice, n=5) was used to evaluate intramuscular injections containing saline, empty microspheres, butyrate-loaded microspheres, or bulk butyrate 24 hours after surgery. Laser Doppler Imaging (LDI) was conducted weekly to assess blood perfusion. Mice were euthanized on day 28 for histological analysis for fat infiltration, muscle fiber diameter, regenerative fiber density, and fibrosis formation. Statistical analysis involved unpaired Student t-test for microsphere characterization, and one-way ANOVA for in vivo data, with a 95% significance level.

Results: SEM imaging indicated that butyrate incorporation did not affect the microsphere surface. Quantitative analysis showed variation in microsphere size, with an average diameter of 50.69±42.8 um. The microspheres had a 54.3±0.65% encapsulation efficiency. Release studies showed 60% of encapsulated butyrate was released within the first 8 hours, and continued to release up to 75% of the encapsulated butyrate in 4 weeks. Injection studies showed the mice tolerated the microsphere formulation well, with no mortalities, signs of infection or extreme pain, and maintenance of healthy body weight throughout the study. LDI data showed increased limb perfusion over time and no significant difference among the treatment groups, indicating butyrate did not significantly impact vascular regeneration. Qualitative analysis showed lower fat infiltration, decreased fibrosis and more regular muscle fiber morphology compared to saline and vehicle-treated groups. Data analysis of muscle fiber area, regenerating fibers, fat infiltration and fibrosis are currently being conducted.

Conclusions: Butyrate was successfully encapsulated, however improvements can be made to achieve a slower, consistent release profile. Additionally, localized delivery of butyrate was well tolerated. With this initial data and future planned analysis, this study will elucidate butyrate’s effects on ischemic muscle tissue and its potential therapeutic capability

Introduction: Assessment of individual patient rejection risk can guide personalized treatment post-transplant. Traditional HLA serologic mismatchfails to account for differences and immune system recognition at the molecular level, which may improve rejection risk stratification.

Methods: We examined the association between molecular HLA mismatch and incidence of acute rejection in the first 2 years post transplant. We performed high resolution HLA typing of 71 kidney donors and recipients enrolled in a clinical trial. We used HLA Matchmaker software to enumerate eplet mismatches and Antibody verified mismatch load for Class I, Class II, HLA-DR and HLA-DQ. Subjects were grouped according to occurrence of acute rejection. TX group (n=38) had no occurrences of acute rejection, subAR group (n=25) had one or more episodes of subclinical rejection (TCMR or ABMR), and the cAR group (n=8) had one or more episodes of clinical acute rejection (TCMR or ABMR). ANOVA test and linear regression were performed to evaluate differences and strengths of association between HLA mismatch and severity of rejection.

Results: Trends towards the association of higher eplet mismatch load and increasing risk of subclinical and clinical acute rejection were seen across all groups, but only the HLA DQ eplet mismatch load was statistically significantly associated with risk of more severe rejection (p=0.049).

Conclusions: We observed a trend towards higher levels of eplet mismatch associating with greater incidence of both sub clinical and clinical acute rejection (with higher mismatch load associating with more severe, clinical acute rejections). The HLA DQ Loci appeared to have the greatest association with rejection episodes. Future work will focus on expanding the cohort size and exploring the associations between eplet mismatch load and other immunologic events such as emergence of de novo antibodies and positive screening molecular biomarkers.

Introduction: Acute kidney injury (AKI) occurs in about 20-50% of patients with TBI, and is associated with increased mortality, length of hospital stay, and delayed neurologic recovery. The development of AKI in patients with TBI has largely been attributed to kidney hypoperfusion, concomitant acute respiratory distress syndrome, and medication side effects. Additionally, there is evidence to suggest that TBI induced inflammatory responses lead to endothelial dysfunction and subsequent development of AKI, with the mechanism of this remaining unknown. This study was performed to prove the development of AKI in a clinically relevant large animal model of isolated TBI and determine if MG53, a protein important in cell membrane repair and tissue regeneration across multiple organs including kidney and brain, would protect the kidney from TBI induced injury.

Methods: Female Yorkshire swine (40-45kg; n=5/group) were subjected to controlled cortical impact TBI and randomized to either 1) recombinant human MG53 (rhMG53) protein (2mg/kg, intravenous) or 2) normal saline control. Animals were monitored for 6 hours after injury. Serum and plasma samples were obtained prior to injury, 1 hour after injury, 4 hours after injury, and 6 hours after injury. Key laboratory values including creatinine were collected. Enzyme-linked immunosorbent assay (ELISA) was used to determine levels of serum neutrophil gelatinase-associated lipocalin-2 (NGAL), a marker of acute kidney injury, between the two groups.

Results: Animals in the control group had a statistically significant increase in creatinine by 6 hours post TBI (p=0.0073). Animals treated with rhMG53 had no significant increase in creatinine values following TBI (p=0.0894). Comparing the two groups, control animals had a statistically significant higher creatinine compared to rhMG53 treated animals at 6 hours post TBI (p=0.0116). On ELISA, control animals had a statistically significant increase in serum NGAL starting 4 hours following TBI (p=0.0141, p=0.002, baseline vs. 4 hours post injury and baseline vs. 6 hours post injury, respectively). Animals treated with rhMG53 did not have an increase in serum NGAL (p=0.1627, p=0.7890, baseline vs. 4 hours post injury and baseline vs. 6 hours post injury respectively).

Conclusion: TBI induces the development of AKI early after injury. Systemic administration of rhMG53 protects the kidney from TBI induced AKI. More research is needed to understand the mechanism by which TBI induces AKI.

Introduction: Endothelial dysfunction is a hallmark of diabetes mellitus, driven by mitochondrial dysfunction and leading to cardiovascular complications. However, existing therapeutic modalities often fail to adequately address this intricate interplay. Mitochondrial transplantation emerges as a promising strategy to address the underlying mitochondrial deficits, potentially enhancing the functionality of diabetic endothelial cells. In this study, we aim to investigate the efficacy of mitochondrial transplantation in restoring the function of endothelial cells isolated from diabetic patients, providing insights into novel therapeutic interventions for diabetic vasculopathy. Methods: Mesenchymal stem cells (MSCs) were differentiated from induced pluripotent stem cells and transduced with Mitochondria Cyto-Tracer (pCT-Mito-GFP) at different multiplicities of infection (MOIs) for 72 hours. Transduction efficiency was assessed with fluorescence microscopy to determine the optimal MOI. GFP+ MSCs were subsequently co-cultured with pre-seeded diabetic human arterial endothelial cells at varying ratios (0:1, 1:1, 2:1, 4:1, and 9:1) for 72 hours. Afterwards, cells were fixed with paraformaldehyde, stained for CD31+ DHAECs, and imaged with fluorescence microscopy. Additionally, DHAECs were treated with freshly isolated mitochondria at a concentration of 1 µg of mitochondrial protein per 5000 cells for 20 hours. Intracellular nitric oxide production was quantified using fluorescent DAF-FM diacetate, while VCAM-1, ICAM-1, and P-selectin expression levels were measured using in-cell ELISA. All measurements were taken in triplicates and normalized to resazurin cell viability assay. Treatment groups were compared to controls with a two-sample t-test (significance p<0.05). Results: Lentiviral transduction of MSCs with Mito-GFP resulted in a decrease in cell viability with increasing MOI (100±7.77% at MOI=0 vs 73±6.31% at MOI=60). The transduction efficiency peaked at MOI=30 (93±2.96%) with cell viability at 81.46±0.93%, establishing this condition as the optimal setting for Mito-GFP transduction in subsequent experiments. Fluorescent microscopy of cocultured cells revealed colocalization of CD31+ DHAECs containing GFP+ mitochondria, indicating successful mitochondrial transfer. Ongoing quantification of mitochondrial transfer efficiency involves flow cytometry analysis to identify the optimal co-culture ratio. Preliminary findings showed no significant differences in endothelial function following treatment with isolated mitochondria. Assessment of endothelial functions will be conducted after the isolation of DHAECs from the co-culture system. Conclusions: Our study demonstrates the feasibility of mitochondrial transfer from MSCs to diabetic endothelial cells, highlighting the potential of this approach as a therapeutic strategy for mitigating endothelial dysfunction in diabetes. The optimized conditions for mitochondrial transfer pave the way for further investigations into the mechanisms underlying this process and its therapeutic implications for diabetic vasculopathy.

Introduction: Presently the only way to restore native ovarian hormone function and fertility is through ovarian tissue transplantation (OTT) after ovarian tissue cryopreservation (OTC). With current techniques, there is an 80% depletion of the ovarian reserve after OTT as a result of increased primordial follicle recruitment (or activation). Additionally, there is a risk of reintroducing metastatic disease with OTT in some patients who underwent OTC for cancer diagnoses. This has led to an interest in developing a bioprosthetic ovary to both optimize long term ovarian reserve survival and minimize the risk of reintroducing malignancy during OTT. The microenvironment of the ovarian extracellular matrix needs to be better understood in order to replicate the native ovary in an engineered matrix. Prior work has shown the ovarian cortex, where primordial follicles are maintained in a quiescent state, is more rigid than the ovarian medulla (8.87 kPa vs. 1.05kPa). Here, we tested an encapsulating hydrogel at different rigidities for its effect on bovine primordial follicles activation on the growth and survival. Methods: Bovine primordial follicles were isolated from ovarian cortex. A mean of 9.9 follicles (range 3-24) were encapsulated per bead in either 1% or 5 % alginate across 4 experiments. The encapsulated follicles were subsequently crosslinked in a calcium sulfate solution. Follicles were then cultured for 8 days with light microscopy imaging taken every other day along with media exchanges. Follicles were then examined using immunofluorescence. Growth and survival curves were constructed and all statistical analyses were performed using Graph Pad Prism 9. Results: A total of 372 follicles were encapsulated across 32 beads (16 in 1% alginate and 16 in 5% alginate). There were no differences in initial follicle size between the two conditions (33.53µm vs. 32.45µm, p=0.47). At the end of 8 days, there was no difference between follicle size (59.55 vs. 56.06, p=0.48). Additionally, there was no difference in survival between 1% and 5% alginate encapsulation (57.75% vs. 52.43%. p=0.40). Immunofluorescence of encapsulated follicles confirmed presence of MSY2, a molecular marker of oocytes, after 8 days in culture. Conclusion: There was no significant difference in growth or survival between primordial follicles cultured in 1% or 5% alginate gels. Immunofluorescent analysis confirmed the presence of viable follicles at the end of 8 days of culture. Future work needs to further explore how factors in the ovarian extracellular matrix impact follicle maintenance and growth.

Introduction: Measuring EMG signals intramuscularly has been proposed for decades as an alternative to using surface EMG signals in regard to providing the signals necessary to allow for accurate and reliable myo-electric control of prosthetics. Intramuscular recording helps alleviate challenges which are encountered from signal attainment via surface level skin electrodes which can produce unreliable signals. The purpose of this study was to evaluate the novel implantable MyoNode sensors to evaluate functionality, feasibility, and implantation/explantation in a long-term large animal in-vivo study for potential use for future myoelectric prosthetic control.

Methods: Using a live American fox hound model, we placed 8 MyoNodes in the hindlimb. A 3-D printed custom fit external coil/base station was constructed for powering and signal acquisition of the myonodes and placed around the hindlimb. Data were recorded in vivo to assess EMG signal quality and wireless power transfer at the 3, 6, and 12 month time-points. Two forms of assessment were performed: 1) assessment to evaluate wireless power, and 2) assessment to evaluate EMG signal recordings. Additionally, X-rays were taken immediately after surgery, and at 1 ,3, 6, and 12 months post-implantation to evaluate migration of the MyoNodes.

Results: Qualitatively, the signal-to-noise level was observed to be low during resting as well as the signals revealing individual motor unit action potential firings indicating that the implants could detect low-level EMG contractions. There were no surgical or wound healing complications. No significant scarring of the muscle pocket was visible, and examination of the explanted MyoNodes using high magnification stereoimaging revealed no damage to the MyoNode casings. Small changes in MyoNode positions on the XR measurements were observed, but were attributed to changes in the limb position of the animal between measurements.

Conclusion: We found that implantation/explantation was performed with relative ease and minimal morbidity with no infections or wound healing complications. Additionally, we demonstrated the feasibility of implantation of multiple MyoNodes with ability to access very large muscles with no damage to the implants upon placement or extraction. We also found that EMG channels of the MyoNode to be fully biocompatible as well as the EMG signal quality to be above accepted thresholds for potential prosthetic control with low crosstalk between channels that would potentially allow functional control of a myoelectric prosthetic.

Introduction: Animal models of nerve injury are important for studying interventions for nerve repair and injury that cannot be studied in humans. However, the vast majority of gait analysis in animals has been limited to univariate analysis despite the fact that gait data is highly multi-dimensional. As a result, little is known about how various spatiotemporal components of the gait relate to each other in the context of peripheral nerve injury and trauma. We hypothesize that a more rigorous multivariate characterization of gait will reveal novel relationships between spatiotemporal components of gait in the context of peripheral nerve injury and trauma. We further hypothesize that legitimate relationships between said components will allow for more accurate classification between distinct gait phenotypes than univariate studies alone.

Methods: Video gait data was collected of mice walking on the DigiGait treadmill system across groups representing increasing degrees of damage to the neuro-musculo-skeletal sequence of healthy gait; that is: (a) healthy controls, (b) nerve damage only via total nerve transection + reconnection of the femoral and sciatic nerves, and (c) nerve, muscle, and bone damage via total hind limb transplantation. Multivariate relationships among the 30+ spatiotemporal measures were evaluated using exploratory factor analysis and forward feature selection to identify the features and latent factors that best described gait phenotypes. The identified features were then used to train classifier models and compared for their accuracy to the results of classifiers trained with features identified using only univariate analysis.

Results: 10-15 features relevant to describing gait in the context of different degrees of peripheral nerve injury and trauma were identified. Factor analysis uncovered relationships among the identified features and enabled the extrapolation of a set of latent factors that further described the distinct gait phenotypes. The latent factors seemed to tie to intuitive biological phenomena characteristic of the different pathologies (e.g. alterations to the anatomical configuration of the limb due to transplantation or aberrant fine motor function due to peripheral nerve injury alone). Models trained using the identified features generated injury scores that could be used to distinguish among pathophysiological states with high statistical significance (p<.001) and accuracy (>80% accuracy) as compared to models trained using features identified from univariate analysis alone.

Conclusion: This is the first system to demonstrate superior performance of a multivariate approach to gait analysis in animals. It is also the first system to use multivariate statistics to characterize and distinguish different etiologies of gait deficit in animals.

Introduction: Despite advances in surgical therapy for arterial occlusive disease secondary to atherosclerosis, up to 50% of procedures to treat atherosclerosis fail due to restenosis from neointimal hyperplasia, a cell proliferative process potentiated by inflammation. Studies have reported the association of gut microbiome with metabolic disorders including cardiovascular diseases. However, there is lack of evidence suggesting their role in neointimal hyperplasia. We hypothesized that neointimal hyperplasia is a phenotype that can be modulated by fecal transplantation and that severity is associated with a distinct microbiome profile.

Methods: We performed fecal transplantation (FT) using stool donors with distinct microbiome profiles and differential susceptibility (High: Sprague-Dawley [SD] rats, Low: Lewis [LE] rats) to neointimal hyperplasia. Cefoperazone (cefo)-treated recipient (C57/BL6) mice underwent carotid artery ligation after three weeks of stabilization time following fecal transplantation. Portal venous blood and tissues were harvested at 1 or 4 weeks after injury. Short chain fatty acids were quantified using gas chromatography and microbial community characterization was performed using 16S rRNA gene amplicon and metagenome shotgun sequencing. Arterial morphometrics were assessed using serial frozen sections. In a follow up experiment with similar experimental time points, germ-free underwent FT using slurries from C57/BL6 donors with and without Akkermansia muciniphila BAA835 (Amuc) prior to carotid ligation.

Results: In contrast to our initial hypothesis, the ratio of the intima to total wall thickness was significantly larger in cefo-LE than cefo-SD recipients (P=0.003). The gut microbial diversity was reduced (P<0.01) after cefo treatment and the composition was altered (P<0.001) in the FT recipient mice. The relative and absolute abundances of A. muciniphila were higher in cefo-SD group than cefo-LE and was the main contributing taxa for the enrichment (P<0.001) of the ADP-heptose biosynthesis pathway in the cefo-SD group. Relative abundances of both the taxa and the pathway showed strong negative correlations (P<0.01) with neointimal hyperplasia. Similarly, the absolute abundance of A. mucininiphila specific bifunction HldE gene in ADP-heptose pathway was higher (P=0.013) in cefo-SD than cefo-LE group. In mice colonized with fecal slurries that differed only by the presence of Amuc, the ratio of intima to total wall thickness was significantly larger in recipients with Amuc than without Amuc (P=0.01).

Conclusion: The presence of specific gut microbiota modulates the formation of neointimal hyperplasia after arterial injury. A. municiniphila and the microbial ADP-heptose biosynthesis pathways are negatively associated with neointimal hyperplasia and are potential targets for novel microbe-based strategies to mitigate restenosis risk.

Introduction: In utero gene therapy (IUGT) aims to treat congenital diseases via the replacement of deficient or nonfunctional transgenes prior to irreversible damage that accumulates in the womb. However, clinical translation of IUGT is hampered by concerns for fetal toxicity and immunogenicity of existing delivery vectors. PEG10, an endogenous retrovirus-like protein expressed in the fetus and placenta, can be isolated and programmed to package specific mRNA within “virus-like” particles (VLPs) and pseudotyped to guide cell tropism. In this study, we hypothesized that PEG10 VLPs are an adaptable system for functional cargo mRNA delivery to fetal stem cell lines in vitro and to the fetus in vivo with minimal fetal toxicity.

Methods: To challenge this hypothesis, PEG10 VLPs encapsulating Cre-recombinase mRNA were isolated from supernatant of transfected HEK293FT cells via differential centrifugation and pseudotyped with VSVg (viral fusogen conferring broad cell tropism), myomaker (endogenous muscle fusogen conferring tropism to myoblasts), or no fusogen (nonpseudotyped). VLPs were co-cultured with murine Lox-STOP-Lox reporter neuroblasts, myoblasts, and embryonic fibroblasts, respectively. Cells were examined via fluorescence microscopy and flow cytometry at 24-96 hours. In vivo, VLPs were delivered to time-dated mT/mG dams, which constitutively express red fluorescence and switch to green fluorescence upon cleavage at LoxP sites. Fetal intrahepatic injection of VSVg-VLPs at various doses or saline (control) was performed at embryonic day 14, and fetal tissues (liver, lung, heart, brain, intestine) analyzed at 24-72 hours after surgery via stereomicroscopy and flow cytometry.

Results: VSVG-pseudotyped VLPs permitted efficient (>40%) transduction of all three fetal cells lines in vitro, whereas myomaker-pseudotyped VLPs mediated specific transduction of C2C12 myoblasts (10.7 +/- 8.7%). Alternatively, nonpseudotyped VLPs did not mediate transduction of any cells in vitro, demonstrating the requirement of fusogen proteins for successful mRNA delivery. In vivo, VSVg-pseudotyped VLPs mediated dose-dependent levels of transduction within the fetal liver and lung, primarily of CD45+ hematopoietic cells, whereas nonpseudotyped VLPs and saline controls did not demonstrate any background recombination. There was no difference in fetal absorption with VLPs compared to saline controls (P=.460).

Conclusion: We conclude PEG10 VLPs mediate functional mRNA delivery with adaptable specificity to various fetal cell lines in vitro and to the fetal liver and lung in vivo. Using the myomaker fusogen, this study demonstrates the first evidence of mRNA delivery to muscle cells entirely via endogenous proteins. As a native fetal/placenta protein, PEG10 may be an ideal delivery vector for IUGT with reduced fetal toxicity.

Introduction: Transdermal deferoxamine patch (DFO) is a novel experimental treatment with potential to reduce radiation-induced skin injury in breast cancer patients undergoing radiation. However, DFO safety in oncological settings has not been extensively explored. Thus, the purpose of this study is to investigate the effects of DFO on mammary tumor cell biology using xenograft murine breast cancer model.

Methods: Experiments were performed on 8 weeks old immunodeficient albino female mice. To support tumor growth estrogen pellet was inserted subcutaneously three days before MCF7-luc breast cancer cells implantation into mammary gland. The in vivo tumor growth was evaluated weekly immediately after luciferin injection using LAGO bioluminescence imaging system until study endpoint. Once tumor reached desired size (bioluminescence intensity ~1x108) DFO patches were applied daily for 30 days. Non-treated mice served as control. Changes in mice weight and activity were monitored. On the day of tissue harvesting size (length and width) and weight of the excised tumor were recorded. Harvested tissue samples (skin and tumor) were preserved in 10% formalin and embedded in paraffin for immunofluorescence (IF) staining. IF for HIF1a and CD31 on 4 mm sections was performed to evaluate the changes in vascularization in DFO-treated mice compared to control.

Results: The IF staining revealed an increase in HIF1a and CD31 expression in skin samples in DFO-treated group compared to control. This suggests that DFO treatment improves skin vascularization in HIF1a-dependant manner. No significant changes were observed in tumor samples, suggesting that DFO applied through transdermal patch works locally and does not penetrate deeper tissue. BA demonstrated steady tumor growth over time, without significant differences between treated and control group.

Conclusions: This study confirms that transdermal deferoxamine patch treatment has potential to improve the regenerative processes in skin, by stimulating blood vessel formation at least partially through HIF1a stabilization. Our data suggest that due to lack of ability to reach deeper tissues, the risk that DFO pro-vasculogenic effect is extended to residual cancer cells is limited. Our next step is to evaluate the effect of DFO on radiation efficacy.

Introduction: Aortic calcification is a significant pathological process associated with various cardiovascular diseases. While the sympathetic nervous system's influence on bone remodeling via β2-adrenergic receptors (β2-ARs) is well-established, its impact on vascular calcification remains relatively unexplored. Previous research suggests β2-ARs play a critical role in regulating valve calcification but have yet to be studied in aortic calcification. Thus, we aim to characterize the expression patterns of both β1-ARs and β2-ARs to elucidate the downstream signaling mediators in human aortic tissue and determine the effects of selective β2-AR agonists on aortic smooth muscle cell phenotype and function.

Methods: Calcified and non-calcified abdominal aortic wall segments from 6 patients with abdominal aortic aneurysm (AAA) were collected during elective open aortic repair at Northwestern Memorial Hospital (Chicago, IL). Samples were fixed, decalcified, and embedded in paraffin. Sections of 5 μm thickness were used for immunostaining of alpha-smooth muscle actin, β1-AR, and β2-AR. Human aortic smooth muscle cells (HAoSMC) were cultured in osteogenic media and treated with one of the following β-AR agonists (1 - 100 μmol/L): salmeterol, isoproterenol, norepinephrine, or an antagonist (0.3 – 30 μmol/L): ICI-118,551 or CGP-20712A. Cells were fixed for immunocytochemical staining of osteogenic markers (osteocalcin, osteopontin, and Runx2) and β-ARs. HAoSMCs were treated with salmeterol (1 μmol/L, β2-AR agonist) in the presence of either growth media or osteogenic media for up to 14 days. Metabolic and alkaline phosphatase (ALP) activity were measured with resazurin (n=8) and ALP assays (n=3), respectively, on days 7 and 14.

Results: Patient abdominal aortic wall segments show stark differential expression of β2-AR between samples, and further investigation to compare to non-calcified aortic tissues is warranted. In HAoSMCs, qualitative immunostaining for β-AR and osteogenic markers showed no obvious difference between treatment groups after 2 days. Longer treatment times (21 days) are currently being investigated. Thus far, ALP-positive staining has qualitatively increased in cells cultured in osteogenic medium by day 7 and day 14 in comparison to growth medium. Resazurin assays show no significant difference in metabolic activities with the current treatments through day 14.

Conclusions: We observed a differential expression of β2-ARs across sections of human calcified aortas, suggesting a role of these receptors in areas subjected to different calcific stresses. Further in vitro experiments with HAoSMCs treated with β-AR agonists and antagonists will help to elucidate the influence of β-adrenergic signaling on vascular calcification pathways.

Introduction: While current cross-matching methodologies are used to detect donor specific antibodies (DSAs), there is no reliable cellular based assay that accounts for direct T-cell alloreactivity. In this study, we performed pre-transplant donor-specific mixed lymphocyte reactions (MLRs) and T-cell receptor (TCR) beta chain sequencing to pre-operatively identify donor reactive T cell clones (DRTC) in kidney transplant recipients. DRTC were then tracked in the post-operative period to evaluate their association with rejection.

Methods: Patient Selection: Transplant recipients undergoing living or deceased donor kidney transplantation were enrolled according to standard center practices. All subjects underwent standard of care (SOC) kidney transplantation with SOC immunosuppression. Identification of DRTC: Peripheral blood mononuclear cells (PBMCs) were obtained from organ donors and recipients pre-operatively. Donor specific MLRs were performed using CSFE-labeled recipient responder cells and irradiated, PKH-26 labeled donor stimulator cells. Proliferating responder cells were then flow-sorted into CD4+ and CD8+ subsets, which were then subjected to TCR-beta chain sequencing using the Adaptive Immunosequencing platform (Adaptive Biotechnologies). DRTC were defined as T-cell clones that i) expanded at least 2 fold in the MLR and ii) were significantly expanded in the MLR by binominal modeling after multiple comparisons corrections. Post-Transplant Tracking of DRTC: Blood, urine, and renal biopsy samples were collected at designated time points post-operatively. DRTC were then detected in post-transplant samples using Adaptive Immunosequencing.

Results: Twenty subjects underwent successful kidney transplantation and sample collection. 9 developed acute rejection or borderline rejection within 3 months of transplant, and 11 remained stable during this time period. In subjects with abnormal biopsy, there were more CD8+ DRTC generated in the pre-transplant MLR and present at baseline in the pre-transplant peripheral blood. Notably, elevated pre-transplant CD8+ DRTC was correlated with increasing number of HLA mismatches. Post-transplant, the elevated number and frequency of circulating CD8+ DRTC persisted in subjects that developed an abnormal biopsy. Furthermore, in some patients there was clear evidence of specific CD8+ DRTC tracking from the blood into the allograft at rejection.

Conclusion: Using a multi-plex PCR based assay, we defined the alloreactive T-cell repertoire in a set of kidney transplant recipients. Increased presence of CD8+ DRTC correlated with number of HLA mismatches and pathological diagnosis of rejection. Methodologies such as this could compliment the current crossmatch protocol to identify high risk donor-recipient pairs.

Introduction: Current literature reporting on atypical ductal hyperplasia (ADH) upstage rates after excisional biopsy analyzed data collected prior to the widespread introduction of tomosynthesis mammography and consistent use of vacuum-assisted core needle biopsies (CNB). To our knowledge, there is no study specifically investigating the upstage rates of ADH with contemporary mammographic and pathologic modalities. The purpose of the study is to determine the upstage rate of ADH detected in patients who underwent screening tomosynthesis mammography, and to identify risk factors that may affect upstaging rates. We hypothesize that current upstage rates are lower than previously reported. Specifically, we hope to identify a subset of women with ADH diagnosed on CNB with a cancer upstage rate of <2% in the surgical excisional biopsy specimen.

Methods: We plan to conduct a retrospective review of all adult patients (> 18 yo) treated within the Northwestern Medicine network hospitals (01/01/2016 - 01/01/2024) diagnosed with at least one ADH lesion on screening tomosynthesis mammography, and subsequently underwent surgical excisional biopsy. Patient with prior or concomitant breast carcinoma, malignant CNB findings, and incomplete charts will be excluded.

Results: We will determine upstage rate of ADH to carcinoma following excisional biopsy, and identify clinical risk factors associated with lesion upstaging, focusing on diagnostic patient-specific, exam-based, imaging, and pathologic factors.

Conclusions: Our project will provide insight into the true upstage rate for patients whose ADH lesions are initially diagnosed on screening tomosynthesis. As most previously published projects on the topic report cases of ADH detected with less sensitive mammographic modalities, our project has the potential to contribute substantial data to determining a more contemporary and clinically relevant upstage rate. If rates are low, this would suggest that surgical excision can potentially be deferred. Thus, our results could limit unnecessary surgery for patients in the future. Furthermore, for the subset of patients opting for surveillance of ADH lesions detected on screening tomosynthesis, the additional benefit of our project will be to provide data to support the feasibility and safety of continued surveillance of these lesions rather than excision.

Introduction: 4,160 heart transplants were performed in the United States in 2023, all using deceased donor allografts. After the stress related to donor cardiac or brain death, it is important to minimize further insults to cardiac allografts, especially ischemia-reperfusion injury (IRI), first encountered by microvascular endothelial cells (ECs). The additive effects of hypoxic cold storage (CS) followed by warm reperfusion (WR) are known to cause endothelial injury and pre-dispose the donor organ to higher immunogenicity. Autophagy, “self-eating”, the process of cellular machinery disposal and recycling, has been implicated in cardiac IRI and graft failure. We aimed to understand the changes in autophagy during CS and IRI and its impact on EC immunogenicity.

Methods: Autophagy in mouse cardiac endothelial cells (MCECs) was investigated using an in vitro model of CS-WR to simulate IRI. MCECs were subjected to cold storage at 4°C in University of Wisconsin preservation solution for 6 hours (h) in a hypoxic chamber followed by warm conditions with culture medium for up to 24 h. MCECs under standard culture conditions served as controls (NT). Immunoblotting and confocal microscopy were performed to characterize autophagy changes. To investigate the role of autophagy in IRI associated immunogenicity, genetic knockout of the autophagy-related gene 5 (ATG5-/-), key in regulating autophagosome formation, as well as pharmacological modulation of the autophagy machinery, was performed in MCECs. MCECs were co-cultured with sensitized CD8+ T cells and T cell activation was determined by measuring the secreted levels of interferon-gamma (IFN-γ) via ELISA.

Results: Immunoblotting of MCEC lysates post-CS-WR compared to NT showed a significant increase (P<0.01) in LC3, an established autophagy marker, indicating more autophagosome formation. Results were verified via confocal imaging. The co-culture experiment using ATG5-/- MCECs and mock transfected controls under CS-WR conditions demonstrated that ATG5-/- MCECs had a 10.5-fold increase in IFN-γ levels, compared to controls. Using pharmacologic autophagy induction with rapamycin in MCECs under CS-WR, there was a 51.5-fold reduction of IFN-γ (pg/mL), compared to untreated controls, whereas inhibition with chloroquine resulted in a 1.82-fold increase of IFN-γ.

Conclusion: We observed that increased autophagic activity may be protective during IRI by mitigating EC immunogenicity. Thus, modulating microvascular EC autophagy in donor hearts during CS prior to transplantation may mitigate insults incurred during IRI, potentially increasing the longevity of cardiac allografts and reducing the burden of systemic immunosuppression.

Introduction: Traumatic brain injury (TBI) afflicts over 3 million Americans every year. Patients over 65 years of age experience increased mortality and greater long-term neurocognitive morbidity compared to younger adults. CD8+ T-cell infiltration after injury has been related to worse neurocognitive outcomes. Aged microglia release ligands for CD8+ T-cells, allowing for selective recruitment into the aged brain. We hypothesize that depletion of microglia will attenuate accumulation of T-cells post-TBI.

Methods: Young adult (14-weeks N=5) and aged (80-weeks, N=3) male C57BL/6 mice underwent microglia depletion via PLX5622, a colony stimulating factor 1 receptor (CSF1R) inhibitor or were kept on a control diet. Depletion was confirmed with flow cytometry. After depletion, mice underwent TBI via controlled cortical impact vs. sham injury. One-month post-TBI, brains were harvested, and flow cytometry was used to assess resident and infiltrating immune cells.

Results: Treatment with PLX5622 significantly reduced microglia with 95% reduction in young mice and 75% reduction in aged mice. Pre-injury, aged mice demonstrated disproportionate T-cell infiltration within the brain as compared to young adult mice. This pre-injury disparity was not influenced by microglial depletion. Post-injury, T-cell infiltration in aged mice was significantly reduced after microglial depletion as compared to aged, injured, mice without depletion (from 2% to 0.5% of live cells, p = 0.03). Upon further inspection, the loss of T-cell infiltration was specific to CD8+ T-cells as CD4+ T-cells were unchanged after injury. Conversely, CD4+ T-cells were increased in the young mice after PLX5622 treatment one-month post-TBI (from 0.2% to 0.7% of live cells, p=0.003).

Conclusions: We hypothesized that depletion of microglia would reduce the infiltration of T-cells after TBI in aged mice. Our data suggests an age-dependent response to TBI that is induced by microglia. Microglia depletion attenuates this response through reduction in CD8+ T-cells in the aged brain post-TBI. Previous studies displayed the negative impacts of CD8+ T-cells in the aged brain after TBI. Thus, microglia depletion may be a promising therapeutic in aged TBI subjects. Ongoing studies are focused on gene signature changes and neurocognitive impacts of microglia depletion in TBI.

Introduction: The role of circulating tumor DNA (ctDNA) to predict outcomes after neoadjuvant chemotherapy (NAC) is unclear in pancreatic ductal adenocarcinoma (PDAC). The objective of this study was to investigate the prognostic value of KRAS mutant ctDNA in PDAC patients after treatment with NAC as assessed by digital droplet polymerase chain reaction (ddPCR).

Methods: Patients with newly diagnosed, localized, PDAC were enrolled in a prospective cohort study. Peripheral blood samples were collected at diagnosis, after NAC, and after local therapy, including surgery and/or radiotherapy. DNA was extracted using a commercially available extraction kit. Each sample was analyzed using ddPCR probes for KRAS G12D, G12V, G12R mutations. Kaplan-Meier and log-rank survival analyses were performed.

Results: 67 patients with localized PDAC were enrolled and underwent NAC (median [IQR] 2.8 [2.3-5.5]). Median (IQR) follow up time was 13.9 (9.1-24.3) months. At initial staging, 41.7% had resectable, 20.2% had borderline resectable, and 17.9% had locally advanced disease. KRAS mutant ctDNA was detectable in 59.7% at diagnosis, 69.6% after NAC, and 67.4% after local therapy. The presence of mutant KRAS ctDNA at diagnosis, after NAC, and after local therapy was not associated with overall survival (OS). For patients who underwent curative-intent surgery, the presence of mutant KRAS G12R was present in 30.3% of patients and was associated with shorter OS (25.1 vs 31.7 months; p<0.05). After NAC and resection, receiving adjuvant chemotherapy was not associated with improved OS (p=0.9149). However, in patients who received adjuvant chemotherapy, lack of mutant KRAS G12R ctDNA, but not G12D and G12V, was associated with improved OS. (25.1 months vs not reached; p<0.05).

Conclusions: In patients with PDAC treated with NAC, KRAS mutations are detectable in ctDNA by ddPCR in the majority of patients over the course of treatment and predict survival. The results demonstrate that KRAS mutant ctDNA predicts prognosis after NAC and resection and may serve as a predictive biomarker to assess benefit from adjuvant chemotherapy.

Introduction: Diaphragm paralysis is a serious complication of both high spinal cord injury and phrenic nerve injury. We hypothesized we could restore diaphragm muscle function by spinal accessory nerve to phrenic nerve transfer. Furthermore, we hypothesized that diaphragm muscle reinnervation and functional recovery would be accelerated by therapeutic electrical stimulation.

Methods: Our therapeutic electrical stimulation protocol consisted of 1-hour of 20 Hz electrical stimulation at 2-4 mA of current delivered immediately after nerve repair surgery. Rats with unilateral phrenic nerve transection were randomly separated into four treatment groups: phrenic-to-phrenic surgical repair with electrical stimulation, phrenic-to-phrenic repair only (sham), spinal-accessory-to-phrenic transfer with electrical stimulation, spinal-accessory-to-phrenic transfer only (sham).

Main Outcome Measures: Our primary outcome measurement was M-Mode ultrasound imaging for diaphragmatic excursion. Other outcome measurements include phrenic motor conduction studies, concentric needle electromyography, and whole cervical spinal cord fluorescent imaging for retrogradely labeled motoneurons. Results: Phrenic nerve transection and direct repair (no stimulation) took 10 weeks to recover normal diaphragmatic excursion. Therapeutic electrical stimulation did not improve phrenic nerve regeneration. However, therapeutic electrical stimulation after spinal accessory nerve transfer significantly improved diaphragm muscle recovery rate compared to non-stimulation groups (phrenic-to-phrenic, and spinal-accessory-to-phrenic) by approximately 50% (5 weeks vs 10 weeks). In addition, electrical stimulation improved synchronicity of diaphragm needle electromyography (right vs. left side) and compound muscle action potential amplitude compared to non-stimulation group (17.4% vs 9.3%).

Conclusions: Spinal accessory nerve transfer is a promising option to re-innervate diaphragm and restore spontaneous synchronized respiratory movement. Brief therapeutic electrical stimulation selectively improves regeneration of spinal accessory axons, but not phrenic axons, for functional restoration of the paralyzed diaphragm.

Introduction: Post-thrombotic syndrome (PTS) is the most common sequelae of deep vein thrombosis (DVT), manifested as venous insufficiency in up to 50% of patients. Unfortunately, none of the existing therapies, including anticoagulants and thrombolytics, have shown definitive effects for PTS, presenting a vital unmet clinical need. Akkermansia Muciniphila (AKK), a gut commensal anaerobic bacterium, has been recognized with pleiotropic benefits in a wide range of diseases including cardiometabolic disorders. However, its therapeutic and mechanistic implications in thromboembolic diseases and the subsequent PTS remain unknown. We hypothesize that probiotic therapy with live AKK after DVT formation could ameliorate venous wall fibrosis, indicative of PTS.

Methods: In male specific-pathogen free c57b6/j mice (10-12 weeks), DVT was experimentally created via partial ligation of inferior vena cava (IVC) as previously established, leading to progressive venous fibrosis. For the probiotic treatment, AKK (strain BAA-835) was cultured using BHI broth in an anaerobic Coy chamber, followed by daily oral gavage to mice beginning from day 1 post DVT induction. At day 14 post procedure, IVC was collected for Masson Trichrome and immunofluorescent staining, followed by quantitation of venous collagen deposition/fibrosis and phosphorylation of endoplasmic reticulum (ER) stress PERK kinase. Endpoint plasma specimens were collected to quantify the circulating levels of trimethylamine N-oxide (TMAO).

Results: When administered post-DVT formation, AKK probiotic therapy effectively mitigated venous wall fibrosis in comparison to BHI culture media gavage alone (fibrotic area: 61.12±6.64 vs 53.17±5.99; p<0.05). TMAO, the only microbial metabolite associated with thromboembolic risk, was significantly reduced in plasma following AKK probiotic therapy. Furthermore, phosphorylation of ER stress PERK, which is directly activated by TMAO and critically contributes to myofibroblast activation and organ fibrosis, was inhibited in the AKK-treated IVC (435.09±26.49 vs 326.86±17.61; p<0.01).

Conclusion: Our current study supports the potential use of AKK probiotic therapy for the treatment of PTS, possibly via modulating the TMAO-PERK axis.

Introduction: Renal ischemic reperfusion injury (IRI), a large increase in cell death and poor function due to cold preservation and subsequent implantation, is the major cause of acute kidney injury (AKI) in the transplantation setting. Tubular epithelial cells (TECs) are the primary target of IRI and a source of the resulting cellular stress and inflammatory response. Targeting the unfolded protein response (UPR) has been implicated in the possible mitigation of this injury. Our previous scRNA-seq analysis showed that XBP1-mediated UPR pathway was robustly activated. We thus aimed to determine the role of XBP1, the vital transcription factor in the UPR pathway, in TECs using a novel in vitro model of TEC IRI.

Methods: Kidneys from wild-type (WT) B6 mice were harvested and TECs were isolated and cultured. These TECs, after reaching confluency, underwent XBP1-siRNA transfection or treatment as usual (control). To mimic IRI in vitro we developed an in vitro model which subjected TECs to hypoxia for 6hr in cold UW solution (4℃, CIT) followed by 24h of reperfusion with fresh media (37℃, IRI). This created 4 separate experimental groups: control, XBP1-siRNA, 24h IRI, and 24h IRI + XBP1-siRNA. We then used qPCR to quantify gene expression differences of injury markers or inflammatory cytokines, as well as live/dead staining to detect the cell death between groups.

Results: Using this well-developed in vitro IRI model, we found that the injury marker NGAL and KIM1 were significantly increased when TECs underwent IRI for 24h. Live/dead staining and Annexin V/PI flow cytometry assays confirmed that TECs exhibited more cell death after IRI. Furthermore, the expression of inflammatory cytokines (e.g. IL-6, TNFα) was also upregulated triggered by IRI. Interestingly, the expression of XBP1s mRNA was robustly elevated in the CIT phase, suggesting that XBP1s-mediated UPR pathway might participate in cellular injury at cold storage time. After knocking down the expression of XBP1s using XBP1-siRNA transfection, we found that XBP1s-deficiency TECs exhibited significantly lower expression of NGAL and less cell death compared to control TECs with IRI.

Conclusions: Our in vitro IRI model exhibited a classic injury phenotype of TECs, which partially mimicked the IRI process in vivo. In addition, TECs displayed the upregulation of XBP1s expression resulting from IRI. Knockdown of XBP1 protected the TECs from IRI-mediated cell death and inflammatory response, which implicates XBP1 as a promising target for therapeutics to alleviate renal IRI and improving kidney transplant survival.