United States

Background:
Thyrotoxic periodic paralysis (TPP) is a rare but reversible cause of acute flaccid paralysis, resulting from thyrotoxicosis-induced upregulation of the Na+/K+ ATPase in skeletal muscle, leading to a rapid intracellular shift of potassium. TPP affects approximately 0.1%–0.2% of hyperthyroid patients in the U.S., with a strong male and Asian predominance. Normokalemic TPP variants are exceedingly rare and frequently overlooked, particularly in non-Asian populations. We present an unusual case of normokalemic TPP in a young Caucasian woman with overlapping endocrine disorders whose presentation mimicked a spinal cord emergency.
Case Presentation:
A 30-year-old Caucasian woman presented with sudden-onset bilateral lower extremity flaccid paralysis, accompanied by perineal numbness and difficulty initiating urination, lasting approximately two minutes. Past medical history included Sheehan syndrome, hypothyroidism and adrenal insufficiency.
Initial concern was for cauda equina or conus medullaris syndrome. Further history revealed that she frequently self-adjusted her medications: levothyroxine (100–300 mcg/day) and hydrocortisone (80 mg/day, divided doses). She had withheld both medications for over 36 hours before arrival, which likely contributed to partial biochemical normalization by the time of evaluation.
On exam, she exhibited persistent perineal numbness, urinary hesitancy, and bradycardia; an unexpected finding in the context of thyrotoxicosis. MRI of the brain and entire spine was unremarkable effectively ruling out spinal cord emergencies, demyelinating processes, transverse myelitis and a spinal abscess. Laboratory testing revealed:
• TSH 0.008 uIU/mL
• Free T4 1.51 ng/dL
• Total T3 0.85 ng/mL
• Potassium 4.4 mmol/L
• Cortisol 68.86 mcg/dL
She reported multiple classic TPP triggers: intense physical activity, heat exposure, a high-carbohydrate meal, and acute emotional stress. Despite normal potassium and borderline thyroid labs, the overall presentation was consistent with normokalemic TPP.
Discussion:
TPP is a hormonally driven channelopathy in which excess thyroid hormone enhances Na+/K+ ATPase activity, shifting potassium intracellularly, hyperpolarizing the resting membrane potential, and rendering skeletal muscle fibers temporarily inexcitable. This hyperpolarization prevents adequate depolarization by inactivating voltage-gated sodium channels, resulting in transient flaccid paralysis.
In hyperthyroid states, elevated T3 and T4 can rapidly decline due to increased type 3 deiodinase activity, masking the biochemical severity. Normokalemia may also reflect post-event normalization of serum potassium.
This patient’s exogenous levothyroxine and hydrocortisone misuse magnified the intracellular shift: thyroxine directly increases ATPase expression, while cortisol enhances β-adrenergic sensitivity. Combined with physiologic triggers, this created a transient but dramatic neuroendocrine storm.
Recognizing TPP, particularly in atypical presentations, is essential to avoid unnecessary invasive workups, inappropriate steroid escalation, and iatrogenic harm.

AMA Research Challenge 2025 

The Paralysis Paradox: A Thyrotoxic Periodic Paralysis Masquerading As A Spinal Cord Emergency

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Background

Human papillomaviruses (HPV) cause most cervical carcinomas and substantial fractions of oropharyngeal, anal, vulvar, penile, and vaginal carcinomas. During infection, HPV DNA initially replicates as circular episomes. However, it becomes integrated into the host genome in most HPV-induced cancers. Following integration, HPV is most commonly found as a subgenomic segment that includes the non-coding regulatory region (URR) and viral oncogenes E6 and E7. In tumors, HPV DNA can exist as extrachromosomal DNA (ecDNA) circles, chimeric HPV-human tandem repeats (heterocatemers), or both. The presence of ecDNA is associated with poor patient prognosis across cancer types. However, distinguishing whether particular amplified DNAs in tumors are ecDNAs and/or intrachromosomal heterocatemers and determining the extent of genomic rearrangement surrounding them, present technical challenges. Characterizing HPV-related integration structures accurately and completely is essential for advancing our understanding of HPV-driven tumorigenesis and the mechanisms behind viral integration.

Methods

We applied a battery of genomic analysis techniques at various size-scales to the HPV16-positive human oropharyngeal squamous cell carcinoma cell line UM-SCC-47. These techniques included a custom hybridization capture assay coupled with short-read Illumina sequencing (HC+SEQ), Nanopore long-read DNA and RNA sequencing, Bionano optical genome mapping, and single-cell fluorescence in situ hybridization (FISH). 

Results

HC+SEQ and long-read DNA sequencing defined the structure of the integrated HPV16 DNA, determined the human-virus DNA junctions, and mapped a repeated 23.5 kb HPV-human heterocatemer at single-base-pair resolution. RNA-seq revealed that these heterocatamers could produce spliced, polyadenylated virus-human fusion transcripts alongside high viral oncogene expression. Optical genome mapping further showed that the 23.5 kb heterocatemer occurred in tandem arrays of various lengths, ranging from one unit to more than 27 tandem units. These large-scale arrays (up to 700 kb) were further rearranged with adjacent human DNA in even larger-scale structures. Additionally, single-cell FISH analysis revealed that every cell had intrachromosomal HPV16 associated with human DNA, with a subset of cells also containing HPV16 ecDNA.

Conclusion

These findings highlight extensive genomic instability, complexity, and heterogeneity of HPV16-human DNA structures in this cell line, including previously undescribed higher-order organizational patterns. They also underscore the value of combining large-scale analyses to resolve complex genomic rearrangements and emphasize the importance of FISH for distinguishing intrachromosomal from extrachromosomal DNA structures. Ultimately, understanding how integrated HPV DNA higher-order structures contribute to tumorigenesis could lead to patient stratification based on individual risk of progression or inform the development of targeted therapeutic strategies.

Defining complex and heterogeneous HPV-human DNA structures through large-scale genomic analyses

Background
Molar–incisor hypomineralization (MIH) is a common developmental enamel defect in children, leading to higher risk of caries, post-eruptive enamel breakdown, and dental pain. Proper enamel formation depends on the expression and function of matrix proteins like ameloblastin (Ambn) and the protease matrix metalloproteinase 20 (Mmp20), which regulates mineral deposition and organic matrix removal during amelogenesis. We hypothesized that enamel hypomineralization caused by Ambn overexpression in mice could be rescued by increasing Mmp20 expression to
restore the correct substrate-to-enzyme ratio essential for healthy enamel.

Methods
Overexpression of Ambn and Mmp20 was achieved in separate mouse lines from transgenes (Tg). Ambn-Tg mice exhibited MIH-like phenotypes, and Mmp20-Tg mice displayed hypoplastic amelogenesis imperfecta. Crossbreeding of Ambn-Tg with Mmp20-Tg generated double-Tg mice overexpressing Ambn and Mmp20. Mandibular incisors of 7-week old mice (n=5/grp) were analyzed from the apical region to the incisal edge in sequential 1.0 mm volumes of interest (5 µm pixels) using isotropic micro-CT. Low, medium, and high thresholds were applied to quantify enamel volume and percent mineral volumes. Enamel mineral density was
calibrated to hydroxyapatite phantoms (.25 and .75 g/cm3). We compared the Ambn-Tg+Mmp20-Tg to the wildtype (WT) controls. We ran a single factor ANOVA test and a two-tailed T-test to determine if there is a significant difference between the groups.

Results
At eruption, the volumes of middle and high-density enamel in the Ambn and Mmp20 Tg’s demonstrated significant differences from the WT. Ambn-Tg had more middle density enamel volume (p=0.007). The Mmp20-Tg had significantly greater middle density (p=0.00001) and less higher density enamel (p=0.0000001) volumes compared to the WT, respectively. Ambn-Tg+Mmp20-Tg middle density and high-density enamel volumes were similar to the WT. Calculated enamel mineral densities were 38% (p=0.00001) and 8% (p=0.0005) lower in the Mmp20-Tg and Ambn-Tg groups compared to the WT, respectively. The Ambn-Tg+Mmp20-Tg
enamel mineral density was similar to the WT. Visual analysis indicated that the incisor enamel of Mmp20-Tg and Ambn-Tg groups was distributed in circular patches containing regions of lower and higher enamel density. A similar visual inspection indicated that the Ambn-Tg+Mmp20-Tg enamel was distributed like the WT.

Conclusion
These results show that enamel mineralization is highly sensitive to the balance of Ambn and Mmp20 expression. Imbalances can cause hypomineralized or hypoplastic enamel, while restoring the substrate-to-enzyme ratio can rescue normal enamel formation. This underscores the importance of maintaining balanced extracellular matrix protein and enzyme levels for healthy, functional enamel development.

Rebalancing the Substrate-Enzyme Ratio in Enamel Rescues Molar Incisor

Background
Despite advancements in genomic research, minoritized populations remain underrepresented, limiting the equitable distribution of benefits and contributing to ongoing health disparities. The Rare Genomes Project (RGP) at the Broad Institute of MIT and Harvard offers free whole genome sequencing to individuals in the U.S. with rare, suspected monogenic conditions who lack a genetic diagnosis. Between 2017 and 2021, enrollment was limited to English-proficient individuals. As a result, >90% of participants had some college education and >60% reported household incomes over $90,000, and no participants spoke a language other than English. Recognizing the lack of demographic diversity, RGP implemented a new diversity-focused study protocol in 2021 to address barriers to participation and increase access among historically underserved populations. 

Methods
To improve accessibility, RGP adopted strategies such as a simplified physician referral form, expanded language access (Spanish materials and interpreters), mobile phlebotomy, phone support, and extended staff hours. From 2021 to 2024, a total of 68 participants were enrolled who met at least one of six criteria for underrepresentation: non-white race, Hispanic/Latinx ethnicity, low income, limited English proficiency, rural residence, or lower educational attainment. Demographic and survey data were collected at baseline and sequencing was completed for all participants.

Results
Post-implementation, RGP saw an 8% decrease in the total proportion of white participants and a 5.5% increase in Hispanic participants. The proportion of non-English speakers increased by 3.3%. However, rural enrollment declined after 2022, despite offering mobile phlebotomy and flexible contact hours. Of 437 total RGP participants, 68 comprised the new underrepresented cohort. This group was more likely to have lower education and income and greater use of government insurance (62% vs. 36%, p<0.001). At baseline, the underrepresented cohort reported higher perceived importance of a genetic diagnosis and slightly higher stress levels. While both groups agreed a diagnosis would aid understanding and preparation, the underrepresented group prioritized its role in identifying better treatments. Challenges such as limited internet or transportation and provider trust remained barriers for rural and minoritized communities.

Conclusion
The revised RGP protocol showed some success in improving access and enrollment for underrepresented populations, especially through multilingual support and flexible engagement strategies. However, limited internet access and reduced rural participation highlighted remaining gaps. Patient-provider trust also emerged as a potential barrier. Future efforts will include participant interviews and follow-up surveys to better understand values and inform long-term engagement strategies. Continued enhancements and community-centered approaches are needed to ensure equitable access to genomic medicine.


The Impact of Enhanced Recruitment and Enrollment Support
on the Diversity of a Genomic Research Study

Background
In the last century, Influenza A viruses have caused the most global pandemics. The global panzootic of Highly Pathogenic Avian Influenza A (H5N1), its expanding mammalian host range, first mammalian-to-human transmissions and rising human infections are ominous. As a leading pandemic risk, early H5N1 detection is crucial to outbreak response but currently remains limited to the CDC and state public health laboratories. Here, we present a simple, rapid, adaptable, high throughput multiplex fragment analysis test for Influenza A (H5N1, H3N2, H1N1) subtyping and Influenza A/B, RSV and SARS-CoV-2 identification.

Methods
From the most up to date viral genomes we designed novel primers targeting conserved pathogenic regions across clinically relevant Influenza subtypes and generated unique gene-specific subtype signatures for H5N1 Influenza, endemic Influenza strains, RSV and SARS-CoV-2 via fragment analysis by capillary electrophoresis. Analytic sensitivity was determined by limit of detection analysis of Influenza A viral RNA in replicate reactions for each input amount of the viral RNA genome. Inter-/intra-run variability was assessed for all gene targets. Analytical specificity was established in evaluations of H5 and non-H5 human and avian Influenza A virus genomic RNA and by in silico analysis of potential primer cross-reactivity with other pathogens using BLAST® suite analysis.

Results
We generated unique fragment size-based signatures for each targeted gene. Targeted conserved regions in the HA gene are conserved in all newly sequenced U.S. Human H5N1 cases. The generated H5 amplicon had CT-values 2-12 lower than CDC and WHO H5 primers. No confounding false positives or over-lapping non-specific amplicons were identified. 100% H5N1 detection sensitivity was observed with a lower limit of detection of 2.5 copies/uL.

Conclusion
We have applied fragment analysis to Influenza A subtyping and respiratory virus detection for the first time. The multiplex target fragment analysis test described here has 100% analytical sensitivity and no false positives. This enables total subtyping capability of all human circulating Influenza A strains in one assay. This platform also proposes an epidemiological tool for detecting possible H5 Avian strain cross-over via dual detection of the H5 and M gene without the N1 amplicon. The use of multiple gene-specific targets for each strain increases test rigor, reproducibility and confidence in the accuracy of results.

Seasonal and Avian (H5N1) Influenza Subtyping by a Multiplex Fragment Analysis Test

Background: 
Dissecting aneurysms of the vertebral artery are life-threatening causes of subarachnoid hemorrhage, with higher rupture rates in the posterior circulation. Their pathogenesis is multifactorial, often involving structural vessel wall abnormalities and hemodynamic stress. While beta thalassemia minor is typically considered a benign carrier state, it can contribute to endothelial dysfunction and vascular injury. We present a case of a ruptured vertebral artery dissecting aneurysm in a patient with beta thalassemia and discuss whether her hematologic
condition could have played a contributing role. 

Case Presentation:
A 50-year-old woman with history of beta thalassemia minor presented to the ED for altered mental status. On arrival, her GCS declined from 12 to 8, prompting intubation. CT head revealed acute subarachnoid hemorrhage within the basilar cisterns and sylvian fissures and EVD was placed. Diagnostic cerebral angiogram showed right vertebral artery V4 segment irregular fusiform dissecting aneurysm at the takeoff of the right PICA, with the aneurysm measuring 7.1mm in length and 3.7mm in width. Patient underwent deconstruction of the right vertebral artery proximal to the dissection. Despite intervention, the patient deteriorated and was
ultimately declared brain dead. 

Discussion:
Several mechanisms raise the question of whether beta thalassemia could have predisposed this patient to vascular pathology. Beta thalassemia is associated with ineffective erythropoiesis, leading to chronic anemia, which appeared in this patient with baseline hemoglobin of 8-9 g/dL. Anemia reduces oxygen carrying capacity of the blood, leading to tissue hypoxia and cerebral hypoperfusion. In response to
hypoxia, mitochondrial respiration is inefficient which leads to formation of reactive oxygen species. Reactive oxygen species causing oxidative stress and altered nitric oxide metabolism can promote endothelial injury. In response to cerebral hypoperfusion, autoregulatory mechanisms trigger compensatory dilation of cerebral blood vessels to increase cerebral blood flow. But because of the persistent anemia in beta thalassemia, the ability to fully normalize cerebral oxygenation is limited, which can promote vessel wall remodeling, leading to a vessel
that is structurally more vulnerable.

Vertebral Artery Aneurysm Rupture in a Patient with Beta Thalassemia Minor: A Case Report

Background: Asthma is one of the most common chronic diseases among children, afflicting around 8.1% of school-aged children in the U.S. and contributing significantly to school absenteeism. Effective asthma management requires consistent medication adherence and education, which can be challenging for young children. Because nearly all children attend school and spend much of their day there, schools represent an ideal setting for health interventions. This study evaluates New York City’s Asthma Case Management Program (ACMP), which placed trained case managers in K–8 community schools to support asthma control, medication adherence, and coordination between families and providers. ACMP began in 2015-16 in one school and was rolled out to 32 schools by 2018-19. Using a triple-differences identification strategy motivated by the staggered rollout of ACMP, we studied the effect of a program placing asthma case managers in schools to educate and support children with asthma on school processes and health outcomes. 

Methods: We used student-level data (n=291,813) from the NYC Office of School Health on NYC elementary and middle schools from 2011 to 2019. We developed a triple differences design to study the causal effect of the program by comparing: 1) students with asthma vs. without asthma, 2) schools with vs. without ACMP, and 3) before vs. after the program’s implementation. Outcomes include student absenteeism, school nurse visits, and the completion of Student Asthma Questionnaires (SAQs). We assume no other policies affected these outcomes simultaneously.

Results: After ACMP implementation, students who ever had asthma had one fewer absence per year, on average. There was also an increase of about two nurse visits for medication administration per year, on average. Further investigation suggests this increase was driven by more asthma controller (preventive) medication. Lastly, after implementation, there was about a 44% increase in completed SAQs returned by students with asthma. 

Conclusion: ACMP shows promise in improving both administrative and health-related outcomes for students with asthma. The program led to greater return of administrative forms, suggesting improved care coordination. The program also benefited student outcomes as seen through reduced absences and increased nurse visits for prophylactic asthma management, pointing to better asthma control. This study provides early causal evidence of the benefits of school-based asthma management programs and supports the broader role of schools in chronic disease care for children. 


Asthma Management in Schools: A Tool for Reducing Absenteeism?

Background
Endovascular aortic repair (EVAR) has emerged as a less invasive alternative to open repair, offering improved outcomes. Non-elective EVAR is also associated with worse outcomes compared to elective EVAR with an in-hospital mortality of 14% and 1.9% respectively. Our study aims to validate that non-elective EVAR outcomes are worse than elective EVAR outcomes in a community setting, reinforcing the importance of early detection and intervention. 
Methods
Patient records were evaluated and any patient who underwent an EVAR for an infrarenal AAA from January 2019 to December 2024 was included. Patients were excluded if they had any trauma, if they died prior to intervention, or if they were under the age of 18. A bivariate analysis was done comparing elective vs urgent/emergent cases to compare comorbidities and outcomes. 
Results
The analysis included a total of 113 patients who underwent AAA procedures. The mean age of the patients was 75.4 years (SD = 7.9) and a majority of patients were male (77.8%) and non-Hispanic (78.8%). Most procedures were elective (88.5%, n=100), while urgent procedures accounted for 11.5% (n=13) of cases. The mean fluoroscopy time was 21.26 minutes (SD = 12.44) and the mean AAA size was 5.36 cm (SD = 1.44). The mean estimated blood loss was higher in urgent cases (548.46 mL) compared to elective cases (164.55 mL). Length of stay (LOS) was significantly longer for urgent cases (7.08 days) compared to elective cases (1.68 days) (p = 0.0268, statistically significant at p < 0.05).
Discussion
The significant difference in length of stay of 6 days shows these non-elective cases require more to recover. Non-elective cases also have a higher estimated blood loss during their procedures and were at higher risk of death. This reinforces the importance of aortic screening for early detection and timely referral to Vascular Surgery for intervention.

Elective vs Urgent Endovascular Aortic Abdominal Aneurysm Repair at a Community Hospital

Background: Hispanic Americans comprise about 25% of the United States population but only 3% of orthopaedic surgeons. Therefore, Spanish-speaking patients facing communication barriers may use tools like large language models (LLMs), such as ChatGPT, for medical information. Given the rising use of LLMs for patient education, it is critical to evaluate whether language-based disparities exist in the quality of information provided. While prior research has evaluated ChatGPT’s performance between languages across various specialties, no study has
examined this in sports surgery. The purpose of this study was to compare the accuracy of ChatGPT responses to patient questions about anterior cruciate ligament reconstruction (ACLR) in English versus Spanish. We hypothesized ChatGPT will provide generally accurate responses overall, but that responses in English will be more accurate.
Methods: Using methodology consistent with prior orthopaedic research, ten commonly asked patient questions related to ACLR were compiled via review of patient education materials from various orthopaedic institutions. Each question was independently submitted to ChatGPT in both English and Spanish using separate chat threads. Responses were evaluated by two
sports medicine fellowship-trained orthopaedic surgeons per language, using a standardized 5-point Likert scale (1 = major errors; 5 = completely accurate). Mean accuracy scores were calculated for each language. Inter-rater reliability was assessed using the intraclass correlation coefficient (ICC[2,k]) for average ratings. A Mann–Whitney U test was performed to compare average accuracy scores between English and Spanish responses.
Results: Inter-rater reliability was acceptable (ICC = 0.727 for both languages). The mean accuracy score was 4.45±0.69 for English responses and 4.65±0.53 for Spanish responses. There was no significant difference in accuracy between the languages (p = 0.460). Qualitatively, raters indicated most responses provided excellent, detailed summaries, but some responses, particularly regarding graft options, provided outdated information.
Conclusion: There was no significant difference in accuracy between English and Spanish responses generated by ChatGPT to patient questions regarding ACLR. This finding is novel, as no prior studies have evaluated language-based differences in ChatGPT accuracy within sports surgery. Given the noted underrepresentation of Hispanic Americans among orthopaedic surgeons in the United States, these results suggest that ChatGPT may be a valid source of information for Spanish-speaking patients facing language barriers when seeking care for ACL injuries. However, regardless of language, some responses appeared to rely on outdated information. As such, LLMs like ChatGPT should continue to be used with caution in clinical practice.

Accuracy of English vs. Spanish ChatGPT Responses to Patient Questions
about ACL Reconstruction

Background
Social determinants of health (SDOH), particularly food insecurity and related socioeconomic factors, are increasingly recognized as critical influences on health outcomes. Extensive evidence highlights their profound role in shaping health disparities across various populations. This study examines associations between food security-related SDOH and cancer mortality and prevalence in the Kansas City Metropolitan Area. 

Methods
Using publicly available census tract-level data from the CDC PLACES, U.S. Census Bureau, and Neighborhood Atlas for the Kansas City Metropolitan Area (Missouri & Kansas) in 2023, linear regressions were performed to assess the relationships between cancer deaths and prevalence and key SDOH variables: total seniors (TractSeniors), housing units receiving SNAP (TractSNAP), population beyond 1/2 mile from a supermarket (lapophalf), and poverty rate (PovertyRate). Multicollinearity was assessed using VIF values; additionally, bivariate Pearson correlations were also performed. The analysis included 131 observations.

Results
Analysis revealed distinct predictors for cancer deaths versus prevalence. For cancer deaths, the model explained 57.8% of the variance, showing significant positive associations with higher proportions of seniors ( β=0.0198, p<0.001), SNAP recipients ( β=0.0211, p=0.001), and limited food access ( β=0.0016, p=0.001). PovertyRate was negatively associated with cancer deaths ( β=−0.1883, p=0.011). Excluding senior population intensified the impact of SNAP recipients ( β=0.0319, p<0.001), limited food access ( β=0.0027, p<0.001), and PovertyRate ( β=−0.3627, p<0.001) on cancer deaths. For crude cancer prevalence, the model explained 18.2% of the variance, with PovertyRate as the sole significant negative predictor (β=−0.0527, p<0.001). Bivariate correlations supported these findings.

Conclusion
Cancer deaths are strongly predicted by age, SNAP participation, and limited food access, underscoring their prominent and independent roles in cancer mortality. In stark contrast, crude cancer prevalence is less directly explained by these variables. The consistent negative association between poverty rate and both cancer deaths and prevalence is a critical and counter-intuitive finding that likely points to significant under-diagnosis or under-reporting in impoverished areas, potentially due to reduced healthcare access, rather than poverty itself being protective. These distinct findings underscore that different underlying drivers influence cancer mortality versus prevalence. Ultimately, this research underscores the profound impact of social determinants on health disparities in cancer outcomes, pinpointing specific census tracts within the Kansas City metropolitan area for targeted public health interventions and policy reform to address the true burden of cancer in vulnerable communities.


Unpacking the Plate: Food Insecurity as a Driver of Cancer Mortality in the Kansas City Urban Core

IL-21 and IL-21R interaction plays a key role in mounting humoral and cell-mediated immune response to adenoviral and adeno-associated viral vectors in lung

Authors: Mina R Hanna,1,2 Robert Clark,1 Alexander J Martin,1 Dong Lin,1 Nnamdi L Onyene,1 Xin Wang,1 and Jay Kolls1,2

1Center for Translational Research in Infection and Inflammation and 2Department of Pediatrics, Tulane School of Medicine, New Orleans, LA, USA

Purpose of Study: Although cystic fibrosis (CF) was discovered ~ a century ago, it remains without an available cure for all mutations. 8-10% of CF patients remain without therapy options despite advancements in targeting misfolded CFTR. Gene therapy is an active area of investigation in hopes of tackling this unmet need; however, the need for re-dosing remains a challenge for any means of viral vector delivery. Thus, mitigating the immune response may be the key to overcoming this challenge. Given the central role of IL-21R in regulating T and B cell immune response, we hypothesized that targeting IL-21R may attenuate B and T cell responses and enhance secondary transgene expression. 

Methods: Naïve or IL21R-/- mice received 4x1010 viral particles and 1x1011 viral particles intratracheally of an Ad5 vector for first and repeat doses, respectively, at days 0 and ~27. Serum anti-vector antibodies were assayed 2 weeks post-dosing. All mice were euthanized 3 days following 2nd viral dose. Lung tissue was harvested for analysis of B cells and CD8 tissue resident memory (TRM) cells as well as secondary transgene expression. Similarly, naïve or IL21R-/- mice received 1x1011 vg of adeno-associated virus 6.2 (AAV6.2) by endotracheal intubation and monitored with In Vivo imaging system (IVIS) to assess for transgene expression. 

Summary of Results: At 2 weeks, serum anti-AdV IgG (P<0.05) and IgG1 (P<0.01) were significantly decreased in the IL21R-/- mice by 2-way ANOVA. Flow cytometry analysis showed a significant reduction of CD8 TRM cells in IL-21R-/- mice (P<0.01) by nonparametric T-test and a reduction in CD19 B cells (P<0.05) by 2-way ANOVA. Moreover, secondary transgene expression was significantly enhanced in IL-21R-/- mice compared to B6 controls (P<0.01). Similarly, in the context of AAV, the generation of anti-AAV IgG requires IL-21R signaling (P<0.0001). IVIS also showed significantly enhanced secondary transgene expression of AAV6.2 in IL-21R-/- (P<0.0001). 

Conclusion: Our results consistently showed how IL-21R and IL-21 play an important role in the development of humoral and CD8 TRM-mediated response to adenoviral and AAV vectors. This suggests how targeting IL-21R signaling may be the key to overcoming challenges for gene therapy using viral vectors as a means of delivery. Future studies will investigate the role of IL-21R in adult mice using antibody blockade. 


IL-21 and IL-21R interaction plays a key role in mounting humoral and cell-mediated immune responses to adenoviral and adeno-associated viral vectors in the lung

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