Faculty Bibliography

Rationale Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with increased Th17 inflammatory phenotype. In this study we evaluated the microbial and host immune response dynamics after aspiration with a oral commensals using a preclinical mouse model. Methods Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of sacrifice. Genetic background of mice included WT, MyD88 knock out and STAT3C. Measurements 16S rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host transcriptome sequencing was used to characterize host immune phenotype. Main Results While MOC aspiration correlated with lower airway dysbiosis that resolved within five days, it induced an extended inflammatory response associated with IL17-producing T-cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration prior to a respiratory challenge with S. pneumoniae led to a decreased in host's susceptibility to this pathogen. Conclusions Thus, in otherwise healthy mice, a single aspiration event with oral commensals are rapidly cleared from the lower airways, but induce a prolonged Th17 response that secondarily decreased susceptibility to respiratory pathogens. Translationally, these data implicate an immuno-protective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower airway pathogens.

Hidradenitis suppurativa (HS) is a severe chronic inflammatory skin disease affecting human apocrine sweat gland-bearing skin regions. The overall prevalence of HS ranges from 0.05-4.1% with higher occurrence among females and African Americans, and strong associations with smoking and obesity. One unique feature of HS is the development of highly immunogenic keratinized sinus tracts that grow deeply in the dermis which further complicate HS pathogenesis and treatment. Using single cell transcriptomic analyses, we finely dissected different epidermal cell types in the HS lesional skin and revealed significant dysregulation of skin barrier function in the sinus tracts. We demonstrated that sinus tract keratinocytes exhibit dual cell fates of surface epidermis and skin appendages, and derived from progenitors in infundibulum of the apocrine-pilosebaceous unit. By analyzing ligand-receptor expressions between different skin appendages and immune cells, we highlighted Th17 and TNF responses at early and late stages during HS progression, respectively. Our work provides unprecedented understanding of pathological epidermal remodeling in human inflammatory diseases and important implications for therapeutics.
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Purpose/UNASSIGNED:To evaluate outcomes for a combined osteoligamentous reconstruction technique for Neer Type IIB clavicle fractures. Methods/UNASSIGNED:Patients with Neer Type IIB clavicle fractures treated with combined clavicular locking plate and coracoclavicular ligament suture reconstruction were identified. Demographics, clinical outcomes, and radiographic outcomes were collected. Results/UNASSIGNED:Twenty-four patients with mean 13 months of follow-up were included. Bony union and normal radiographic coracoclavicular relationship were achieved in 23 (96%) patients. The mean UCLA Shoulder score was 33.3. Three (13%) complications occurred. Discussion/UNASSIGNED:The combined osteoligamentous reconstruction approach as described is a successful option for treating Neer Type IIB clavicle fractures.

OBJECTIVE:Endovascular abdominal aortic aneurysm repair (EVAR) is preferred to open surgical repair (OSR) for the treatment of abdominal aortic aneurysm (AAA) in high-risk patients. We sought to compare perioperative and long-term outcomes for EVAR in patients designated as unfit for OSR using a large national dataset. METHODS:The Vascular Quality Initiative database collected from 2013 to 2019 was queried for patients undergoing elective EVARs for AAA > 5cm. The patients were stratified into two cohorts based on the suitability for OSR (FIT vs. UNFIT). Primary outcomes included perioperative (in-hospital) major adverse events, perioperative mortality, and mortality at 1 and 5 years. Patient demographics and postoperative outcomes were analyzed to identify predictors of perioperative and long-term mortality. RESULTS:Of 16,183 EVARs, 1,782 patients were deemed unfit for OSR. The UNFIT cohort was more likely to be older and female, with higher proportions of HTN, CAD, CHF, COPD, and larger aneurysm diameter. Postoperatively, the UNFIT cohort was more likely to have cardiopulmonary complications (6.5% vs. 3%; P<.001), with higher perioperative mortality (1.7% vs. 0.6%; P<.001) and 1 and 5-year mortality (13% and 29% UNFIT vs. 5% and 14% FIT; P<.001). Subgroup analysis within the UNFIT cohort revealed those deemed unfit due to hostile abdomen had significantly lower 1 and 5-year mortality (6% and 20%) compared to those unfit due to cardiopulmonary compromise and frailty (14% and 30%; P=.451). Reintervention-free survival at 1 and 5-years was significantly higher in the FIT cohort (93% and 82%) as compared to the UNFIT cohort (85% and 68%; P<.001). Designation as unfit for OSR was an independent predictor of both perioperative (OR 1.59; 95% CI, 1.03-2.46; P=.038) and long-term mortality (HR 1.92; 95% CI, 1.69-2.17; P<.001). Advanced age (OR 2.91; 95% CI, 1.28-6.66; P=.011) was the strongest determinant of perioperative mortality while ESRD (HR 2.51; 95% CI, 1.78-3.55; P<.001) was the strongest predictor of long-term mortality. Statin (HR 0.77; 95% CI, 0.69-0.87; P<.001) and ACE inhibitor (HR 0.83; 95% CI, 0.75-0.93; P<.001) were protective of long-term mortality. CONCLUSION/CONCLUSIONS:Despite low perioperative mortality, long-term mortality of those designated by the operating surgeons as unfit for OSR was rather high in patients undergoing elective EVARs, likely due to the competing risk of death from their medical frailty. Unfit designation due to hostile abdomen did not confer any additional risks after EVAR. Judicious estimation of the patient's life expectancy is essential when considering treatment options in this subset of patients deemed unfit for OSR.

Hidradenitis suppurativa (HS) is a prevalent inflammatory skin disease. HS causes deep, painful, recurrent abscesses. African Americans and females are at an increased risk. A lack of effective therapies and limited knowledge about HS pathogenesis contribute to unmet needs. Unlike other common inflammatory skin diseases, there has never been a genome-wide association study (GWAS) conducted for HS. Here, we performed a first GWAS for HS using data from the eMERGE network of electronic health record linked biorepositories (project NT227). We used HS diagnosis codes to identify cases and controls. We estimated ancestry with principal component analysis using a set of 40,156 SNPs. Our final cohort consisted of 600 HS cases and 82,611 controls with comparable multi-ethnic ancestry (lambda=1.005). Our cohort recapitulated HS race and gender predilections with genetically African female participants accounting for 35% of cases, but only 10% of controls. Genotype data for 6 million variants was tested for association, adjusting for five principal components. No locus exceeded our threshold for statistical significance. Importantly, there was no evidence for HLA association supporting classification of HS as inflammatory rather than autoimmune. Several loci approached the significance threshold, suggesting that an expansion in cohort size is needed to provide adequate power to detect associations. Interestingly, the lead SNP at one of the most significant loci (rs11075745; p=8x10^-7) is an eQTL for NFAT5, a mediator of NOTCH signaling whose expression is downregulated in HS lesional skin relative to patient-matched nonlesional skin. The risk allele influences expression in tissue specific manner. Our group is constructing multi-ethnic replication cohorts that will allow us to expand this study in the near future.
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A balanced intake of macronutrients-protein, carbohydrate and fat-is essential for the well-being of organisms. An adequate calorific intake but with insufficient protein consumption can lead to several ailments, including kwashiorkor¹. Taste receptors (T1R1-T1R3)² can detect amino acids in the environment, and cellular sensors (Gcn2 and Tor)³ monitor the levels of amino acids in the cell. When deprived of dietary protein, animals select a food source that contains a greater proportion of protein or essential amino acids (EAAs)⁴. This suggests that food selection is geared towards achieving the target amount of a particular macronutrient with assistance of the EAA-specific hunger-driven response, which is poorly understood. Here we show in Drosophila that a microbiome-gut-brain axis detects a deficit of EAAs and stimulates a compensatory appetite for EAAs. We found that the neuropeptide CNMamide (CNMa)⁵ was highly induced in enterocytes of the anterior midgut during protein deprivation. Silencing of the CNMa-CNMa receptor axis blocked the EAA-specific hunger-driven response in deprived flies. Furthermore, gnotobiotic flies bearing an EAA-producing symbiotic microbiome exhibited a reduced appetite for EAAs. By contrast, gnotobiotic flies with a mutant microbiome that did not produce leucine or other EAAs showed higher expression of CNMa and a greater compensatory appetite for EAAs. We propose that gut enterocytes sense the levels of diet- and microbiome-derived EAAs and communicate the EAA-deprived condition to the brain through CNMa.

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.

Rationale:Malignant pleural mesothelioma(MPM) has a poor prognosis with median survival of 12-24 months. We are not aware of prior studies examining the immune microenvironment in tumor draining lymph nodes (TDLN) in MPM. Our aim is to compare the tumor microenvironment(TME) and the microenvironment of TDLN. We hypothesize that the TME will display an immunosuppressive phenotype reflected in the TDLN.
Method(s):We performed digital spatial profiling(DSP) using the GeoMx (NanoString) platform on stored primary tumor and nodal biopsy specimens from 3 patients from our tumor bank. Samples from both primary tumor and lymph nodes were sectioned and labeled with pancytokeratin (CK). Tissue was then classified as "tumor" or "nontumor" using semi-automated segmentation based on pan-Cytokeratin (panK) labeling. The slides were then labeled with antibodies to 58 selected markers, with each unique antibody attached to a respective oligonucleotide. The tissue was exposed to UV light separately for tumor and non-tumor regions, cleaving the oligonucleotides from the attached antibodies. The oligonucleotides from the separate tumor and non-tumor regions were quantified using nCounter (NanoString).
Result(s):The non-neoplastic regions of the primary tumor contained higher expression of proteins associated with inflammatory cells including helper T-cells, cytotoxic T-cells, B-cells, macrophages, neutrophils, natural killer cells(Table 1). Furthermore, there was greater expression of immune checkpoint proteins, PD-L1 and CTLA-4, and CD163 and CD14, proteins associated with immunosuppressive macrophages, in the non-neoplastic region compared to the neoplastic region of the tumoe(Table 1). TDLNs contained similar levels of expression of lymphocyte markers, including those delineating cytotoxic T-cells and helper T-cells, as the primary tumor(Table 1). Despite this, TME expressed higher levels of T-cell exhaustion and immunsupression markers (FOXP3, LAG3, PD-1, CTLA-4) than TDLN(Table 1).
Conclusion(s):DSP is feasible in Formalin-fixed paraffin embedded (FFPE) mesothelioma specimens, providing a method for using quantitative immunopathology to study corresponding immune microenvironments. In our study, the non-tumor region of the primary tumor contained macrophages, lymphocytes, natural killer cells, and cancer-associated fibroblasts consistent with prior descriptions of the mesothelioma TME. Increased expression of immune checkpoint molecules in the non-tumor region suggests an immunosuppressive TME. TDLNs demonstrated similar lymphocyte markers, but without corresponding immune checkpoint expression of t suggesting the immunosuppressive phenotype of the TME may not be reflected in TDLNs. This pilot study is the first to use DSP to preliminarily characterize TDLNs in mesothelioma. We plan to apply this approach to stored additional MPM and NSCLC specimens to gain an in-depth understanding of the relationship between TME and TDLN