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

Background: SARS-CoV-2 or (CoV2) utilizes the receptor binding domain (RBD) of its surface SPIKE protein to infect human cells that express ACE2 receptor. Infected persons can be asymptomatic or progress to a potentially fatal pneumonia-associated illness. Antibodies that prevent SPIKE/RBD/ACE2 interaction can result from infection or vaccination. However, their quantity, function, and cross-reactive protection requires serological testing. We obtained 50 deidentified serum samples, and were blinded to CoV2 vaccination (Vax+; Vax-) or CoV2 positive test (CoV2+; CoV2-). Serum IgG antibodies to SPIKE and RBD were quantified and efficacy in preventing CoV2 infection in vitro determined. We hypothesized that high titer RBD IgG would be efficient at preventing viral infection and be effective against a later CoV2 variant.

Methods: ELISA was used to quantify SPIKE and RBD antibodies in sera. Neutralizing capability of antibodies was determined by mixing sera with a firefly luciferase lentivirus psuedotyped with Wuhan CoV2 SPIKE. The mixture was applied to ACE2 expressing cells and luciferase activity used to calculate infection rates. Samples were unblinded to determine correlation between in vitro results and CoV2 exposure or vaccination. Cross-protection was determined by repeating neutralization assays with the Omicron BA.2 variant.

Results Thirteen participants reported no CoV2 vaccine or CoV2 positive test (Vax-/CoV2-) yet 7 had anti-SPIKE and -RBD titers and neutralizing function suggesting prior asymptomatic CoV2 infection. The remaining 37 participants reported Vax-/CoV2+ (n=8), Vax+/CoV2- (n=27), or Vax+/CoV2+ (n=2). Titers to RBD were comparatively lower than titers to SPIKE in most patient samples. Average RBD antibody titers were higher for the Vax+/CoV2- group (1:2600) versus Vax-/CoV2+ cohort (1:900). Highest RBD antibody titers were found in Vax+/CoV2+ patients (1:5400). Finally, sera with RBD titer ≥1:2700 and neutralization capacity had equal neutralizing function on Omicron BA.2, a variant with 28 amino acid changes in SPIKE and 16 in the critical RBD region.

Conclusion High-titer RBD sera correlated with greater viral neutralization activity highlighting the functional importance of this domain in infection. High-titer anti-RBD IgG to the initial Wuhan CoV2 virus also provided potent neutralization activity against the BA.2 variant despite 16 amino acid changes in its RBD region. This information could be used in future vaccination efforts and passive immunization, as well as provide a greater level of confidence to healthcare workers, first responders, and other high-risk populations regardless of the CoV2 strain. Serial immune studies in high risk populations could provide indications for booster vaccines.

Functional Serologic Testing and Cross Reactivity of Serum Antibodies to SARS-CoV-2 SPIKE Protein

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

Background: Recurrent urinary tract infections (rUTI) are common in postmenopausal women with type 2 diabetes mellitus (T2DM). However, little is known about the composition of the urinary microbiome in this population and how it contributes to rUTI susceptibility. We aimed to identify key differences in urinary microbiome composition and function between women with and without T2DM through shotgun metagenomic sequencing of their urine.

Methods: Following IRB approval, urine samples were collected from consenting postmenopausal women with T2DM across 3 groups: rUTI remission (rUTI history, no active infection), rUTI relapse (active infection), and no history of UTI. Groups were age and estrogen-matched and DNA was purified from 20 mL of urine per patient. Sample concentrations were standardized and 20 pg of spike-in DNA of a rare bacterial genome was added. Shotgun metagenomic sequencing was performed using the Illumina NextSeq2000 platform. Reads were quality controlled and purified of human-mapping sequences using CLC Genomics Workbench. All microbial reads were taxonomically analyzed using MetaPhlan3. For further controls, data for non-diabetic women in the same groups were obtained.

Results: Fifteen TD2M and 15 non-T2DM samples were compared for this study. The mean genomic DNA yield across diabetic samples was 18.5 ng/uL. We observed an average of 75.8% human mapping reads, which were censored from further analysis. The remaining dataset contained an average of 5.1 million microbial reads. In the No UTI group, common urogenital flora microbes dominated across all samples in both T2DM and non-T2DM groups. In the rUTI Remission group, urogenital flora microbes were the most common again in non-diabetics, while common UTI pathogens such as Escherichia coli, Staphylococcus spp. and Pseudomonas aeruginosa were present in diabetic samples. For the rUTI Relapse group, both T2DM and non-T2DM were infected with common UTI pathogens (Escherichia coli, Klebsiella pneumoniae, Anaerococcus vaginalis, Enterococcus faecalis), however, 5/5 diabetic samples showed multi-strain infections while only 2/5 non-diabetic samples did.

Conclusion: We aim to identify differences in urinary microbiomes between women with T2DM with rUTI relapse, rUTI remission, and no history of UTI. By comparing our results with a published study on women without T2DM, we found that our cohort had ~10% more human mapping reads. Our pilot study suggest that T2DM patients may have higher rates of multi-strain infections compared to their non-T2DM counterparts. An expanded study (n = 60) is currently in progress to further understand the differences in urinary microbiomes in women with and without T2DM. 


Metagenomic Sequencing of the Urinary Microbiome of Postmenopausal Women with Recurrent Urinary Tract Infection and Type 2 Diabetes

Background
Leukemia, lymphoma and myeloma accounted for nearly 10% of new cancer cases and more than 50,000 deaths in 2022. Although multiple treatment modalities exist, Chimeric Antigen Receptor (CAR) T cells have been particularly useful, using genetic modification of a patient’s harvested T cells to better target cancer cells. Current methods of altering antigen specificity and targeting, however, have proven to be inadequate over time, requiring additional genes to boost the immune response. One such gene is OX40, a co-stimulatory cassette member of the Tumor Necrosis Factor Receptor (TNFR) superfamily that is thought to boost T cell migration, proliferation and survival. Given these inherent qualities of OX40, we hypothesized that incorporating OX40 variants into CAR T cells could enhance their duration of response and tumor-targeting capabilities.

Methods
We combined variants of the OX40 gene with our 2nd generation CAR T cells, adding a costimulatory cassette to CD19 CARs. We started with transformations of plasmid cloning vectors containing OX40 genes, using these CD19 CAR/OX40 constructs to transiently transfect retroviral cell lines. The transient virus produced was used to transduce donor human T cells, inducing uptake of the CD19 CAR/OX40 vectors. Once transduced, T cell reactivity and longevity were assessed using ELISA assays, flow cytometry and cell counts.

Results
We successfully used our CD19 CAR/OX40 T cells to transduce various human donor T cells, demonstrating a 1:1 linear relationship between OX40 and CAR T cell expression via flow cytometry. We also found that the constructs produced markedly higher levels of OX40+ expression compared to endogenous levels, particularly in regulatory T cells. When tested against a Burkitt’s lymphoma line, our
constructs showed reactivity above the standard deviation. With regards to longevity, our highest proliferating construct was a truncated version that survived over 60 days in culture.

Conclusion
We were able to produce CD19 CAR/OX40 donor T cells that had increased longevity and enhanced hematologic tumor targeting capabilities over time, likely due to an increased regulatory T cell response. Surprisingly, our truncated construct survived and proliferated the most, suggesting that the missing signaling domain could be key to balancing OX40’s enhanced growth effects versus over-proliferation.
With the endogenous signaling domain in place, OX40 is a promising addition to our CAR repertoire, boosting the in vitro antitumor response while balancing sustained proliferation and longevity. Our next steps will include translation into in vivo mouse models before bringing it to patients’ bedsides as a novel immunotherapy treatment.

Driving CARs Forward: An OX40 Tale of Immunotherapy

Background: Life adversities, such as neighborhood disadvantage and lower household incomes, are associated with well-established impairments on language development, which predict academic difficulties throughout school. These language delays are observable by age 2, but it remains unclear if adversities experienced in utero affect neurological underpinnings of language development before delays emerge. Of interest are 3 white matter tracts implicated in early language skills: the corpus callosum (CC), inferior fronto-occipital fasciculus (IFOF), and uncinate (Unc).

Methods: A longitudinal cohort of 160 term-born (>36 weeks gestation) neonates were recruited from the eLABE study at Washington University, which oversampled St. Louis families exposed to poverty. Structural equation modeling was used to create 2 latent constructs of prenatal adversity. The first, “social disadvantage”, aggregates neighborhood disadvantage, family income, insurance status, maternal education, and diet. The second, “maternal psychosocial stress”, models maternal depression, stress, life adversities, and perceived discrimination. At birth, diffusion MRI data were collected, and parameters for white matter tract development were created using probabilistic tractography in FSL. In-person follow-up at age 2 evaluated child language skills. To assess the independent and interactive effects of prenatal adversities and neonatal white matter microstructure on language skills, stepwise linear regression models were conducted after adjustment for child sex, gestational age, and postmenstrual age at scan. 


Results: Prenatal social disadvantage (p=.00) and maternal psychosocial stress (p=.00) predicted significantly lower language scores by age 2. However, in a joint model, only the effects due to social disadvantage remained significant. Lower CC fractional anisotropy predicted significantly lower language scores (p=.02) independently of social disadvantage (p=.00). IFOF and Unc were not predictive of language ( p > .05). To examine the relationship between CC maturation and social disadvantage, a moderation analysis was conducted after dichotomizing families’ income into above and below 200% the federal poverty line (FPL), as most federal assistance programs use this value as a threshold for benefit eligibility. The interaction term was not significant (p= .11), but upon plotting results by income group, the slope for infants above 200% FPL was somewhat stronger. 

Conclusion: This study suggests that prenatal adversity, particularly social disadvantage, and immature CC myelination are strong predictors of language delays by age 2. These data can be used as empirical evidence to advocate for increased funding of federal assistance programs that benefit pregnant people (ex., WIC) and downstream fetal brain development. By continuing to explore the relationship between prenatal adversities on fetal brain development and emerging language skills, future studies will continue to identify specific neurological markers of early language development to allow clinicians to intervene before delays emerge.

The Effects of Prenatal Adversity and Neonatal White Matter Microstructure on Child Language Skills at Age 2

Background: Research shows that increased severity of congenital heart disease (CHD) is associated with increased frequency of neurological deficits in children with structural cardiac abnormalities. Young children with more complex CHDs who require cardiopulmonary bypass (CPB) have a significantly higher incidence of developmental abnormalities and neurological deficits, including academic difficulties and behavioral abnormalities. Microglia, the resident immune cells in the central nervous system, play a key role in neural immune function. Mesenchymal stromal cells (MSCs) are multipotent, non-hematopoietic cells that possess immunomodulatory and regenerative properties and regulate microglia activation. It is hypothesized that intra-arterial MSC delivery through CPB in a survival porcine model is neuroprotective by modulating systemic and brain-specific inflammatory responses.

Methods: To mimic the effects in young CHD patients, two-week old female piglets were randomly assigned to one of three treatment groups: control, CPB, and CPB+MSC. The CPB+MSC group consisted of a CPB surgery followed by MSC administration before weaning. Microglia undergo morphological changes from a naïve, ramified to an amoeboid shape under activated condition. To analyze the effects of MSCs on microglia activation, changes in the length of microglial processes and number of branch points from the nuclei were assessed.

Results: Four weeks post-surgery, analysis using Imaris software showed that CPB reduced both the length of processes and number of branch points compared to control in three different cortex regions. CPB+MSC revealed significantly longer processes (p=0.0281) and more branch points (p=0.0232) than CPB alone, indicating that morphological changes in microglia were normalized after MSC delivery.

Conclusion: MSCs regulate microglial activation and participate in the phenotypic switch from a pro-inflammatory state to a repair-permissive state. In contrast to their ramified morphology under normal conditions, activated microglia undergo structural remodeling and adopt an amoeboid morphology with highly retracted processes, a hallmark of brain inflammation, as shown in the CPB group. Microglia routinely migrate toward sites of neuronal degeneration induced by excitotoxic injury. In MSC-treated animals, there are significantly fewer amoeboid microglia with an associated increase in the length of processes and number of branch points to mitigate the effects of CPB, indicating attenuated activation of the microglia. MSC delivery during CPB is highly effective and shows translational potential to minimize CPB-induced systemic inflammation and microglial activation in children with CHD.

Effect of mesenchymal stromal cell delivery through cardiopulmonary bypass in a piglet model

Background
The reimbursement landscape for infectious diseases is constantly evolving. In recent years, there have been significant changes in Medicare reimbursement for infectious disease procedures. These changes have had a major impact on infectious disease specialists, who may be disproportionately affected by changes in reimbursement in certain regions, which could have implications for access to care. Previous research has not examined these fluctuations especially in different geographic regions. Thus, our study analyzed Medicare reimbursement changes from 2013 to 2023 for all U.S. states and territories. We also explored the relationship between these changes and select social determinants of health (SDOH).
Methods
Medicare Administrative Contractor codes for the top 50 infectious disease procedures were used to identify states using the Centers for Medicare and Medicaid Services’ Physician Fee Schedule. Each price was adjusted for inflation using the consumer price index and weighted using an index of codes. A total percent change was generated for each state or territory. State-based data for various SDOH were used to calculate a correlation coefficient with geographic changes in
reimbursement (statistical significance factored with α of 0.05).
Results
From 2013 to 2023, the inflation-adjusted Medicare reimbursement for the top-grossing infectious disease procedures for U.S. states and territories decreased by a mean and median of 24.8% and 25.0%, respectively. The greatest total decrease was experienced by Illinois (-29.0%), Wyoming (-26.8%), Kansas (-26.8%), and Wisconsin (-26.6%). Massachusetts (-22.5%), New York (-22.3%), Washington (-19.8%), and Puerto Rico (-14.2%) were states that had the lowest change over the ten-year period. Some of the top grossing procedures (Current Procedural Terminology code) and their yearly percent change included critical care for the first 30-74 minutes of instance(99291, -197.2%), debridement of subcutaneous tissue for the first 20 cm2 or less(11042, -197.0%), transitional care management with high medical decision complexity(99496, -197.0%), and removal of central venous catheter without subcutaneous port or pump (36589, -197.0%).
Conclusion
Our findings suggest that over the past ten years, Medicare reimbursement for infectious disease procedures has decreased in many regions of the United States. These findings have important implications for access to care. Decreased reimbursement may make it more difficult for infectious disease specialists to provide care in certain regions, which could lead to longer wait times and decreased
access to care for patients. Policymakers should consider and develop strategies to address these geographic disparities. This could include providing financial assistance to infectious disease practices or expanding access to telehealth services.

The Impact of Geography on Medicare Reimbursement for Infectious Diseases: A 10-Year Analysis

Introduction: Hand-foot-mouth disease (HFMD) is a common childhood infectious disease. Atypical skin findings of HFMD, often associated with coxsackievirus A6 (CVA6), were first reported in 2008, with increasing reports worldwide since. Atypical lesions of HFMD often involve sites beyond the palms and soles and tend to have unusual, polymorphic morphology. 
Methods: A systematic review was conducted on clinical features and outcomes of pediatric HFMD with atypical cutaneous manifestations.
Results: Eighty-five studies were included, representing 1,359 cases with mean age 2.4 years and a male predominance of 61%. The most reported morphologies were vesicles (53%), papules (49%), and bullae (36%). Other morphologies included eczema herpeticum-like (19%), purpuric/petechial (7%), and Gianotti Crosti-like (4%). Common atypical sites included the arms and/or legs (47%), face (45%), and trunk (27%). CVA6 was identified in 63% of cases. Symptoms resolved in a mean of 10 days. Overall, 16% of cases received treatment, most commonly with acyclovir, intravenous antibiotics, or topical steroids. The most common complications were nail changes (21%) and desquamation (4%) which occurred a mean of three and two weeks after symptoms, respectively. 
Conclusion: Due to unusual morphologies resembling other conditions, HFMD with atypical cutaneous findings may be misdiagnosed, leading to inappropriate and unnecessary investigations, hospitalization, and treatment. Greater awareness of atypical presentations of HFMD is warranted to improve patient care and counseling on infection control precautions.

Atypical Cutaneous Findings of Hand-Foot-Mouth Disease in Children: A Systematic Review

Background
Breast cancer related lymphedema effects one in five breast cancer survivors. This debilitating complication is due to transection and obstruction of lymph flow during surgery. Although lymphedema has been met with new surgical and clinical treatments, little is known about the anatomy of the lymphatic system. Diagnostic dyes have become a vital tool in surgery and lymphatic mapping. Microsurgeons utilize florescent Indocyanine green (ICG) distally in extremities to identify afferent lymphatic pathways. In immediate lymphatic reconstruction (ILR) following mastectomies, ICG helps microsurgeons locate transected channels for anastomosis to a neighboring vein, restoring lymph flow. Isosulfane blue (ISB) is an easily visible dye favored by breast surgeons. In axillary reverse mapping, surgeons inject ISB into the proximal upper extremity, allowing them to locate and preserve lymphatic channels wherever possible. The benefit of utilizing both ICG and ISB during axillary dissection and ILR is reliable visualization of lymphatics intraoperatively. Using dual-dye technique, we aim to differentiate afferent lymphatic drainage from the distal and proximal upper extremity into the axilla.

Methods
To identify the distal and proximal lymphatic zones in breast cancer patients receiving ILR following axillary dissection, ICG and ISB were injected in the ipsilateral arm. ICG was injected intradermally at the first and third webspace and volar wrist, while ISB was injected at the same depth into the upper medial arm. Operative reports were reviewed to determine how often each dye was visualized during axillary dissection. Details of the anastomoses were also documented.

Results
Twenty-six breast cancer patients undergoing ILR following axillary lymph node dissection were reviewed. Twenty-one patients met the criteria of dual-dye technique. ICG was seen in 100% of axillary dissections. ISB was seen with ICG in 61.9% of procedures, indicating a shared lymphatic pathway. ISB was never seen alone. The average lumen size of all lymphatic channels was 1.12mm. An average of 2.46 channels were bypassed to an average of 1.65 veins per procedure. Venous lumen size ranged from 1 to 3 mm. 

Conclusion
Dual-dye technique allows for visualization of axillary lymphatic drainage by the breast and plastic surgeons intraoperatively. Utilizing dyes with different visual properties enabled us to determine if lymphatic flow originated from the distal or proximal upper extremity. This technique could provide useful anatomic data in correlating the development of lymphedema to regional lymphatic drainage within the arm, allowing potential anastomotic connections and clinical treatments. 


Differentiating Afferent Lymphatic Pathways Using Dual-Dye Technique During Immediate Lymphatic Reconstruction

Our study addressed COVID-19 vaccine knowledge and attitudes among medical students at the University of New England College of Osteopathic Medicine (UNECOM). Medical students recruited to complete an anonymous online survey assessed backgrounds, experiences, knowledge, and attitudes toward the COVID-19 vaccine. The correlation analysis utilized gender, physical health rating, close connection to someone with COVID-19, and work in the medical field during March 2020 as potential factors affecting students' attitudes. Respondents included 139 medical students aged 20-39 years old, with 99.3% having received at least one dose of the COVID-19 vaccine under institutional mandate. Every student obtained vaccine information from any combination of medical professionals, scientific journals, or review articles. Almost all correctly identified the components of the vaccine, its benefits, and its potential side effects. We found that 85% of medical students expressed little hesitancy toward receiving the vaccine; about 90% of participants supported a vaccine mandate for students. The marked enthusiasm expressed by UNECOM medical students to receive the vaccine contrasts surveys conducted before any vaccine mandate, although students' accurate knowledge of the vaccine and its potential benefits may have influenced acceptance. Overall, consideration of societal and individual implications is paramount. Access to scientifically validated information is essential when implementing public health policy at both institutional and federal levels.




Medical Students' Attitudes and Knowledge on the COVID-19 Vaccine and Mandate

Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 expression is normally repressed in most adult organs but is reactivated in a multitude of solid and blood cancers and upon treatment with hypomethylating agents (HMA). There is outstanding clinical need for the development and validation of a less invasive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders.

Using the Simoa (Single Molecule Array) platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations, using the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. Quantitative assay development further included creating recombinant protein standards, antibody pair screening and validation, and pre-analytical optimization. Upon completion of these steps, a retrospective cohort analysis via patient peripheral blood samples is currently being conducted to validate assay accuracy and precision. Additionally, we are aiming to conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring.

Thus far, for quantitative assay development, we have successfully expressed and purified full-length SALL4 which will be used to calibrate the standard curve and determine the assay’s limit of quantification. We also optimized multiple pre-analytic steps, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. The best antibody pair can detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform. For the retrospective cohort validation, we have obtained samples for analysis for liver cancer, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) with plans to expand to breast cancer and lung cancer.

In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy.

Underline digital video library

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

Functional Serologic Testing and Cross Reactivity of Serum Antibodies to SARS-CoV-2 SPIKE Protein

Investigation of HIV-1 Antisense Expression In Vivo

Metagenomic Sequencing of the Urinary Microbiome of Postmenopausal Women with Recurrent Urinary Tract Infection and Type 2 Diabetes

Driving CARs Forward: An OX40 Tale of Immunotherapy

The Effects of Prenatal Adversity and Neonatal White Matter Microstructure on Child Language Skills at Age 2

Effect of mesenchymal stromal cell delivery through cardiopulmonary bypass in a piglet model

The Impact of Geography on Medicare Reimbursement for Infectious Diseases: A 10-Year Analysis

Atypical Cutaneous Findings of Hand-Foot-Mouth Disease in Children: A Systematic Review

Differentiating Afferent Lymphatic Pathways Using Dual-Dye Technique During Immediate Lymphatic Reconstruction

Medical Students' Attitudes and Knowledge on the COVID-19 Vaccine and Mandate

Ultrasensitive detection of SALL4 oncoproteins for the use in therapeutic monitoring

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