Detailed survey of an in vitro intestinal epithelium model by single-cell transcriptomics.

The co-culture of two adult human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an in vitro gut mimic, yet the translatability of insights from such a system to adult human physiological contexts is not fully characterized. Here, we used single-cell RNA sequencing on the co-culture to obtain a detailed survey of cell type heterogeneity in the system and conducted a holistic comparison with human physiology. We identified the intestinal stem cell-, transit amplifying-, enterocyte-, goblet cell-, and enteroendocrine-like cells in the system. In general, the co-culture was fetal intestine-like, with less variety of gene expression compared to the adult human gut. Transporters for major types of nutrients were found in the majority of the enterocytes-like cells in the system. TLR 4 was not expressed in the sample, indicating that the co-culture model is incapable of mimicking the innate immune aspect of the human epithelium.

Consuming Mushrooms When Adopting a Healthy Mediterranean-Style Dietary Pattern Does Not Influence Short-Term Changes of Most Cardiometabolic Disease Risk Factors in Healthy Middle-Aged and Older Adults.

BACKGROUND: Mushrooms are a nutritious food, though knowledge of the effects of mushroom consumption on cardiometabolic risk factors is limited and inconsistent. OBJECTIVE: We assessed the effects of consuming mushrooms as part of a healthy United States Mediterranean-style dietary pattern (MED) on traditional and emerging cardiometabolic disease (CMD) risk factors. We hypothesized that adopting a MED diet with mushrooms would lead to greater improvements in multiple CMD risk factors. METHODS: Using a randomized, parallel study design, 60 adults (36 females, 24 males; aged 46 ± 12 y; body mass index 28.3 ± 2.84 kg/m2, mean ± standard deviation) without diagnosed CMD morbidities consumed a MED diet (all foods provided) without (control with breadcrumbs) or with 84 g/d of Agaricus bisporus (White Button, 4 d/wk) and Pleurotus ostreatus (Oyster, 3 d/wk) mushrooms for 8 wk. Fasting baseline and postintervention outcome measurements were traditional CMD risk factors, including blood pressure and fasting serum lipids, lipoproteins, glucose, and insulin. Exploratory CMD-related outcomes included lipoprotein particle sizes and indexes of inflammation. RESULTS: Adopting the MED-mushroom diet compared with the MED-control diet without mushrooms improved fasting serum glucose (change from baseline -2.9 ± 1.18 compared with 0.6 ± 1.10 mg/dL; time × group P = 0.034). Adopting the MED diet, independent of mushroom consumption, reduced serum total cholesterol (-10.2 ± 3.77 mg/dL; time P = 0.0001). Concomitantly, there was a reduction in high-density lipoprotein (HDL) cholesterol, buoyant HDL2b, and apolipoprotein A1, and an increase in lipoprotein(a) concentrations (main effect of time P < 0.05 for all). There were no changes in other measured CMD risk factors. CONCLUSIONS: Consuming a Mediterranean-style healthy dietary pattern with 1 serving/d of whole Agaricus bisporus and Pleurotus ostreatus mushrooms improved fasting serum glucose but did not influence other established or emerging CMD risk factors among middle-aged and older adults classified as overweight or obese but with clinically normal cardiometabolic health. TRIAL REGISTRATION NUMBER: https://www. CLINICALTRIALS: gov/study/NCT04259229?term=NCT04259229&rank=1.

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super-enhancers in maintaining EBV latency.

We probed the lifecycle of Epstein-Barr virus (EBV) on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350- LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350+ LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+ LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV-associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

IL-1ß promotes IL-9-producing Th cell differentiation in IL-2-limiting conditions through the inhibition of BCL6.

IL-9-producing CD4+ T helper cells, termed Th9 cells, differentiate from naïve precursor cells in response to a combination of cytokine and cell surface receptor signals that are elevated in inflamed tissues. After differentiation, Th9 cells accumulate in these tissues where they exacerbate allergic and intestinal disease or enhance anti-parasite and anti-tumor immunity. Previous work indicates that the differentiation of Th9 cells requires the inflammatory cytokines IL-4 and TGF-ß and is also dependent of the T cell growth factor IL-2. While the roles of IL-4 and TGF-ß-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the Il9 locus remains less clear. We show here that murine Th9 cells that differentiate in IL-2-limiting conditions exhibit reduced IL-9 production, diminished NF-kB activation and a reduced NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is required for optimal NF-kB activation in Th9 cells. Interestingly, both IL-9 production and the NF-kB transcriptional signature could be rescued by addition of the NF-kB-activating cytokine IL-1ß to IL-2-limiting cultures. IL-1ß was unique among NF-kB-activating factors in its ability to rescue Th9 differentiation as IL-2 deprived Th9 cells selectively induced IL-1R expression and IL-1ß/IL-1R1 signaling enhanced the sensitivity of Th9 cells to limiting amounts of IL-2 by suppressing expression of the Th9 inhibitory factor BCL6. These data shed new light on the intertwined nature of IL-2 and NF-kB signaling pathways in differentiating Th cells and elucidate the potential mechanisms that promote Th9 inflammatory function in IL-2-limiting conditions.

Granzyme-Producing CD4 T Cells in Cancer and Autoimmune Disease.

CD4 T cells play important roles in promoting protective immunity and autoimmune disease. A great deal of attention has been given to the differentiation and function of subsets of cytokine-producing CD4 T cells (i.e., Th1, Th2, and Th17 cells) in these settings. However, others have also observed the accumulation of granzyme-producing CD4 T cells in tumors and in autoimmune patients that are distinct from their cytokine-producing counterparts. Despite the relatively large numbers of granzyme-producing cells in diseased tissues, their roles in driving disease have remained enigmatic. This review will focus on the phenotype(s) and roles of granzyme-producing CD4 T cells in cancer and autoimmunity. We will also examine how granzyme-producing cells interact with current therapeutics and speculate how they may be targeted during disease.

Repair of Rotator Cuff Tears in the Elderly: Does It Make Sense? A Systematic Review.

BACKGROUND: The indications and outcomes for rotator cuff repair (RCR) among patients ≥70 years old are not widely reported. Many active patients in this age range desire a joint-preserving option, and several small series reported successful clinical outcomes after RCR among patients aged ≥70 years. PURPOSE: To systematically review the literature on the outcomes of RCR among patients ≥70 years old. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: A systematic review of the literature was performed following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The electronic databases of PubMed and Cochrane were used for the literature search. The quality of the included studies was evaluated according to the Coleman Methodology Score. Studies in English evaluating repair of full-thickness rotator cuff tears among patients aged ≥70 years were included. RESULTS: Eleven studies were reviewed, including 680 patients (694 shoulders) who were treated with arthroscopic and/or open RCR with a mean follow-up of 24.2 months (range, 12-40.8 months). Forty patients were lost to follow-up, leaving 654 shoulders with outcome data. This age group demonstrated a significant increase in clinical and functional outcomes after RCR with high satisfaction. American Shoulder and Elbow Surgeons scores showed an improvement from 44.2 (range, 35.4-56) preoperatively to 87.9 (range, 84-90.3) postoperatively, while Constant scores improved from 41.7 (range, 22.6-53.6) to 70.8 (range, 58.6-76). Postoperative imaging evaluation was performed on 513 shoulders, revealing a retear rate of 27.1% (139 shoulders). There were 45 retears after open repair and 94 after arthroscopic repair. The difference in retear rate among patients receiving arthroscopic repairs was not significantly different than open repairs (P = .831). Pain according to a visual analog scale improved from 5.5 (range, 4.6-6.4) preoperatively to 1.3 (range, 0.5-2.3) postoperatively. CONCLUSION: RCR among patients ≥70 years old shows high clinical success rates with good outcomes and overall excellent pain relief. Although patients in this age group have a high potential for retear or persistent defects on imaging studies, RCR offers a joint-preserving option with significant functional and clinical improvement for the appropriately indicated patient.

Biomechanical and Histopathological Analysis of a Retrieved Dermal Allograft After Superior Capsule Reconstruction: A Case Report.

The purpose of this study was to evaluate biomechanical and histopathological results of a retrieved acellular human dermal allograft (AHDA) after superior capsule reconstruction (SCR). A 67-year-old man with pseudoparalysis was treated with SCR for an irreparable posterosuperior rotator cuff tear. The patient failed clinically 4.5 months postoperatively and elected to undergo reverse total shoulder arthroplasty (RTSA). At the time of RTSA, the AHDA was harvested. Biomechanical and histopathologic analyses were performed and compared to native grafts. Failure loads for the explanted graft and native grafts 1 and 2 were 158, 790, and 749 N, respectively. The stiffness values were 20.2, 73, and 100.5 N/mm. The displacement at failure for each graft was 10.1, 27.9, and 17.0 mm. Hematoxylin and eosin and Masson's trichrome staining revealed the presence of cells in all portions of the AHDA. The medial portion presented extensive cellular infiltration, the middle portion moderate, and the lateral portion the least infiltration. Although the only identifiable cells in the lateral portions were found in pockets on the interior of the graft, cells were mainly localized on the exterior. Postoperative cell incorporation could be found in acellular dermal allograft after SCR. However, biomechanical properties in the early postoperative phase were inferior compared with unimplanted allografts.

Operative treatment of torsional humeral shaft fractures in throwers leads to an earlier return to sport: a survey of expert shoulder and elbow surgeons.

PURPOSE: To determine the management of torsional humeral shaft fractures in a group of expert shoulder and elbow surgeons and analyse the rate of return to sport of these throwing athletes. METHODS: A survey was sent to all physician members of two prominent sports medicine professional associations: the American Shoulder and Elbow Surgeons and the Herodicus Society. Due to the rare nature of this injury, a historical survey of management and return to play was performed to allow analysis of trends in treatment and return to play after both non-operative and operative management. RESULTS: The survey was emailed to 858 physician members. Out of the 95 respondents, 35 surgeons indicated they had treated ≥ 1 torsional humeral shaft fractures in throwing athletes (average 1.7 per surgeon). A total of 72 fractures were recorded with an average age of 20.4 years and the majority being male (68/72). Eighty-one percent (58/72) of the fractures were classified as simple spiral. Sixty-one percent (44/72) of the fractures were treated non-operatively, while 35% (25/72) of the fractures were treated by open reduction and internal fixation (ORIF). Patient age, return to sport rate and level, type of fracture, and fracture healing time did not significantly differ based on treatment type. Average time to return to sport was significantly shorter for patients who underwent ORIF compared to non-operative treatment (p = 0.001). Overall, 48 (92.3%) of the 52 athletes returned to sport, with 84% (36/43) returning to the same level of play. CONCLUSION: Torsional humeral shaft fractures in throwers are most commonly seen in young men and can be treated both operatively and non-operatively with overall similar results for healing time, rate of non-union, and return to sport. The only significant difference in the groups was an earlier return to sports in those fixed surgically, however, operative intervention also yielded a higher complication rate. Regardless of the treatment method, the overall rate of return to play was moderate. These finding are clinically relevant and can assist physicians with decision making for treatment and can help when advising throwers of appropriate expectations for recovery after this injury. LEVEL OF EVIDENCE: IV.

Conjoint Tendon Tenotomy for Glenoid Exposure in the Setting of Previous Coracoid Transfer.

Surgical exposure of the glenoid after previous coracoid process transfer is technically challenging as a result of distorted anatomy, obliterated soft-tissue planes, and adhesive scar tissue, which poses additional risk to adjacent neurovascular structures. The purpose of this article is to present a technique for glenoid exposure following coracoid transfer that involves tenotomy of the conjoint tendon to minimize the risk for neurovascular injury while leaving the well-healed coracoid bone graft in place.