HMS Theses and Dissertations

Background: The extent of variation in analgesic prescribing following musculoskeletal injury between countries is poorly understood. Understanding how analgesic prescribing varies between countries and cultural contexts can inform both domestic prescribing and future policy. Our survey study aimed to evaluate how opioid and non-opioid prescribing by orthopedic residents varies by geographic context. Methods: Orthopedic residents in 3 countries in which residents are the primary prescribers of postoperative analgesia at academic medical centers (Haiti, the Netherlands, and the U.S.) responded to surveys utilizing vignette-based musculoskeletal trauma case scenarios. The residents chose which medications they would prescribe for post-discharge analgesia. We standardized opioid prescriptions by conversion to Morphine Milligram Equivalents (MMEs), and we quantified the likelihood and dose of acetaminophen or a non-steroidal anti-inflammatory drug (NSAID) prescription. We constructed multivariable regressions with generalized estimation equations to describe differences in opioid and non-opioid prescription between countries based on prescriber and test case characteristics. Results: Compared to residents from the Netherlands and Haiti, U.S. residents prescribed more total MMEs per case overall (in MMEs, U.S.:383 [CI:331-435]; Netherlands:229 [CI:160-297]; Haiti:101 [CI:52-150], p<0.0001) as well as when analyses were stratified by anatomical site of injury. Similar results were obtained when examining MMEs prescribed per day, opioid prescription duration, and likelihood of opioid prescription. Residents from Haiti were more likely to prescribe non-opioids (OR 3.22 [CI:1.94-5.34], p<0.0001) compared to U.S. residents, and residents from the Netherlands nearly always prescribed non-opioids. When prescribing opioids, providers co-prescribed a non-opioid (acetaminophen or NSAID) in 345/603 (57.2%) of U.S., 152/152 (100%) of Dutch, and 69/97 (71.1%) of Haitian cases (Fisher's exact test p-value <0.0001). Finally, providers prescribed only non-opioids for pain control in 3/348 (0.86%) of U.S., 32/184 (17.4%) of Dutch, and 107/176 (60.8%) of Haitian cases (Fisher's exact test p<0.0001). Conclusions: Our results demonstrate greater post-operative discharge opioid prescribing in the U.S. compared to at least 2 other countries, one low-income and one high-income. Additionally, U.S. prescribers prescribed non-opioid analgesics less frequently, either in isolation or in conjunction with opioids. Our findings highlight the high domestic reliance on opioid prescribing for post-operative pain in orthopedic trauma.

Over the last decade, thousands of United States service members have sustained limb injuries from blast exposure, largely attributed to the rising use of improvised explosive devices (IEDs) by terrorist and insurgent groups. These blasts destroy large amounts of tissue causing peripheral nerve injuries and amputations in many cases. The objective of this research was to develop a device to improve functional recovery among these patients by providing in vivo monitoring of neuromuscular regeneration, maintenance of acetylcholine receptors for reinnervation and prevention of muscle atrophy. An implantable, flexible microelectrode array (MEA) was engineered that provides stimulation and recording during acute denervation of muscle distal to nerve injuries and muscle grafts for creation of regenerative peripheral nerve interfaces (RPNIs). One issue that has prevented the development of muscle surface electrodes is the immune-mediated foreign body response resulting in fibrotic capsule formation around the device. To inhibit fibrosis, the electrode was embedded with crystal formulations of a drug called GW2580, a Colony Stimulating Factor 1 (CSF1R) inhibitor allowing for long-term release of the drug from the surface of the MEA. The MEA uses a flexible polyimide elastomer and an array of gold-based microelectrodes featuring Peano curve motifs, which together maintain electrode flexibility. A new surgical technique was developed utilizing a biceps femoris muscle flap along the dorsal surface of the electrode to prevent wound breakdown during implantation. The devices were implanted along the denervated gastrocnemius muscles of rats. These rats underwent therapeutic stimulation using the MEA daily beginning on post-operative day 2. Tissues were harvested on post-operative day 14 and evaluated for quantification acetylcholine receptors and muscle fiber area using immunofluorescence and histological staining. Additionally, the MEAs were implanted along the dorsal surface of free flap grafts to serve as a RPNI for 6 weeks in rats. Using electrophysiological and histological assessments, we compare with unstimulated control grafts. The Young’s modulus was 1.67 GPa, which is comparable to native tendon and muscle. The devices successfully recorded electromyogram data when implanted in rats. When compared to untreated denervated muscles, MEA therapy attenuated atrophy by maintaining larger muscle fiber cross-sectional areas. Furthermore, the acetylcholine receptor areas were markedly larger with MEA treatment. The postoperative course of one animal without the biceps femoris flap (control) was complicated by complete wound dehiscence requiring euthanasia of the animal on postoperative day 4; the remaining control animals showed evidence of ulceration at the implant. The animals that did have the biceps femoris flap did not have ulcerative lesions on postoperative day 7. Expression of specific markers for acute inflammation (TNF-alpha), innate immune cell macrophages (CD68) and fibrosis (alphaSMactin) were also decreased in the flap group by qPCR analysis. Drug-loading with crystalized formulations of GW2580 successfully prevented fibrotic capsule formation and improved therapeutic efficacy of the SMEA. When the MEAs were implanted along the surface of muscle grafts for RPNIs, we found significantly delayed reinnervation and abnormal electromyographic (EMG) signals, with significantly more polyphasia, lower compound muscle action potentials and higher fatigability in stimulated animals. These metrics are suggestive of myopathy in the free flap grafts stimulated with the electrode. Active inflammatory processes and partial necrosis were observed in grafts stimulated with the MEA. This work demonstrates the ability to combine conformability, tensile strength-enhancing metal micropatterning and drug loading for long-term release into a functional implant for both epimysial stimulation and recording during acute denervation injury. However, under the same treatment protocol, implanted epimysial electrodes and electrical stimulation to deinnervated and devascularized flaps during the early recovery phase may be detrimental to regeneration.

A wide variety of skin conditions can arise in cancer patients, whether from cancer therapy, underlying genetic syndromes, paraneoplastic processes, or immunosuppression. This thesis examines five skin conditions that typically arise in oncologic patients: atypical post-radiation vascular proliferation (APRVP), skin neoplasms in Lynch syndrome, nonmelanoma skin cancers in pediatric patients, dermatomyositis in a patient with EGFR exon 20 mutation non-small cell lung cancer, and atypical erythema multiforme in a patient after bone marrow transplant. The first three studies are multi-institutional retrospective reviews that characterize the demographic and clinical characteristics of the conditions. The last two studies are case reports that suggest a possible pathogenetic mechanism and highlight the importance of recognizing atypical presentations in cancer patients. Through these five studies, this thesis provides insight into risk factors, preventative screening, management, and prognosis of rare cutaneous conditions that may enhance dermatologic care of patients with cancer.

Bell’s palsy is an idiopathic mononeuritis of the facial nerve thought to result from viral reactivation, causing inflammation and subsequent compression of the nerve within the rigid confines of the temporal bone. While most affected patients eventually recover normal facial function, aberrant neural regeneration occurs in a subset of Bell’s patients who present with severe facial weakness at onset, resulting in permanent facial synkinesis and disfigurement. Neural decompression surgery to alleviate pressure on the facial nerve in the acute setting of severe Bell’s palsy has been advocated as means to reduce risk of development of long-term sequelae, but use of this procedure has been limited and its efficacy is not well understood. Described is the design and validation of a rat model for unilateral compressive neuropathy of the facial nerve with the ability to examine long-term functional recovery associated with various durations and degrees of neural compression. Animals were implanted with vascular occlusion cuffs around branches of the facial nerve controlling whisker movements on one side, and cuffs inflated to various pre-set pressures for five to seven days. Whisking activity was quantified daily to characterize the threshold extrinsic pressure required to induce palsy. Cuffs were then be deflated and explanted, and whisking activity tracked and compared between sides over the ensuing 10 weeks. Functional nerve recovery and histological analysis of compressed nerves were then evaluated to determine the pressure range in which a Bell’s-like phenomenon is observed in this rat model. This work is the first to characterize the relationship between neural compression pressure and long-term recovery of facial function in highly quantitative and controlled fashion in a live animal model. Future work will seek to characterize the relationship between the timing of neural decompression and long-term facial function, providing evidence to support or refute the utility of mechanical decompression of the facial nerve in the setting of acute Bell’s palsy.

Background: Access to surgical care for cleft lip/palate (CL/P) has improved in low- and middle-income countries (LMICs). After surgery, however, children with cleft palate often have speech deficits such as velopharyngeal insufficiency (VPI) that significantly impact quality of life and development. Speech therapy is therefore required but often lacking in low-resource settings due to limited staff and healthcare infrastructure. Innovative programs that improve cleft care delivery in low-resource settings and tools to measure quality of life among patients with cleft palate are desperately needed. Objectives: 1) Translate and validate the VPI Effects on Life Outcomes (VELO) instrument in Nepali; 2) Evaluate the impact of task-shifted speech therapy on a standardized speech score; 3) Describe the experiences of families and associated barriers towards achieving cleft care; 4) Explore cleft care optimization informed by the experiences of families whose children had nominal improvements after task-shifted speech therapy. Methods: For objective 1, the VELO instrument was translated to Nepali, piloted, and modified using patient cognitive interviews. It was then administered to children aged 8-18. Internal consistency was evaluated using Cronbach’s α coefficient. Concurrent validity and discriminant validity were assessed using a two-sample t-test, assuming unequal variances. For objectives 2-4, we evaluated the experience of children and families who received speech therapy from lay workers (i.e., in a task-shifted model) with a convergent parallel mixed-methods study. Standardized speech scores were compared by a blinded speech-language pathologist before and after the speech intervention. Semi-structured interviews (SSIs) and focus groups with families evaluated cleft care experiences and barriers. Qualitative and quantitative data were merged and analyzed. Results: The VELO-Nepali demonstrated excellent internal consistency, with Cronbach’s α coefficients of 0.93, 0.94, and 0.90 for VPI cases, guardians of VPI cases, and non-VPI controls, respectively. VELO-Nepali exhibited strong discriminant validity between VPI cases (x̅=45.4, SD 22.1) and non-VPI controls (x̅=84.9, SD 12.3), (p<0.001). VELO-Nepali showed strong concurrent validity with similarities in VPI case scores (x̅=45.4, SD 22.1), and guardian scores (x̅=52.9, s=22.8), (p=0.473). Thirty-nine children with speech deficits after palatoplasty (ages 3-18) underwent task-shifted speech therapy and demonstrated significant improvements in composite speech scores (p<0.0001) and weakness (p=0.0002), with borderline improvements in misarticulation (p=0.07) and glottal stop (p=0.05). Forty-seven SSIs demonstrated that the greatest barriers to follow-up were family responsibilities (62%), travel/distance (53%), and work (34%). In five focus groups, families expressed a desire to improve their child’s speech and seek formal speech therapy. The speech intervention was found to be beneficial because of the compassionate staff, free lodging/food, and ability to socialize with other patients and families. After merging quantitative and qualitative data, we noted that younger children between 3-5 years old and families who traveled greater distances for healthcare access benefited less from the speech therapy intervention. Conclusion: The translated VELO-Nepali demonstrates strong internal consistency, discriminant validity, and concurrent validity, and can assess quality of life for Nepali patients with VPI. This instrument represents the first VPI quality-of-life assessment validated in a low- or low-middle-income county; it supports the feasibility of its validation and implementation in other such countries, especially those in South Asia where the burden of CL/P is significant. Task-shifted speech therapy has the potential to improve cleft lip/palate speech in LMICs. Multiple biosocial issues limit access to appropriate post-operative care.

Background: The University of Malawi College of Medicine (COM) launched a new Master’s in Global Health Implementation (MSc-GHI) in 2017 in collaboration with Partners in Health (PIH) Malawi . The program employs the Teaching Case Method, a widely used model in professional schools, to facilitate in-depth discussions. Initial experience from this method identified an unmet need for new teaching cases that may have contextual implications for trainees in Malawi and beyond. While global health cases abound in other resources, this project sought to generate a locally inspired teaching case to support training at COM and other resource-limited settings. Method: In addition to an extensive literature review, semi-structured interviews were conducted to collect firsthand experiences from 17 policymakers and early adopters in Malawi. Employing a narrative approach, a teaching case was generated from these interviews, to reflect some of the ‘lived moments’ behind policy discussions and provide contextual insights for class-room discussions. Results: Against the realities of a struggling HIV prevention program in the early 2000s, Malawi pioneered Option B+ in 2010, an unpopular policy that expanded life-long HIV treatment for all affected pregnant and breastfeeding women. The World Health Organization (WHO) rapidly embraced this initiative within two years, making it the cornerstone moment that emboldened other initiatives in HIV policy. Conclusion: Here I present a teaching case with an accompanying discussion guide, in response to an unmet need for locally inspired cases and toward capacity-building in Malawi and elsewhere. Acknowledging the contributions that low-income nations make to health delivery science, this case invites its audience to examine the political and socioeconomic factors that influence policies like Option B+ and broader global health initiatives in low-resource settings.

Background: The optimal timing of adjuvant radiotherapy (RT) in the management of atypical meningiomas remains controversial. We compared the outcomes of atypical meningiomas managed with upfront adjuvant RT versus postoperative surveillance. Methods: Patients with intracranial atypical meningiomas who underwent resection between 2000-2015 at a single institution were identified. Patients receiving adjuvant RT (n=51), or RT within the first year of surgery before tumor progression/recurrence (P/R), were compared to those undergoing initial surveillance (n=179). The primary endpoints were radiographic evidence of P/R and time to P/R from surgery. Results: A total of 230 patients were identified. Fifty-one (22%) patients received upfront adjuvant RT, while 179 (78%) underwent surveillance. Compared to the surveillance group, patients who received adjuvant RT had larger tumors (5.2 cm versus 4.6 cm; p=0.04), were more likely to have undergone subtotal resection (65% versus 26%; p<0.01), and more often had bone invasion (18% versus 7%; p=0.02). On multivariable analysis, receipt of adjuvant RT was associated with a lower risk of P/R compared to surveillance (HR=0.21 [95% CI 0.11-0.41]; p<0.01). Patients who initially underwent surveillance and then received salvage RT at time of P/R had a shorter median time to local progression following RT compared to patients who developed local P/R following upfront adjuvant RT (19 versus 64 months, respectively; p<0.01).
Conclusion: Upfront adjuvant RT was associated with improved local control in atypical meningiomas irrespective of extent of initial resection compared to surveillance. Early adjuvant RT should be strongly considered following gross total resection of atypical meningiomas.

Background: Although lower-complexity cardiac malformations constitute the majority of adult congenital heart disease (ACHD), the long-term risks of adverse cardiovascular events and relationship with conventional risk factors in this population are poorly understood. We aimed to quantify the risk of adverse cardiovascular events associated with lower-complexity ACHD that is unmeasured by conventional cardiovascular risk factors. Methods: A multi-tiered classification algorithm was used to select individuals with lower-complexity ACHD and individuals without ACHD for comparison amongst >500,000 British adults in the UK Biobank (UKB). ACHD diagnoses were sub-classified as “isolated aortic valve (AoV)” and “non-complex” defects. Time-to-event analyses were conducted for primary endpoints of fatal or non-fatal acute coronary syndrome (ACS), ischemic stroke, heart failure (HF), and atrial fibrillation (AF), and a secondary combined endpoint of major adverse cardiovascular event (MACE). Maximum follow-up time for the study period was 22 years using retrospectively and prospectively collected data from the UKB. Results: We identified 2,006 individuals with lower-complexity ACHD and 497,983 unexposed individuals in the UKB (median [IQR] age at enrollment 58 [51,63]). Of the ACHD-exposed group, 59% were male; 51% were current or former smokers; 30% were obese; 69%, 41%, and 7% were diagnosed or treated for hypertension, hyperlipidemia, and diabetes respectively. After adjustment for 12 measured cardiovascular risk factors, ACHD remained strongly associated with the primary endpoints, with hazard ratios (HR) ranging from 2.0 (95% confidence interval [CI] 1.5-2.8, p<0.001) for ACS to 13.0 (95% CI 9.4-18.1, p<0.001) for HF. ACHD-exposed individuals with less than or equal to 2 cardiovascular risk factors had a 29% age-adjusted incidence rate of MACE in contrast to 13% in non-ACHD individuals with greater than or equal to 5 risk factors. Conclusions: Individuals with lower-complexity ACHD had a higher burden of adverse cardiovascular events relative to the general population that was unaccounted for by conventional cardiovascular risk factors. These findings highlight the need for closer surveillance of patients with mild to moderate ACHD and further investigation into management and mechanisms of cardiovascular risk unique to this growing population of high-risk adults.

Through the analysis of large-scale human genetic datasets, I identify five therapeutic targets and risk factors for cardiometabolic disease. First, using Mendelian randomization, I demonstrate that body fat distribution is a causal risk factor for coronary artery disease and type 2 diabetes, with a similar magnitude of effect on disease risk as body mass index. Second, exploiting the genetic association between body fat distribution and type 2 diabetes, I identify a series of damaging variants in the receptor ALK7 that reduce abdominal adiposity and protect against type 2 diabetes. These findings suggest that pharmacologic ALK7 antagonism may be useful in the treatment of type 2 diabetes. Third, I show that genetic nitric oxide signaling protects against cardiovascular disease and improves renal function, suggesting that nitric oxide signaling agents such as PDE5A inhibitors could be repurposed for the treatment of cardiovascular and renal disease. Fourth, through the analysis of rare predicted loss-of-function variants in UK Biobank, I identify that deficiency in GPR151, a G-protein coupled receptor, and PDE3B, an intracellular enzyme, protects against obesity and coronary artery disease, respectively. These findings suggest that pharmacologic GPR151 inhibition may be a novel therapeutic approach to weight loss. Finally, I show that healthy lifestyle can mitigate inherited genetic predisposition to risk of cardiovascular disease, identifying a non-pharmacologic method of reducing genetic risk for coronary artery disease.

Glioblastoma (GBM) is an aggressive malignant brain cancer with rapid progression from diagnosis to death even with standard of care (SOC) therapy. Novel therapeutics are under investigation to help increase overall and progression free survival in patients. One area of investigation is the use of gene mediated cytotoxic immunotherapy (GMCI). With GMCI, the enzyme or protein for a therapeutic gene is inserted into the cell using an engineered virus. For GBM, the use of GMCI involves a non-replicating adenovirus (AdV) that expresses the herpes simplex virus thymidine kinase (HSV-tk) gene. In clinical trials, AdV-HSV-tk is injected into the resection cavity intraoperatively after resection of the GBM. The HSV-tk is required for activation of an anti-herpetic prodrug given systemically. The anti-herpetic prodrug acts as a toxic nucleotide that induces cell death by blocking DNA polymerase. The tumor antigens released from death of the cell promotes an immune response. As immunosuppression is thought to significantly contribute to the malignant nature of GBM, therapies that promote a robust anti-tumor immune response, such as GMCI and immunotherapy, hold great promise. GMCI combined with immunotherapy is now in a clinical trial for patients with GBM. Biomarkers that measure tumor responsiveness to treatment are under investigation. Extracellular vesicles (EVs) are mediators of intercellular communication through the transmission of biological information. Tumor derived EVs that are secreted into biological fluid, such as blood and CSF, can be isolated from patient blood and CSF samples. These EVs represent a good source of biomarkers. The purpose of this study is to identify changes in EVs secreted from virally infected cells compared to non-virally infected cells, and to assess the functional role of EVs in GMCI therapy.
For cultured GBM cells infected with AdV-HSV-tk, the maximal expression of HSV-tk occurred at 24 hours after viral infection. Although no plateau of HSV-tk expression at increasing multiplicity of infection (MOI) was reached, a MOI of 240 was decided upon given the amount of viral stock available. Cell viability was assessed after infection with AdV-HSV-tk and treatment with the anti-viral drug ganciclovir (GCV). GCV had strong cytotoxicity at 96 hours with an IC50 of 0.1 uM. Differences in EV size was measured using the NanoSight particle analyzer. EVs from virally infected cells were significantly larger than EVs from non-virally infected cells. However, virally infected cells produced significantly less EVs than non-virally infected cells. EVs produced by virally infected cells contained HSV-tk mRNA. EVs from virally infected cells were able to merge with non-virally infected cells and transfer HSV-tk. When GCV was given to non-virally infected cells co-incubated with EVs from virally and non-virally infected cells, increased killing of the GBM cells incubated with EVs from virally infected cells occurred compared with GBM cells incubated with EVs from control cells.
Our results suggest that GMCI therapy may alter the characteristics of EVs. EVs derived from virally infected GBM cells may serve as an important biomarker to monitor the response to treatment using GMCI. EVs may also play a functional role in the spread of viral mRNA to other cells in the local tumor microenvironment.

Background: Local control of spinal metastases is an important issue for patients who may live long enough to experience cancer recurrence. However, the literature on patterns of radiographic local failure among patients with spinal metastases receiving conventional radiotherapy (RT) is sparse. The purpose of this study was to evaluate the rates, consequences, and predictors of radiographic local failure in patients with spinal metastases managed with palliative conventional RT alone, in order to guide clinical decision-making and counsel patients treated with this modality of RT. Methods: We retrospectively reviewed 296 patients with spinal metastases treated with palliative conventional RT at the Brigham and Women’s Hospital/ Dana-Farber Cancer Institute. Of these patients, 182 had follow-up computed tomography (CT) or magnetic resonance (MR) imaging and were included in local failure analyses. We assessed the presence of and time to development of radiographic local failure, defined as radiological progression within the treatment field. We also evaluated consequences of and interventions for local failure. Kaplan-Meier analyses determined overall survival (OS) estimates. Cumulative incidence estimates determined local failure rates, with death considered a competing risk. Cox regression analyses tested predictors of local failure. Results: The median OS for all 296 patients was 4.0 months. The median OS was 1.0 month for the 114 patients without follow-up imaging versus 7.7 months for the 182 patients with follow-up imaging. Patients received a median of 30 Gy in 10 fractions to a median of 4 vertebral bodies. Overall, 74 of 182 patients (40.7%) experienced local failure. The 6-month, 12-month, and 18-month local failure rates were 26.5%, 33.1%, and 36.5%, respectively, while corresponding rates of death were 24.3%, 38.1%, and 45.9%. The median time to local failure was 3.8 months. Of those with local failure, 51.4% had new compression fractures, 39.2% were admitted for pain control, 28.4% developed neurological symptoms, and 17.6% developed cord compression; the median time from RT to each of these events were 3.0 months, 5.7 months, 10.5 months, and 9.0 months, respectively. Interventions for local failure included reirradiation (35.1%), invasive interventions for pain control (14.9%), salvage surgery (10.8%), and vertebroplasty (6.8%). On multivariable analysis, independent predictors of local failure included single-fraction RT (8 Gy) (HR = 2.592 [95% CI 1.437-4.675], p = 0.002), lung histology (HR = 3.568 [95% CI 1.532-8.309], p = 0.003), and kidney histology (HR = 4.937 [95% CI 1.529-15.935], p = 0.008). Conclusions: Patients experience a >30% rate of radiographic local failure by 1-year after conventional RT and often require admissions for pain control and re-treatment. Single-fraction RT and lung or kidney histology predicted local failure. Given the high rates of local failure for patients with favorable prognosis, assessment of the risk of death and progression versus risk of local failure is important for guiding clinical decisions regarding treatment modality and fractionation.

Human Immunodeficiency Virus-1 (HIV-1) entry into cells is mediated by the envelope (Env) trimer of gp120 and gp41 heterodimers. Sequential binding to the target cell receptors, CD4 and CCR5 or CXCR4, triggers the metastable Env to undergo entry-related conformational changes. PF-68742 was recently identified as a small molecule that inhibits infection of a subset of HIV-1 strains. We found that of the four PF-68742 diastereomers, only one, MF275, inhibited the infection of Cf2Th-CD4-CCR5 and Cf2Th-CD4-CXCR4 cells by some HIV-1 strains. Unexpectedly, MF275 activated the infection of Cf2Th-CCR5 cells by several HIV-1 strains resistant to the compound’s inhibitory effects in Cf2Th-CD4-CCR5 cells. In both cases, the strain susceptibility profiles were unique from those of other entry inhibitors. Washout of MF275 prior to addition of the virus mixture to target cells was able to abrogate its inhibitory effect in Cf2Th-CD4-CCR5 cells, whereas abrogation of the MF275 activating effect in Cf2Th-CCR5 cells was strain-specific. Sensitivity to other entry inhibitors in the presence of MF275 indicated that MF275-activated virus entry requires State 1-to-State 2 transitions, CCR5 binding, and 6-helix bundle formation and can be enhanced by pre-activation by a CD4 mimetic, whereas MF275 inhibition occurs regardless of CD4 mimetic pre-activation. In contrast to CD4 mimetic compounds, MF275 binding and activity did not depend upon availability of the gp120 Phe43 cavity, a known target of some other HIV-1 entry inhibitors. MF275 inhibition did not demonstrate a preference for State 1 versus State 2 Env conformations, but MF275 activation was abrogated by gp120 inner domain and gp41 ectodomain changes that favor the State 1 Env conformation. Changes in the gp120 C5 and gp41 fusion peptide (FP) and disulfide loop (DSL) regions conferred resistance to MF275 inhibition but not MF275 activation or CD4 mimetic activity. Finally, MF275 induced distinct conformational changes (as determined by a panel of broadly-neutralizing antibodies) in an Env susceptible to inhibition versus an Env susceptible to activation, with the former demonstrating conformational changes similar but not identical to those induced by CD4 or CD4 mimetics. Alterations in the gp120 C5, gp41 FP, and gp41 DSL desensitized Envs to broadly-neutralizing antibodies recognizing the FP and gp120-gp41 interface and abrogated the MF275-induced conformational changes associated with inhibition, but did not affect activation. Overall, MF275 is unique among HIV-1 entry inhibitors, mediating strain-specific inhibition or activation by two distinct mechanisms. We propose that MF275 binds and activates susceptible Envs in a manner similar to that of the CD4 receptor and CD4 mimetics; in addition, the Envs of some HIV-1 strains are susceptible to inhibition of a post-receptor binding step in virus entry.

Background: Immune checkpoint inhibitors (CPIs) have revolutionized oncologic therapy but can lead to immune-related adverse events (irAEs). Corticosteroids are first-line treatment with escalation to biologic immunosuppression in refractory cases. CPI-related gastroenterocolitis (GEC) affects 20-50% of patients receiving CPIs and carries significant morbidity and mortality. The severe form of disease is not well-described. In addition, patients with a histopathologically defined subset of CPI-related GEC, which we term CPI-related microscopic colitis (MC), may benefit from first-line budesonide administration. We present the retrospective clinical characterization of CPI-related GEC requiring admission at a single institution (inpatient GEC cohort) and a retrospective preliminary evaluation of a pilot cohort of CPI-related MC treated with budesonide (MC cohort). Methods: For the inpatient GEC cohort, clinical, laboratory, radiographic, and endoscopic data were extracted from charts of all melanoma patients ≥18 years of age admitted to one institution for CPI-related GEC, from 2/05/2011 to 12/13/2016. Patients were followed until 12/31/2017 for further admissions. Survival, outcomes, and pharmaceutical-use analyses were performed. For the MC cohort, clinical, laboratory, and endoscopic data were extracted from charts of all patients ≥18 years of age with prior CPI exposure and prior flexible sigmoidoscopy performed from 3/1/2017 to 12/31/18 for evaluation of possible colitis. CPI-related MC was defined as clinical and histopathologic evidence of colitis without endoscopic evidence of inflammation (Mayo Endoscopic Score 0). Diagnoses were confirmed by two reviewers, one with expertise in CPI complications. Survival and outcomes analyses were performed. Results: Median time-to-admission from initial CPI exposure for the overall cohort was 73.5 days. Median length of stay was 4.5 days. 50.0% required second-line immunosuppression. Readmission for recrudescence occurred in 33.3%. Common Terminology Criteria for Adverse Events (CTCAE) grade was not significantly associated with outcomes. Hypoalbuminemia (p=0.005), relative lymphopenia (p=0.027), and decreased lactate dehydrogenase (p=0.026) were associated with second-line immunosuppression. There was no difference in PFS or OS (p=0.367, 0.400) for second-line immunosuppression. Subgroup analysis showed that early corticosteroid administration (p=0.045) was associated with decreased PFS. There was no significant difference in average age or sex distribution between patients with MC and patients with non-MC GEC. Exposure to potential MC triggers tended to occur more often in the MC than non-MC cohort. Symptoms tended to start a median of 84 days later in the MC cohort, with borderline significance (p=0.064). Budesonide administration tended to result in faster symptom resolution (p=0.070). 10/12 (83.3%) patients with MC received additional cycles of immunotherapy after budesonide. Univariate Cox regressions showed that budesonide administration was significantly protective against treatment failure (HR 0.33, 95% CI 0.14-0.81) and borderline protective against progressive disease (HR 0.27, 95% CI 0.04-0.91). Conclusions: Severe CPI-related GEC typically manifests within 3 months of immunotherapy exposure. Rates of second-line immunosuppression and readmission for recrudescence were high. CTCAE grade did not capture heterogeneous degree of severity in our cohort. Second-line immunosuppression was not associated with poorer oncologic outcomes; however, early corticosteroid exposure was associated with decreased PFS. CPI-related MC, defined as clinical and histopathologic evidence of colitis with Mayo Endoscopic Score 0, is a clinical subset of CPI-related GEC that tends to present later in the course of immunotherapy. Budesonide is an effective treatment for CPI-related MC that controls symptoms and prolongs time on immunotherapy. Further prospective investigation is warranted in both cohorts.

Objectives: To understand the types of coping strategies reported by young women with breast cancer in the first 2 years following diagnosis and to evaluate the relationship between coping strategies and anxiety in early survivorship. Background: Young women with breast cancer are thought to be vulnerable to age- specific stressors, and this group’s use of coping strategies has not been clearly defined in the literature. A better understanding of coping strategies used by this group and the relationship between specific coping strategies and anxiety could help to inform the development of effective interventions to promote healthy coping, with the goal of improving quality of life in this population. Methods: Helping Ourselves, Helping Others: The Young Women’s Breast Cancer Study (YWS) is a multi-center, prospective cohort that enrolled 1297 women with newly diagnosed breast cancer at age < 40 years between 2006 and 2016. YWS participants complete serial surveys that include information on demographics (baseline), coping strategies (6 months and 18 months), and anxiety and depression (baseline, 2 years). We performed descriptive statistics, tabulated hospital anxiety and depression (HADS) scores at baseline and 2 years, tabulated coping strategies used at 6 and 18 months. We used univariable and multivariable logistic regression models to examine the relationship between specific coping strategies used at 18 months and anxiety at 24 months. We also performed an exploratory analysis of utilization of coping strategies among those participants with anxiety at 24 months. Results: After excluding women who had not completed the 6 month and/or 18 month surveys and those with metastatic disease at diagnosis, 833 women remained in the analytic sample. The median age at diagnosis was 37 (range: 17-40) years. The cohort was 87% white non-Hispanic, 86% college-educated, 80% married or living as married, and 81% able to pay bills without cutting back. 86% were diagnosed with stage 0, I, or II disease, and over 70% received a bilateral (46%) or unilateral (25%) mastectomy. Social supports were the most commonly reported coping strategies at both 6 and 18 months, with greater than 80% reporting at least moderate use of support from partner, parents, family, and friends. Greater than 40% of women reported partner support as the most helpful coping strategy. The reported use of alcohol/drugs, exercise, and diet changes to cope increased from 6 to 18 months. In multivariable logistic regression, those with moderate or greater reliance on family support and moderate or greater use of exercise for coping at 18 months were less likely to have anxiety at 24 months, while those with moderate or greater reliance on alcohol/drug use as well as reporting taking care of others to cope were more likely to have anxiety. Conclusion: Young breast cancer survivors rely heavily on social supports from family and friends, and they employ more health-related coping strategies during survivorship compared to early after diagnosis. There is a relationship between specific coping strategies (less social support, less exercise, more alcohol/drug use, more taking care of others) and moderate or greater anxiety in early survivorship. Our findings may help inform the development of psychosocial interventions for young breast cancer survivors and their caregivers.

Background: Glaucoma, a progressive disease of the optic nerve in the eye, is the leading cause of irreversible blindness worldwide. A better understanding of the mechanisms involved in the onset and progression of glaucoma is essential for the development of improved diagnostic and therapeutic options for this condition. Glaucoma is thought to result from mechanical, ischemic, and other types of insult to the retinal ganglion cells and their axons, but the pathophysiology of ischemia in glaucoma remains unclear. Optic nerve head (ONH) and peripapillary retina microvasculature pathology as well as abnormalities of systemic microcirculation, specifically the nailfold capillaries, have independently been found in patients with glaucoma compared to control subjects. These findings suggest that measurements of nailfold capillary vasculature may correlate with those of the microvasculature in the eye in patients with primary open-angle glaucoma (POAG). Methods: POAG patients and control subjects underwent swept-source optical coherence tomography angiography (SS-OCTA) and nailfold video capillaroscopy of the fingers. SS-OCTA was used to measure microvascular vessel density and Integrated OCTA by ratio analysis Signal (IOS), a surrogate marker for blood flow, of different layers of the ONH and peripapillary region of the eye with worse functional loss indicated by Humphrey Visual Field mean deviation (HVF MD) in POAG patients and of one randomly selected eye in control subjects. Nailfold capillary density and blood flow were also measured. SS-OCTA parameters (vessel density and IOS) from the eye were plotted against nailfold capillary density and blood flow respectively, and Pearson’s correlation (r) was calculated. Results: 22 POAG patients (HVF MD: -3.5 ± 2.8 dB) and 12 control subjects were included. POAG patients were younger than control subjects (64 ± 10 years vs 70 ± 7 years, p = 0.03) but similar in gender and ethnicity (42% male, 88% Caucasian overall, p > 0.72). Superficial ONH vessel density and flow (IOS) were not significantly different in POAG vs control subjects (p > 0.20). Deep ONH vessel density and flow and peripapillary vessel density and flow were lower in POAG patients (39.1 ± 3.5%, 37.8 ± 5.3%, 37.9 ± 2.6%, 44.1 ± 3.1%, respectively) than control subjects (43.8 ± 5.7%, p = 0.02; 46.0 ± 7.8%, p = 0.005; 43.4 ± 7.6%, p = 0.03; 46.8 ± 3.9%, p = 0.05, respectively). Nailfold capillary density and blood flow were also lower in POAG patients compared to control subjects (8.8 ± 1.0 capillaries/mm vs 9.8 ± 0.9 capillaries/mm, p = 0.009; 19.9 ± 9.4 pL/s vs 33.7 ± 9.8 pL/s, p = 0.0007). There were significant correlations between deep ONH flow and nailfold capillary blood flow (r = 0.43, p = 0.01), peripapillary and nailfold capillary density (r = 0.43, p = 0.01), and peripapillary flow and nailfold capillary blood flow (r = 0.50, p = 0.003). Subgroup analysis was performed to assess the correlation between nailfold capillary and ophthalmic microvasculature measurements in POAG patients (n = 22) and control subjects (n = 12) separately, with significant correlation between nailfold capillary density and deep ONH vessel density in control subjects (r = -0.80, p = 0.002) but no other significant correlations. Additional subgroup analysis of POAG patients divided into high-tension and normal-tension glaucoma groups based on maximum intraocular pressure demonstrated statistically significant correlations between nailfold capillary density and deep ONH vessel density (r = 0.61, p = 0.044) and between nailfold capillary density and flow with peripapillary vessel density and flow respectively in high-tension glaucoma patients (r = 0.60, p = 0.049; r = 0.63, p = 0.04, respectively) but no significant correlations in normal-tension glaucoma patients. Conclusions: POAG patients demonstrated decreased vessel density and flow in the deep ONH and peripapillary region of the eye compared to control subjects; POAG nailfold capillary density and blood flow were also significantly decreased. More importantly, measurements of microvasculature in the deep ONH layer and peripapillary region of the eye significantly correlated with those in terminal nailfold capillaries. These results suggest that there is an association between ophthalmic and systemic microvasculature, which may help to elucidate the pathophysiology of glaucoma as well as potentially provide new biomarkers and novel therapies for this condition in the future.

Abdominal pain is a common symptom that greatly impacts patient quality of life and can be due to a variety of underlying gastrointestinal (GI) diseases. Sensory neurons that are intrinsic and extrinsic to the GI tract play a central role in mediating pain signals in response to various stimuli and yet little is known about their functional properties. My goal is to establish the functional role of the extrinsic innervation of the distal colon and to ultimately develop therapies to treat colonic pain. Here, I began to characterize the extrinsic innervation of the colon. I used mouse genetic tools that have previously been reported to be specific for dorsal root ganglia (DRG) sensory neurons but have not been well defined in the GI tract. I found that genetic tools employing the calcitonin gene-related peptide (CGRP-alpha) gene are specific for labeling the peptidergic DRG neuron extrinsic innervation of the GI tract, whereas Advillin or Phosphoinositide Interacting Regulator of Transient Receptor Potential Channels (Pirt) genetic labeling tools are not specific. I also established behavioral assays in the lab for colonic pain as well as an optogenetic approach to manipulate colon-innervating DRG neuron subtypes, and these are being used to study the functional properties of the CGRP-alpha+ colon innervating DRG neurons and their roles in pain associated with inflammation and distension of the colon. Thus, we begin to characterize the morphology and anatomy of the extrinsic sensory neurons with cell bodies within the DRG that innervate the distal colon, establishing the basis for the future study of sub-populations of these neurons. These studies will be crucial for understanding the perception of different etiologies of abdominal pain, including colitis, distension, and ischemia, and thus hopefully uncovering therapeutic targets.

Background and Objectives: As immune checkpoint inhibitors (ICIs) continue to revolutionize the treatment of countless advanced malignancies, the immune-related adverse events (irAEs) they provoke are likely to increase in prevalence and complexity. Dermatologic irAEs – though among the most common of these unique, multisystem toxicities – nevertheless remain among the most poorly understood. Particularly notable knowledge gaps exist regarding the ways that dermatologic irAEs differ in presentation and therapeutic responsiveness from their classic autoimmune counterparts, the incidence and implications of co-occurring complications from ICI use, and the role and responsibility of dermatology consultation in the management of severe skin toxicities. The underlying goal of the studies presented within this thesis is to build a foundational understanding and provide an evidence-based framework that can begin to fill these knowledge gaps. Methods and Results: To appraise clinically relevant differences between dermatologic irAEs and their idiopathic counterparts, we focused on bullous pemphigoid (BP) and Stevens-Johnson syndrome (SJS) as independent case studies that may offer insight into the broader question. After matching ICI-induced BP (ICI-BP) cases to idiopathic BP (iBP) controls, we found that ICI-BP presents unique diagnostic challenges – prolonged pruritic prodrome (medians, 28 vs7 days, P < .001) and non-bullous presentation – compared to classic iBP that may lead to diagnostic delays as well as a disproportional need for immunosuppression to manage ICI-BP compared to iBP (odds ratio [OR], 9.4; P = .04). Reviewing our multicenter cohort of a severe, generalized bullous eruptions mimicking SJS, we proposed a new terminology for this atypical toxicity pattern - progressive immunotherapy-related mucocutaneous eruption (PIRME). We found that PIRME is notably distinct from SJS in its delayed onset (median, 63 days from ICI initiation), mild initial presentation, rare ocular involvement, benign clinical course, and favorable treatment response. To better understand co-occurring ICI toxicities, we undertook a retrospective cohort study of ICI-induced colitis – the next most common irAE – patients with or without dermatologic irAE. We found that co-occurring ICI-induced rash and colitis is common, may indicate increased risk of developing additional irAEs (OR, 18.5; P = .001), and could be among the earliest clinical markers of improved outcomes, compared to patients with colitis alone, in progression-free survival (hazard ratio [HR], 0.32; P = .001) and overall survival (HR, 0.32; P = .018). Lastly, to quantify the value of involving an inpatient dermatology service, we identified all patients admitted to our institution with dermatologic irAEs. We found that patients who received dermatology consultation were significantly less likely to experience disruptions in their oncologic management (exposure to systemic immunosuppression or need for ICI discontinuation) due to dermatologic irAE, compared to patients without dermatology consultation (OR, 0.03; P = .015). Conclusions: Our research indicates that while dermatologic irAEs may represent uniquely challenging entities that are distinct from their classic autoimmune counterparts, multidisciplinary collaboration and involvement of expert subspecialists remain crucial to the prompt recognition, adequate management, and responsible counseling of these medically complex and increasingly prevalent patients.

Background: Social and economic disparities in the United States have occupied significant media and political attention since the election of 2016. In particular, the country has focused on the lack of understanding of the struggles of Americans who live outside its predominantly liberal cities. In the academic literature, there remains little objective data on the differences between urban and rural Americans, and how these differences have changed over time. The rural-urban divide within health and health care is especially poorly understood. Filling in this knowledge gap would bring evidence-based data to our political discourse, and also potentially better target policy and public health interventions. This study has three aims: a) to describe the demographic and social differences of rural and urban counties; b) to understand differences in health between rural and urban counties along major causes of death; and c) to explore differences in health care access and health delivery systems between urban and rural counties. Methods: This study uses the U.S. Census classification of all 3,142 counties as either urban or rural (defined as mostly rural or all-rural). Data from several sources were gathered to compare demographic characteristics of urban and rural counties, including the racial composition and socioeconomic status. Data from the Institute of Health Metrics and Evaluation was gathered to compare the mortality rates from all-cause mortality, cardiovascular disease, neoplasms, chronic respiratory disease, substance use disorder, and self-harm/interpersonal violence over the period of 1980-2014. Regression analysis was used to assess for differences in outcomes between rural and urban counties, adjusted for observed covariates. Data from the Area Health Resource file was used to investigate measures of access including physician density, hospital bed density, and insurance access. Two-tailed t tests were used to assess the significance of differences. Results: Rural counties on average were less diverse and poorer than their urban counterparts. In unadjusted analysis of mortality data, rural counties had higher mortality rates both overall and in most disease categories; these differences generally widened over the study period. In adjusted analyses, differences in mortality between rural and urban counties were statistically significant in overall mortality and every cause of death measured except for substance use disorder/mental health. Moreover, significant differences were found between urban and rural counties across all measures of physician density (Total physicians, primary care physicians, subspecialists, and psychiatrists) with rural counties consistently having fewer providers per capita. Conclusion: This study found differences across the various dimensions of health and health care between urban and rural counties. In the case of mortality, disparities widened over time and regression analyses demonstrated continued increasing mortality over time in all causes of death. The potential causes of this apparent disparity are unclear and likely multifactorial, ranging from socioeconomic characteristics to issues of access. The data on lower physician density in rural areas provides some understanding of the lack of availability and access to the delivery system in rural areas. Further work should be conducted to better understand these differences and policy/public health interventions should be tailored to address these health disparities if further validated.

Inducing production of fetal hemoglobin (HbF) is a promising therapeutic approach to ameliorate disease severity in beta-thalassemia and sickle cell disease. While studies have characterized individual genetic factors affecting fetal hemoglobin levels and begun to elucidate some underlying mechanisms, a complete understanding of how these elements interact to influence overall fetal hemoglobin expression levels has yet to be achieved. We hypothesize that varying range of fetal hemoglobin expression in the human population is the result of complex genetic architecture involving the interaction between multiple common and rare genetic variants. To interrogate the underlying genetic architecture of this complex and clinically-relevant trait, we have performed large genome-wide association study (GWAS) from two distinct study populations ascertained in different ways: a Thai population and a Swedish population. We genotyped all samples and implemented standard quality-control measures. From the samples and genotypes that passed quality control, we performed an association study for HbF levels using a linear mixed model instantiated through the BOLT-LMM tool. Our initial results have replicated known loci above genome-wide significance levels, including BCL11A, HBS1L-MYB, and HBB. Moreover, several novel loci and rare variants, including unique structural variants, appear to be present in our study. We are integrating whole genome sequencing on a subset of samples and in general population controls to better define these loci using imputation approaches, and we will account for the aggregate contribution of rare variants with large effects, including the structural variants we have identified. This work has tremendous promise to improve our understanding of how HbF levels can vary in populations, characterize underlying mechanisms by which this clinically important factor is regulated, and more generally elucidate how a range of allelic variants can collectively contribute to the genetic architecture of a complex trait.

Objective: Rett syndrome (RTT) is a neurodevelopmental disorder caused by a mutation in the X-linked MECP2 gene. Individuals with RTT typically develop normally until around 18 months of age before undergoing a developmental regression, and the disorder can lead to cognitive, motor, sensory, and autonomic dysfunction. Understanding the mechanism of developmental regression represents a unique challenge when viewed through a neuroscience lens. Are circuits that were previously established erased, and are new ones built to supplant old ones One way to examine circuit-level changes is with the use of electroencephalography (EEG). Using EEG, it is possible to study neural oscillations, and power spectral analysis allows for the decomposition of the EEG signal into frequency components linked to various neurological processes. Previous studies of the EEG in RTT have focused on morphological characteristics, but few have explored spectral power, including power as an index of brain function or disease severity. This study sought to determine if EEG power differs in girls with RTT and typically developing (TD) girls and among girls with RTT based on various clinical characteristics in order to better understand neural connectivity and cortical organization in individuals with this disorder. Methods: Resting state EEG data were acquired from girls with RTT (n=57) and typically developing children (n=37). Clinical data such as disease stage, performance on the Mullen Scales of Early Learning (MSEL), and Clinical Severity Score (CSS) were also collected for girls with RTT. EEG power across several brain regions in numerous previously defined frequency bands was then compared between girls with RTT and TD controls, and power in girls with RTT was compared based on scores on the previously collected clinical and cognitive measures. Additionally, power in all participants was related to participant age, and for a subset of participants, power was compared between a first study visit and a second visit approximately one year later. Results: Girls with RTT demonstrate significantly lower power in the middle-range alpha and beta frequency bands across multiple brain regions. Additionally, girls with RTT that are postregression demonstrate significantly higher power in the lower frequency theta band in the frontal region. EEG power was not found to change significantly over one year in girls with RTT or TD controls. Increased power in these lower frequency bands in girls with RTT trended with lower scores on the MSEL in all domains, and delta and theta power were observed to increase with age in girls with Rett syndrome but decrease with age in TD controls. Interpretation: Increased power in the lower frequency bands, as well as decreased power in the middle frequency bands, is consistent with previous studies demonstrating a “slowing” of the background EEG in RTT and is consistent with EEG patterns seen in other progressive neurological disorders. This increase, particularly in the delta band, could represent abnormal cortical inhibition due to dysfunctional GABAergic signaling or circuitry and could potentially be used as a marker of disease severity due to its associations with more severe RTT phenotypes and worsening with disease progression.