United States

Background
The Missouri Board of Registration for the Healing Arts (“the Board”) publishes information on all physicians who have been disciplined by the Board, as well as information on the medical schools, board certifications, and business locations of all active physicians. However, this information has not been publicly analyzed through formal research. Such analyses would help patients and other stakeholders contextualize disciplinary information. With this objective in mind, this study analyzed the characteristics and context of physician discipline in Missouri from the year 2000 to 2022.

Methods
Information on the disciplined physicians was collected using the Board’s published disciplinary action list and the Missouri Division of Professional Registration’s licensee search tool. IBM SPSS Statistics was used to calculate the frequencies of different types and reasons for discipline, the median time between license issuance and discipline, the median time between revocation and reinstatement, and the discipline rates among various physician subgroups.

Results
Less severe disciplinary actions were more common than more severe actions. Inappropriate prescribing practices were the most common reason for discipline. Tax delinquency was also a common reason for discipline, although tax-related discipline has declined in recent years. The median time between license issuance and discipline was 17.5 years, and the median time between license revocation and reinstatement was eight months. Physicians in surgical specialties tended to have higher rates of discipline, while physicians in specialties with limited patient interaction (pathology, radiology) tended to have lower rates of discipline. Osteopathic physicians were subjected to discipline at higher rates than allopathic physicians. Discipline rates by county were generally unreliable due to small sample size.

Conclusion
Physicians most often face discipline in the middle of their careers. The prescribing habits of disciplined physicians may contribute to drug misuse. Recent legal requirements to discipline physicians for tax delinquency appear to be effective. Frequent patient interaction and procedure-heavy specialties appear to be risk factors for discipline. Osteopathic physicians and medical schools are associated with higher rates of discipline compared to their allopathic counterparts, although further research is needed to identify the reasons for this pattern.

2023 AMA Research Challenge

Disciplinary Patterns for Missouri Physicians 2000-2022

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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

Background:
Atherosclerosis is a vascular inflammatory disease that is the leading cause of morbidities and mortalities worldwide, with late-stage plaque instability as the major pathology in myocardial infarction. The pathogenesis of atherosclerosis consists of lipid deposits in the inner wall of vasculature and chronic inflammation induced by oxidized lipoproteins. Efferocytosis is a process that contributes to the clearance of apoptotic cells in the plaque but is impaired in the late stage of the disease. Mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP-5) is known to regulate tissue fibrosis and the dampening of inflammatory responses. MKP-5 deficiency in late-stage atherosclerosis contributes to greater plaque instability through thinner fibrous caps and increased necrosis in the atherosclerotic plaque by impaired efferocytosis. This project aimed to conduct an RNA sequencing (RNAseq) analysis and to identify pathways and genes of interest, highlighting the regulatory role of MKP-5 in late-stage atherosclerotic plaque stability. 
Methods:
Bone-marrow-derived macrophages (BMDMs) isolated from wild-type (WT) and MKP-5 knockout (MKP-5-/-) mice were stimulated with ox-LDL for varying durations (0 hours, 6 hours, and 24 hours). Bulk RNAseq was performed on these cells generating a binary alignment mapping (BAM) and subjected to principal component analysis (PCA) to validate the data. Volcano maps were then generated for each marker of stimulation, highlighting genes of interest. Gene set enrichment analysis (GSEA) was then conducted to identify the most enriched gene sets. A heatmap was used to visualize the expression levels of several enriched gene sets and known gene pathways, namely efferocytosis. To study efferocytosis capability directly, WT and MKP-5-/- BDMDs were incubated with apoptotic Jurkat cells and subsequently analyzed with confocal microscopy and flow cytometry at different time points (0 hours, 30 minutes, 1 hour, 2 hours). 
Results: 
Compared to WT BMDMs, we identified 40720 differential expressed genes (p < 0.05) in MKP-5-/- cells, including 1267 upregulated genes (FC > 2), and 1460 downregulated genes (FC < 0.5) at basal conditions. In 6 hours of ox-LDL stimulation, we found 4050 differential expressed genes including 303 upregulated and 484 downregulated genes in MKP-5-/- BMDMs. In 24 hours of stimulation, we found 37195 differential expressed genes including 892 upregulated and 477 downregulated genes. Through GSEA analysis on these significant genes, we found the top 15 WT enriched and MKP-5-/- enriched gene sets (q > 0.25). The MKP-5-/- enriched gene sets include protein tyrosine kinase binding and small GTPase-mediated signal transduction. The WT enriched gene sets include intracellular transport and stress-activated protein kinase signaling cascade. The efferocytosis pathways were a particular focus of our research, and corresponding heat maps show that efferocytosis was significantly inhibited in MKP-5-/- BMDMs. Furthermore, in-vitro assays indicate that the loss of MKP-5-/- impairs efferocytosis. 
Conclusion: 
Through bioinformatics analysis and biological assays, our results show that MKP-5 is essential for efferocytosis in atherogenic conditions.

Bioinformatics analysis identifies the crucial role of MAP Kinase Phosphatase-5 in regulating macrophage functions in atherogenesis

Background:
Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer with few treatment options. Oncolytic viruses (OV) can induce anti-tumor immunity by exploiting immuno-inflammatory responses. Emerging concepts suggest cooperative immune evasion between dsDNA Vaccinia (VACV) and other OVs, like ssRNA Vesicular Stomatitis Virus (VSV) can enhance anti-tumor immunity. This project tests the hypothesis that VACV attenuation of interferon-α/β (IFN) enhances VSV oncolysis and immunogenicity.
Methods: 
To understand iCCA susceptibility to select OVs, we infected iCCA cells with VACV +/- VSV. To elucidate OV efficacy as a function of IFNα/β stimulation, we measured cytotoxicity and viral replication via MTT assay and 50% tissue culture infectious dose (TCID50) +/- IFNα stimulation. To study cooperative immune evasion (VACV-induced IFNα/β suppression enhancing VSV oncolysis), we quantified proteins from pathways that activate IFNα/β. Western blots (WB) of STING (DNA pathogen sensing) & RIG1 (RNA pathogen sensing) were performed to evaluate the impact of infection on IFN-stimulating cell-signaling. 
To assess efficacy of VACV+VSV in vivo as a function of IFNα/β response, we infected patient-derived iCCA xenograft (PDX) with PBS, VACV, VSV, or VACV+VSV. Tumor size and IFNα/β signaling (RIG1/STING) were assessed. Tumor growth abrogation was evaluated with repeated measures, one-way ANOVA, and post hoc multiple comparison was made with Dunnett’s test between groups.
Results: 
In vitro, VACV & VSV both show dose-dependent viral replication and cytotoxicity against iCCA measured through TCID50 and MTT cell viability assays. IFNα pretreated iCCA cells showed significant and robust upregulation of both RIG1/STING following viral infection with VACV (p=0.003/0.03, respectively), slightly less intense upregulation was seen in VACV+VSV combination (p=0.02/0.03, respectively), and no change was seen with VSV infection (p=0.89/0.78, respectively).
In vivo, iCCA PDX growth was significantly abrogated in the VACV group (p=0.007) & combination group (p=0.04) compared to control (figure 1). ANOVA results showed significant effect of treatment on tumor volume changes [F(1.385, 12.46)=11.97, p=0.0025, η²=0.5708]. No significant differences observed between the PBS & VSV groups (p=0.5452). WB quantification showed upregulated RIG1 in the combination group (p=0.03) and no significant difference between groups for STING expression (p=.4304).
Conclusions: 
The VACV and VACV + VSV combination treatments resulted in significant tumor growth abrogation compared to VSV treatment and PBS control. However, the RIG1 and STING signaling suggests that this does not occur via immune evasion or rescue of VSV by VACV in this model. These findings underscore the therapeutic potential of combination OV in iCCA and warrant further investigation to elucidate the underlying mechanism.


Treating intrahepatic cholangiocarcinoma with combination viroimunnotherapy

Background: Cholangiocarcinoma (CCA) is a malignancy of the bile ducts with five-year survival rates of less than 10%. For those with early-stage disease, resection or liver transplantation can be curative. Unfortunately, existing diagnostic tools fail to enable definitive diagnosis, as CCA is notoriously difficult to distinguish from fibrotic or inflammatory lesions. Therefore, novel tools are needed to enable early and accurate diagnosis of CCA, especially in those with indeterminate lesions. Proteases are enzymes that are frequently dysregulated in cancer, including CCA, playing diverse roles in angiogenesis, invasion, and metastasis. We have previously developed a class of peptide-based diagnostic agents, activatable zymography probes (AZPs), which bind to cell membranes after proteolytic cleavage and can be used for ex vivo and in vivo cancer imaging. In this work, we sought to develop AZPs to distinguish CCA from fibrosis.

Methods: We induced CCA in the Mdr2-/- mouse model of biliary fibrosis via hydrodynamic injection of plasmids encoding for AKT, YAP1, and the Sleeping Beauty (SB) transposase. We synthesized a panel of 26 AZPs, applied them to liver tissue sections from CCA and Mdr2-/- mice, and performed whole-slide fluorescence imaging. Select AZPs were co-stained with antibodies against vimentin and cytokeratin 7 and validated in a mouse model of FBXW7/AKT-mutant CCA. Finally, AZPs were PEGylated to increase solubility, intravenously injected into CCA and Mdr2-/- mice, and livers were imaged with in vivo imaging system (IVIS) to assess tumor uptake in vivo. 

Results: We found that 14 AZPs, most notably AZP6, exhibited significantly increased binding to CCA tumors (padj¬ < 0.05, fold change > 1.5) relative to fibrotic bile ducts. In CCA tumors, AZP6 bound significantly more strongly to cells with higher expression of vimentin (p < 0.01) and lower expression of cytokeratin 7 (p < 0.001), suggesting that AZP6 is cleaved by mesenchymal cells. AZP6 also specifically labeled tumors from an FBXW7/AKT-mutant CCA model in a pattern similar to that seen in the YAP1/AKT model, demonstrating its generalizability. Finally, after in vivo administration, AZP6 demonstrated significantly more fluorescence in explanted livers from mice with CCA than mice with fibrosis (p < 0.01), enabling perfect discrimination of malignant and benign disease (AUC = 1, p = 0.009).

Conclusion: AZPs detect and localize dysregulated protease activity ex vivo and in vivo in mouse models of CCA, without false positives from benign biliary fibrosis. With further refinement, AZPs may aid in the diagnosis of CCA.

Detection of cholangiocarcinoma with protease activity probes

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

The role of Lcn2 in Triple Negative Breast Cancer Organoids

The kappa-opioid receptor (KOR) has been a target of recent interest in the treatment of alcohol use disorder, due to its role in negatively reinforced ethanol drinking, whereby aversive stimuli can promote ethanol intake. The KOR is densely expressed in the nucleus accumbens (NAc) on terminals of afferent neurons from the ventral tegmental area (VTA), where their activation has been found to suppress dopamine release such as is observed during alcohol withdrawal. We have previously found that KOR agonism differs along the rostro-caudal axis of the NAc shell, and that there may be a sex-related difference in baseline levels of ethanol intake. In the present study, we sought to determine if KOR stimulation in the rostral NAc shell could reverse effects of dopamine afferent activation on ethanol drinking. In Experiments 1 and 2, we used Long-Evans rats, as well as female transgenic tyrosine hydroxylase (TH)-Cre rats on a Long-Evans background in Experiment 3, which have a TH promoter directing expression of Cre recombinase in dopamine-containing cells, and we allowed them to drink under an intermittent access model, which establishes binge-like ethanol drinking. After the establishment of stable drinking, we bilaterally infused an excitatory designer receptors exclusively activated by designer drugs (DREADD) virus in the VTA of Experiment 2 groups, and a Cre-dependent excitatory DREADD in the VTA of the Experiment 3 group. After time for transduction, we injected into the NAc shell (1) the DREADD agonist, clozapine-N-oxide (CNO) (Experiments 2-3), (2) the KOR agonist, U50,488H (Experiments 1-3), (3) CNO + U50,488H (Experiments 2-3), and (4) saline vehicle (Experiments 1-3), counterbalanced in a Latin-Square design, and measured subsequent ethanol drinking. Surprisingly, we found that both U50,488H and CNO, independently and combined, enhanced ethanol drinking in high-level drinkers, and that these effects were not additive. Furthermore, the opposite trend was seen in low-level drinkers, suggesting a phenotype-dependent effect. These results suggest that both the DREADD activation and the KOR agonist microinjection have a similar net effect on dopamine release into the NAc shell, and that these effects depended on the phenotype of the drinker. The findings support the use of KOR antagonism as a therapy for alcohol use disorder, despite its potential unconventional effects on the dopamine system, and the overall use of individualized medication that considers an individual’s sex and phenotype.

Effects of Dopamine and Kappa Opioid Receptor Activity in the Rostral Nucleus Accumbens Shell on Ethanol Drinking in Rats

Background

Numerous studies have explored COVID-19 in adults and pediatric patients with chronic illnesses, but limited research has focused on previously healthy children. This study reports results from a COVID-19 screening program implemented at two pediatric clinics in central California, focusing on the clinical presentation of COVID-19 in this population.

Methods

Patients seeking care at the clinics completed a screening questionnaire to identify symptoms consistent with COVID-19. Temperature and oxygen saturation were measured, and nasal swabs were collected for antigen testing. Chief complaints, final diagnoses, and test results were recorded. The severity of COVID-19 illness was categorized using the CDC's severity index.
1) Asymptomatic/ pre-symptomatic - patient reported no symptoms and vital signs were normal.
2) Mild disease - respiratory or GI symptoms were present with or without fever.
3) Moderate disease; evidence of lower respiratory tract involvement was present.
4) Severe disease; oxygen saturation was < 94%.
5) Critical disease; the patient was transferred to the ER for further treatment.

Results

Between 11/2020 and 12/2022, 11,649 COVID-19 antigen screening tests were performed. Patients ranged in age from 0.1 to 17.0 years (mean 8.7, SD 4.5). 47.7% of patients identified as female, 50.5% as male, and 1.8% as other. In total, 1,560 pts. (13.4%) tested positive. Among these, 665 (43%) were asymptomatic/pre-symptomatic, 560 (36%) had mild disease, 318 (20%) had moderate disease, and 17 (1%) had severe disease. No critical cases or transfers to the emergency room were reported. A higher body mass index (BMI) was associated with increased severity of illness. 4,446 patients reported no symptoms at the time of screening. Among these, 85% tested negative, and 15% tested positive. Demographics were similar between these 2 groups. 7,203 patients report symptoms consistent with COVID-19. Among these, 87.6% tested negative and were presumed to have an infection other than COVID-19, while 12.4% tested positive. Median severity of illness did not differ significantly between the 2 groups.

Conclusion

The screening program effectively detected pediatric COVID-19 cases, including asymptomatic patients. In healthy children, COVID-19 was mild, with severity similar to other viral infections. Higher BMI is linked to greater illness severity. These findings enhance understanding of acute COVID-19 in outpatient pediatric populations, emphasizing the need for proper screening to ensure safe delivery of care.

Clinical Manifestations of COVID-19 in Previously Healthy Pediatric Patients Presenting to a Primary Care Office.

Background
With the increased acceptance of transgender individuals, so has the number of gender-affirmation surgeries. Transgender individuals face elevated depression rates, leading to an increase in suicide ideation and attempts. This study evaluates the risk of suicide or self-harm associated with gender affirmation procedures.

Methods
This retrospective study utilized de-identified patient data from the TriNetX database, involving 56 US healthcare organizations and over 90 million patients. The study involved three cohorts: Cohort A, adults aged 18-60 who had gender-affirming surgery and an emergency visit (N=1,501); Cohort B, control group of adults with emergency visits but no gender-affirming surgery (N=15,608,363); and Cohort C, control group of adults with emergency visits, tubal ligation or vasectomy, but no gender-affirming surgery (N=142,093). Propensity matching was applied to Cohorts A and C. Data from February 4, 2003, to February 4, 2023, was analyzed to examine suicide attempts, death, self-harm, and PTSD within five years of the index event. A secondary analysis with pharyngitis controls was conducted to validate Cohort C.

Results
Individuals who underwent gender-affirming surgery had a 12.12-fold higher suicide attempt risk than those who didn’t (3.47% vs. 0.29%, RR 95% CI 9.20-15.96, p<0.0001). Compared to the tubal ligation/vasectomy controls, the risk was 5.03-fold higher before propensity matching and remained significant at 4.71-fold after matching (3.50% vs. 0.74%, RR 95% CI 2.46-9.024, p<0.0001) for the gender affirmation patients with similar results with the pharyngitis controls.

Conclusion
Gender-affirming surgery is significantly associated with elevated suicide attempt risks, underlining the necessity for comprehensive post-procedure psychiatric support.

Risk of Suicide and Self-Harm following Gender-Affirmation Surgery

Background
We present a Quality Improvement study on a community exercise program to determine if attending community walking events provides participants sense of comfort to exercise individually. We define comfort with exercise as experiencing increased motivation and support to exercise. This study was conducted through Walk With A Future Doc Lake County, whose goal is to help promote behavioral changes that can help community members live healthier lifestyles by addressing the racial and socioeconomic disparities within cardiovascular health.

Methods
Before this event, we identified a healthcare professional available to speak at the event or virtually, located a local walking trail for the one-hour walk, and contacted Roberti Community House to gauge community interest. We organized a pre-walk meeting with members and asked participants to complete a survey indicating their agreement with statements regarding comfort with exercise on a scale of 1 (Strongly Disagree) - 5 (Strongly Agree). After completing that survey, participants watched a video created by a healthcare professional. Volunteers and participants then completed a one-hour walk with breaks for stretching and hydration. After the walking event, participants completed an additional survey indicating their agreement with statements regarding comfort with exercising on a scale of 1 (Strongly Disagree) - 5 (Strongly Agree). 

Results
After attending the community event, members felt more comfortable exercising independently. The community members reported their comfort with individual exercise following the walk as 2.81 out of 5, their motivation to exercise as 4.13 out of 5, their understanding of the health benefits of exercising as a 4.69 out of 5, the encouragement to attend community events in the future as a 4.56 out of 5, and the support they felt from the event to stay active as a 4.31 out of 5. 


Conclusion
Members of the Roberti Community House experienced how community engagement and education can encourage individuals to make healthier lifestyle changes in the future. Members learned where the local walking trail was, how to stretch before and after walking, and connect with community members. This increased community involvement and support has been missing since the COVID-19 pandemic. Our current goal is to follow up with the Roberti Community House every two months, schedule events 3-4 times yearly, and maintain a relationship with Rosalind Franklin University of Medicine and Science. Some challenges during the study were finding a healthcare professional, communicating with adults who speak a language other than English, and publicizing the event.



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Disciplinary Patterns for Missouri Physicians 2000-2022

Next from 2023 AMA Research Challenge

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