United States

Background: Ovarian cancer (OC) is a deadly disease, with the 3rd highest mortality to incidence ratio of all cancers. OC’s asymptomatic nature early in the disease causes identification at advanced stages in 70+% of patients, at which point the 5-year survival rate can be as low as 20%. To date, no single biomarker has been able to detect early OC with high sensitivity and specificity. While elevated levels of Cancer Antigen 125 (CA-125) have been used to monitor patients pre-/post-operatively, CA-125 is limited in detecting early OC, and other benign ovarian or nongynecological conditions also have shown elevated CA-125. There is a critical need for novel biomarkers that can be used for screening, early diagnosis of OC and distinction of benign vs malignant masses. Our objective is to develop a novel sensitive and specific biomarker panel for OC early diagnosis where prognosis can be improved.

Methods: 288 plasma samples from women with OC (at various stages), benign gynecological conditions, other cancer types, and healthy non-cancer controls were analyzed with a curated panel of 8 biomarkers: CA-125, Growth Differentiation Factor 15 (GDF-15), Human Epididymis Protein (HE-4), Hepatocyte Growth Factor (HGF), Interleukin-6 (IL-6), Leukemia Inhibitory Factor (LIF), Osteopontin (OPN), and Prolactin (PRL). Receiver-operating characteristic (ROC) analysis was performed to assess sensitivity and specificity of single and multi-biomarkers amongst various subgroups.

Results: Univariate ROC analysis identified HE-4 and GDF-15 as having the highest area under the curve (AUC) (AUC=0.868, AUC=0.850, respectively) when comparing all stages of OC to healthy controls, a value greater than CA-125 (AUC=0.762). GDF-15 had the highest AUC when differentiating between early OC and healthy controls (AUC=0.772), a value greater than CA-125 (AUC=0.607). Amongst univariate markers, LIF was able to best differentiate between several other types of cancer (peritoneal, pancreatic, lung, and stomach) and OC. Multivariate analysis identified the panel containing CA-125, GDF-15, HE-4, IL-6, OPN, and PRL as having the highest AUC when comparing OC to healthy controls (AUC=0.956 for all stages, AUC=0.896 for early stages) and to benign conditions (AUC=0.826 for all stages). Adding LIF to this model allowed for best differentiation between OC and other cancer types (AUC=0.805 for all stages).

Conclusion: These results validate a novel biomarker panel for early detection of OC as well as distinction of benign masses and other malignancies from OC. Our results are expected to have an important positive impact by accurately detecting premalignant changes or early-stage OC in asymptomatic individuals.

2023 AMA Research Challenge

Novel Biomarker Panels for Ovarian Cancer Screening

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Purpose: Age-related macular degeneration (AMD) is characterized by pathologic extracellular lipid-rich deposits below and above the retinal pigment epithelium (RPE), respectively termed drusen and reticular pseudodrusen (RPD). Given apolipoprotein E (apoE) is one of the major components in drusen and RPD, the polarity of apoE secretion by the RPE likely contributes to whether RPD, drusen, or both dominate in an AMD patient. The mechanism dictating the polarity of apoE secretion in the RPE remains unknown. However, ATP-binding cassette A1 (ABCA1), a lipid transporter expressed in the RPE, is required for maintaining extracellular apoE levels in hepatocytes, where ABCA1 and apoE are both polarized to the sinusoidal side of the cell. We therefore investigated whether localization of ABCA1 in the RPE dictates the polarity of apoE secretion and if modulation of ABCA1 levels or function can alter extracellular apoE levels.

Methods: Human fetal (hf) RPE were plated on Transwells to allow for separate manipulation of the apical and basolateral sides of the RPE. To induce preferential apical apoE secretion, we applied two treatments we have shown will alter apoE secretion polarity: the addition of serum to only one chamber and aging cultures for more than a year. Immunocytochemistry (ICC) was then used to assess the localization of ABCA1 in hfRPE. ABCA1 was colocalized to phalloidin, an apical marker, and MCT3, a basolateral marker. Accumulation of secreted apoE was evaluated by Western Blots of the apical and basolateral media. ABCA1 expression was determined by Western Blot after treatment with Liver X Receptor (LXR) agonist, which upregulates ABCA1 expression, and after treatment with probucol, a ABCA1 inhibitor.

Results: ICC of hfRPE under conditions that favored apical apoE secretion did not show a corresponding shift in apical localization of ABCA1. In general, whether apoE secretion was polarized or not, ABCA1 had a fairly even distribution between the apical and basolateral RPE membranes. Treatment of hfRPE with LXR agonist upregulated ABCA1 but did not increase or switch the polarity of apoE secretion. Treatment with probucol caused a decrease in ABCA1 expression but no consistent effects on apoE secretion.

Conclusions: We demonstrated that the localization of ABCA1 in hfRPE is not related to the polarized secretion of apoE. Further experiments will probe other candidate mechanisms governing the polarity of apoE secretion in the RPE.

ABCA1 is Not Responsible for Polarized ApoE Secretion in the RPE

Purpose: To test the hypothesis that rod photoreceptor mitochondria function in vivo progressively declines over time.

Methods: 2, 12, and 24 mo dark- and light-adapted C57BL/6J (B6J) mice were examined by OCT. We measured i) an index of mitochondrial configuration within photoreceptors measured from the profile shape aspect ratio (MCP/AR) of the hyperreflective band posterior to the external limiting membrane (ELM)], ii) a proxy for energy-dependent pH-triggered water removal, the thickness of the ELM-retinal pigment epithelium (ELM-RPE), and its correlate iii) the hyporeflective band (HB) signal intensity at the photoreceptor tips. Visual performance was assessed by optokinetic tracking.

Results: In 2 mo and 24 mo mice, MCP/AR in both inferior and superior retina were smaller in light than in dark; no dark-light differences were noted in 12 mo mice. Dark-adapted inferior and superior, and light-adapted superior, ELM-RPE thickness increased with age. The dark-light difference in ELM-RPE thickness remained constant across all ages. All ages showed a decreased HB signal intensity magnitude in dark relative to light. In 12 mo mice, the dark-light difference in HB magnitude was greater than in younger and older mice. Anatomically, outer nuclear layer thickness decreased with age. Visual performance indices were reduced at 24 mo compared to 2 mo mice.

Conclusions: Whilst the working hypothesis was not supported herein, the results raise the possibility of a mid-life adaptation in rod mitochondrial function during healthy aging in B6J mice, a plasticity that occurred prior to declines in visual performance. 


Do Multiple OCT Biomarkers Indicate Age-related Decline in Rod Mitochondrial Function in C57BL/6J Mice? 

Purpose:
Multipotent adult stem cells (MASCs) isolated from human neonatal foreskin have been previously shown to differentiate into multiple mesodermal phenotypes. In vivo bone regeneration studies indicated that MASCs differentiated to osteoclasts, which are derived from monocytes. We tested the ability of MASCs isolated from human neonatal foreskin to differentiate into hematopoietic phenotypes in culture.

Methods:
Human tissues were obtained from the National Disease Research Institute under an IRB. MASCs isolated from human neonatal foreskin were isolated using an established protocol, enriched by freeze thawing, and expanded in culture. The MASCs were obtained by trypsinizing confluent adherent cultures, then aliquoting these cells into suspension or adherent cultures. The cells were fed with media with logarithmic concentrations of dexamethasone, a non-specific differentiator, from 0 M to 10-6 M, for 4 weeks. One culture method was the use of 15 mL centrifuge tubes. The centrifuge tubes were placed in the incubator after feedings at a 45 degree angle. Another method was suspending the MASCs into methylcellulose. Adherent cultures were done in 24 well plates. The presence of hematopoietic phenotypes was tested by immunohistochemical staining with primary antibodies to CD45, CD11b, CD33, and CD4. Alexa Flour 488 conjugated goat anti-mouse was used as a secondary antibody, and DAPI was used to stain nuclei. Staining was then visualized and images were captured.

Results:
MASCs from human neonatal foreskin differentiated into hematopoietic phenotypes. Nucleated hematopoietic cells (anti-CD45), myeloid lineages (anti-CD33), leukocytes (CD11b), and lymphocytes (CD4) were visualized in the final suspension cultures. Phenotypes were absent in control cultures (no dexamethasone), and found in greater numbers cultures treated with 10-8 to 10-6 M dexamethasone. 

Conclusion:
These preliminary results indicate that human MASCs are capable of differentiating into cells of the hematopoietic lineage. CD45 is a marker for all nucleated hematopoietic cells, CD33 is specific for monocytes/macrophages, CD11b is specific for leukocytes, and CD4 is specific for lymphocytes. Further research is needed to confirm these in vitro results, and then test the ability of MASCs to differentiate to hematopoietic phenotypes in vivo.

Differentiating Human Multipotent Adult Stem Cells toHematopoietic Phenotypes in Vitro

Background
Our group has shown that high density mesenchymal stromal cell (MSC) sheet implantation improves bone healing in a critical-sized bone defect model; however, these cells exhibit increased aging and cellular senescence in prolonged sheet culture, diminishing their therapeutic potential. Promisingly, rapamycin has emerged as a potential agent to rejuvenate aged MSCs. This study aimed to confirm rapamycin’s ability to prevent senescence and isolate specific pathways/genes responsible.

Methods
hPMSCs were seeded at high density (65,000 cell/cm2) and treated with either rapamycin (10 nM) or control vehicle (DMSO) for up to 7 days. Senescence and autophagy were assessed by senescence-associated 𝛽-Galactosidase (SA 𝛽-Gal) staining and DAPI nuclear staining, respectively. Additionally, cell culture media was extracted at different time points to assess senescence-associated cytokines IL-6 and IL-8 via ELISA. Finally, total RNA was extracted for differential gene analysis via GeneChip Clariom S Human Arrays.

Results 
Rapamycin significantly decreased SA-B Gal staining at all time points, with 67.7% and 72.9% less area when compared to control at day 4 and 7, respectively. Senescent-associated cytokine IL-6 levels were significantly reduced when compared to control, with 614.3 pg/ml and 286.6 pg/ml decreases at day 2 and 4 of treatment, respectively. Interestingly, IL-8 expression had increased significantly at all time points, with a 979.9 pg/ml difference at day 4 of treatment. Autophagy associated vacuoles-stained area had a 3.94-fold increase at day 4 of treatment compared to vehicle. Microarray gene analysis identified a total of 429 genes with a 2-fold or greater change in expression following treatment. FoxO, cellular senescence, and cell cycle pathways are some of many effected with significantly altered expression of FBXO32, PSAT1, CXCL8, and MYC, acting as potential players in rapamycin’s anti-aging effects.

Conclusion 
Rapamycin has proven effective in prolonging pMSC lifespan. The speculation and understanding of genes enacting this function may aid in isolating this specific role for usage in prolonging sheet cultures to enhance bioengineering applications. Numerous pathways identified in this study may explain rapamycin’s anti-aging functionality and lead to the creation of therapeutics specifically targeting relevant genes. The presence of enhanced IL-8 may prove beneficial in preserving stem cell functionality, specifically for wound healing applications. These findings prove rapamycin is a candidate for improving stem cell preparation and support previous studies indicating IL-8 may enhance stem cell viability, results useful in advancing stem cell therapy usage for bone remodeling/bioengineering.


Rapamycin Prevents Senescence and Functional Decline of High-Density Placental Mesenchymal Stem Cells

Background
Osteoporosis is one of the major causes of fractures in adults. As of 2004, it was estimated that approximately 10 million Americans >50 years have osteoporosis with annual cost of $25 billion. Osteoporosis can lead to many fragility fractures, especially hip fractures. Hip fractures are often associated with high levels of mortality. The cumulative mortality that is associated with one-year post hip fracture is between 20-40% with an additional risk of refracture. Although there are non-pharmacologic and pharmacologic treatment options available for osteoporosis, their use post hip fracture is low. Of the patients who present with fragility fractures, only 9-20% of the patients are treated for osteoporosis, increasing the chance of re-fracture in untreated patients.

Methods
A literature search was performed using PubMed and the following key words: “Osteoporosis” and “Hip fractures” in Title/Abstract. Initial search led to 3345 results. Studies on only humans were included. Literature after 2013 was included. Only Clinical Trials, Meta-Analyses, Randomized Controlled Trials, Reviews, Systematic Reviews were included. After an extensive two-tier review process, 14 articles are included in this review. 

Results
Fracture Liaison Service (FLS) and Orthogeriatric Service (OGS) are multidisciplinary team approaches that provide care for patients post-fragility fractures. FLS is shown to increase the rates of DEXA evaluations, improved prescription initiation rates for osteoporosis medications, and reduction in re-fracture rates. When comparing the effectiveness of FLS at tertiary hospitals, the hospital with FLS approximately 30% reduction in any refractures and 40% reduction in new major fractures over a three-year period. FLS is shown to be cost-effective as it prevented more fractures and increased quality-adjusted life years (QALYs). OGS is also shown to decrease length of stay at the hospital, lower risk of in-hospital mortality, and delirium. In addition, patients receiving OGS had improved mobility at 12 months post fracture when compared to usual orthopedic care. Implementation of FLS and OGS also shows decrease in mortality within 30 days and within one year in patients with hip fracture.

Conclusion
The introduction of FLS and OGS models changed the field of post fracture osteoporosis management. Initiation of pharmacological and nonpharmacological treatments post fracture has the potential to improve bone health, decrease re-fracture rates, decrease mortality and morbidity, and improve quality of life. The costs of follow up medical care have been shown to be reduced these services These models have been effective at providing the care that fragility fracture patients need.

The Importance of Fracture-Liaison Services and Orthogeriatric Services for Osteoporotic fractures

Background
Cartilage damage is challenging to treat due to its limited self-healing capacity caused by its avascular nature. However, 3D tissue engineering approaches that utilize biomaterials, additives, and cells offer a promising solution to regenerate functional cartilage. These innovative techniques have the potential to restore native cartilage properties, prevent the development of osteoarthritis, and restore joint function. In this study, we investigate a novel alginate-based 3D bioink composed of human articular cartilage for cartilage tissue engineering.

Methods
The bioink was developed by encapsulating C20A4 human articular chondrocytes in a mixture consisting of 20% medium viscosity sodium alginate (Alg), 5% polyvinyl alcohol (PVA), 5% Gum Arabic (GA), and 5% human articular cartilage. The human articular cartilage was obtained from cartilage shavings of distal femurs and ground into a powder for incorporation into the bioink. The bioink was printed into concentric discs using a BIO-X 3D Bioprinter, and the resulting 3D-biofabricated constructs were cross-linked with calcium chloride, rinsed, and cultured in chondrogenic media. Thirty-six total constructs were fabricated. Immediately after 3D printing, a portion of constructs were visualized with SEM (n=1) and underwent compression testing (n=5) to examine the microstructure and mechanical strength respectively. The remaining constructs were placed in culture for 1,7 or 14 days and subsequently analyzed for cellular viability (n=5) and gene expression (n=5) at each time point.

Results
SEM imaging revealed that the 3D-printed constructs exhibited a porous structure. The average elastic modulus of the constructs was 0.48 MPa. Post-printing, the cellular viability was 87.2% which decreased significantly to 76.4% at day 7 (p<0.001) but recovered to 85.9% at day 14 in culture. Furthermore, gene expression analysis showed a significant increase in transcriptional activity of chondrogenic genes Col2a1 (p=0.008) and Sox9 (p=0.021) at day 14 compared to day 1.

Conclusion
This study explored the use of an innovative alginate-based 3D bioink composed of human articular cartilage for cartilage tissue engineering. The bioink successfully formed porous 3D constructs that withstood the printing process. Additionally, the elastic modulus of these engineered constructs approached that of native cartilage (0.45-0.8MPa). Although cellular viability initially decreased, it gradually recovered over a two-week culture period. Moreover, the upregulation of chondrogenic genes indicates the potential of the bioink to promote cartilage regeneration. These findings suggest this bioink holds promise as an effective approach for cartilage tissue engineering, offering possibilities for future therapeutic interventions in cartilage repair and regeneration.

Novel 3D Extrusion Bioink Containing Processed Human Articular Cartilage for Application in Cartilage Tissue Engineering

Cerebrospinal fluid (CSF) homeostasis in the brain relies on a delicate balance in CSF production, circulation, and excretion. When this balance is compromised, patients develop a spectrum of disorders, including hydrocephalus and neurodegenerative diseases. Emerging studies suggest that the lymphatic system, referred to as the glymphatic system in the brain, may have a previously unknown role in CSF homeostasis. Interestingly, it is unknown whether the glymphatic system has any role in embryonic CSF regulation. Frog Xenopus has been widely used to study fundamental neurodevelopmental processes and is a unique model to investigate CSF dynamics. In Xenopus, unlike any other model system, whole brain CSF circulation can be visualized in real-time and manipulated easily, providing unprecedented insight into CSF hemostasis. In this work, we asked whether frogs have a glymphatic system and its role in CSF regulation. Using the frog Xenopus model system, we show that the glymphatic system is essential in CSF drainage during embryonic brain development. We used post-fertilization day 4-Xenopus tadpoles, where we injected conjugated ovalbumin into the brain ventricle and simultaneously into the tadpole's heart. Then we used confocal imaging to visualize CSF circulation and systemic cardiac circulation in parallel. We showed that there was no overlap between the two circulations. However, when we disrupted lymphatic formation, we showed that CSF excretion was disrupted, suggesting that Xenopus CSF excretion relies on the glymphatic system rather than cardiac circulation. In further studies, we will look at how CSF excretion is conserved among species and relate to our patients with hydrocephalus, Alzheimer’s, and other neurodegenerative diseases where abnormal CSF excretion is critical for disease progression.

The glymphatic system in Xenopus tropicalis – a model system for Congenital Hydrocephalus

Background
Migraines represent one of the leading causes of disability worldwide. While the pathophysiology of migraine pain is complex, the sensitization of the trigeminovascular pathway is implicated. Osteopathic manipulative treatment (OMT) has been used clinically to supplement pharmacologic treatment for patients suffering from migraines, but wide-spread adoption of these treatments has been limited by a lack of physiological and mechanistic evidence supporting its application. We aim to determine if OMT is an effective method of treating the pain associated with an acute migraine, abortively or prophylactically, and investigate its potential mechanisms of action.

Methods
Rats were randomly divided into treatment groups and assessed by behavioral testing and immunohistochemical (IHC) staining. Migraines were induced by a two-hit method. Animals were primed with Complete Freund’s Adjuvant (CFA) via injection and were exposed to umbellulone, a human migraine trigger, via inhalation on day 6. The presence of cutaneous allodynia, indicating a sensitized pain response, at the periorbital and hindpaw areas was determined using von Frey filaments. Subjects receiving OMT were given 2 osteopathic treatments for 1 minute each either before inhalation (prophylactic) or after inhalation of umbellulone (abortive). For the IHC studies, subjects were sacrificed and brain tissue was collected. The trigeminal nucleus caudalis (TNC) and the trigeminal ganglion (TG) were analyzed to determine the activation of the second-order sensory neurons by measuring expression of calcitonin gene-related peptide (CGRP), a neuropeptide implicated in migraine pathophysiology, and c-fos, an immediate early response gene. The experimenter was blinded for the treatment groups.

Results
Our behavioral testing showed that CFA + umbellulone induced significant allodynia. OMT employed as an abortive measure partially blocked the development of cutaneous allodynia. Multiple prophylactic episodes of OMT prevented the development of cutaneous allodynia. IHC analysis showed a significant increase of c-fos expression in TNC samples from subjects treated with CFA + umbellulone, which was blocked by prophylactic OMT. The expression of CGRP positive neurons in the TG samples increased significantly in subjects treated with CFA + umbellulone, which was normalized by prophylactic OMT.

Conclusion
Two osteopathic treatments demonstrated effectiveness in mitigating migraine pain as both a prophylactic and abortive measure. These findings provide insight into the efficacy of OMT for migraines and support its potential as a non-pharmacological approach for migraine management. Future studies will assess other forms of OMT as well as treatment directed to other regions associated with the trigeminovascular pathway.

OMT alleviated migraine-like pain via blockade of trigeminal activation

Background
Ex-vivo perfusion (EVP) is an emerging technology used to limit irreversible tissue damage and reduce failure in organ transplantation. Assessment of the viability of vascularized-composite allografts (VCA) during EVP is challenging because of lack of organ-specific viability parameters.
Infrared thermography (IRT) provides a non-invasive method of measuring skin temperature. We hypothesize that skin temperature changes are correlated with edema formation and weight gain during perfusion and can be used to assess VCA viability.
Methods
Eleven superior epigastric artery perforator flaps were procured from Yorkshire pigs and preserved by EVP utilizing a normothermic colloid perfusate with a hemoglobin-based oxygenated carrier (HBOC)-201 (HbO2 Therapeutics, Souderton, PA) for 12 and 24 hours. IRT images were recorded hourly (Fluke TiS Thermal Imager, Everett, WA) and mean skin temperature and percent temperature changes were analyzed (SmartView3.4 Software, Everett, WA). Skin biopsies were collected every 6 hours and analyzed after H&E staining. Indocyanine Green (ICG) angiography (Stryker SPY Elite System, Munich, Germany) was performed before pedicle division and at perfusion endpoint to assess peripheral perfusion. Median pixel brightness (MPB) was measured (ImageJ, Bethesda, MD) and normalized to account for variations in recording conditions. Flap weight and temperature changes from baseline were analyzed with paired T-tests. Correlation of flap temperature changes and MPB were with endpoint weight change (edema formation) was assessed using Pearson correlation tests.
Results
Flap weight did not change significantly from baseline to either 12 hours of EVP (TP0=188+/-5 g, TP12=198+/-5 g, p=0.6763) or to 24 hours (TP24=195+/-5 g, p=0.6412). Median percent flap weight change from baseline was determined: 12hr EVP -1.12% [-4.54-0.93%] versus 24hr EVP 2.0% [0-3.01%]. There was no significant difference between flap weight at 12 hours versus 24 hours (p=0.905). Normalized MPB at endpoint was not significantly different after 12hr EVP (0.47 [0.38-0.57]) compared to 24 hr EVP: 0.82 [0.7-094] (p=0.09524). Skin temperature was not significantly different at 12 hours (28.09°C, [27.56-29.1]) versus 24 hours (28.1°C, [26.9-29]) (p=0.4923). H&E staining of flaps showed preserved skin architecture compared to baseline. Skin temperature change significantly correlated with percent weight change (r=-0.7399, p=0.009) and and with MPB( r= -0.8335, p=0.019).
Conclusion
Flap skin temperature during EVP correlates with flap edema. This finding suggests that a decline in temperature could serve as a non-invasive real time indicator of flap injury during EVP.

Infrared Thermography as a Viability Measure During  Ex Vivo Preservation of Vascularized Composite Allografts

Background:
Psychiatric disorders, including anxiety, depression, schizophrenia, and substance use disorders, impose a significant burden on global public health. Conventional treatments targeting dopamine receptors often have limitations in terms of efficacy and tolerability. Therefore, there is an urgent need to explore and discover new active molecules that can effectively modulate dopamine receptors with improved therapeutic properties. Cannabidiol (CBD) and CBD-like molecules have emerged as potential candidates due to their diverse pharmacological effects and favorable safety profiles.


Methods:
A comprehensive review of existing literature, encompassing Computational Chemical Approach (in silico), was conducted to elucidate the mechanisms of action and biological effects of CBD on dopamine receptors. for this goal, an in silico approximation was carried out. Basically, Several CBD and CBD-like were docked against Dopamine receptors and compared with dopamine molecules. In order to estimate the dopamine receptor-ligands stability, molecular dynamics simulation was applied. Then molecular analyses were employed to explore the impact of CBD and its analogues on dopamine receptor signaling and endocannabinoid system regulation and to suggest new potential ligands.

Results:
The Findings from literature indicate that CBD and CBD- Like molecules modulates dopamine receptor activity and neurotransmission, suggesting potential therapeutic effects on psychiatric disorders associated with dopamine dysregulation. Additionally, CBD and CBD- like molecules interacts with the endocannabinoid system, specifically targeting cannabinoid receptors and influencing endocannabinoid signaling pathways involved in mood regulation, stress response, and neuroinflammation.The in silico results allow to suggest new molecules with dopamine receptor activity potential. 

Conclusion:

This comprehensive study highlights the therapeutic potential of CBD and CBD- Like molecules on dopamine receptors. The modulation of dopamine receptor activity and signaling pathways may contribute to a beneficial effect. The investigation by using computational chemical approach (in silico) enhances our understanding of the binding modes and interactions between these compounds and dopamine receptors. Continued research in this area may lead to the discovery of new active molecules with enhanced therapeutic efficacy and improved treatment strategies for individuals with psychiatric disorders


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Next from 2023 AMA Research Challenge

ABCA1 is Not Responsible for Polarized ApoE Secretion in the RPE