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**Abstract:**



AMA Research Challenge 2023

The role of the triglyceride-glucose index (TyG) in obesity and its progression to Type 2 diabetes

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Various growth factors have been shown to affect the proliferation, migration, and adhesion of keratinocytes in the face of wound healing. The expression of wound repair genes is suspected to be regulated by circadian rhythm. How wound repair functions over the course of a circadian rhythm has yet to be fully described. Moreover, much is unknown about the impact of bacteria on the circadian expression of wound healing genes. Here, we intend to use periodontal disease as a model to describe the effect of a septic environment on expression of wound healing genes by epithelial cells. Furthermore, we aim to demonstrate that stimulation of epithelial cells with pathogenic bacteria alters the circadian patterns of wound repair gene expression. In the experiment, OKF4 cells were seeded, and artificial wounds were created in each well. The cells were challenged with pathogenic oral bacteria, and cell lysates were collected at T=0, T=8, and T=22 hours. PCR quantified gene expression levels. Microscopically, increased cell spreading was observed after T=22 hours in the wounded cells. Impaired cell spreading was observed in the wells challenged with pathogenic bacteria. The expression of EGF and FGF-7 appeared to follow a circadian rhythm. The presence of pathogenic bacteria downregulated the expression of genes involved in keratinocyte migration. These findings support the hypothesis that genes involved in wound healing follow a circadian rhythm and are altered in the face of infection. Continued research into this topic can lead to the development of therapies targeted towards restoring the expression of genes that aid in wound repair complicated by a septic environment or overlying infection. Further elucidating the circadian pattern of expression of these genes can also result in optimization of the timing that therapies are administered to maximize their therapeutic effect thereby improving patient outcomes. 


Altered Wound Healing Response after Challenge with Pathogenic Bacterium

Background: Neuroinflammation has been implicated in the development and progression of numerous neurodegenerative disorders and studies suggest systemic inflammation may precede central inflammation. Chronic low-grade systemic inflammation is a hallmark of metabolic disorders like Type II diabetes mellitus (T2DM), which is a risk factor for cognitive dysfunction. In fact, researchers have proposed the term “Type III diabetes” for Alzheimer’s disease due to the shared molecular and cellular mechanisms among diabetes and cognitive decline. For both of these pathologies, diet has been recognized as a modifiable risk factor, yet the specific impact of diet on neuroinflammation remains poorly understood. Current literature suggests that a high intake of saturated fats and processed foods may promote neuroinflammation and worsen neurodegeneration, while a ketogenic diet has been linked to cognitive improvement in dementia via regulation of neuroinflammatory pathways. This ongoing work seeks to uncover how diet composition influences neuroinflammation, specifically how diet modulates the activity of the NLRP3-inflammasome.

Methods: To address this question, we utilized a novel biosensor mouse developed in our laboratory that allows us to measure caspase-1/NLRP3-inflammasome activity in vivo. Due to the strong association of dementia and T2DM, and the established abnormalities in blood glucose levels in T2DM, this study focuses on high glycemic index (HGD) and ketogenic (KD) diets. These diets will allow us to determine whether rapid spikes in blood sugar seen with the HGD increase NLRP3-inflammasome neuroinflammation, potentially contributing to neurodegeneration, and if the extremely low carbohydrate load in the KD has the opposite effect.

Results: Long term administration of a KD promotes glucose intolerance but does not increase neuroinflammation, which a HGD increases NLRP3-inflammasome-dependent neuroinflammation without altering glucose regulation in wild-type male mice.

Conclusion: We found that the ketogenic diet impairs glucose metabolism, but does not lead to increased neuroinflammation, while the HGD showed the opposite phenotype. Future experiments will assess inflammatory cytokines systemically and centrally, determine levels of pro-inflammatory transcripts and cleaved caspase-1 protein levels in the brain, and evaluate behavior deficits to probe at the mechanism behind these unexpected results.


The Impact of Diet on Neuroinflammation

Background
On March 11, 2020, the WHO declared the COVID-19 outbreak a pandemic. Multiple aspects of healthcare were interrupted, as individuals were instructed to stay home, protect other citizens, and avoid potential exposure. Individuals with HIV heavily rely on healthcare providers for essential medical treatment and critical testing. They are particularly susceptible to the upheaval in healthcare services and access. We undertook this study to evaluate the impact of the COVID-19 pandemic on diverse aspects of HIV patient care. 
Methods
Using the Cascades analysis template provided by the New York State Department of Health populated with our patient data, we compared the effects of the COVID-19 pandemic on established clinic patients on Antiretroviral Therapy (ARV) and whether they received viral load testing (VL) or kept viral load suppression (VS). Established clinic patients had two or more visits to our Infectious Disease clinic for HIV management before COVID-19. We also highlighted differences between viral load testing and suppression status within groups when comparing 2019 and 2021 against 2020. Data were stratified by demographics, including; age 20 to 60 years or older, race (Black, White, Asian), and Ethnicity (Hispanic or Non-Hispanic). The relationship between these outcomes was analyzed via the Chi-square test of independence and logistic regression. A p-value < 0.05 indicated statistical significance. 1,498 patients were seen in our Infectious disease clinic and on antiretroviral therapy during 2019-2021.
Results
A significantly higher percentage of patients did not receive testing in 2020 compared to 2019 & 2021 (OR 0.427, CI [0.237- 0.75], p-value = 0.0035, Chi-square p-value = 0.004). During 2019 & 2021, we observed a higher percentage of detectable patients; however, the year was unlikely to be related to decreased suppression rates (p-value= 0.79). Age is a significant predictor of the outcome for viral testing and suppression (F-value < 0.049, F-value < 0.0001) with an average of 55-56 years. Interestingly, we observed no significant difference in the odds or probability between patient race or ethnicity in obtaining VL testing or achieving suppression.
Conclusion
The observation during our analysis that stood out most prominently was the impact of the COVID-19 pandemic in 2020, likely influenced by nationwide medical facility closures and patient reluctance to leave their homes. Telemedicine appointments were a significant response to COVID-19 and were a tool to combat interruption of care. There is further emphasis on the advantages of home delivery of ARV provided by specialty pharmacies, especially under extreme circumstances. Home medication deliveries, telemedicine visits, and considering age-related factors in HIV-care planning during the earliest part of the pandemic may have prevented deterioration in monitoring these at-risk populations. These findings underscore the imperative of adaptive healthcare delivery methods to navigate the consequences of unforeseen medical emergencies effectively.

Disruptions in the HIV Continuum of Care During COVID-19: Lessons from a Clinic-Based Study

Objective: Since the COVID-19 pandemic began, the number of individuals suffering from mental health disorders has increased substantially without an equal increase in the number of mental health workforce members. This has created difficulty in creating safe discharge plans for hospitalized patients, discouraged patients from seeking necessary help when suffering and can deter patients from completing treatment due to scheduling and financial barriers. The goal of this project is to evaluate availability and cost of outpatient psychiatric and psychological services in the Phoenix, Arizona area. 

Methods: Psychiatry and psychology practices were identified via an online search in the Phoenix, AZ area and contacted by phone or email to enquire about the services they provide. Ten psychiatric and ten psychological practices were contacted. Information was obtained including type of services (psychiatry, psychology, neuropsychiatric testing), type of provider (MD/DO, NP, PA, PhD/PsyD, master’s level counselor, social worker), soonest available appointment and cost of appointment (cash pay vs accepted insurance). Wait times to reach a representative were also recorded. 

Results: For psychiatric services, the average wait time to speak to a representative over the phone was 5 minutes. The average soonest available appointment was 12 days ranging from same day to 5 weeks from date of call. The most common provider available for the soonest available appointment was a physician psychiatrist or NP. Neuropsychiatric testing was offered at 3/10 practices. Insurance was accepted at 8/10 contacted locations, with cash pay costs ranging from $225-450 for intake and $100-300 for each follow up appointment. For psychological services, the average wait time to speak to a representative was 48.5 minutes. The average soonest available appointment was 18 days from date of call ranging from same day to 2 months. The most common provider available for the soonest available appointment was a Psychologist (PsyD/PhD) or master’s level counselor. Neuropsychiatric testing was offered at 4/10 practices. Four practices accepted insurance, all practices accepted cash pay with costs ranging from $75-350 for an intake appointment and $75-265 for each follow up appointment.


Conclusion: In the Phoenix area, access to psychological services including talk therapy and neuropsychiatric testing is more difficult to obtain than psychiatric services. Barriers to these services include longer wait times to speak to representatives for scheduling and information, which can discourage patients from making appointments, longer wait times to the soonest available appointment which can be detrimental to those who are struggling and need help quickly and lack of services covered by insurance. A comprehensive mental health treatment plan often involves both therapy and medication management. This information suggests a focus of increasing both the volume of psychological and psychiatric providers and providers willing to accept insurance to meet the collaborative needs of appropriate mental health care in this location.

Accessibility of Outpatient Mental Health Resources in the GreaterPhoenix, Arizona Area

Background: 
Metabolic syndrome (MetS) is characterized by interrelated metabolic risk factors that increase the risk of cardiovascular disease. With a global prevalence of about 25% in the adult population, prevalence differs across different racial/ethnic populations and is higher in non-Hispanic White (NHW) than non-Hispanic Black (NHB) Americans. Epidemiologic studies have indicated that poor diet quality is associated with fluctuations in blood glucose and dyslipidemia, two essential components of MetS. The study examined the associations between metabolic syndrome (MetS), self-rated dietary quality (SRDQ), and self-rated health (SRH) in non-Hispanic White (NHW) and non-Hispanic Black (NHB) American adults. 


Methods: 
Data (n=3008) from the 2017-2018 US National Health and Nutritional Health Survey were used for this study. Race/ethnic-specific prevalence odds ratio (POR) from multivariable logistic regression analysis was used to evaluate the cross-sectional associations between MetS, SRPDQ, and SRPH. Statistical adjustments were made for age, sex, education, annual household income, alcohol use, smoking, and lack of exercise. 


Results: 
Approximately 39% and 31.2% of NHW study participants with MetS rated their dietary quality and health status as poor compared to 44.7% and 40.8% of NHB with MetS, respectively. SRH was positively correlated with SRDQ in NHW (r=0.281, p <.001) and NHB (r=0.495, p<.001) study participants with MetS. SRPDQ was associated with increased odds of MetS [POR=1.78;95% CI:1.16-2.66] and SRPH [POR=3.73;95% CI: 2.87-4.84] in NHW individuals, and with MetS [POR=1.45;95% CI:1.07-2.83] and SRPH [POR=4.35;95% CI:3.17-5.98] in NHB individuals, controlling for covariates. MetS was associated with increased odds of SRPH in NHW [POR=2.28;95% CI:1.04-3.68] and NHB [POR=2.56;95% CI:1.28-5.12] individuals. SRPDQ was found to mediate the association between MetS and SRPH and increased the association by 25.9% and 46.1% in NHW and NHB individuals, respectively. 


Conclusions: 
This study shows a positive association between SRPDQ and MetS, and that MetS is associated with SRPH in NHW and NHB persons. Longitudinal studies are needed to investigate whether MetS accelerates declines in overall health in NHW and NHB adults and inform clinical and public health interventions to promote optimal dietary quality. Race/ethnic-specific dietary intervention may help prevent and control MetS and its subsequent sequelae, including poor health outcomes.

Self-rated Dietary Quality, Metabolic Syndrome, and Self-rated Health in non-Hispanic ​Black and non-Hispanic White Americans.

Purpose: To explore the effects of recalibration on a cardiac surgery risk of mortality (STS-ROM) model in a majority-minority population (25% white). 

Methods: 3,721 patients from 2009-20 were identified in a cardiac surgery registry at a tertiary care center in Hawai'i. Recalibration was performed by training a new model built on only this local data, with in-hospital mortality as a dependent variable, and the following three independent variable strategies: (1) only STS-ROM score (add a flat value), (2) the same independent variables as the original STS-ROM (same model, new coefficients), and (3) entirely new variable selection (local-only model). In keeping with STS methodology, logistic regression models were created with a 70:30 test:train split and optimism-adjusted LASSO variable selection when warranted. Models were evaluated via observed/expected (O/E) ratios and area under the curve (AUC). Clinical impact was evaluated in the test set by comparing classifications of the new models compared to the original STS-ROM risk classification (Low/Medium/High). During this timeframe, STS-ROM was a primary determinant of receiving surgical aortic valve replacement vs transthoracic aortic valve replacement. Thus we also calculated the percentage of aortic valve patients who would have qualified for a different procedure had we used the local model. 

Results: The STS-ROM model underestimated risk overall (O/E: 1.14). Adding a flat value (model 1) performed marginally better (O/E: 1.07) but building a new model (2) using local data and STS-ROM variables performed the best (O/E: 0.99). Model 3, which used only local data, significantly overestimated risk (O/E: 0.82). All models aside from Model 3 had similar AUC (0.82-0.83). If the best model (2) was used instead of the STS-ROM, 3.72% of all patients and 4.1% of non-white patients would have moved up in risk category. Of those already at intermediate risk, 15.6% would have been elevated to high risk. In the aortic valvular population, where STS-ROM was an especially important variable in clinical decision making, 16.7% of surgical replacements may have qualified for less-invasive procedures instead. 

Conclusion: Recalibrating national risk models using local data from our majority-minority population was superior in all models, but entirely new models without national input studies performed worse. Given that these risk models are major factors in surgical decision making, recalibration can have a significant impact on patient care. 


Surgical Risk Models in a Majority-Minority Population: The Clinical Effect of Local Recalibration

Background: The long-term sequelae of undiagnosed and untreated obstructive sleep apnea (OSA) are well-established. Current research supports the existence of racial disparities in prevalence, risk factors, and treatment utilization in OSA. However, most studies focus on adult patients or sample from limited cohorts. The aim of this study is to investigate possible racial disparities in the diagnosis and treatment of OSA in patients aged 0-20 years.
Methods: The TriNetX national database was utilized to identify patients under the age of 22 who were diagnosed with OSA in this retrospective cohort study. The incidence of surgical treatment (tonsillectomy, adenoidectomy, or adenotonsillectomy) and Positive Airway Pressure (PAP) treatment, as well as polysomnography (PSG) rates were examined across multiple races with sub-analyses performed on demographics including age, ethnicity, sex, BMI class, and comorbidities.
Results: We identified 143,111 patients with a diagnosis of OSA using ICD-10 codes across 71 healthcare organizations. 96,181 patients were Caucasian, 41,463 were African American (AA), 4,002 were Asian, 1,127 were American Indians or Alaska Natives (AI/AN), and 338 were Pacific Islanders/Hawaiian Natives (PI/HN). Rates of surgical treatment differed by race (p<0.001), with AA (37.4%), Caucasian (39.5%), AI/AN (37.5%), and PI/HN (34.9%) compared to Asian patients (27.2%). AA (26.3%) and Caucasian (26.4%) children were more likely to obtain a PSG compared to AI/AN (23.8%), Asian (20.2%), and PI/HN (16.9%), p<0.001. In addition, more Caucasian patients (36%) received surgical treatment before 12 years of age compared to AA (34.1%), Asian (24.6%), AI/AN (32.7%), and PI/HN (31.4%) groups (p<0.001) despite AA patients receiving PSG in young children. The adjusted odds of CPAP use were 1.6 times greater for AI/AN vs. Caucasian (95%CI 1.06-2.4) and 1.2 times greater for AA vs. Caucasian (95%CI 1.1-1.3). Significant differences remained after controlling for demographics, BMI, and comorbidities.
Conclusion: There are differential rates of PSG and OSA treatment utilization when analyzed by race in this national sample of pediatric OSA patients. Given the importance of diagnosing and treating OSA early to prevent undesirable outcomes, more studies should be done to confirm these differences and investigate the factors contributing to them.

National Trends in Pediatric Obstructive Sleep Apnea Diagnosis & Treatment Utilization Among Various Racial Groups

Examining the influence of Demographic Factors and Socioeconomic Variables on Bariatric Surgery outcomes at an Urban Academic Center.

Human Immunodeficiency Virus 1 (HIV-1) is a retrovirus that primarily infects human CD4+ T cells, a key cell-type in the adaptive immune system. The virus establishes a latent reservoir in the early phases of infection that persists in people living with HIV-1 (PLWH) despite effective antiretroviral treatment (ART). Designing strategies to target the HIV-1 reservoir is critical to achieve progress towards a cure. In eukaryotic and prokaryotic cells, and in viruses, antisense transcripts have been shown to exist as long, non-coding RNAs (lncRNA) or coding RNAs (mRNA). Some viruses, like Human T-cell Leukemia Virus Type 1, encode antisense genes to regulate viral expression and replication. HIV-1 antisense transcripts (AST) have been detected in in vitro and ex vivo studies and have been shown to induce viral latency through methylation of histone 3 lysine 27 by recruiting the polycomb repressive complex 2 (PRC2). Our previous studies demonstrated the presence of HIV-1 AST in vivo at levels of 1-3 copies in a median of 5% infected peripheral blood mononuclear cells (PBMC), suggesting a potential role in inducing viral latency in vivo. To determine if HIV-1 AST expression affects levels of plasma viremia, we designed an assay to measure AST expression in PLWH with high viral loads (non-controllers, viral load 5,000-200,000 copies/mL), with low viral loads (viremic controllers, 50-2,000 copies/mL), and with undetectable viral loads (elite controllers, <40 copies/mL). The assay is a limited-dilution, digital PCR approach that targets HIV-1 AST in donor blood cells using donor-specific primers. First, nucleic acid is extracted and subjected to thorough DNase treatment. The remaining antisense RNA is reverse transcribed using a primer targeting the AST region. Detection is achieved with a fluorescent probe and reported as the number of HIV-1 AST molecules per 100 infected cells. We measured AST levels using ACH-2 cells (a model of HIV-1 latency) as a positive control with an average of 3.18 copies of AST per 100 infected cells, as expected. Negative controls included no reverse transcriptase and no template to ensure complete DNA digestion and no mis-priming nor contamination. Our preliminary data includes 4 donor samples in which HIV-1 AST was detected at 1-53 per 100 infected cells. Future studies will determine if AST functions to control HIV-1 replication and induce viral latency in vivo.

Investigation of HIV-1 Antisense Expression In Vivo

Mucosal immunity to SARS-CoV-2 is crucial in attenuating viral replication. While intramuscular mRNA vaccination has been shown to produce durable SARS-CoV-2 specific antibody responses in the systemic circulation, there are few studies pertaining to local responses in the nasal mucosa. The extent to which vaccination may recall nasal antibody responses primed by prior infection or if nasal antibody responses to infection are primed by prior vaccination remains unelucidated. In the present study, we compared SARS-CoV-2 specific nasal antibody responses among unvaccinated infected, vaccinated infected, infection-experienced vaccinated, and infection-naïve vaccinated participants.

Between June 2020 to February 2023, nasopharyngeal swab (NPS) samples were collected from participants as part of an ongoing longitudinal surveillance study. Samples collected within 14-89 days after either confirmation of an acute SARS-CoV-2 infection via RT-PCR or vaccination with a third mRNA dose were selected for the present study. Secretory-IgA (SIgA) and IgG responses to the Spike antigen of ancestral (B.1 lineage) SARS-CoV-2 were measured via enzyme-linked immunosorbent assay. SIgA was normalized to total-IgA concentration. Nonparametric continuous variables were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Paired groups were compared using Wilcoxon signed-ranked test. This study was approved by the Children’s Hospital Los Angeles Institutional Review Board.

Of 429 NPS samples, 176 (41.0%) were from male participants, and the mean age at collection was 27.7 years (range, 0.1-81.3 years). Median SIgA concentrations (x10 3 AU/mL) were 39.2 (unvaccinated infected), 230.2 (vaccinated infected), 22.1 (infection-experienced vaccinated), and 5.3 (infection-naïve vaccinated). Median IgG concentrations (x10 3 AU/mL) were 2.2 (unvaccinated infected), 16.0 (vaccinated infected), 5.4 (infection-experienced vaccinated), and 4.4 (infection-naïve vaccinated).

SIgA and IgG were significantly higher in vaccinated infected participants compared to unvaccinated infected (p<0.001), infection-experienced vaccinated (p<0.001), and infection-naïve vaccinated (p<0.001) participants.
SIgA was significantly lower in infection-experienced vaccinated participants compared to unvaccinated infected (p=0.045) participants whereas the opposite trend was observed for IgG (p<0.001).

Acute COVID-19 infection robustly induces SARS-CoV-2 specific nasal SIgA and IgG
responses in vaccinated participants to a greater extent than infection alone. Infection-experienced vaccinated individuals elicit a weaker SIgA response compared to infection alone, indicating that vaccination cannot completely recall nasal SIgA responses. Acquiring a better understanding of SARS-CoV-2 specific nasal antibody responses is an important step towards developing vaccines that can elicit strong mucosal immunity.

Next from AMA Research Challenge 2023

Altered Wound Healing Response after Challenge with Pathogenic Bacterium