2021 Graduating Senior Scholar Abstracts
Class of Summer 2021
Graduating Scholar Abstracts
Tau Uptake in Microglia via Endocytosis
Paulina J. Esquinca-Moreno, Kristian Odfalk, Sarah Hopp, PhD
Microglia, immune cells of the central nervous system, take up debris in the brain via different forms of endocytosis. Microtubule associated protein tau, hereafter known as tau, misfolds and creates intraneuronal aggregates that can cause damage and even death in neurons, a characterizing trait of tauopathies such as AD. These aggregate are then taken up by microglia to prevent further damage to surrounding neurons. My experiments support the larger study focused on determining the contribution macropinocytosis to tau aggregate uptake in BV2 cells. I conducted a validation experiment to observe the impact of macropinocytosis-targeting drugs on phagocytocisis, an off-target endocytic mechanism. Phagocytosis is a mechanism of endocytosis cells use to engulf solid materials with a diameter >0.5µm. At their optimized concentration, I measured the effect of Cytochalasin D, an actin polymerization inhibitor, Dyngo 4a, a dynamin dependent endocytosis inhibitor, PMA, a protein kinase C activator, Wortmannin, a PI3K inhibitor, and U73122, a PLC inhibitor on 2 um bead phagocytisis. The effects of vehicles DMSO and DMF were also quantified. The BV2 cells seeded at a 5,000 cells/well, serum starved for 3 hours at 37 °C, then were incubated with 50µl of the effector solutions for 30 minutes at 37 °C before adding 50µl of 2µm fluorescent beads diluted 1:2000 and left to incubate for an hour at 37 °C. After the incubation period the cells were washed, fixed, and marked with 10 µl/ml WGA, and washed one final time before imaging. The images were then analyzed by quantifying the number of beads in the cells, the number of cells with beads, and the total number of cells. We observed that most inhibitors of macropinocytosis had some nonspecific effects on phagocytosis. These results allowed for a better understanding of the pharmacology of these compounds in BV2 microglia.
Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer.
Avani Gunuganti, Henriette Balinda, PhD, Andrew J. Brenner, MD, PhD; UT Health San Antonio, San Antonio, TX; UT Health Science Center at San Antonio, San Antonio, TX
Background: The past decade has seen significant advancement in increasing survival estrogen receptor alpha (ERa) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) and cyclin-dependent kinase inhibitors aided to extend overall survival of breast cancer patients. However, resistance to endocrine therapy is a common occurrence and patients will eventually succumb to their disease. Fatty acid synthase (FASN), a key enzyme in lipid biosynthesis, is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease ($16 weeks). Methods: We studied FASN inhibitor, TVB-3166 and its effects on MCF7 and ZR75 cell lines. We generated therapy-resistant cells by long term exposure to tamoxifen (MCF7/ TamR cells), and fulvestrant (MCF7/FR cells). Palbociclib-resistant (MCF7/RB1Crispr and ZR75/ RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor in these cells on proliferation, viability, cell cycle, apoptosis, ERa expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate gene expression. Subcellular localization of ERa was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERa were assessed by immunoprecipitation. ERa and p-eIF2a protein levels were analyzed by western blot after treatment with TVB with or without the addition of palmitate or BAPTA. Results: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERa positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERa levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERa expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress. Conclusions: Our preclinical data provide evidence that FASN inhibition presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.
Mindfulness Practice Among Veterans with Posttraumatic Stress Disorder (PTSD):
A Systematic Literature Review
Annabel Guthrie, M. Danet Lapiz Bluhm, PhD, RN, MSCI, ANEF, FAAN
Introduction: Posttraumatic stress disorder (PTSD) is a significant health issue among veterans and active-duty service members. There are evidence-based treatments for PTSD; each with associated issues related to adherence and efficacy. Mindfulness practice has potential to support PTSD treatment. This review aimed to evaluate most recent evidence on the effects of mindfulness among veterans with PTSD as a potential replacement therapy or adjunct treatment to existing therapy.
Methods: PubMed, CINAHL, and Scopus databases were systematically searched for relevant articles following the Preferred Items for Systematic Review and Meta-analyses (PRISMA) using the keywords: mindfulness, PTSD, and veterans. All study participants must have had a diagnosis of PTSD or subthreshold PTSD.
Results: Of the 366 records identified, 20 met inclusion criteria. All studies examine the effects of used mindfulness-based therapies to address PTSD among veterans including, yoga, “mantram” (sacred word) repetition, compassion meditation, acceptance and commitment therapy, mobile resilience apps, mindfulness-based interventions, sailing, cognitive therapy, and mindfulness exercise programs. Most studies (n=20) reported improvements in PTSD symptom severity with other treatments. Mindfulness interventions showed potential in improving retention rates in every clinical study examined. Limitations of the studies include small sample sizes.
Conclusion: Mindfulness-based interventions were shown to improve veterans’ PTSD symptom severity with higher retention rates than traditional PTSD treatment options. Future studies with larger sample sizes are recommended.
Keywords: Veterans, posttraumatic stress disorder, PTSD, mindfulness
TRIM5α Ubiquitylation by Various E2-Ub Conjugates With and Without Presence of Free Ubiquitin
Lukesh Hornick, Vi Dougherty, Corey H. Yu, PhD, Dmitri N. Ivanov, PhD
Ubiquitylation is the post-translational modification of proteins by the addition of ubiquitin (Ub) or poly-ubiquitin chains and is integral in the regulation of proteins in eukaryotic organisms. The ubiquitylation process is carried out by three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). In the ubiquitylation process, E3 functions by catalyzing ubiquitin transfer from E2~Ub conjugates onto a selected substrate. Tripartite motif (TRIM) proteins are one of the largest subfamilies of RING (Really Interesting New Gene) E3 ligases, which TRIM5α belongs to. Two RINGs form a dimer that activates the E2~Ub conjugate, which then assists the antiviral function of TRIM5α which binds to the capsid of immunodeficiency viruses and blocks viral replication. Previous studies have shown the formation of a trimer increases the rate of both the E2~Ub conjugate and E3 steps in the ubiquitylation process. In our TRIM5α ubiquitylation assays, we used two different RING trimers, R3-WT and Ub-R3-WT (a fusion chemically identical to monoubiquitylated R3-WT). The E2s used in making the different E2-Ub conjugates are UBE2D3, UBE2W, and UBE2N. In our ubiquitylation reactions, UBE2N worked best when formed a heterodimer with UBE2V2. To monitor ubiquitylation reactions, we used Förster resonance energy transfer (FRET). The Ub and E2 that comprise the conjugates are labeled with fluorescent dyes that form a FRET pair. When Ub is transferred from the E2-Ub conjugate onto a RING trimer or free unlabeled Ub, fluorescence decreases. The different E2~Ub conjugates, E3s, and free unlabeled ubiquitin were mixed for 28 combinations to examine differences in TRIM5α ubiquitylation products. Ubiquitylation reactions were first tested using a Biotek plate reader to monitor a decrease in fluorescence due to the transfer of Ub from the E2~Ub conjugate. Various time points were taken during the ubiquitylation assays and then loaded onto SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) gels. Gels were imaged on a Typhoon Trio scanner to examine the fluorescent ubiquitylation products. In ubiquitylation assays containing only UBE2D3~Ub conjugate, we observed a faster decrease in fluorescence when compared to the other E2 conjugates. In assays using Ub-R3-WT as our substrate, we saw higher molecular weight bands which may indicate the attachment of a single Ub to form Ub2-R3-WT or two Ub to form Ub3-R3-WT. We did not observe Ub3-R3-WT in assays containing R3-WT as our substrate. In the combinations with multiple E2~Ub conjugates, we see formation of di-Ub. Overall, the use of any of the E2~Ub conjugates does produce Ub2-R3-WT, with Ub3-R3-WT being more prevalent the longer the reaction goes when Ub-R3-WT is used as substrate.
Examining the Effects of Simple Context on Predictive Reading Processing Among Monolinguals
William Hoswell, Antonio Allevato, Ava Romero, Tara Flaugher, and Nicole Wicha Ph.D.
Neurolinguistic researchers often rely on available linguistic databases to create language stimuli for their research studies. These databases, which might purport to measure features like the frequency of a word or the likelihood of words co-occurring in text, are often created by analyzing written text, such as published newspapers and literature. One challenge with this is knowing if these databases have psychological validity in everyday language use. We conducted a study to measure the psychological validity of the co-occurrence of adjective-noun pairs from the Corpus of Contemporary American English, which uses a corpus-based mutual information (MI) score that depends on word frequency in texts.
In Connecting and Considering: Electrophysiology provides insights into comprehension, Federmeier (2021)1 argues that the brain uses context while listening or reading to make predictions about upcoming stimuli. Using event-related potentials (ERP), which are derived from continuous electroencephalograms (EEG) that are time-locked to events of interest, we examine how simple context and task demands impact predictive mechanisms during language comprehension. The N400 is one of the most well-studied and replicated brain responses elicited by anything meaningful. The N400 response is a negative going deflection in ERPs that peaks around 400 milliseconds and has been shown to be a reflection of access to semantic memory. As context is developed through a sentence, facilitation of more predictable words increases, resulting in decreased amplitudes of the N400.
We examined the N400 response to nouns in adjective-noun pairs of variable cloze probability (high, low, and incongruous). Cloze probability were previously established through norming, which asked participants to state the first noun that comes to mind after reading an adjective. The word pairs either matched or mismatched between cloze and MI scores, with some word pairs having high MI but low cloze scores. The word pairs were presented in either a rapid serial visual presentation (RSVP) or self-paced by participants who pressed a button to advance from one word to the next. EEG was recorded using scalp electrodes, and was time stamped in real time based on the variable cloze condition. The raw EEG was then processed by averaging multiple trials in each condition to extract ERPs to each stimulus type and measure the amplitude and latency of that ERP.
We predict that high-cloze word pairs (word pairings with high predictability) will produce decreased N400 amplitudes compared to low-cloze word pairs. We also predict that low-cloze word pairs (adjectives not strongly predictive of the upcoming nouns) will produce increased N400 amplitudes compared to the high-cloze constraint, and furthermore, that incongruous word pairs (adjective-noun pair creates a semantic violation) will elicit the largest N400 amplitude among the three conditions. Critically, we predict that the N400 response to words that conflict between MI and cloze will show amplitudes that align with cloze, revealing that the brain relies more on psychologically valid statistical information.
1 Federmeier K. D. (2022). Connecting and considering: Electrophysiology provides insights into comprehension. Psychophysiology, 59(1), e13940. https://doi.org/10.1111/psyp.13940
The Implementation of Wrap-Around Services Leads to Improvements of Health and
Wellbeing of Foster Youth and the Community
Payton James, Karen Schwab, PhD, APRN, CPNP-PC , Debbie Jennings DNP,
MSN, RN, CENP
Youth in foster care face insurmountable challenges including poor health outcomes, higher rates of teen pregnancy, lower educational attainment, and high rates of incarceration. Our research consisted of development and implementation of wrap around services for youth touched by the foster care system and their families. The wrap around approach emphasizes a strict based, individualized, and community driven approach.
The pediatric clinic on the campus of UT Health provides care to youth with a lived experience of foster care and their families in urban and rural communities. Their reach on college campuses include Palo Alto College and San Antonio College, along with homeless youth who are part of University of Texas at San Antonio’s Housing First Program. They provide in person and mobile health clinics for these populations. Most recently they have been awarded a $4 million Health Resources & Services Administration grant to provide rural health hubs in the 27 counties around Bexar County. Their recent partnership with Respite Care San Antonio provides care to youth in the custody of CPS and assists with needs for their community daycare.
In addition to providing healthcare services it is important to offer upstream solutions, so we partnered with the daycare at Respite Care San Antonio to provide community classes for parents and guardians. These classes will focus on financial literacy, caregiver fatigue, stress reduction, and community resources. By implementing upstream solutions, through increased awareness and understanding, our goal is to decrease child abuse and break generational cycles.
Housing First sees youth ages 18-24 who have experienced homelessness, which is prominent for those with a history of foster care. When partnering with Housing First San Antonio, we support this population by offering monthly mobile clinics and identifying resources. Many of the youth in this program are parents and it is important to provide them with wrap-around support systems to give them the stable foundation needed for future success.
As these programs evolve and scale, it is imperative to continuously create sustainable and impactful wrap-around services and resources ensuring upstream and downstream impact for foster youth and their community.
Utilizing AI-Powered Neuroscoring for Accurate Disease Onset Detection in ALS Mice
Michael Jeffords, Qing Zhou, Shujie Zhaos, Barath Sivasubramanian MD, Senlin Li, MD
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that typically manifests progressive motor deficit midway through one’s life. This progressive loss of motor neurons causes an inability of the central nervous system (CNS) to control motor function. Current treatments prolong the lifespan of the patient minimally but do not prevent the disease progress. To protect these vital motor neurons to prevent the disease progress, multiple methodologies have been tested. One such avenue is the use of glial-cell-derived-neurotrophic factor (GDNF) as it protects motor neurons from degeneration. Our lab found that in SH-SY5Y cells, a type of brain cell that is sensitive to damage, those treated with human and rat GDNF had an increased cell viability of 92.0% and 59.9%, respectively, compared to a control, emphasizing the beneficial neuroprotective effects of GDNF. However, effective delivery of GDNF to the affected areas of the brain remains a challenge due to the formidable blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) blockage.
To overcome the delivery obstacle, our lab is developing a novel method in which pre-genetically engineered hematopoietic stem cells after transplantation serve as a source to provide macrophages through differentiation capable of crossing the BBB (and BSCB). These macrophages infiltrate into the CNS and become microglia or microglia-like cells that express and release GDNF to surrounding neurons. The highly selective and effective delivery of GDNF in the diseased areas will result in potent neuroprotective effects and lead to the mitigation of neuronal dysfunctions. Our lab utilized this approach in treating mouse models of Parkinson’s, another neurodegenerative disease, and obtained up to 80% infiltrations of donor cells into the CNS. Yet, when employing that same procedure in a mouse model of ALS, our results were inconsistent, which may have been partially confounded by the uniform number of days (98) as disease onset used to initiate the date of treatment. Therefore, the existing method to define disease onset may need to be improved.
To confront this issue, we implemented a combination of a neuro-scoring system and bodyweight measurements, along with Hang-wire and Rotarod tests to find the best method to determine disease onset. Both neurological score and peak body weight exhibited promise for untreated mice, with a correlation coefficient of 0.932. However, we experienced challenges with the treated mice, as irradiation and transplantation created a notable difference in body weight, leading to a correlation coefficient value of 0.695 and 0.714 for male and female mice, respectively. To mitigate this challenge, we developed the groundwork protocol for an AI model that would automate the neuro-scoring process while still increasing accuracy in effectively determining ALS onset in mice by reducing observer subjectivity. Our lab will capture video segments of mice and label them through a pose estimation software known as Social LEAP Estimates Animal Poses (SLEAP). The labeled data would then run through our AI model which analyzes the labeled segment and monitors for biomarkers such as tremors in the hind legs present in the neuroscoring system. By aggregating pose data from SLEAP, our model will assess new mice and determine if they exhibit those characteristics, notifying the user when a mouse displays onset.
Identifying cognitive-behavioral factors contributing to Marijuana use and the development of Marijuana use disorder among adolescents from families with a history of substance use disorder
Maria Jimenez
Background
Marijuana is the most frequently used illicit drug among Texas adolescents: 22% reported marijuana at least once in their lifetimes, and 14% reported Marijuana use in the past 30 days. Through a longitudinal analysis of adolescents in Bexar county, this study identifies both protective (i.e., resilience) and risk factors (e.g., stress, impulsivity, sensation-seeking) influencing marijuana use and the development of marijuana use disorder (MUD) among adolescents.
Significance
Substance use including marijuana use is among the leading causes of morbidity and mortality during adolescence. Earlier onset of substance use is associated with a greater risk of development of dependence and worse outcomes including psychiatric disorders and transition to other substance use. In particular, parental history of substance use disorder increases the susceptibility to the development of SUD among children.
Methods
Data for this study came from a longitudinal cohort of 386 adolescents with initial assessments at 10–12 years old. Follow-ups occurred every 6 months thereafter. At each assessment, adolescents completed self-report questionnaires and structured interviews to assess. Out of 386 cohorts, 217 adolescents reported a parental history of substance use, and those were included in the analysis.
Findings
The number of stressful events at baseline predicted increased the odds of using marijuana use by 31.3% (95% CI [1.15 – 1.51], p < .001) and developing MUD by 20.6% (95% CI [1.05 – 1.39], p < .01) at age 18. Per each unit increase in sensation-seeking scale, the odds of using marijuana use increased by 57.3% (95% CI [1.10 – 2.24], p < .05) and developing MUD increased by 63.0% (95% CI [1.03 – 2.58], p < .05) at age 18. Resilience at baseline, however, reduced the odds to use marijuana by 43.1% (95% CI [0.36 – 1.05], p < .10) and to develop MUD by 63.6% (95% CI [0.17 – 0.70] p < .01). Baseline impulsivity had no impacts on marijuana use and the MUD.
Conclusion
Early-life stress and sensation-seeking trait were risk factors aggravating adolescent’s marijuana use and MUD. However, resilience protected adolescents from using marijuana and developing MUD. Further research needs to explore how to enhance resilience among adolescents.
Improvement of Mental Health Symptoms in Response to Ketamine Therapy in Medicaid-Eligible Patients
Olivia Lopez, Carl J Bonnett, Rakesh Jain, Andrew Stabbert, and Casey Sayre, Jasmine Bonder
Abstract: Since 2020 there has been an expansion in the number of studies highlighting the positive effect that Ketamine for Non-Anesthetic Indications (KNAI) can have on patients suffering with mental health disorders. Medicaid programs cover a large community of patients that face significant health disparities and suffer more frequently from mental health conditions. Many of these conditions are exacerbated due to socioeconomic stressors, lack of access to care, and poor support systems. Our case study focuses on 289 Medicaid-eligible patients and the effect ketamine treatment had on their mental health symptoms. These participants had an established diagnosis of Generalized Anxiety Disorder (GAD), Major Depressive Disorder (MDD) and/or Post Traumatic Stress Disorder (PTSD) prior to beginning treatment. The GAD-7, PHQ-9, and PCL-5 surveys were utilized for assessment of symptomatology in this cohort of patients. These instruments were all deployed prior to beginning treatment and at the end of the six-session Induction Series of ketamine treatments. The comparison of scores on these screening instruments pre-and post-treatment demonstrated a clinically significant reduction in symptomatology in the three diagnosis cohorts of patients listed above. The results indicated in the depression sub-group (PHQ-9), the mean score showed a 51% reduction from an average of 17.3 to 8.4 (F=174.5, P<0.001) . In this group, 137 patients (91%) had an improvement in their scores, 11 patients (7%) had a worsening of their score, and two patients (2%) had no change in score. In the PTSD sub-group (PCL-5), patients showed a decrease in mean score from 48.7 pre-treatment to a score of 23.4 after treatment; this represents a 52% reduction (F=186.4, P<0.001). In this group 137 (92%) patients had an improvement in score, 12 patients (8%) had a worsening in their score. The generalized anxiety disorder subgroup showed a mean score of 14.5 pre-ketamine, which decreased to a mean score of 6.9 after ketamine treatment, representing a 52% decrease (F=262.6, P<0.001). In this group, 191 (91%) had an improvement in their score, 18 patients (9%) had a worsening of their score, and two patients (1%) had no change in score. A follow up study is in progress to analyze long-term benefits of Ketamine therapy in this subgroup. Further studies on cost saving logistics in state-sponsored ketamine therapy and general mental health services are necessary.
Development of New Tests and Technology to Improve the Evaluation of Cognitive Function Among Patients Experiencing Cognitive Decline
Emma Majors, Gabriella Hromas, Summer Rolin, Anthony Rios, Kellen Morgan, Jeremy Davis
Performance validity tests (PVTs) and other neuropsychological tests are necessary in the evaluations of cognitive function in order to accurately make a diagnosis. The accuracy of performance validity tests among bilingual examinees is unknown, and was the focus of the first research study (Project 1). The second research study (Project 2) focused on creating an AI program that could allow for over the phone diagnostic test administration.
Project 1 involved secondary analysis of a deidentified dataset (N = 22,688) from the National Alzheimer’s Coordinating Center (NACC). Exclusion criteria were diagnosis of mild cognitive impairment (MCI; n = 5,160), dementia (n = 5,550), and impairment that was not MCI (n = 1,194). The initial sample included 10,319 participants identified as cognitively normal by a NACC grouping variable. A subset was identified with a primary language other than English who were evaluated in English (n = 225). Propensity score matching was conducted to match bilingual to monolingual, English
speaking, participants on age, education, gender, and Mini-Mental State Examination (MMSE) score. The final sample included 450 participants. Failure rates on five embedded PVTs in the NACC cognitive test battery were examined in language groups: Coding age-corrected scaled score, Digit Span age-corrected scaled score (DS), Logical Memory raw score (LM), Semantic Word Generation raw score (animals); (SWG), and Trail Making Test -B to -A Ratio (TMT B/A). For Project 2, we recorded a series of scripts that had been written based on different patient experiences, and ran them through an AI program to examine if the AI could detect the inconsistencies in speech and dialogue.
Project 1 showed that age (M = 68.2, SD = 10.5), education (M = 16.1, SD = 3.2), gender (68.2% female), and MMSE score (M = 28.8, SD = 1.4) were not significantly different by group. 4.9% of bilingual and 2.2% of monolingual participants failed two or more PVTs (n.s.). Failure rates on the DS, Coding, LM, and TMT B/A were relatively low within both the monolingual (0.4%, 0%, 4%, and 5.3%, respectively) and the bilingual (0.9%, 0.4%, 6.7%, and 3.6%, respectively) groups. Failure rates on SWG were significantly higher in the bilingual group (14.2%) than in the monolingual group (4.9%), Χ 2 (1) = 11.3, p = .001, r = 0.16. Project 2 has yet to be completed, so there are currently no results.
Project 1 demonstrated that overall PVT failure rates were not significantly different between bilingual participants evaluated in English and monolingual participants. However, failure rates on a single verbal-based PVT were significantly higher and above a common false positive threshold of 10% in bilingual participants. Project 2 is still in the research phase, so there are no conclusions.
Role of LUBAC in antibody and autoantibody response
Shahan Mohammad, Maria Fernandez, Zhenming Xu.
Background of Systemic Lupus Erythematosus:
Systemic Lupus Erythematosus (SLE) is an autoimmune disorder caused by the attack of tissue by the immune system, causing tissue damage and inflammation, Inflammatory response is typically caused through B-cell dysfunction, resulting in the abundance of pro-inflammatory cytokines. The hyperactivity of germinal center B-cells prevents the production of autoantibody productions, as well as potential scarring through excessive inflammation.
Proposed effects of LUBAC on inflammatory response
The linear ubiquitin chain assembly complex (LUBAC) is a series of three individual components, HOIP, HOIL-1 and SHARPIN. These components are crucial to catalyzing linear ubiquitination and keeping cells in a healthy state through enabling the NF-κβ transcription factor to promote cell growth. Apoptosis, while crucial in regard to mediation of antibody and autoantibody production as well as response to infection, can lead to premature cell death and inhibit the production of antibodies due to stress, while also increasing the risk of autoantibody development due to the antigen release following cell death. Analyzing the effects of LUBAC in germinal center B-cells, we can aim to maximize the production of antibodies while minimizing cell death and the production of autoantibodies. This will dampen the effects of SLE and eventually provide insight into potential treatment.
Methods:
To analyze the immune and autoimmune response and investigate the role of germinal center B-cells, the abrogation of LUBAC components will provide the necessary insight. Mice models with B-cell Sharpin deficiency were generated through bone marrow extraction of muMT donor mice and littermate donor mice, upon which extracted cells were transferred into C57 mice intravenously. HOIPIN-8 will be used to analyze apoptosis in germinal center B-cells. Mouse B-cells were treated with molecule HOIPIN-8, a 40 uM solution, which was then further diluted to 20 uM for in vitro studies. Mice were injected in two-day intervals, with 40 mg/kg.
Additionally, SHARPIN deficient mice have been proven as an effective mouse model to study LUBAC function and will be used to analyze germinal center B-cells. The analysis of B-cell deficiency will be performed by generating bine marrow chimera mice. The bone marrow chimera mice were generated by mixing muMT bone marrow cells with Sharpinᶜᵖᵈᵐ or Sharpin+/+ bone marrow mice with respective same sex Sharpinᶜᵖᵈᵐ or Sharpin+/+ donor mice at a 4:1 ratio. Bone marrow B-cells were transported intravenously to two month old C57 recipient mice.
It has been accepted that pristane treatment in mice results lupus-like symptoms such as in lupus-associated autoantibodies, organ malfunction and butterfly rash. By using pristane to simulate symptoms in mice models, the effects of the presence of LUBAC within affected mice can be measured through the severity in appearance of symptoms, particularly the inflammation and damage of skin, the presence of autoantibodies, and the potential damage to internal and external tissue.
The mice serum was collected weekly from these mice, and enzyme linked immunosorbent assay (ELISA) was performed to analyze antibody levels, as well as anti-double-stranded deoxyribonucleic acid antibodies (dsDNA). These markers assist in establishing a rough estimate of LUBAC deficiency and antibody and autoantibody production. The mouse B-cells collected from these mice were further analyzed using flow cytometry to determine the percentages of germinal center B-cell undergoing apoptosis in untreated and treated mice. Apoptosis was measured by staining B-cells with 7-Aminoactinomycin D and Annexin V.
Potential significance of LUBAC within autoantibody response:
Utilization of HOIPIN-8 to regulate the NF-kB transcription factor to prevent B-cell hyperactivity is crucial to the development of effective treatment of SLE. No large scale treatment or research has been effectively conducted in regards to the severity of SLE in the last fifty years, and previous studies have been limited in efficacy. Through the usage of LUBAC pathways, morbid symptoms of SLE can be suppressed through prevention of B-Cell signaling, and therefore inhibit the detrimental effects of SLE and autoantibody attacks upon tissue. Future pharmaceutical development in vivo will assist in diminishing the harmful mechanisms of SLE in human models.
Understanding the Role of RPS3 in Breast Cancer Growth and progression
Abigail Montemayor, Manjeet K Rao, Panneerdoss Subbarayalu
Greehey Children’s Cancer Research Institute, Department of Cell Systems & Anatomy,
UT Health San Antonio
Despite improvements in overall survival, many breast cancer patients still struggle to survive the disease. There are about 297,790 new diagnoses of women found in the U.S. Identification of new breast cancer-specific proteins or biomarkers and safe therapeutic targets are urgently needed to improve the overall clinical outcome of breast cancer patients. In this study, we examined the role of Ribosomal Protein S3 (RPS3), a multifunctional protein involved in DNA repair mechanisms and gene expression regulation as a potential player in breast cancer progression and metastasis. To understand the role of RPS3 in breast cancer, we used small interfering RNA (siRNA) to knock down RPS3 expression then conducted a series of in vitro assays, including cell viability (short term), migration, invasion, colony formation (long term), stemness, and apoptosis assays. Downstream mechanisms and targets were validated using gene/protein expression analysis. We discovered RPS3 to be a novel tumor promoting gene, whose expression is significantly altered in breast tumors. Depletion of RPS3 using two different siRNAs in breast cancer cells significantly reduced the short-term viability, migration, invasion, and long-term viability. Concurrently, RPS3 depletion led to increased apoptosis in breast cancer cells. In addition, silencing RPS3 significantly reduced breast cancer stem cell self-renewal, as evidenced by reduced number and size of mammospheres. Our preliminary results with qPCR analysis showed DNA damage/repair related genes were significantly decreased in the RPS3 silenced breast cancer cells when compared to the control. Overall, these findings indicate that RPS3 plays a significant role in breast cancer cell proliferation, stemness, DNA repair, and survival. Targeting RPS3 could be a potential therapeutic strategy for the inhibition of breast cancer.
Acknowledgement: We would like to thank Dr. Manjeet Rao lab members, collaborators, and the Voelcker summer research program and my VBRA colleagues/fellows.
Retention of Imitative Scratch in the Mouse Model of Fragile X Syndrome
Abbey Muenchow, Eric Oh, Nandhini Pakala, Izumi Vazquez, Hye Young Lee, PhD
Imitation is a crucial part of cognitive development that plays a large role in one’s ability to learn. Patients with autism spectrum disorders are commonly seen to have a deficit in imitative behavior, which impacts their ability to learn important skills like speech, social interaction, and joint attention. Though significant, imitative deficits are underexplored in pre-clinical autism mouse models. Fragile X Syndrome is an X-linked genetic disorder that is characterized by intellectual disabilities and is associated with an increased risk for developing autism spectrum disorders. In 2020, our lab demonstrated that Fmr1 KO mice, the mouse model of Fragile X Syndrome, display a deficit in imitative scratch behavior (Gonzales-Rojas et al. 2020, Scientific Reports). In continuation of this, we started the Retention Project to better understand the prolonged impacts of imitative behavior in the mouse model. We sought to learn to what extent mice with Fragile X Syndrome can learn an observed scratching behavior after being shown a scratching demonstrator for one day. In our initial methods, the mice were recorded for only three days, but we soon realized that we needed at least four days and adjusted our recording schedule for the next mice. This meant that on the first day (day -1) mice were shown an empty beaker, then the second (day 0) day they were shown the scratch demonstrator, and finally on the third and fourth day (day 1, 2) they were once again shown an empty beaker. We counted the total number of scratches each day in order to observe if there was a trend. In our initial data, we didn’t find a significant difference in the number of scratches between days, but we did see a significant difference between genotype and strain. Between the two strains of mice we used, the BL6 mice scratched more, making them the better model to use in the future. In our next round of recording, we used solely wildtype BL6 mice and added an extra day where the mice are shown the demonstrator. We hypothesize that by adding this extra day of exposure to the demonstrator video we will see an increase in scratching behavior on days 1-2, or, in other words, an improvement in retention. We hope that collectively our research will bring to light the important role imitation plays in learning and how largely it can affect patients with autism spectrum disorders. We also hope that through our exploration of imitation in a preclinical autism mouse model we will enable the development of therapeutic treatments in pre-clinical models.
Significance of KDM1A in Triple Negative Breast Cancer Brain Metastases
Ifigenia Omiridis, Jessica D. Johnson, Gangadhara R Sareddy PhD
Background: Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer that represents a disproportionate fraction of breast cancer mortality. TNBC accounts for approximately 15% of all breast cancers, is associated with young age at diagnosis (<40 years), aggressive clinical course, high relapse rate, and a worse prognosis. TNBC has the highest propensity of distant metastasis to the brain, lung, liver, and bone and is associated with the shortest overall survival. TNBC patients are often diagnosed with metastatic disease in the brain and the median survival of TNBC brain metastases (TNBC-BM) patients can be as short as 5 months. The standard of care for TNBC-BM often consists of surgical resection, whole-brain radiotherapy, and stereotactic radiosurgery and depends on the size, location, and number of metastatic lesions. Further, there are no FDA-approved treatments specifically for TNBC brain metastasis. Therefore, there is an unmet need for the development of new therapeutic agents that effectively treat TNBC brain metastasis. Epigenetic modifier KDM1A regulates gene expression programs by changing the epigenetic histone marks at the gene prompters. Emerging studies have shown that KDM1A is overexpressed in TNBC. However, little is known about the mechanistic basis of KDM1A effects on TNBC-BM and whether KDM1A inhibitors have clinical utility in treating TNBC-BM.
Methods: The expression of KDM1A in breast cancer subtypes was examined in TCGA patient databases. To study the role of KDM1A in TNBC brain metastases, brain-tropic human MDA-MB-231-BrM2 and mouse 4T1-BR cells were transduced with KDM1A-specific gRNA to generate KDM1A knockout (KO) cells. The effect of KDM1A-KO or KDM1A inhibitor (NCD38) on cell viability and cell survival and was determined using MTT cell viability and colony formation assays respectively.
Results: TCGA analysis of breast cancer data sets showed that KDM1A is highly expressed in TNBC compared to normal and other breast cancer types. Western blot analysis confirmed the KO of KDM1A in TNBC-BM cells. The cell viability assay results showed that knock out of KDM1A or its pharmacological inhibition with NCD38 significantly reduced the cell viability of both human and mouse TNBC-BM cells. Further, knockout or NCD38 treatment significantly reduced the cell survival of TNBC-BM cells when compared to controls.
Conclusion: Our results demonstrated that KDM1A-KO or KDM1A inhibitor treatment is highly efficacious in reducing TNBC-BR growth.
Vitamin B12 and Folic Acid mediate the association between the gut microbiome structure and cognition in middle-aged individuals
Sophia Ramirez, Jazmyn Muhammad, Yannick Wadop, Tiffany Kautz Ph.D., Claudia
Satizabal Ph.D., Jayandra Himali Ph.D, Sudha Seshadri MD, Bernard Fongang Ph.D.
Background: Brain and gut health are intricately connected via the gut-microbiota-brain axis. Studies have shown that gut dysbiosis is associated with neurodegenerative diseases, including Alzheimer’s disease1-2. However, how cognitive changes affect the gut microbiome structure is currently understudied. We aimed to assess the association between the gut microbiome and the global cognitive score (GCS) and evaluate the role of medication intake on microbiome in the Framingham Heart Study (FHS).
Method: Our sample included 1,014 participants of the third generation FHS cohort with available stool samples, cognitive assessments, and no history of dementia or stroke.3 We quantified the gut microbiome composition using 16S rRNA sequencing and performed multivariable association and differential abundance analyses, adjusting for age, sex, education, BMI, and other confounders. The GCS contains neuropsychological assessments of four cognitive domains: Executive function (trails-making B); Processing speed (visual reproduction immediate and delayed); Language (similarity test); and Memory (logical memory immediate and delayed). Participants were additionally stratified by GCS with lower and higher scores indicating poor and normal cognition, respectively. To evaluate the association between medication intake and the gut microbiome in relation to GCS, metanalysis using the average causal mediated effect (ACME) was evaluated for all medications significantly associated with the identified genera.
Result: Our results showed that individuals with poor cognition have a decreased abundance of genera Clostridium (OR = 0.69, 95% CI [0.55, 0.86]) and Ruminococcus (0.933, [0.927, 0.941]). Meanwhile, the genus Alistipes, previously connected to anxiety, chronic fatigue syndrome, depression, and hypertension5-6, was more abundant (1.058, [1.054, 1.063]) in the poor cognition group. Moreover, the genus Pseudobutyrivibrio, a butyrate-producing bacteria from the rumen, was also found to be highly abundant (1.122, [1.109, 1.135]) in the poor cognition group. Furthermore, there was no difference in alpha and beta diversity between cognitive groups. Finally, ACME metanalysis showed that the genera Clostridium (pval < 2e^-16) was associated with Vitamin B12 and Folic Acid.
Conclusion: Our study suggests that the abundance of several genera, including Pseudobutyrivibrio, Alistipes, Ruminococcus, and Clostridium is associated with cognition. Additionally, Vitamin B12 and Folic Acid were shown to be associated with Clostridium and GCS. Clostridium was previously proposed as novel probiotics for human health, and increasing its abundance was viewed as an effective strategy to regulate the homeostasis of the gut microbiota. 4 As all these bacteria have neuroprotective effects, thus manipulating their abundance through diet and pre/pro-biotics could be a research path for preserving global cognition in the future.
1. Bullich, C., Keshavarzian, A., Garssen, J., Kraneveld, A. & Perez-Pardo, P. Gut Vibes in Parkinson’s Disease: The Microbiota-Gut-Brain Axis. Mov Disord Clin Pract 6, 639-651 (2019).
https://doi.org:10.1002/mdc3.12840
2. Long-Smith, C. et al. Microbiota-Gut-Brain Axis: New Therapeutic Opportunities.
https://doi.org/10.1146/annurev-pharmtox-010919-023628(2020). https://doi.org:10.1146/annurevpharmtox-010919-023628
3. Tsao CW, Vasan RS. Cohort Profile: The Framingham Heart Study (FHS): overview of milestones in cardiovascular epidemiology. Int J Epidemiol. 2015 Dec;44(6):1800-13. doi: 10.1093/ije/dyv337. PMID:26705418; PMCID: PMC5156338.
4. Petra AI, Panagiotidou S, Hatziagelaki E, Stewart JM, Conti P, Theoharides TC. Gut-microbiota-brain axisand its effect on neuropsychiatric disorders with suspected immune dysregulation. Clin Ther. (2015) 37:984–95. doi: 10.1016/j.clinthera.2015.04.002
5. Parker, B.J., Wearsch, P.A., Veloo, A.C. and Rodriguez-Palacios, A., 2020. The genus Alistipes: gut bacteria with emerging implications to inflammation, cancer, and mental health. Frontiers in immunology, 11, p.906.
6. Naseribafrouei, A., Hestad, K., Avershina, E., Sekelja, M., Linløkken, A., Wilson, R. and Rudi, K., 2014. Correlation between the human fecal microbiota and depression. Neurogastroenterology & Motility, 26(8),pp.1155-1162.
Investigating the N400 Response and Semantic Memory Elicited by Variable Cloze Probability
Ava Romero, Antonio Allevato, Nicole Wicha, Ph.D.
The N400 is one of the most well-replicated and described event-related potential (ERP) components and has been shown to be elicited in response to anything with meaning. The N400 response is a negative going deflection peaking around 400 milliseconds. The N400 reflects semantic memory and facilitates comprehension through context-based prediction after being exposed to auditory, visual, or other sensory stimuli. The brain utilizes context to make predictions about upcoming stimuli, see Federmeir 2021 for a full review. We are investigating how simple context from adjective-noun word pairs affects this predictive mechanism.
Three categories of manipulated cloze predictability word pairs are presented to participants through a self-paced paradigm. These word pairs consist of an adjective and a proceeding noun that are either high cloze, low cloze, or incongruous. Cloze probability is the predictability of the noun following the adjective (cloze prompt), thus affecting the amplitude of the elicited N400 response. We use an electroencephalogram (EEG) to record electrical brain activity with high temporal resolution. Trials of the same condition are averaged offline to generate ERPs, which identifies common activity following exposure to a specific type of stimuli. Within the word pairs, high cloze endings are those that are expected following the context of the introduced adjective, like “rainy day.” High cloze probability includes more facilitation and access to the semantic memory, therefore allowing for the production of a lower N400 response. Low cloze probability, however, has less facilitation of semantic memory, therefore producing a higher or stronger N400 response. This is primarily since high cloze word pairs are both expected and specific to a semantic memory producing less activity, as opposed to low cloze and incongruous pairs. Low cloze allows room for various endings, where more activity is present, and incongruous pairs produce the largest N400 response as it is a full semantic violation.
Reference Section:
Federmeier K. D. (2022). Connecting and considering: Electrophysiology provides insights into comprehension. Psychophysiology, 59(1), e13940. https://doi.org/10.1111/psyp.13940
Amantadine’s Efficiency to Reduce Restrictive Repetitive Behavior in BTBR Mice
Anja Shakocius, Susan M. Greene, Gloria-Andrea Alcala, Angela Ontanon, Ian Pak, Elle Reede, Alexia Johnson, Isabela Bustamante, Megan Schillerstrom, Brett C. Ginsburg, Georgianna G. Gould
Restrictive-repetitive behaviors are treatment-resistant core symptoms of autism spectrum disorders (ASD). To lessen these symptoms, the drug amantadine, usually used to control certain viral infections and dyskinesia in Parkinson’s disease, was tested to see if it could reduce these stereotypical behaviors. The study hypothesis was that sub-chronic amantadine treatment would be more effective than acute treatment, and higher doses would be more effective than lower doses to reduce marble burying. Marble burying is a 30-minute test wherein mice are placed into cages lined with a 6×3 grid of marbles. The number of marbles buried by the mice is considered indicative of the severity of restrictive-repetitive behavior displayed. The black and tan brachyury mouse strain (BTBR) exhibits parallels of core ASD behaviors, so it was used as an autism mouse model and C57BL/6J strain was used as a control. Both male and female mice were used as subjects and the drug was injected daily at a dose of 50 or 150 mg/kg for 6 days. These doses resulted in steady-state serum levels of (mean ± S.E.M.) 260 ± 58 and 693 ± 40 ng/ml amantadine, respectively. Ten male and female mice per dose of amantadine were tested for marble burying on day 1 and again on day 6. On Day 1, male BTBR buried more marbles than female BTBR mice, but on day 6 this difference was not evident because control females were burying more marbles. Importantly, however, in both males and females, amantadine at a dose of 150 mg/kg/day reduced marble-burying significantly relative to the control-treated groups. Overall, amantadine may be useful to ameliorate restrictive-repetitive behaviors that occur in autism and other psychiatric disorders, a purpose different from its current use to reduce dyskinesia. The knowledge gained from this study could lead to new strategies to address the core symptoms of ASD and improve the lives of individuals living with the condition.
The Effects of APOBEC3A on Tumor Microenvironment, Immunotherapy Efficacy
Anusha Soni1,4, Sofia Henderson4, Tommy Kalantzakos2,4, Harshita Gupta2, Reuben Harris2,3,4
1 Voelcker Biomedical Research Academy
2 South Texas Medical Scientist Training Program
3 Howard Hughes Medical Institute
4 University of Texas Health San Antonio, San Antonio, Texas.
The APOBEC3A (A3A) is an innate immune defense mechanism against viral infections that deaminate DNA cytosine to uracil. These mutations assist in viral death. However, their aberrant activation in host cells serve as leading causes of tumor mutagenesis. Breast cancer is one of the most common forms of cancer and second leading cause of cancer death amongst women. The current therapies have shown limited success, and thus there is an urgent need to find novel therapies to target breast cancer.
Immunotherapy is a revolutionary anti-tumor therapy, which works by reactivating anti-tumor T cells to recognize and kill tumor cells that express neo-antigens (foreign protein fragments), generated due to mutations in the host cells. A3A is highly expressed in many tumor types, including breast cancer, and is a leading cause of mutations. Therefore, we hypothesized that A3A mutations, and resulting tumor neoantigens can modify immunotherapy. We generated a stable breast cancer cell line MM006.2L.2R from our novel MMTV-Cre+p53fl/+ mice. Wildtype (WT) and immunocompromised mice were injected and tumor growth assessed. Immunocompromised mice showed rapid growth, and the wildtype mice had less rapid, yet steady growth curves over three weeks. In cell culture, MM006 was PD-L1+ (Programmed death ligand-1), an immune co-signaling molecule that inhibits T cells by binding T cell PD-1 receptors. Thus, we are currently treating tumors in WT mice with anti-PD-1 immunotherapy. In the future, we plan to characterize MM006.2L.2R, express tunable A3A, and investigate effects of A3A on immunotherapy outcomes. We hope that this research can translate into future drug therapies and treatment options for patients suffering from breast cancer, and other resistant forms of cancer.
Evaluating AMD3100 and Maraviroc in a Preclinical Model for the Treatment of Cocaine Use Disorder
1Thomas, Anona; 2,3Mason, Briana M.; 2,3Shi, Yonggong; 2,3Collins, Gregory T.
1BASIS San Antonio Shavano, San Antonio, Texas; 2Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX, U.S.; 3South Texas Veterans Health Care System, San Antonio, TX, U.S
Stimulant misuse is a persistent and growing issue in the U.S. that contributes to over 20,000 deaths annually, yet there are no FDA-approved pharmacotherapies for stimulant use disorders. Drugs of abuse, including cocaine, can increase dopamine levels and, thusly, increase their reinforcing properties. Similarly, cocaine also seems to enhance cytokines and chemokine (“chemoattractant cytokine” receptors) in plasma and mRNA levels in preclinical rodent models. Antagonism of two chemokine receptors, CXC chemokine Receptor 4 (CXCR4) and CC Chemokine Receptor 5 (CCR5), has been shown to reduce cocaine-induced locomotion in a rodent behavioral assay as well. However, it is unclear whether this reduction in cocaine-induced locomotion could be due to sedating effects. Moreover, the effects of chemokine antagonism on the reinforcing effects of cocaine are unknown. To test the whether antagonism of CXCR4 and CCR5 can influence the reinforcing effects of cocaine without decreasing response rates, adult Sprague Dawley rats (n =6 – 8/group) were trained to respond on two levers under a fixed-ratio 5 schedule of reinforcement for cocaine (0.032-1 mg/kg/inf) or 1 food pellet in a choice procedure. Once stability was achieved, rats were pretreated 15 min before the session with AMD3100 (3.2-17.8 mg/kg, intraperitoneally [IP]), a Food and Drug Administration (FDA) approved CXCR4 antagonist, or Maraviroc (3.2-17.8 mg/kg, IP), an FDA-approved CCR5 antagonist, to determine their effectiveness at reducing cocaine self-administration. Concurrently, a locomotor activity (LMA) study was conducted with a separate group of adult male Sprague Dawley rats (n=8) to further characterize the effects of AMD3100 and Maraviroc on cocaine sensitization and motor effects. After habituation in LMA assessment chambers, rats were pretreated with AMD3100 (10 mg/kg, IP) or Maraviroc (10 mg/kg, IP) prior to injections of cumulative doses of cocaine (saline, then 0.032 – 17.8 mg/kg, IP) and tested once a week to determine the effects of chemokine antagonism on cocaine-induced LMA. During the last week of testing (week 11), all rats were tested only under vehicle conditions. In the choice assay, antagonism of CXCR4 with 17.8 mg/kg AMD3100 diminished cocaine choice. Maraviroc was ineffective in reducing responding for cocaine. Antagonism of CCR5 via Maraviroc modestly decreased cocaine-induced locomotor activity, but did not have the same duration of effect as AMD3100 as testing continued over 10 weeks. Antagonizing CXCR4 with AMD3100 significantly attenuated cocaine-induced locomotor activity throughout testing. These findings illustrate the potential for chemokine receptor antagonists to be useful in the treatment of cocaine misuse or substance use disorder. Additionally, because both pretreatments used are FDA-approved, they are presumably safe for clinical treatment in the context of HIV. These data suggest their potential multipurpose use as candidate medications for cocaine-use disorder.
Expression of Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6) in Type II Epithelial Cells
Iman Zakaria, Elizabeth Buell, Richa S. Varughese, Ann Friesenhahn, Dr. Shamim B. Mustafa
Department of Pediatrics, The University of Texas Health Science Center at San Antonio
Bronchopulmonary dysplasia (BPD) is a respiratory disorder that causes significant morbidity and mortality in premature babies. BPD is a multifactorial chronic lung disease that affects neonatal preterm infant alveolar tissue and disrupts growth and angiogenesis. The stress caused by infection and oxygen toxicity due to prolonged supplemental oxygen therapy on preterm lungs of infants is proposed to be the main trigger for the onset of BPD.
Tumor necrosis factor-stimulated gene-6 (TSG-6) is a reparative, anti-inflammatory mediator released by several cell types. TSG-6 exerts its beneficial and protective effects by interacting with the cell surface CD44 receptors on a variety of different cells found in the lung. Treatment of preclinical rodent models with BPD with TSG-6 have shown improvements in lung structure, indicating the reparative properties of TSG-6. Our lab has shown that TSG-6 is expressed in the lung tissue of newborn experimental animals.
An important function of alveolar type II epithelial cells (AECII) is the production of pulmonary surfactant, which prevents alveolar collapse and plays a role in pulmonary defense mechanisms. It has also been suggested that AECII also secrete factors that promote repair of the developing lung following injury. Our lab has shown that A549 cells, a commercially available human alveolar type II epithelial cell line, constitutively express and secrete TSG-6 when cultured under normal conditions.
We then investigated whether the expression of TSG-6 is altered following injury. This was achieved by exposing the cultured A549 cells to hyperoxia (40%) and lipopolysaccharide endotoxin (LPS, 1mg/ml) for 24h. Brightfield imaging showed changes in cell morphology and decreased cell counts following treatment with LPS and hyperoxia. We found fewer cells were present following injury using immunofluorescence staining for Ki67, a marker of cellular proliferation. However, cells cultured in normal and injurious conditions appeared to express the same of TSG-6 protein. Using a western blot analysis and ImageJ analysis, we confirmed quantitatively that the TSG-6 expression was consistent under normoxic conditions and treatment with LPS, hyperoxia, and both LPS and hyperoxia. In addition, the secreted TSG-6 was elevated from A549 cells treated with LPS and hyperoxia.
After conducting the experiments, we found that cellular TSG-6 expression remains present when cells are exposed to normoxia and treated with LPS and hyperoxia. We hypothesize that TSG-6 secreted into the airspaces by AECII during injurious events is upregulated and acts in a manner to foster the repair of neighboring cells and contribute to mitigating lung tissue damage.