Neurogenic cough is a type of chronic cough related to hypersensitivity of the nerves in the larynx. There are several treatments for neurogenic cough, including neuromodulating medications, procedures, and cough retraining therapy. Superior laryngeal nerve (SLN) block is an emerging treatment that has yet to be well studied. This procedure involves injection of a steroid, along with a local anesthetic, into the neck trans-cervically, anatomically targeting the main nerve responsible for transmitting laryngeal sensation. It is thought to interrupt or dampen the neural pathways that simulate coughing. We aimed to assess factors that are predicative for a positive response to an SLN block via a retrospective chart review cohort study. We examined a variety of factors, such as history of cough, including triggers and type of cough, history of reflux and allergies, and prior neuromodulator use. We reviewed physical exam and testing findings, such as unilateral vocal cord paresis, which has been associated with neurogenic cough. We have identified 33 patients who are eligible for this study, have nearly completed chart review, and we will perform statistical analysis to associate factors with positive responses. Our series will report on the infrequently studied (2 existing publications) SLN block as a treatment for neurogenic cough patients. Our results will help us understand whether SLN block is a feasible treatment for neurogenic cough.

COVID-19, the viral disease caused by the novel coronavirus SARS-CoV-2, is associated with cardiovascular complications such as arrhythmias, myocarditis, and even cardiac arrest. There are two possible mechanisms of SARS-CoV-2 entry into human cells; the endosomal-mediated pathway which requires intracellular processing by intracellular proteases, and the membrane fusion pathway mediated by secreted proteases. Importantly, SARS-CoV-2 entry relies on the expression of the transmembrane receptor ACE2, which interacts with the viral spike protein. It’s still unclear if ACE2 is required for both viral entry pathways. Cardiomyocytes express ACE2, thus SARS-CoV-2 can enter heart tissue; however, the mechanism by which this occurs and how it may lead to cardiac dysfunction is unknown. We previously demonstrated that SARS-CoV-2 significantly impairs mechanical and electrical function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our goal is to understand if ACE2 is required for viral entry into the heart, using hiPSC-CMs as a model, in order to better understand COVID-19 pathology affecting the heart. Using a CRISPR/Cas9 system, we targeted the ACE2 gene at three loci to effectively knockout (KO) gene expression from WTC11 iPSCs. Two KO clones were selected and isolated after sequencing. Wild type (WT) and KO iPSCs were directly differentiated into CMs over a 17-day period. Preliminary results confirmed the absence of ACE2 protein expression in both KO clones by western blot. Fluorescent imaging of CMs infected with GFP-tagged SARS-CoV-2 showed severe infection and cell death at varied time points and multiplicities of infection (MOI) in WT WTC-CMs, while ACE2 KO-CMs showed absence of prominent infection and cell death. These data indicate that the lack of ACE2 markedly prevents SARS-CoV-2 entry into CMs, and understanding if blocking viral entry is sufficient to prevent functional impairment will provide key insights into the development of cardiomyopathies in COVID-19 patients.

Human Immunodeficiency Virus type 1 (HIV-1) is currently a major cause of morbidity and mortality worldwide. HIV-1 isolates are classified into genetic groups M, N, O, and P. Group M is the most widespread and is further divided into at least nine genetic subtypes and over 100 circulating recombinant forms, the prevalence of which vary by geographic locations. Subtype B is the most common form in Europe and North America and as a result, receives the majority of funding and research. Because of this, most of what we know about antiretroviral drug resistance in HIV-1 is based on studies of subtype B. However, the majority of HIV-1 infections worldwide involve other “non-B” subtypes, and much less is known about drug resistance in these subtypes. Knowledge of resistance to integrase inhibitors (INIs) is particularly important, since INI-based regimens are now recommended as first-line treatment for HIV-1–infected individuals globally. My objective is to genetically alter a full-length HIV-1-encoding plasmid (HIV-1 pNL4-3) that can used as a “cassette” for cloning non-B, patient-derived integrase sequences. To do this, I have identified restriction sites flanking the integrase region that can be used to remove and replace the integrase region with a patient-derived sequence, but site-directed mutagenesis must be used to knock out additional restriction sites for the same enzymes within the plasmid to avoid undesired enzymatic activity. This will result in only the desired sites, without changing the amino acid sequence. This vector will enable us to generate recombinant viruses from patients who are failing INI-based treatment, which can be tested for resistance to INIs in cell culture. The resulting data will be used to improve prediction of drug resistance in non-B HIV-1 based on sequence information alone. These studies will help guide treatment recommendations for both individuals and populations infected with non-B-HIV-1.

 The herpesvirus Epstein-Barr Virus (EBV) targets B-cells and epithelial cells. The virus is transmitted by saliva and commonly associated with infectious mononucleosis. After initial infection, the virus enters a latent stage. The first oncogenic virus identified in humans, it contributes to 1.5% of all cases of human cancers worldwide, specifically cancers of B-cells and epithelial cells, and roughly 140,000 deaths/year. Currently, no treatment is available for EBV-related cancers. Due to the widespread impact of EBV on populations, including a drainage of resources in parts of the world where EBV-associated cancer rates are disproportionately high, an effective treatment will be greatly beneficial. The gH/gL complex and gB, glycoproteins necessary for virus fusion to the host cell, are conserved among herpesviruses, including EBV. Recently isolated, the monoclonal antibodies AMMO1 and AMMO5 inhibit EBV infection by preventing fusion to the host cell. AMMO1 binds to the gH/gL complex and interferes with both epithelial and B-cell infection, while AMMO5 binds to gB and can prevent epithelial cell infection. Emerging evidence suggests certain tumors express these glycoproteins, thus they may be readily targeted for immunotherapy. In this project I created Jurkat leukemic cell line T-cells which express chimeric express chimeric antigen receptors (CARs) using AMMO1 and AMMO5. This CAR should target EBV viral antigens on tumors. First, I used mutagenesis to insert DNA encoding for the AMMO1 or AMMO5 scFV, into a CAR expression plasmid and verify integration by Sanger sequencing. I then used lentiviral delivery to transduce CAR constructs into Jurkat T-cell genomic DNA. Flow cytometry was used to confirm transduction of the T-cells. Future directions with this project include increasing transduction efficiency and transducing the constructs into cytotoxic T-cell so T-cell killing assays can be used to determine the efficacy of the AMMO1 and AMMO5 CAR-T cells.

Spinal cord injuries can result in devastating health consequences and impair voluntary muscle control. Animal models are invaluable for the development of new treatments to restore hand and arm function. In rats, we are developing a novel targeted, activity-dependent spinal stimulation (TADSS) therapy that promotes plasticity in spared pathways with a neuroprosthetic device after a C4-C5 spinal cord injury (SCI). Recovery is measured through training the rats in reaching tasks and comparing performance during therapy to the pre-injury scores. We have found that females show robust functional recovery with TADSS treatment, but males do not. One potential explanation for the difference in recovery is that females are more motivated to perform the behavioral tasks. Thus, we examined reaching performance of males and females prior to injury to see if there is any evidence of preexisting sex differences in reaching performance. Within groups of uninjured animals learning the reaching task, females on average had more trials than males, though both males and females had similar success rates. This may be evidence of greater motivation to perform the reaching task in females. Little research has been done on the role of sex and motivation in reaching tasks; however, other groups have shown that sex differences in performance of behavioral assays may be due to differences in motivation. We hypothesize differences in motivation may influence the level of functional recovery from SCI’s with TADSS therapy, as the intraspinal stimulation is dependent on muscle activity in impaired forelimbs. The aim of our future experiments is to determine the effect of sex on rodents’ motivation to perform reaching tasks before and after injury. By continuing to investigate the effect of sex-related motivation differences on motor tasks associated with SCI recovery, we hope to optimize TADSS therapy for clinical use in humans.

Arrhythmogenic cardiomyopathy (AC) is a devastating inheritable heart disease characterized by lethal heart rhythms and abnormal contractile function that can lead to sudden cardiac death or congestive heart failure. More than 70% of AC cases are due to mutations in desmosomal proteins, which are essential for maintaining structural integrity and intercellular junctions in the heart. Frameshift mutations in desmoplakin (DSP), a desmosomal protein, are a common cause of AC, therefore my project aims to use human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to model AC due to premature truncating DSP variants and to determine if the mechanism results from haploinsufficiency of DSP protein. From two unrelated patients with AC carrying different pathogenic DSP variants, p.Leu463Serfs*22 and DSP p.Arg941*, we generated patient-specific iPSC-CMs from each patient. With these cells, we created engineered heart tissues (EHTs) and found preliminary data that suggests DSP L463Sfs*22 EHTs generate less active twitch force compared to wild type, indicating that this human iPSC model can recapitulate the salient clinical phenotype. Protein analysis of these cells revealed that patients with premature truncating mutations in desmoplakin have lower desmoplakin levels when compared to wild type cells (DSP L463Sfs*22 iPSC-CMs have 38% less DSP protein levels compared to wild type iPSC-CMs). There was no evidence of the predicted truncated protein to suggest a dominant negative mechanism. Furthermore, we found a significant reduction in DSP transcript in the DSP L463Sfs*22 iPSC-CMs that was partially rescued by knocking down UPF1, a key regulator of the nonsense-mediated decay (NMD) pathway, suggesting a NMD-mediated clearance of DSP transcripts that results in haploinsufficiency. Should we also find that increasing desmoplakin protein rescues the phenotypes observed in patients, there is potential to find a novel treatment option for patients with desmoplakin-associated cardiomyopathy.

Epilepsy is one of the most significant neurological diseases in the world. Although there have been numerous advances in the study of epilepsy, there is still much we can learn about it. This is supported by the fact that one third of the patients with epilepsy are resistant to their anti-seizure drugs. With the mechanisms of pharmacoresistant seizures still unresolved, this project utilizes the mouse 6 Hz model of pharmacoresistant focal seizures to help answer some of its questions. Specifically, we hypothesize that the extent of brain region activation is directly correlated with increasing stimulation intensity used to invoke a seizure. Using cFOS, a marker for neuronal activity, we characterized the effect of varying stimulation intensities on cFOS immunoreactivity and its recruitment of nearby brain regions at various stimulus intensities. Using the established 6 Hz model we determined the convulsive current that caused a seizure in 97% of the population (CC97) in adult male mice. Ninety minutes after stimulation, brains were extracted, and processed for immunohistochemical labeling of the early activation gene, cFOS. Pending results will determine whether increasing stimulation intensity will result in greater cFOS labelling as well as greater recruitment of surrounding brain regions. These data will inform our future studies investigating the effects of several antiseizure drugs on blocking the recruitment and activity of different brain regions at varying stimulus intensities in the 6 Hz model.