United States

Background
The serotonin transporter (SERT, encoded by SLC6A4) is downregulated in patients with inflammatory bowel disease (IBD) and in pre-clinical IBD models. While serotonin (5-HT) and SERT are known to play crucial roles in gut health and the microbiome, the therapeutic potential of targeting SERT in intestinal inflammation remains unexplored.
Methods
SERT knockout (KO) and wild-type (WT) mice were treated with dextran sulfate sodium (DSS) to induce chronic colitis. Fecal and serum samples were subjected to metabolomic analysis. Bioinformatic tools identified enriched metabolic pathways and microbial-related metabolites. Disease severity was assessed via myeloperoxidase (MPO) activity, RT-PCR, immunoblotting, and immunohistochemistry. Tight junction integrity was evaluated using immunofluorescence, Western blotting, RT-PCR, and serum endotoxin levels. Spearman correlation coefficients were calculated to determine the relationship between metabolite abundance and disease activity.
Results
SERT KO mice with chronic colitis exhibited increased disease severity. Baseline metabolomic profiles of SERT KO mice resembled those of WT mice with chronic colitis, indicating a predisposition to metabolic dysregulation. Metabolomic analysis revealed distinct fecal and serum profiles associated with chronic colitis, mirroring human IBD, including variations in purine and plasmalogen metabolites. SERT deficiency alone led to altered tryptophan metabolism, oxidative metabolism changes, and dysregulation of phosphatidylcholines and plasmalogens. Mechanistically, SERT deficiency in an inflammatory context resulted in reduced ileal tight junction expression and increased serum endotoxin levels, exacerbating disease. Additionally, several microbial metabolites in feces and serum correlated with disease activity. Specifically, indolepropionate levels were reduced in the serum of both chronic colitis and SERT KO mice, with significant negative correlations with histology scores and MPO activity. Conversely, tyrosol excretion was increased in feces of both chronic colitis and SERT KO mice, showing significant positive correlations with histology scores and MPO.
Conclusion
SERT deficiency exacerbates DSS-induced chronic inflammation, disrupts intestinal barrier function, and causes microbially influenced metabolic changes corresponding to disease activity. This study highlights novel changes in host-derived and bacteria-derived metabolites in serum and feces due to SERT loss. Our findings suggest that SERT deficiency leads to a loss of beneficial anti-inflammatory and antioxidant compounds. Future research will explore the therapeutic potential of fecal supernatants containing these beneficial compounds for treating intestinal inflammation. These findings underscore the disruptive role of SERT in metabolic pathways associated with IBD and suggest potential therapeutic targets for intestinal inflammation.

AMA Research Challenge 2024 

Global serotonin transporter deficiency induces a metabolomic signature resembling chronic intestinal inflammation

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Gastrointestinal microbes modulate peristalsis and stimulate the enteric nervous system (ENS), whose development, as in the central nervous system (CNS), continues into the murine postweaning period. Given that adult CNS function depends on stimuli received during critical periods of postnatal development, we hypothesized that adult ENS function, namely motility, depends on microbial stimuli during similar critical periods. We gave fecal microbiota transplantation (FMT) to germ-free mice at weaning or as adults, then measured gastrointestinal transit time either 4 weeks post-FMT or age-matched at 16 weeks. We performed RNAseq of colonic muscularis propria to compare gene expression patterns between mice with or without early life microbiota exposure. We found that only the mice given FMT at weaning recovered normal transit, while those given FMT as adults showed limited improvements. RNAseq of colonic muscularis propria revealed enrichments in neuron developmental pathways in mice exposed to gut microbes earlier in life, while mice exposed later – or not at all – showed exaggerated expression of inflammatory pathways. These findings highlight a microbiota-dependent sensitive period in ENS development, pointing to potential roles of the early life microbiome in later life dysmotility.

Microbiota-dependent early life programming of gastrointestinal motility

Background
Trauma patients with hemorrhagic shock (HS) accounts for 40% of preventable death. Shock-induced endotheliopathy has garnered interest recently and one aspect of this, endothelial glycocalyx shedding (EGX), has shown to promote coagulopathy following hemorrhage. Ferroptosis is an iron dependent, non-apoptotic form of controlled cell death triggered by the oxidation of membrane phospholipids. Liproxstatin-1 is an inhibitor of this process and can protect against oxidative damage to the endothelial cell membrane and subsequent shedding of glycocalyx. Our goal is to determine if administration of Liproxstatin-1, a ferroptosis inhibitor can reduce glycocalyx shedding in hemorrhaged rats providing evidence of its therapeutic value in the setting of hemorrhagic shock. 

Methods
A rat model of hemorrhagic shock and resuscitation was used to assess glycocalyx disruption in the lungs via fluorescent-labeled wheat germ agglutinin staining in frozen tissue and by syndecan-1 ELISA on plasma samples. A control group (n=5) resuscitated only with Lactated Ringer’s solution, following hemorrhage and resuscitation (H/R) was compared against an experimental group (n=1) that received Liproxstatin-1 following hemorrhage and before resuscitation.

Results
Glycocalyx shedding (assessed by an increase in plasma syndecan-1 levels) was significantly lower in the Liproxstatin-1 treated group compared to control group. Additionally, Liproxstatin-1 treated animals were able to sustain a higher mean arterial pressure (MAP), and oxygen saturation throughout the resuscitation period measured at 15- and 30-minutes following Liproxstatin-1 infusion. 

Conclusion
Ferroptosis inhibitor Liproxstatin-1 can reduce morbidity acutely during hemorrhagic shock and protect endothelial cell membrane from oxidative damage due to its ability to slow the generation of reactive oxygen species (ROS) and lipid hydroperoxides. 

Further experiments will be performed to complete the Liproxstatin-1 experimental group (n=5) as well as a control group using Cyrene (n=5), a DMSO-like solvent used to dissolve Liproxstatin-1 for intravenous administration. Additionally, tissue staining of lung vasculature will be performed to evaluate the glycocalyx disruption in the vascular beds of the lungs in the Cyrene, and Liproxstatin-1 groups as well as a group resuscitated with Lactated Ringers solution (H/R) alone. 


Can Inhibiting Ferroptosis Mitigate Vascular Injury and Subsequent Glycocalyx Shedding in the Setting of Hemorrhagic Shock?

Background 

Known to be one of the four major anastomoses between the median (MN) and ulnar (UN) nerves, the Martin-Gruber Anastomosis (MGA) has drawn much attention. Although previous studies have reported data regarding its prevalence, pattern and associated function, there is much variation within the given data. This present study’s aim is to dive further via dissections to further enhance our understanding of both the prevalence and patterns of the MGA. 

Methods 

Human cadaveric forearms (n = 66) were carefully dissected in the anatomy labs of Tilman J. Fertitta Family College of Medicine (TJFFCOM) and Baylor College of Medicine (BCM) for identification of various MGA nerve pathways. MGA patterns (Type I, Type II Type III) were photographed, documented, and stored. They were then compared to previous literature and studies for assessment of prevalence and pattern types. 

Results 

The MGA prevalence found within this study was 42%. Type I pattern anastomosis was measured to have approximately 54% prevalence. Type II showed the lowest prevalence in pattern type at 14% prevalence. Type III was found to be at 32% prevalence. It was also noted that unilateral MGA distribution was at 60% (n = 12) compared to bilateral at 40% (n = 8). Furthermore, bilateral distribution often showed congruence within pattern type. 

Conclusion 

It was noted that the prevalence found within this study seemed significantly higher compared to the reported median within previous literature (42% vs 20%). A notable highlight that resembled previous literature was the high occurrence of MGA buried within the flexor digitorum profundus (FDP) muscle. 

The MGA has significant clinical implications for iatrogenic injuries. Exploring the connectional relationship between MGA and associated structures may provide further insights in both prevention and treatment of related injuries including Carpal Tunnel Syndrome, peripheral neuropathies, and nerve transplant. 

However, it is noted that Type I pattern is often fibrous due to its embedding nature within the muscle. Often, there were findings that seemed to resemble Type I or II pattern types but could not adequately be confirmed as true MGA incidence. Due to this interesting finding, underlying nerve connections and structures can be further researched to provide additional explanation.

Delving Deeper into the Martin-Gruber Anastomosis – A Pilot Cadaveric Study

Background
Protein-based therapeutics are limited by their route of administration and inability to confine them to their site of action. One innovative approach would be to chemically bind therapeutics to medical devices, allowing localized and concentrated delivery to the site of action. This study aims to evaluate if GLP-2 can be covalently bound to our Vaginal Expansion Sleeve (VES) and Intestinal Expansion Sleeve (IES) in measurable quantities.

Methods
Expansion sleeves were coated and crosslinked with polyvinyl alcohol (PVA) making a chemically reactive surface capable of binding amine-containing therapeutics such as GLP-2. A standard curve was created by adding 250ug, 100ug, 50ug, 25ug, and 0ug of GLP-2 into respective wells with PVA of a 24-well plate. A rabbit anti-GLP-2 antibody followed by a goat anti-rabbit IgG alkaline phosphatase secondary antibody was added to the wells to allow the addition of SeraCare KPL BluePhos Microwell Phosphate Substrate System. Once added, the color changes from yellow to blue depending on the concentration of GLP-2 bound antibodies, allowing the absorbance to be read at 620nm to generate the standard curve and calculate the concentration of GLP-2 on the PVA-coated sleeves. 


Results
Addition of 50ug of GLP-2, each IES and VES device bound an average 22.69 ± 9.32 ug/cm2 of GLP-2 after adjustment for an external surface area of 9.425 cm2, allowing for 44% of added GLP-2 to remain fixated to the PVA coated sleeves. 



Conclusion
Current GLP-2 dosing in humans in 0.6mg/70Kg. With an external surface area of 9.425 cm2, each sleeve is capable of giving a localized delivery of 213.85ug of GLP-2, which is 25.2x greater dose than systemic dosing. This methodology makes it possible to add dramatically lower doses of therapeutic agents to get the same effect as systemic administration of the GLP-2 drug while also avoiding systemic effects. 


Preliminary results of Polyvinyl Alcohol Coated Medical Device Therapeutic Conjugation

Background: Liver transplantation (LTx) is the only curative treatment for end-stage liver disease. However, due to the shortage of high-quality deceased donor (DD) organs, 2,000 patients die annually while waiting for suitable livers. DD marginal livers (i.e. prolonged ischemic damage or fatty livers) could address this disparity, though it is associated with primary graft non-function and decreased recipient survival post-LTx. Advances in Normothermic Machine Perfusion (NMP) technology allow for extended preservation and ex-situ assessment of DD livers before transplant to guide decisions on liver quality and transplantability. Understanding the molecular mechanisms of injury, repair, and recovery during NMP is crucial for identifying safe transplant organs and potential pharmaceutical intervention targets to optimize marginal organ quality before transplant. This study aims to evaluate these mechanisms by analyzing temporal gene expression signatures using whole genome RNA sequencing in transplantable and non-transplantable livers during NMP.

Methods: Human donor livers without significant steatosis (n=6) and fatty donor livers (n=5) were subjected to 12 hours of NMP. Livers were categorized based on predefined criteria for demonstrating adequate hepatocellular function (transplantable vs. non-transplantable). Core needle biopsies were taken immediately before NMP and after 3 and 6 hours of NMP for bulk RNA sequencing. Bioinformatic analysis evaluated temporal changes in gene expression between functional and nonfunctional lean and fatty livers during NMP.

Results: Principal component analysis (PCA) revealed that gene expression changed for both fatty and lean livers during NMP. PCA effectively differentiated functional lean and fatty livers from non-functional fatty livers when all four categories were examined together. However, PCA was less sensitive in distinguishing functional lean and fatty livers from non-functional lean livers. The transcriptomic profile of biopsies from functional lean and fatty livers showed strong activation of mTORC1 signaling (growth/regeneration pathway) and protein secretion throughout NMP, correlating with improved hepatocellular function. Conversely, nonfunctional fatty livers exhibited a gradual decrease in the expression of both pathways during NMP.

Conclusion: We demonstrated that temporal overexpression of genes associated with liver regeneration and normal synthetic function during NMP can differentiate between transplantable and non-transplantable lean and fatty livers. Targeting the mTORC1 and protein secretion pathways may represent viable therapeutic interventions for rehabilitating the function of marginal livers, thus improving their quality and making them safe for transplantation. This can potentially reduce organ shortages. Our findings provide a foundation for future research aimed at predicting the recovery of marginal livers and enhancing liver transplantation outcomes through targeted therapies.


Studying the Molecular Pathways Involved in Hepatocellular Recovery During Normothermic Machine Perfusion to Identify Livers Safe for Transplantation

Background: Colorectal Cancer (CRC) ranks as the third leading cause of cancer-related deaths in the United States. Current guidelines recommend initiating CRC screening at age 50; however, there has been a concerning rise in CRC incidence among younger populations. Genetic predisposition, oxidative stress, and inflammation are believed to contribute to CRC pathogenesis. However, when treated via conventional methods like chemotherapy and bowel resection, some patients are met with undesirable side effects, such as cardiotoxicity associated with agents like anthracyclines. Vialinin A, an extract from the Chinese mushroom Thelephora Vialis, demonstrates both antioxidant and anti-inflammatory properties.

Moreover, we propose that Vialinin A has potential anticarcinogenic mechanisms that may be utilized in the treatment of CRC cell proliferation and metastasis.
Methods: To assess the impact of Vialinin A on both SW480 and CaCO-2 CRC cell lines, we exposed them to increasing concentrations of Vialinin A. Viability of these cells after 24 and 48 hours was evaluated using an MTT assay. Subsequently, specific assay kits were employed to measure caspase-3 activity and apoptosis induction. Additionally, an RD Systems Antibody Multiplex Array was utilized to ascertain the expression levels of diverse apoptosis markers in Vialinin A-treated CRC cell lysates. Moreover, a mitochondrial reactive oxygen species (MT-ROS) assay was performed to measure the ROS production of CaCO-2 cells pre and post-treatment with Vialinin A. Furthermore, a trans-well migration, scratch assay, and multiplex array are slated for use to identify the antimetastatic potentials of Vialinin A, as well as the oncogenic, inflammatory markers expressed in its presence. Ultimately, in vivo experiments using nude mice xenografts will further allow us to examine the potential chemopreventive role of Vialinin A.

Results: Our findings indicate that Vialinin A inhibits CRC cell growth in a concentration-dependent manner. Additionally, Vialinin A suppresses reactive oxygen species production, activates caspase-3, and triggers apoptosis in growth factor-stimulated cancer cells. Moreover, Vialinin A modulates the expression of various apoptotic markers in CRC cells. Ongoing investigations aim to elucidate the underlying mechanisms by which Vialinin A hinders CRC growth.



An Antioxidant, Vialinin A, Extracted from An Edible Chinese Mushroom Prevents Colon Cancer Cell Proliferation.

Abstract Title
BRCA1 Is Required for DNA2 Deficiency-Induced Replication Stress Response at the ALT Telomeres

Background
Cancer cells bypass senescence and achieve replicative immortality by elongating their telomeres. They accomplish this through two primary mechanisms: telomerase activation and the Alternative Lengthening of Telomere (ALT) pathway. Approximately 85-90% of cancers utilize telomerase (TEL+), while 10-15% of cancers employ ALT (ALT+). Notably, about 60% of osteosarcomas rely on ALT for telomere maintenance. There are currently no targeted therapies for ALT+ cancers, highlighting the need for research into specific molecular targets for the ALT pathway. FDA-approved PARP inhibitors are effective for treating the BRCA1/2 mutated cancers, highlighting the importance of understanding BRCA1-deficient cells in ALT contexts. Previously, the Zhang lab showed that depletion of FANCM induces a robust replication stress response at the ALT telomeres, which requires a functional BRCA1, emphasizing BRCA1's critical function in this pathway. Most recently, the Zhang lab found that depletion of DNA2 also induces a robust replication stress response at the ALT telomeres. This study aims to elucidate the functional interplay between BRCA1 and DNA2 at the ALT telomeres.

Methods
U2OS cancer cells underwent two rounds of siRNA transfection. The transfection was followed by immunofluorescent staining where the cells were seeded onto coverslips. The cells were then fixed with 3% paraformaldehyde containing 2% sucrose for 10 minutes, followed by treatment with Triton X-100 solution on ice for 5 minutes. Subsequently, the cells were stained with primary antibodies and the respective Alexa-488 and Alexa-546 conjugated secondary antibodies. Colocalization of DNA damage markers and TRF2 was quantified to determine telomere DNA damage in BRCA1 and DNA2 depleted U2OS cell lines.

Results
Our study utilized siRNA transfection to investigate the roles of BRCA1 and DNA2 in ALT+ cancer cells. Depletion of BRCA1 and DNA2 followed by immunofluorescence staining revealed the recruitment of critical cell cycle regulator proteins - pChK1, pRPA, BLM, and yH2AX - to telomeres. Our results demonstrate an increase of pChK1, pRPA, BLM and yH2AX to the telomere in DNA2-depleted cells, however the recruitment does not occur in BRCA1-DNA2 co-depleted cells. 

Conclusion
These findings highlight BRCA1 and DNA2 as crucial regulators of telomeric replication stress in ALT+ cancers. BRCA1 is necessary for the DNA damage response in DNA2-depleted ALT+ cells. Understanding BRCA1 and DNA2-mediated pathways may provide a promising approach to disrupt ALT-specific telomere maintenance, potentially advancing therapeutic strategies tailored to ALT+ cancers and improving treatment outcomes in these malignancies.

BRCA1 Is Required for DNA2 Deficiency-Induced Replication Stress Response at the ALT Telomeres

Melanoma is the second leading invasive cancer with a greater capacity to invade and metastasize. Increased oxidative stress has been shown to be involved in melanoma growth and spread. Several antioxidants, vitamins, and other phytochemicals have been shown to prevent melanoma cancer growth. However, the chemopreventive efficacy of vialinin-A, an edible mushroom-derived p-terphenyl antioxidant, on cancer growth and invasion is unknown. We, therefore, hypothesized that with its potent anti-oxidative and anti-inflammatory actions, vialinin-A could prevent melanoma cell growth and invasion. B16-F10 melanoma cells were treated without or with various concentrations of vialinin-A. The cell viability was determined by MTT assay and apoptosis by annexin-V staining. Our results suggest that vialinin-A prevents melanoma cell growth in a dose-dependent manner. Vialinin-A also prevents the invasion and migration of melanoma cells. Further, vialinin-A prevents the generation of reactive oxygen species and promotes apoptosis of B16-F10 cells. In addition, vialinin-A increases the activation of caspase-3 and cleavage of PARP protein. Vialinin-A also regulated the expression of various anti-apoptotic, pro-apoptotic, and inflammatory markers in melanoma cells. In conclusion, our results suggest that vialinin-A, through regulating oxidative stress-modulated apoptotic pathways, could prevent melanoma cell growth and invasion and has potential chemopreventive properties. 


Edible mushroom-derived antioxidant, vialinin-A, prevents melanoma growth and invasion


Abstract Title
Harnessing the anti-neoplastic potential of novel mannose-binding lectins on human urothelial and breast cell carcinomas.
Authors and Affiliation: 
Julie Marchioni, BS; Alec Skomo, BS; and Prerna Sharma, PhD*
Geisinger Commonwealth School of Medicine, Scranton PA 18509

Background
Mannose-binding lectins (MBLs) are a class of carbohydrate-binding proteins that are important in modulation of viral infections, inflammation, and cancer progression through interactions with glycosylated regions. High grade cancer cell lines have their membranes decorated with oligomannosidic sugars that can be harnessed to develop anticancer drugs. The model MBL, cyanovirin-N (CVN-N) has shown promise as an antiviral. However, its widespread use is limited due to difficult isolations, poor stability, and limited applicability to cancer targets. This led us to identify novel MBLs and test their anti-neoplastic effects on human cancer cell lines.
Methods
The novel proteins were expressed with the pET23a/BL21(DE3) Escherichia coli expression system and purified using Nickel-NTA chromatography. The purified proteins were assessed for their structure, folding, thermal, and chemical stability using circular dichroism (CD) spectroscopy. Mannose-binding studies using model glycoproteins were performed with electrophoretic mobility shift assay, EMSA. Functional studies to assess the antineoplastic effects were performed on human urinary bladder carcinoma, T24, and breast cell adenocarcinoma, MDA-MB-231, using MTS and WST-1-based assays.
Results
We identified two novel fungal lectins, LC-3 (17 kDa) and LC-4 (13 kDa) with proposed mannose binding ability by data mining using BLAST search. Their predicted secondary structure aligned well with the model structure of CVN-N. The far UV-CD spectra of LC-3 and LC-4 proteins displayed a well folded structure with single negative peaks at 215 and 219 nm respectively. Both the proteins were found to be thermostable up to 90°C. Chemical denaturation studies of LC-3 with guanidinium hydrochloride reveal a Cm of 2.75 M and a ΔG of unfolding to be 7241.7 J/mol. Chemical denaturation studies for LC-4 were significant for a ΔG of unfolding of 1458.4 J/mol and for cooperativity of unfolding of two sub-domains. Positive shifts with Fetuin A, a sialylated ligand, and RNase B, a mannosylated ligand were observed for both proteins in EMSA. Significant inhibition of cell proliferation by both the proteins were observed in low micromolar concentrations (1-5 µM) for the mannose expressing cell lines, T24 and MDA-MB-231, when compared to control groups.

Conclusion
The identified fungal lectins, LC-3 and LC-4 are thermochemically stable and have high specificity for mannosylated ligands. The high yield, thermochemical stability, and significant anti-proliferative effects of LC-3 and LC-4 against urinary bladder and breast cell carcinomas make them a potential candidate for clinical applications.

Harnessing the Anti-Neoplastic Potential of Novel Mannose-Binding Lectins on Human Urothelial and Breast Cell Carcinomas.

Immunosuppressive effects of Transforming Growth Factor Beta (TGFβ) on cytotoxic function of human natural killer cells are reversible

Anjali Patel 1, Giuseppe Maiocco 1, Catherine Mirto 1, Donald S. Torry 1,2,3, and Andrew Wilber 1,3

1 Medical Microbiology, Immunology and Cell Biology, 2 Obstetrics and Gynecology, and 3 Simmons Cancer Institute, SIU School of Medicine, Springfield, IL 62702

Background: Natural killer (NK) cells are innate immune cells that are CD56pos/CD16pos and have anti-tumor function. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg and poorly cytotoxic. These features are analogous to human decidual NK (dNK) cells, which are found at the maternal-fetal interface and support development of the placenta development during embryo implantation. The decidua and kidney tumor environment have high levels of transforming growth factor beta (TGFβ), which is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell cytotoxic function is transient and therefore reversible.

Methods: pNK cells were isolated negative selection from blood of healthy donors (HD) or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess levels of TGFβ receptors (R1, R2, and R3) mRNA and protein, respectively. NK cells were cultured for 4-5 days with the proliferative cytokine IL2 without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). For this, cells were mixed at 1:1, 1:2, and 1:5 target to effector ratio and cultured overnight. Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant.

Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed equivalent levels of all three TGFβ receptors and were potently cytotoxic against K562 target cells. NK cells treated with TGFβ retained only 15% cytotoxic function compared with untreated cells (p≤0.05). Cytotoxic function was completely restored when TGFβ treatment was discontinued and NK cells cultured in standard conditions for 4-5 days.

Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective cancer therapy. TGFβ limits this potential in the tumor environment by converting pNK cells to non-cytotoxic dNK-like cells. Importantly, NK regained complete cytotoxic function when they were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment and have implications for anti-cancer therapies. Efforts to explore this possibility using selective inhibitors or genome editing technology are underway. 



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