United States

Introduction 
Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia.

Methods 
We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&amp;E we validated the explants to be &gt;80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells.

Results 
Through H&amp;E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for &lt;6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ~4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects.

Conclusion 
We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta.

AMA Research Challenge 2023

Validation of a Novel Placenta Explant Protocol Allowing
Screening of Drugs Modulating Circadian Rhythms in the Mouse Placenta

AAMAS is the leading scientific conference for research in autonomous agents and multi-agent systems. The AAMAS conference series was initiated in 2002 as the merging of three respected scientific meetings: the International Conference on Multi-Agent Systems (ICMAS), the International Workshop on Agent Theories, Architectures, and Languages (ATAL), and the International Conference on Autonomous Agents (AA). The aim of the joint conference is to provide a single, high-profile, internationally-respected archival forum for scientific research in the theory and practice of autonomous agents and multi-agent systems.

Browse keynotes, discussions, panels and over 300 presentations.


AAMAS 2020

AAMAS is the leading scientific conference for research in autonomous agents and multi-agent systems. The AAMAS conference series was initiated in 2002 as the merging of three respected scientific meetings: the International Conference on Multi-Agent Systems (ICMAS), the International Workshop on Agent Theories, Architectures, and Languages (ATAL), and the International Conference on Autonomous Agents (AA). 

Introduction 
Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia.

Methods 
We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&E we validated the explants to be >80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells.

Results 
Through H&E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for <6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ~4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects.

Conclusion 
We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta.

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Objective / Hypothesis: Cell replacement therapy is rapidly emerging as a viable cure for Type 1 Diabetes Mellitus; however, the supply of insulin-producing cells for transplantation is severely limited due to a shortage of pancreatic islet donors. Current strategies include generating stem-cell derived pancreatic islets (SC-islets) for eventual implantation, but current methods generate functionally immature cells (mostly due to lack of vascularization and the importance of endothelial cell proximity), which complicates clinical translation. Our goal with this project is to create functionally competent insulin-secreting cells with a built-in vascular network. We hypothesize that the orthogonal differentiation of human induced pluripotent stem cells (h-iPSCs) into beta cells and vascular cells will enable the development of SC-islets with a built-in vasculature.

Approach: We developed an orthogonal differentiation strategy using doxycycline-inducible transgenes encoding key transcription factors to induce differentiation of h-iPSCs into endothelial cells (iECs) and mural cells (iMCs). We are using our differentiation protocol that generates SC-islets from human embryonic stem cells (hESCs) through a multi-stage procedure reminiscent of pancreatic development to generate islet endocrine cells. In the initial phases of the experiment, we used INS1 insulin-secreting rat insulinoma cells in place of SC-islet cells due to their ease of use (no need for >31-day differentiation protocol) and direct translatability to the behavior of SC-islet cells in vitro. These cells were co-cultured and studied by functional assays, imaging, and flow cytometry.

Results: Both transcription factor-based differentiation methods enabled highly efficient differentiation of human iPSCs into competent vascular cells (iECs and iMCs) – h-iPSCs were rapidly and robustly differentiated by simple exposure to doxycycline (4-day process, >95% efficiency). These cells were phenotypically and functionally validated, with the iECs staining positive for CD31, CD144, and VE-Cadherin, and iMCs staining positive for Calponin1, Vimentin, and SM22. These results were confirmed with flow cytometry. Using the INS1 cell line, co-culture of these cells in different ratios of INS1:iEC:iMC did show integrated tube formation reminiscent of vasculature at day 8 (80:18:2 and 91:9:1). In contrast, other ratios (such as 50:50 and 66:33 INS1 to iEC) did not show tube formation, and instead formed segregated cell clusters in which iECs, iMCs, and INS1 cells did not integrate.

Summary / Conclusions: This data shows promising results and suggests an in vitro model for creating an intra-islet vasculature may be achievable, as we did see endothelial/mural cell tube formation in our experiments when using INS1 cells, iECs, and iMCs. Future studies may include additional functional testing and analysis, such as using GSIS to quantify insulin secretion and sc-RNA-seq to study maturation markers pre- and post-differentiation. Once the orthogonal differentiation protocol shows significant success, transplantation studies into animal models could demonstrate how these islets function in vivo.


Generation of stem-cell derived, vascularized islets for transplantation in Type 1 Diabetes Mellitus therapy.

Introduction: While the harmful effects of exogenous glucocorticoids (GC) on the skeleton are well understood, the role of endogenous GC and their glucocorticoid receptor (GR)-mediated signaling in bone are not as clear. Although we originally predicted that GR knockout would be beneficial to the skeleton, both constitutive and adult-onset conditional knockout (CKO) of the GR in osteoprogenitors promoted low bone mass in young female mice that was further exacerbated with age. The goal of this study was to explore the role of the GR in the male skeleton.

Methods: The GR-CKO male mice and wildtype (WT) littermates were created by crossing GRfl/fl and Osx1-Cre+ mice. Constitutive [RD] GR-CKOosx mice were maintained on regular chow until six months of age. Adult onset [Doxy] GR-CKOosx mice were maintained on doxycycline chow until three months of age to negate developmental defects during rapid skeletal growth. Orchidectomy (ORX) or sham procedures were performed aseptically on six-month-old male GR-CKOosx mice and wildtype littermates. In the fourth week post-surgery, the mice underwent DXA scans to assess bone density and body composition. After sacrifice, femurs were harvested and microarchitectural parameters were determined using microCT.

Results: GR-CKO [RD] mice that underwent ORX surgery had a significant loss of body mass, comprised mainly of lean mass, compared to WT-ORX mice. This effect was not seen in GR-CKO ORX [Doxy] mice. Femur microCT analysis revealed that bone mass in male GR-CKOosx was not different from WT mice under sham conditions, but CKO mice had less trabecular bone mass than WT after ORX in both RD and Doxy models.

Conclusion: Male GR-CKOosx mice did not present with a skeletal phenotype until after ORX surgery. ORX induced the same low bone mass phenotype vs. WT mice, as seen in female GR-CKOosx animals. This suggests an important role of sex hormones in mediating bone GR signaling.

The Role of Sex Steroids in Mediating the Effects of Osteoblast-Lineage Glucocorticoid Receptor Signaling in Skeletal Health

Background:
Hypoglycemia poses a significant risk of irreversible neurological damage and mortality, particularly among elderly with diabetes. It is also the most preventable cause of morbidity and mortality in this patient population. The urgent need for heightened awareness and proactive management of hypoglycemia by deprescribing is crucial in order to reduce hospitalization rates and prevent poor outcomes.

Case Presentation:
An elderly male with a history of diabetes and recent hospitalization for acute chronic obstructive pulmonary disease exacerbation arrived at the emergency department (ED) complaining of generalized weakness and urinary retention upon evaluation, he was diagnosed with a urinary tract infection (UTI) and acute kidney injury. His blood glucose level was 100 mg/dL. Intravenous fluids were administered, and a Foley catheter was inserted. The patient's condition improved and was discharged home. However, three days later, the patient returned to the ED in an unresponsive state. His glucose levels were undetectable, and he required intubation for airway protection. A thorough review revealed that he had been on long-acting sulfonylurea and biguanide for the past 30 years and his recent Hemoglobin A1C was 5.8. Despite extensive evaluations, no alternative cause was identified and he was diagnosed with hypoglycemic coma. A week later, with no improvement in his mental status, the patient's family made the difficult decision to pursue a do-not-resuscitate order. The patient was extubated and subsequently passed away.

Discussion:
Hypoglycemic coma is a serious adverse event that can lead to irreversible brain damage and death. It is also the most preventable complication across all healthcare settings including ambulatory, ED, hospital, skilled or long-term care, and assisted living facilities. Providers in these care settings should 1) carefully monitor risk factors like advancing age, new illness, new poor oral intake, and kidney injury that can decrease the clearance of diabetic medications. 2) Closely monitor glucose checks, and provide timely diabetic and diuretic medication dose reductions in acute illness or deprescribe to prevent adverse events or death from hypoglycemia in older diabetic patients.


Hypoglycaemic Coma Manifested by Urinary Tract Infection - A Case report and Comprehensive Review

Background
This study aimed to investigate the impact of COVID-19 on perioperative adverse outcomes in patients undergoing Per Oral Endoscopic Myotomy (POEM) procedures. As the COVID-19 pandemic has presented unique challenges in healthcare, understanding its association with adverse outcomes during POEM is crucial for optimizing patient care and management. 

Methods
The study utilized data from the Nationwide Inpatient Sample (NIS) 2020, a comprehensive and nationally representative database. 464% (p<0.001) more likely to experience pulmonary embolism during POEM.
Results
The cohort included 5,776 adults who underwent POEM, with 118 individuals diagnosed with COVID-19. Demographic variables, comorbidities, and perioperative adverse outcomes were examined. Univariable and multivariable logistic regression analyses were conducted to assess the relationship between COVID-19 and adverse outcomes, while controlling for potential confounding factors. Among the cohort, individuals with COVID-19 had higher rates of complications, including pneumonia, DVT or other thromboembolism, pulmonary collapse or atelectasis, pulmonary embolism, and portal vein thrombosis, compared to those without COVID-19 (p<0.001). In the univariable analysis, individuals with COVID-19 254% (p<0.001) more likely to develop pneumonia and 515% (p<0.001) more likely to develop pulmonary embolism during POEM. In the multivariable analysis, after adjusting for age, gender, race, hospital characteristics, region of the country, insurance, and comorbidities, individuals with COVID-19 were 230% (p<0.001) more likely to develop pneumonia and 464% (p<0.001) more likely to experience pulmonary embolism during POEM. 
Conclusion
This study reveals a significant association between COVID-19 and increased risk of perioperative adverse outcomes, particularly pneumonia and pulmonary embolism, in patients undergoing POEM. These findings emphasize the importance of considering COVID-19 status when evaluating perioperative risks and implementing appropriate preventive measures to improve patient outcomes. The results underscore the need for tailored approaches to mitigate the impact of COVID-19 in the perioperative period and enhance patient safety.

Perioperative Outcomes in Patients Undergoing Per Oral Endoscopic Myotomy (POEM) During the COVID 19 pandemic, a Nationally Representative Study

Background: Intraductal Papillary Mucinous Neoplasms (IPMNs) are the most common subset of cystic neoplasms and are currently only managed by surgery and surveillance; however, because pancreatic surgery proves to be an unnecessary risk in benign cases, an alternative treatment plan is crucial. Previous experimentation showed that IPMNs exhibited higher levels of MAP kinase, a signaling cascade that regulates cell proliferation and differentiation, than pancreatic ductal adenocarcinoma (PDAC). We hypothesized that IPMNs would be more susceptible to MEK (upstream regulator in the MAP kinase pathway) inhibition than conventional PDAC cells due to the significant increase of MAPK phosphorylation in IPMNs.

Methods: 4619 cells were obtained from a mouse PDAC tumor carrying mutations in Kras and Trp53; 4838 cells were obtained from a mouse IPMN tumor carrying mutations in Kras and Gnas (doxycycline-inducible). Cells were treated with 1 ug/ml doxycycline for 24hr prior to treatment. Gene expression was determined by RT-qPCR at 24hr of 100nM Trametinib or vehicle (DMSO) normalizing to Pmm1 levels. Western blotting for phospho-ERK 1/2, total ERK1/2, and GAPDH was used to control for trametinib efficiency. Growth was determined by MT Viability Assay, following indicated concentrations of Trametinib at the indicated timepoints.

Results: Trametinib significantly inhibited expression of Aqp5 in both PDAC and IPMN cells. Mki67 fold change showed a significant decrease in both lines with Trametinib whereas Pcna fold change was significant between PDAC Trametinib vs. vehicle but not in the IPMN line. No significance was noted for Gnas, Spdef, Cdkn2a, and Cdkn1a. Growth and proliferation curves showed the highest concentration of Trametinib (100 nM) prohibiting growth the most in both cell lines.

Conclusion: MEK inhibition had similar effects on PDAC and IPMN cells. Trametinib decreased expression of proliferation markers similarly in both cell lines, consistent with a similar inhibition of growth. Expression of growth arrest markers was unaffected by trametinib. The role of MEK in regulating Aqp5 is novel and will be pursued in future experiments. These experiments were performed in 10% fetal bovine serum which is highly growth stimulatory. Future experiments could treat cells in low or no serum to determine if Trametinib has more of an effect under nutrient-limited conditions. Overall, IPMNs need to be studied more regarding their differences from PDAC and the MAP Kinase pathway.

Role of MAP Kinase in IPMN and PDAC

Background
SLC6A4 (SERT) is downregulated in IBD patients and pre-clinical models of IBD. Despite the established roles of serotonin (5-HT) and SERT in maintaining the gut and its microbiome, the mechanisms by which decreased SERT contributes to intestinal inflammation remain unclear.

Methods
SERT KO and WT mice administered Dextran sulfate sodium (DSS) induced chronic colitis underwent fecal and serum metabolomic analysis. Bioinformatic analysis identified enriched metabolic pathways and microbial-related metabolites. Disease severity was evaluated with myeloperoxidase (MPO) activity, RT-PCR, immunoblotting, immunohistochemistry. Tight junction perturbation was assessed by immunofluorescence, Western blotting, RT-PCR and serum endotoxin levels. We computed Spearman correlation coefficients between the abundance of the altered microbial metabolites and disease activity.

Results
SERT knockout (SERT KO) mice induced with chronic colitis revealed increased disease severity. The baseline metabolomic profiles of SERT KO mice resembled those of WT mice with chronic colitis, suggesting a predisposition to metabolic dysregulation. Metabolomic analysis demonstrated distinct profiles in feces and serum associated with chronic colitis in mice mimicking human IBD such as variations in purine and plasmalogen metabolites. SERT deficiency alone led to altered tryptophan metabolism, changes in oxidative metabolism, and dysregulation of phosphatidylcholines and plasmalogens in feces and serum. Mechanistically, SERT deficiency in an inflammatory context led to diminished ileal tight junction expression and increased serum endotoxin levels worsening disease. Furthermore, we identified several microbial metabolites in the feces and serum correlating with disease activity. 

Conclusion
SERT deficiency augmented chronic inflammation induced by DSS, perturbed intestinal barrier dysfunction, and caused microbially influenced metabolic alterations which corresponded to disease activity. The results of this study highlight novel changes in both host-derived and bacteria-derived serum metabolites and fecal metabolites occurring due to the loss of SERT. These findings demonstrate the functionally disruptive role of SERT in metabolic pathways associated with IBD and suggest potential therapeutic targets for intestinal inflammation.


Serotonin transporter deficiency-driven metabolomic derangements drive intestinal inflammation

There is a role for the gut virome in numerous dysbiosis-associated states including obesity, inflammatory bowel diseases, and C. difficile infection. These findings are likely related to the ecological function of bacteriophages (phages) in regulating the structure, composition, and diversity of the gut microbiome. Dysregulation in the gut phage community may therefore lead to microbiome dysbiosis. To study whether exogenous phage administration may have therapeutic potential as a treatment for dysbiosis, our lab purified phages from lactic acid bacteria-fermented sauerkraut to test the effect of sauerkraut phages in a mouse model for stress-induced dysbiosis. Stress is strongly associated with dysbiosis, decreased microbiome diversity, and the growth of pro-inflammatory bacteria in the gut microbiome. Sauerkraut and other fermented foods have been shown to promote microbiome diversity and our lab hypothesized that phages may have a role in mediating this effect.

Bacteriophages derived from fermented food increase cecal microbiome diversity in the setting of stress-induced dysbiosis

Background: From neoangiogenesis to tissue regeneration, the greater omentum has been used in a variety of unlikely, but innovative ways in surgical research. Despite its surgical importance, the greater omentum’s anatomy has not been systematically characterized. Previously collected cadaveric omenta revealed statistical differences between sex and omental mass and volume without any available information on BMI. This research will continue to collect greater omenta metrics and further investigate the role of BMI in biometrics using cadaveric omenta and estimations of stature and body mass.  

Methods: Greater omenta were collected and measured from 30 embalmed cadavers. Maximum femoral length (MFL) and femoral head diameter (FHD) were collected from each respective cadaver to estimate height and weight, respectively. Statistical analysis using R compared anatomical parameters with age, sex, body mass, stature, and BMI using a student’s t-test and linear regression.   

Results: Sex differences in omental mass, volume, and surface area (P < 0.05). Sex differences in estimated body mass, stature, and BMI (P < 0.01). Age negatively correlated with estimated BMI, stature, and body mass (P < 0.001). Age negatively correlated with omental length (P < 0.05). Estimated stature positively correlated with omental biometrics, body mass, and BMI (P < 0.001). 

Conclusion: Recent studies claim that BMI plays a role in omental biometrics. Since height, weight, and BMI were unavailable from the cadavers, this research used published equations to estimate body mass and stature. Omental mass, volume, and surface area demonstrated significant differences between males and females, correlating well with the significant differences between anthropometrics between sexes. Omental mass and volumes were positively correlated with estimated cadaver height. Omental length, estimated stature, body mass, and BMI decrease with increased age. Future studies aim to validate cadaveric height with estimated stature as well as increase sample size. Delineating the factors that influence omental biometrics will improve predicted omental size prior to operation.

Gaining Momentum on the Greater Omentum’s Biometric Correlation with Human Size

The neuropeptide hormone, endothelin (specially, ET-1) plays an important role in the invasiveness of breast cancer. High ET-1 levels are associated with decreased disease-specific survival. Very few studies highlight the differential expression of ET-1 and its receptors ETAR and ETBR among triple negative breast cancer (TNBC), non-TNBC, and normal phenotypes. The purpose of the present study was to find out whether the expression of ET-1, ETAR and ETBR is higher in TNBC cells versus non-TNBC and normal cells. An association between higher expression of ET-1 axis and promotion of aggressive TNBC phenotype could represent ET-1 axis as useful biomarkers of TNBC prognosis and potential therapeutic targets. Our hypothesis is that expression levels of ET-1 and its receptors might play an important role in the development and progression of TNBC cells as compared to non-TNBC and normal cells. We aimed to assess the possible prognostic and predictive values of ET-1, ETAR and ETBR by studying their mRNA expression and protein levels in TNBC and non-TNBC via formalin fixed paraffin embedded breast tissue from 4 subjects with TNBC and 4 normal subjects, representing non-TNBC. Western blot, immunofluorescent, and immunohistochemical analysis served to quantify the differential expression. Results from Western Blotting showed ET1 levels were 81.6% higher, ETAR levels were 35% higher, and ETBR levels were 30% higher in TNBC vs non-TNBC cell lines. Immunofluorescence revealed greater intensity of ETBR in all cell lines compared to ETAR, greater expression of ET-1 and ETBR in TNBC cell lines, HCC-1806 and MDA- 231, and greater expression of ETBR in the nucleus of all cell lines and of all proteins in MDA- 231. Immunohistochemistry visually depicted greater staining of ET1, ETAR, and ETBR and quantitatively showed approximately 20% higher ET1, 40% higher ETAR, and 20% higher ETBR positive cells in TNBC vs normal breast tissue. Due to the similar results obtained from Western blot analysis with cell lines and immunohistochemical analysis with breast tissue, our hypothesis that expression of ET-1 and its receptors is higher In TNBC than non- TNBC and control is validated and thus could guide subsequent investigations for targeted pharmaceuticals against these biomarkers in TNBC.

Differential Expression of Endothelin-l (ET1) and its receptors, Endothelin Receptor Type A (ETAR) and Endothelin Receptor Type B (ETBR) between subjects with Normal, Triple

Background: Decidua natural killer (dNK) cells accumulate at the maternal-fetal interface during implantation and placentation. These cells are CD56brightCD16dim/neg, poorly cytotoxic, anti-inflammatory, and proangiogenic; characteristics shared by tumor infiltrated NK cells. We, and others, found that peripheral blood NK cells (pNK; CD56dimCD16bright) can be converted to dNK-like cells in vitro by exposure to hypoxia (1% O2) or TGFβ. However, it is unknown whether mechanisms driving these alterations are analogous or independent. We hypothesized that hypoxia inducible factor (HIF)1α, a known driver of VEGF, also induces TGFβ receptors to suppress NK function.

Methods: Healthy donors (HD) and kidney cancer (KCa) patient pNK were purified by negative selection. Flow cytometry, RT-qPCR, and ELISA assessed expression of NK markers (CD56, CD16), TGFβ receptors (R1, R2), and VEGF. HD pNK cells were cultured in 21% or 1% O2 and in presence or absence of TGFβ (2ng/mL). Functional conversion of HD pNK to dNK-like cells was gauged by VEGF and cytotoxicity. The impact of hypoxia on TGFβ receptors was assessed for pNK exposed to 1% O2 versuses 21% O2, and HIF1α-specific effects determined for cells transduced with lentivirus to overexpress HIF1α. Statistics (ANOVA/t-test) were performed using GraphPad Prism with p0.05 considered significant.

Results: Purity of HD/KCa pNK cells was ≥87%. Compared to HD pNK (n=3), RCC pNK (n=4) had significantly higher expression of TGFβ-R1 and TGFβ-R2, and 40% lower cytotoxic activity. HD pNK cells cultured under 21% O2 were potently cytotoxic, but hypoxia or TGFβ decreased cytotoxicity by 55% and 61%, respectively. Only hypoxia upregulated VEGF mRNA (10-fold) and protein (3-fold). The combination of hypoxia and TGFβ abolished cytotoxicity by ≥88%, but VEGF expression was not increased further. In both cohorts, hypoxia upregulated all TGFβ receptors, but particularly TGFβ-R2. Finally, HIF1α overexpression increased TGFβ-R2 levels (3.5-fold).

Conclusions: Hypoxia and TGFβ independently suppressed pNK cytotoxicity, but induction of VEGF and TGFβ receptors, particularly TGFβ-R2, was unique to hypoxia. In combination, hypoxia and TGFβ potently inhibited cytotoxicity, with no additive effect on VEGF or TGFβ-R2 expression. HIF1α overexpression confirmed these hypoxia-specific effects. These studies support dual roles for hypoxia and TGFβ in dNK-like conversion. While both independently and cooperatively impact cytotoxicity, hypoxia-induced HIF1α specifically drives VEGF and TGFβ-R2 expression changes. These cellular conversions are conceivably beneficial for placentation, but exploited to support tumor growth. Thus, regulation of HIF1α and consequential NK sensitization to TGFβ likely has implications both for healthy pregnancy and cancer therapy.

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Generation of stem-cell derived, vascularized islets for transplantation in Type 1 Diabetes Mellitus therapy.

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