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  Gemcitabine Resistant PDAC Cell- Neutrophil Interaction Regulates their Proliferation and Survival

  Lauren Abrahams, Reegan Sturgeon, Michael Maher, Parker Tinsley, Ester Johnson, and Rakesh K. Singh

  Malignant tumors are inherently pro-inflammatory, and infiltrating leukocytes are thought to be critical for tumor maintenance and progression. Infiltrating cells and molecules driving tumor-associated inflammation have considerable potential as therapeutic targets, yet this area remains relatively under-explored. Neutrophils are the most prevalent leukocyte in the innate immune system and have been shown to play an essential role in cancer progression. Previously our lab has reported that as pancreatic ductal adenocarcinoma (PDAC) disease progresses, there is an increase in the infiltration of neutrophils. The specific objective of this study is to determine the role of neutrophil-PDAC cell interaction in therapy resistance. We analyzed whether direct neutrophil-PDAC cell interaction modulates their survival and whether it is dependent on therapy resistance. We used two human and murine neutrophil cell line models (MPRO and HL60) and two isogenic parent CD18/HPAF (C) and gemcitabine-resistant variant CD18/HPAF-R (CGR). Neutrophil and PDAC cells were co-cultured for 24 h, and their proliferation/survival was determined using WST and MTT assays. We observed that PDAC cancer cell proliferation increased when co-cultured with neutrophils. Moreover, cell free-neutrophil conditioned media modulated PDAC cell proliferation in a concentration-dependent (v/v) manner. We observed significantly higher proliferation in therapy-resistant CGR cell lines. Furthermore, our data suggest neutrophils co-cultured with therapy-resistant CGR cells had higher survival compared to neutrophils co-cultured with parent C cells. Together our data demonstrate that neutrophil-PDAC cell interaction modulates their proliferation and survival.

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  Determining Schistosoma haematobium Population Structures in Ethiopia

  Ava Butz, Sarah Uhm, Tigist Mohammed, Berhanu Erko, Mulugeta Aemero, Abraham Mengist, Joseph R. Fauver, and Kaylee Herzog

  Schistosomiasis, also known as bilharzia, is a Neglected Tropical Disease caused by parasitic helminths that affects over 240 million people around the world. Praziquantel has been used to treat individuals infected with schistosomiasis through Mass Drug Administration (MDA) programs but a recent reduction in its efficacy has been observed, creating concern that the parasite will evolve to become resistant to the chemotherapy drug. Monitoring changes in the population structure of Schistosoma haematobium using microsatellite markers can be a useful metric to determine praziquantel efficacy since variations in the repeat sequences of microsatellites indicate genetic diversity. Since little is known about the population structure of S. haematobium–despite urogenital schistosomiasis being a pressing issue in Ethiopia–18 microsatellite multiplex assays developed in a prior study were tested on stock DNA to validate them for use to study the effects of praziquantel on parasitic S. haematobium in Ethiopia. Through a combination of bioinformatic analysis, PCR, and Next Generation Sequencing on the MinION, 13 of the 18 microsatellite markers were validated, highlighting the importance of developing microsatellite multiplex assays not only based on length distribution, but also based on Next Generation Sequencing data.

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  Identifying Risk Factors for Significant Hyponatremia during Vasopressin Administration in Neonates

  Hailey Cheek, Elizabeth Lyden, and Eric Peeples

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  Stored Blood Transfusion Cause Hyper Inflammatory Response in Monocytes of Neonatal Mouse

  Ashritha Chiguluri, Marie Amalie Balamurugan, Balamurugan Ramatchandirin, and Mohan Krishnan

  Background: Neonatal anemia is nearly universal in preterm infants and is associated with increased morbidity and mortality worldwide. When anemia is severe enough to be treated with RBC transfusion, clinicians must be aware of the risk of critical adverse effects such as necrotizing enterocolitis (NEC), an inflammatory bowel necrosis characterized by monocyte infiltration, and a leading cause of mortality in those born before 28-weeks gestation. We have recently elucidated the connection between anemia and NEC, specifically, the “leaky gut” phenotype that leads to monocytes infiltration and RBC transfusion-associated activation of these monocytes, and the resulting intestinal mucosal injury.

  Hypothesis: Blood transfusion-associated intestinal mucosal injury is unique to neonates

  Objective: To identify the inflammatory response in monocytes of neonate vs adult mouse due to stored RBC-transfusion products by ex-vivo.

  Methods: Monocytes were isolated from liver of C57BL/6 mouse pups (day 10) and adults (6 week) by enzymatic digestion immediately of liver dissection to avoid alterations of cell properties. Positive selection of Ly6C+ monocytes from liver suspension was then carried out using Miltenyi microbeads according to the manufacturer’s protocol. The monocytes are treated with leukoreduced, 7-day refrigerator-stored packed RBCs derived from allogeneic (FVB) adult mice donors for overnight at CO2 incubator. The control cells were treated with media alone. Cells were subjected to qRT-PCR for quantifying inflammatory cytokines (IL-1β, TNF-α, IFN-γ) and M1/M2 polarization marker genes.

  Results: We identified an interesting finding that stored blood products contribute hyper-inflammatory activation in the monocytes derived from mouse neonates than adults which was evident from significant increased mRNA expression of IL-1β, TNF-α, IFN-γ. These findings were of interest because neonatal monocytes are more vulnerable than adults to foreign antigens due to limited antigenic exposure and to the premature status of adaptive immunity in newborns. We have also found that upon exposure to stored blood products, neonatal mouse liver monocytes polarized to M1 phenotype by increased the mRNA expression of CD86, ARG1, IL12A, IL12B, IL23A, CD11B and CD11C. Whereas, M2 polarization phenotype was noted in adults’ liver monocytes during treatment with blood products which is obvious from YM2 mRNA expression. These results raise important concern about a proper understanding of patient’s age-dependent for the re-evaluation of current transfusion guideline.

  Conclusions: Stored blood-derived products induce hyperinflammatory signature in neonatal monocytes than adults and newborn monocytes display M1 (pro-inflammatory) phenotype.

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  Factors related to tracheostomy timing in moderate to severe bronchopulmonary dysplasia

  Carla M. Figueroa-Gomez and Eli M. Rhoads

  Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, occurs in infants born at less than 32w gestational age (GA) who require respiratory support at 36w GA. Infants with moderate BPD are considered G2 (above LFNC oxygen support) and severe is G3 (ventilator support) [3]. A small portion of infants will require tracheostomy placement for long-term respiratory support. The diverse clinical courses and inter-center variations have contributed to the uncertainty of tracheostomy timing [4]. According to previous studies, early tracheostomy placement may be associated with improved developmental outcomes and lower mortality [1,2]. This study follows 19 patients who had their tracheostomy placed during birth hospitalizations and factors related to tracheostomy timing.

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  Effects of Lula Mae Virtual Training Modules on Occupational Therapy Students

  Emma Gardner, Madison McKeever, Leah M. Stade, and Jana L. Wardian PhD

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  A Novel Approach to Sequencing West Nile Virus Genome using IDT xGen and Illumina MiniSeq

  Dikchha Gurung, Joseph R. Fauver, and Kaylee Herzog

  The West Nile virus (WNV) has been identified as a cause of mosquito-borne illness in the continental United States (CDC, 2023). It is a member of the Japanese encephalitis antigenic complex of the Flaviviridae family. WNV is predominantly maintained in an enzootic transmission cycle between Culex species mosquitoes and birds as vertebrate hosts. Humans and horses are considered WNV incidental hosts (a.k.a dead-end hosts) and cannot be transferred to another host. Within the last five years (2018-2022), 8,386 WNV human infection cases have been recorded. In addition, 90 WNV human infection cases have been reported in 17 different states this year. Nebraska has recorded the 4th highest total number of cases since WNV was introduced into the US. Monitoring the genetic variability of WNV will allow researchers to elucidate transmission patterns and ultimately incorporate WNV genomics into estimates of human risk. Understanding virus evolution through time requires an in-depth understanding of genomics. This research project aims to develop a more efficient and effective method for sequencing WNV genomes to better understand evolution. In addition, a novel approach was adopted to sequence WNV from mosquito pools collected in Nebraska. The results of this research suggested that the IDT xGen could potentially be used to sequence WNV from mosquito pools.

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  Tryphostin (AG 879): A Potential Drug to Treat Glioblastoma

  Nathan K. Jobalia, Indumati Ramireddy, Poonam Yadav, Raghupathy Vengoji, Surinder K. Batra, and Nicole Shonka

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  Analyzing Mitochondrial Bioenergetics to Improve Organ Hibernation Methods.

  Ryan Johnson, Marian Urban, Shaheed Merani, and Andrew Trease

  β-hydroxybutyrate is a molecule synthesized in the liver that can be used as an alternative to glucose in cellular metabolism. BHB metabolism does not produce lactic acidosis which makes it an optimal source of energy for tissues in an ischemic state. Melatonin is a hormone produced in the pineal gland that aids in slowing the metabolic rate. Both BHB and Melatonin have antioxidant properties that assist in relieving oxidative stress due to an increased metabolic rate during post-ischemic reperfusion.

  Testing consisted of infusing donor pigs with either BHB/M or a vehicle control. The pigs were then made brain-dead and their hearts were harvested under standard conditions and placed in a UW solution or a UW+BHB/M solution under static cold conditions. Tissue samples were then analyzed immediately after harvest, 2 days, and 8 days post-harvest using a Seahorse XFe96 Flux Analyzer. This test analyzed the oxygen consumption rate in order to show mitochondrial function.

  We hypothesize that the BHB/M solution will aid in mitochondrial function while the tissues are in an ischemic state. We believe this will prolong the viability of harvested organs in order to increase the amount of available organs for transplantation. We also believe that future testing will show that the BHB/M will relieve the effects of oxidative stress during reperfusion of the tissues which will decrease the risk or organ failure post-transplantation.

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  Comparative Immunohistochemical Analysis of EHD1 Expression in Adjacent, Metastatic, and Normal Thyroid Tissue

  Oscar D. Juvera, Bhopal C. Mohapatra, Anupam Kotwal, Haitao Luan, Robert Bennett, Sukanya Chakraborty, Aaqib Bhat, Mariam Zahid, Matthew Storck, Vimla Band, and Hamid Band

  The discovery of prognostic biomarkers plays a crucial role in enhancing the treatment and care of individuals with differentiated thyroid cancer (DTC) who are at risk of disease progression. A significant breakthrough came with earlier research, which revealed higher levels of the EHD1 protein in papillary DTC when compared to the surrounding healthy tissue. This exciting finding served as the driving force behind the initiation of a more extensive investigation aimed at validating EHD1 as a potential biomarker and exploring its connection with clinical outcomes. By unraveling the potential implications of EHD1 in DTC cases, this study holds the promise of advancing our understanding and approach to managing this type of cancer effectively.

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  S100A14 Protein Expression in Prostate Cancer Cell Lines

  Benjamin Kinkor, Fangfang Qiao, Katelyn O'Neill, Jackie Hollinger, Raymond Bergan, and Weining Chen

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  Metastatic PDAC Cell – Neutrophil Interaction Regulates their Proliferation and Survival

  Michael Maher, Reegan Sturgeon, Lauren Abrahams, Parker Tinsley, Esther Johnson, and Rakesh Singh

  Tumor-promoting inflammation is a hallmark of cancer that contributes to tumor cells' survival and proliferation. Infiltrating leukocytes and pro-inflammatory cytokines released into the tumor microenvironment (TME) often cause this inflammation, which is often pro-tumorigenic. Neutrophils are one of the most abundant types of leukocytes found in circulation. Enhanced neutrophil infiltration into the TME with disease progression was previously observed in my lab. Neutrophils are a pro-tumorigenic and pro-metastatic part of the TME. It has been shown in previous research that neutrophils aid in PDAC progression and metastasis. Although, exact mechanisms of this neutrophil-PDAC interaction remain relatively unknown. The specific objective of this project is to determine the role of tumor-associated neutrophils in PDAC progression and metastasis. Our working hypothesis is that neutrophil-PDAC interaction increases PDAC proliferation, survival, and metastasis. The two neutrophil cell lines that were used in this study were mouse neutrophils, MPRO, and human neutrophils, HL-60. The two human pancreatic cancer cell lines that were used are L3.3 and L3.6. The pancreatic cancer cell line L3.3 is a low metastatic cell line while L3.6 is a high metastatic line. Cancer cells were either treated with neutrophil-conditioned media (indirect interaction), or co-cultured with the neutrophil (direct interaction). MTT assays were performed to analyze proliferation of the cancer cell lines, and a WST assay was performed to analyze the survival of neutrophils. We observed concentration-dependent increase in PDAC cell proliferation following treatment with neutrophil-conditioned media. Similarly, co-culture of PADC cell with neutrophils enhanced their proliferation. We did not observe any difference in neutrophil survival when co-cultured with low-metastatic L3.3 cell. However, neutrophil survival was significantly reduced when co-cultured with L3.6 cells (high metastatic). Together, our data suggest that PDAC-neutrophil interaction differentially modulates of neutrophil and PDAC cells survival/proliferation.

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  Preclinical Activity of a Novel Triple-Axis Inhibitor in B-cell Malignancies

  Rolando Martinez-Rico Jr, Audrey Smith, and Dalia El-Gamal

  Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are characterized by CD5+ B-cell accumulation in secondary lymphoid tissues. MCL and CLL B-cells depend upon B-cell receptor (BCR) signaling, tumor microenvironment support, and defective apoptosis mechanisms for expansion and survival. Targeted inhibitors of key proteins involved in proliferation/survival signaling (BTK, PI3K, BCL2) are commonly used in patients. However, multi-therapy resistant MCL and CLL underscores the need for novel therapeutic approaches. Recent studies have demonstrated pre-clinical activity of triple-axis inhibitors capable of concomitantly disrupting PI3K and BTK kinase activity and BRD4-mediated oncogene expression in B-cell malignancies. Here, we demonstrate the enhanced efficacy of a next-generation triple inhibitor (TRPi) in incurable, aggressive (MCL) and indolent (CLL) B-cell malignancies.

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  Effects of HIV Proteins on Macrophage Response to MAI

  Grace E. Michael; Joel Frandsen PhD; Zhihong Yuan PhD; and Ruxana Sadikot MD, MRCP

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  Investigating the Role of TP53 in Peripheral T-Cell Lymphoma-GATA3 Subtype

  Zaina W. Nasser, Dylan T. Jochum, Waseem G. Lone, Alyssa C. Bouska, Sunandini Sharma, and Javeed Iqbal

  Introduction: Non-Hodgkin Lymphoma (NHL) accounts for 4.1% of all cancers in the United States. Peripheral T-Cell Lymphoma (PTCL) consists of ~10-15% of all NHL in the Western world. 30-50% of these PTCLs are not classifiable/diagnosed and are instead designated as PTCL-Not Otherwise Specified (PTCL-NOS). The two major molecular subgroups within PTCL-NOS are PTCL-TBX21 and PTCL-GATA3, determined by their distinct T-helper (TH) transcriptional programs. GATA3 and TBX21 are the master-transcriptional regulators of TH2- and TH1-cell differentiation, respectively. The overall survival analysis of PTCL-NOS cases illustrates the clinical outcome of PTCL-GATA3 cases are significantly lower than PTCL-TBX21 cases over a broad timeframe. Thus, the need for understanding the underlying mechanism and finding therapeutic targets is at the utmost importance.

  Background: TP53 mutations and/or TP53 loss deletions are frequent in PTCL-GATA3 cases, compared to PTCL-TBX21. TP53 is a protein that is essential in cycle regulation but also acts as a tumor suppressor. It stops cells from dividing if they have mutated or damaged DNA. Due to the high mutation rates observed in this subtype, we believe TP53 could play a major role in this mechanism. Therefore, it was important to focus on the TP53-GATA3 interaction at the genomic level. Prior studies using chromatin immunoprecipitation (ChIP)-qPCR on the intron 3 full GATA3 region suggested there was more TP53 binding in this intron region compared to other regions. Therefore, we designed a research strategy to determine the specific binding regions of TP53-GATA3 interaction and the function of the TP53 binding.

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  Preliminary Assessment of HABIT for Children with Unilateral Cerebral Palsy Using Fidelity Measures

  Julia Wegiel, Allie Boothe, James Gehringer, and Andrea Baraldi Cunha

  Purpose/ Hypothesis: The purpose of this study was to behaviorally code participants’ behaviors of a Hand Arm Bimanual Intensive Training (HABIT) camp. It was hypothesized the HABIT program would implement high levels of motor and social behaviors using behavioral coding as a measurement of fidelity.

  Number of Subjects: Five children (Mean age=8.8 years, SD=1.6 years), three females, diagnosed with unilateral cerebral palsy (CP), right-side impairment. Participants were classified as Manual Ability Classification System (MACS) levels I-III.

  Materials and Methods: The HABIT camp took place over a two-week period, ten days of intervention, four hours daily for a total of 40 hours. Oversight of daily intervention was directed by two therapists assisted by seven volunteers trained on HABIT key principles. A fidelity measurement was implemented to establish if participant behaviors were congruent with the intervention principles of HABIT through behavioral coding. Video footage was collected at random intervals throughout the intervention to measure the following behaviors’ duration: right/left contact, right/left object manipulation, tasks [i.e., therapist-provided activities that either do (complex tasks) or do not (simple tasks) cognitively challenge the subject], social engagement with peers, and focused attention (i.e., when subject focuses on an object while object exploration occurs). This preliminary report contains three random videos per participant, averaging approximately 30 minutes per video (total of 6.75 hours). Datavyu software was used to code behaviors [interrater reliability =85.4% 8.6]. The variables were summed as durations and normalized as percentages.

  Results: On average, the percentage of the duration of contacts was relatively equal between left (M= 63.8, SD=11.7) and right (M=46.1, SD=12.5) hands. The percent of the duration of object manipulation varied between the left (M=20.0, SD=10.9) and right (M=5.7, SD=5.8) hands. Children were engaged in simple tasks (e.g., playing with play dough) (M=34.9, SD=12.4) more often than complex tasks (e.g., target game) (M=20.7, SD=12.8), but varied by participant. Children were socially engaged with their peers (M=51.0, SD=12.9), alongside focused on an object while exploring it (M=34.8, SD=13.3).

  Conclusions: Both hands performed a similar duration of contact. Manipulations differed greatly between hands, favoring the unaffected left hand. It may be due to MACS classification systems and the use of their affected hand primarily for support. Simple tasks were performed more often than complex tasks, and social engagement with peers occurred most of the time. Clinical Relevance: This preliminary report of the 2022 HABIT camp suggests the intervention accomplishes its established high-intensity and engagement principles of intervention but may be limited to meeting challenging task goals. This study adds to existing research testing HABIT’s methodological approach to physical therapy intervention and to fidelity use in clinical settings relating to HABIT programming.

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  EHD2 Promotes Store-Operated Calcium Entry (SOCE) and Cellular Migration in Ovarian Cancer Cells

  Mariam Zahid, Haitao Luan, Bhopal C. Mohapatra, Aaqib Bhat, Sukanya Chakraborty, Oscar D. Juvera, Matthew Storck, Vimla Band, and Hamid Band

  Ovarian cancer (OC) ranks as the 5th most common cause of cancer deaths of women, reflecting late diagnoses and lack of targeted therapies. EHD2, a member of the Eps15 homology (EH) domain containing (EHD) proteins family, regulates cell surface expression of Orai1, the mediator of store-operated calcium entry (SOCE) in breast cancer. Disrupting the EHD2-Orai1 axis in OC could provide novel targeted therapies against metastatic disease.