Faculty Bibliography

Background/Purpose : T and B cells come together in ectopic lymphoid aggregates in myositis, suggesting that local T:B cell interactions could play a role in disease. T follicular helper cells are increased in circulation in patients with active myositis. We studied circulating Tfh cells from myositis patients using single-cell RNA-sequencing and examined the proximity of Tfh cells to B cells in patient biopsies. Methods : Tfh cells [CD3 + CXCR5 + PD-1 + CXCR3 neg and CD3 + CXCR5 + PD-1 + CXCR3 pos ] cells were sorted from peripheral blood and subsets were identified by chemokine markers to designate Tfh1 and Tfh2/17 cell subsets. RNA sequencing was performed on individual cells of (3 controls and 3 myositis patients) using Fluidigm C1 HT platform, and data were analyzed using a pseudo-temporal ordering using Monocle. Biopsies were stained using the OPAL standardized sequential immunofluorescence method for PD-1, CXCR5, CD19, and CD4 in human muscle, and machine learning was used to map proximity of B cells to all T-cells compared with Tfh cells. Results : We found various subsets within the Tfh pool, corresponding to Tfh1 and Tfh2/17 cells and some cells that looked to be transitioning between states. Tfh2/17 were enriched in myositis patients vs. controls. The Tfh2/17 cells demonstrated a type I interferon signature, while the Tfh1 cells had a type II interferon and proteasome signature. In tissue, we demonstrate Tfh cells in close proximity to B cells in lymphoid aggregates. Conclusion : Tfh cells are present in myositis biopsies juxtaposed to B cells, suggesting productive T:B interactions in the tissue. Tfh subsets in blood from patients demonstrate distinct pathological signatures when compared to controls

As with most cancer types, there remains a subset of B-cell acute lymphoblastic leukaemia (B-ALL) patients who will relapse and succumb to therapy-resistant disease. It is believed that tumour heterogeneity underpins therapy failure leading to a Darwinian model of clonal evolution, however, such studies do not account for the role of the bone marrow microenvironment in supporting leukaemia survival, progression and escape from treatment. Here, we perform single-cell RNA-Sequencing (scRNA-Seq) to generate a comprehensive map of the primary human B-ALL bone marrow immune microenvironment throughout three distinct stages of the human leukemic disease process: diagnosis, remission and relapse. These studies show extensive re-modelling of the immune microenvironment composition and cell-to-cell interactions throughout the course conventional chemotherapy, and uncover a role for inflammatory leukaemia-associated monocytes in promoting B-ALL pathogenesis in vivo. These monocytic subsets are predictive of Ph+ B-ALL patient event-free survival and when targeted in B-ALL animal models, lead to prolonged disease remission. Our profiling of the human B-ALL bone marrow immune microenvironment provides a greater understanding of the potential extrinsic regulators of B-ALL survival and may highlight previously unknown environmental factors influencing immune-based treatment approaches to high-risk B-ALL.

Imiquimod is a topical toll-like-receptor-7 agonist currently used for treating basal cell carcinoma. Recently, imiquimod has demonstrated tumor regression in melanoma and breast cancer skin metastases. However, the molecular perturbations induced by imiquimod in breast cancer metastases have not been previously characterized. Here, we describe transcriptomic profiles associated with responsiveness to imiquimod in breast cancer skin metastases. Baseline and post-treatment tumor samples from patients treated with imiquimod in a clinical trial were profiled using Nanostring technology. Through an integrative analytic pipeline, we showed that tumors from patients who achieved a durable clinical response displayed a permissive microenvironment, substantiated by the upregulation of transcripts encoding for molecules involved in leukocyte adhesion and migration, cytotoxic functions, and antigen presentation. In responding patients, Imiquimod triggered a strong T-helper-1 (Th-1)/cytotoxic immune response, characterized by the coordinated upregulation of Th-1 chemokines, migration of Th-1 and cytotoxic T cells into the tumor, and activation of immune-effector functions, ultimately mediating tumor destruction. In conclusion, we have shown that topical imiquimod can induce a robust immune response in breast cancer metastases, and this response is more likely to occur in tumors with a pre-activated microenvironment. In this setting, imiquimod could be utilized in combination with other targeted immunotherapies to increase therapeutic efficacy.

Sonic Hedgehog (SHH) signaling, a developmental pathway promoting lung mesenchymal expansion and differentiation during embryogenesis, has been increasingly recognized as a profibrotic factor in mature lung, where it might contribute to the pathogenesis of lung fibrosis. Pathway inhibition at the level of the downstream Gli transcription factors Gli1 and Gli2 (by GANT61) ameliorates lung fibrosis in the bleomycin model, whereas inhibition proximally at the level of HH ligand (by anti Hh antibody 5E1) or Smo (by GDC-0449) of the canonical pathway does not, implicating Gli1 and/or Gli2 as a key target. The fact that both the Gli1-labelled cell lineage and Gli1 expressing cells expand during fibrosis formation and contribute significantly to the pool of myofibroblasts in the fibrosis scars suggests a fibrogenic role for Gli1. Therefore to further dissect the roles of Gli1 and Gli2 in lung fibrosis we evaluated Gli1 KO and control mice in the bleomycin model. Monitoring of Gli1^+/+ (n = 12), Gli1^lZ/+ (n = 37) and Gli1^lZ/lZ (n = 18) mice did not reveal differences in weight loss or survival. Lung evaluation at the 21-day endpoint did not show differences in lung fibrosis formation (as judged by morphology and trichrome staining), Ashcroft score, lung collagen content, lung weight, BAL protein content or BAL cell differential count. Our data suggest that Gli1 is not required for bleomycin-induced lung fibrosis.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Background/UNASSIGNED:Mammalian cells express a single functional heparanase, an endoglycosidase that cleaves heparan sulfate and thereby promotes tumor metastasis, angiogenesis, and inflammation. Malignant mesothelioma is highly aggressive and has a poor prognosis because of the lack of markers for early diagnosis and resistance to conventional therapies. The purpose of this study was to elucidate the mode of action and biological significance of heparanase in mesothelioma and test the efficacy of heparanase inhibitors in the treatment of this malignancy. Methods/UNASSIGNED:The involvement of heparanase in mesothelioma was investigated by applying mouse models of mesothelioma and testing the effect of heparanase gene silencing (n = 18 mice per experiment; two different models) and heparanase inhibitors (ie, PG545, defibrotide; n = 18 per experiment; six different models). Synchronous pleural effusion and plasma samples from patients with mesothelioma (n = 35), other malignancies (12 non-small cell lung cancer, two small cell lung carcinoma, four breast cancer, three gastrointestinal cancers, two lymphomas), and benign effusions (five patients) were collected and analyzed for heparanase content (enzyme-linked immunosorbent assay). Eighty-one mesothelioma biopsies were analyzed by H-Score for the prognostic impact of heparanase using immunohistochemistry. All statistical tests were two-sided. Results/UNASSIGNED:Mesothelioma tumor growth, measured by bioluminescence or tumor weight at termination, was markedly attenuated by heparanase gene silencing (P = .02) and by heparanase inhibitors (PG545 and defibrotide; P < .001 and P = .01, respectively). A marked increase in survival of the mesothelioma-bearing mice (P < .001) was recorded. Heparanase inhibitors were more potent in vivo than conventional chemotherapy. Clinically, heparanase levels in patients' pleural effusions could distinguish between malignant and benign effusions, and a heparanase H-score above 90 was associated with reduced patient survival (hazard ratio = 1.89, 95% confidence interval = 1.09 to 3.27, P = .03). Conclusions/UNASSIGNED:Our results imply that heparanase is clinically relevant in mesothelioma development. Given these preclinical and clinical data, heparanase appears to be an important mediator of mesothelioma, and heparanase inhibitors are worthy of investigation as a new therapeutic modality in mesothelioma clinical trials.

PURPOSE/OBJECTIVE:The purpose of this study was to quantify the regional histology of the long head of the biceps tendon (LHBT) and compare the histopathology present to clinical findings in patients with rotator cuff tears and SLAP lesions. METHODS:Prospectively enrolled patients undergoing an open subpectoral LHBT tenodesis in the setting of a rotator cuff (RTC) tear or SLAP lesion. Perioperative data were collected and the excised LHBT was analyzed by a fellowship trained pathologist. Tendons were sectioned into proximal (biceps anchor), middle (bicipital groove), and distal (myotendinous junction) portions. Sections were stained with Movat's pentachrome stain and digitized for analysis. Comparisons were made between the histologic findings present in the setting of a rotator cuff tear with those seen in the setting of a SLAP tear. RESULTS:39 tendons were analyzed: 20 from patients with SLAP lesions (mean age of 44.7 years, range 23-60 years) and 19 from patients with rotator cuff tears (mean age of 58.7 years, range 43-71). Patients with the most pathologic tendons in the bicipital groove were significantly older (59.4 vs. 50.4 years; p < 0.05), reported higher pre-operative VAS scores (6.6 vs. 5.0; p < 0.02), and demonstrated lower pre-operative ASES scores (41.6 vs. 50.7; p < 0.05). The RTC group showed significantly more mucinous degeneration at both the proximal (p < 0.03) and the middle (p < 0.01) tendon portions compared to the SLAP group. In both groups, the portions of proximal tendon showed significantly (p < 0.05) more mucinous degeneration than distal portions. CONCLUSION/CONCLUSIONS:Regional histologic differences exist in the LHBT. Rotator cuff patients showed the most degenerated tendon in the bicipital groove and these patients tended to be older and have higher VAS and lower ASES scores. Surgeons should consider performing a subpectoral biceps tenodesis as the bicipital groove portion of the tendon may be very degenerated, especially in patients with rotator cuff disease. Additional research is warranted to distinguish whether treating the biceps differently in distinct geographic regions affects patient outcomes. LEVEL OF EVIDENCE/METHODS:II.

Sonic Hedgehog (Shh) signaling regulates mesenchymal proliferation and differentiation during embryonic lung development. In the adult lung, Shh signaling maintains mesenchymal quiescence and is dysregulated in diseases such as IPF and COPD. Our previous data implicated a role for Shh in postnatal lung development. Here we report a detailed analysis of Shh signaling during murine postnatal lung development. We show that Shh pathway expression and activity during alveolarization (P0-P14) are distinct from those during maturation (P14-P24). This biphasic pattern is paralleled by the transient presence of Gli1+;alpha-smooth muscle actin (aSMA)+ myofibroblasts in the growing alveolar septal tips. Carefully-timed inhibition of Hedgehog (Hh) signaling during alveolarization defined mechanisms by which Shh influences the mesenchymal compartment. First, interruption of Hh signaling at earlier time points results in increased lung compliance and wall structure defects of increasing severity, ranging from moderately enlarged alveolar airspaces to markedly enlarged airspaces and fewer secondary septa. Second, Shh signaling is required for myofibroblast differentiation: Hh inhibition during early alveolarization almost completely eliminates Gli1+;aSMA+ cells at the septal tips, and Gli1-lineage tracing revealed that Gli1+ cells do not undergo apoptosis after Hh inhibition, but remain in the alveolar septa and are unable to express aSMA. Third, Shh signaling is vital to mesenchymal proliferation during alveolarization, as Hh inhibition decreased proliferation of Gli1+ cells and their progeny. Our study establishes Shh as a new alveolarization promoting factor that might be affected in perinatal lung diseases that are associated with impaired alveolarization.