Assessment of the inherent chondrogenic potential of human articular cartilage-derived chondroprogenitors in pellet culture using a novel whole pellet processing approach.

Purpose: Cartilage-derived chondroprogenitors have been reported to possess the biological potential for cartilage repair. However, its inherent chondrogenic potential in pellet culture needs evaluation. In-vitro cartilage regeneration models based on pellet cultures have been employed to evaluate the chondrogenic potential of stem cells. Evaluation of the degree of differentiation routinely involves paraffin embedding, sectioning, and immunohistochemical staining of the pellet. However, since chondrogenic differentiation is commonly non-uniform, processing random sections could lead to inaccurate conclusions. The study aimed at assessing the inherent lineage bias of chondroprogenitors with and without chondrogenic induction, using a novel whole pellet processing technique. Methods: Human chondroprogenitors (n=3) were evaluated for MSC markers and processed in pellet cultures either with stromal medium (uninduced) or chondrogenic differentiation medium (induced) for 28 days. The whole pellets and the conventional paraffin-embedded sectioned pellets were subjected to Collagen type II immunostaining and assessed using confocal laser microscopy. The staining intensities of the whole pellet were compared to the paraffin sections and revalidated using qRT-PCR for COL2A1 expression. Results: Uninduced and induced pellets displayed Collagen type II in all the layers with comparable fluorescence intensities. COL2A1 expression in both pellets was comparable to confocal results. The study demonstrated that uninduced chondroprogenitors in pellet culture possess promising inherent chondrogenic potential. Confocal imaging of whole pellets displayed different degrees of chondrogenic differentiation in the entire pellet, thus its probable in-vivo behavior. Conclusion: The novel approach presented in this study could serve as an efficient in-vitro alternative for understanding translational application for cartilage repair.

Migratory chondroprogenitors retain superior intrinsic chondrogenic potential for regenerative cartilage repair as compared to human fibronectin derived chondroprogenitors.

Cell-based therapy for articular hyaline cartilage regeneration predominantly involves the use of mesenchymal stem cells and chondrocytes. However, the regenerated repair tissue is suboptimal due to the formation of mixed hyaline and fibrocartilage, resulting in inferior long-term functional outcomes. Current preclinical research points towards the potential use of cartilage-derived chondroprogenitors as a viable option for cartilage healing. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CP) and migratory chondroprogenitors (MCP) exhibit features suitable for neocartilage formation but are isolated using distinct protocols. In order to assess superiority between the two cell groups, this study was the first attempt to compare human FAA-CPs with MCPs in normoxic and hypoxic culture conditions, investigating their growth characteristics, surface marker profile and trilineage potency. Their chondrogenic potential was assessed using mRNA expression for markers of chondrogenesis and hypertrophy, glycosaminoglycan content (GAG), and histological staining. MCPs displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), with normoxia-MCP exhibiting significantly higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), thus showing superior potential towards cartilage repair. Upon chondrogenic induction, normoxia-MCPs also showed significantly higher levels of GAG/DNA with stronger staining. Focused research using MCPs is required as they can be suitable contenders for the generation of hyaline-like repair tissue.

Racial disparities in surgical outcomes for benign thyroid disease.

INTRODUCTION: Previous studies have shown racial disparities in surgical outcomes in malignant thyroid disease. We hypothesize that minority groups have a higher incidence of postoperative complications following surgery for benign thyroid disease. METHODS: Using NSQIP (2016-2017), patients (>17 years) undergoing thyroid surgery for benign disease were identified. Outcomes included neck hematoma, recurrent laryngeal nerve (RLN) injury, and hypocalcemia. Multivariate analysis was performed controlling for patient factors. RESULTS: 6817 patients were identified. Postoperative outcomes were neck hematoma (2.0%), RLN injury (5.2%), and significant hypocalcemia (4.9%). Compared to White patients, Black patients had higher chance of neck hematoma (OR 2.32, 95% CI 1.51-3.55) and RLN injury (OR 1.97, 95% CI 1.53-2.55) while Asian patients had significantly greater odds of RLN injury (OR 1.88, 95% CI 1.15-3.06). CONCLUSION: Minority compared to White patients are more likely to have significant postoperative complications which indicates racial disparities in the surgical treatment for benign thyroid disease.

Preliminary Studies on Immune Response and Viral Pathogenesis of Zika Virus in Rhesus Macaques.

Zika Virus (ZIKV) is primarily transmitted through mosquito bites. It can also be transmitted during sexual intercourse and in utero from mother to fetus. To gain preliminary insight into ZIKV pathology and immune responses on route of transmission, rhesus macaques (RMs) were inoculated with ZIKV (PRVABC59) via intravaginal (IVAG) (n = 3) or subcutaneous (sub Q) (n = 2) routes. Systemic ZIKV infection was observed in all RMs, regardless of the route of inoculation. After 9 days postinfection (dpi), ZIKV was not detected in the plasma of IVAG- and sub-Q-inoculated RMs. Importantly, RMs harbored ZIKV up to 60 dpi in various anatomical locations. Of note, ZIKV was also present in several regions of the brain, including the caudate nucleus, parietal lobe, cortex, and amygdala. These observations appear to indicate that ZIKV infection may be systemic and persistent regardless of route of inoculation. In addition, we observed changes in key immune cell populations in response to ZIKV infection. Importantly, IVAG ZIKV infection of RMs is associated with increased depletion of CD11C hi myeloid cells, reduced PD-1 expression in NK cells, and elevated frequencies of Ki67⁺ CD8⁺ central memory cells as compared to sub Q ZIKV-infected RMs. These results need to interpreted with caution due to the small number of animals utilized in this study. Future studies involving large groups of animals that have been inoculated through both routes of transmission are needed to confirm our findings.

Automated low flow pump system for the treatment of refractory ascites: a multi-center safety and efficacy study.

BACKGROUND & AIMS: Refractory ascites (RA) affects 10% of patients with advanced cirrhosis and ascites. Usual therapy includes large volume paracentesis, and in selected patients, a transjugular portosystemic shunt (TIPS). These therapies may be associated with increased morbidity: paracentesis may induce circulatory dysfunction and impair quality of life and TIPS may induce encephalopathy and is associated with increased mortality in patients with severe liver dysfunction. We present the results of a multicenter, non-randomized trial to assess the safety and efficacy of a new automated pump system for treatment of RA. METHODS: Forty patients at 9 centers (February 2010-June 2011) received an implanted pump for the automated removal of ascites from the peritoneal cavity into the bladder, from where it was eliminated through normal urination. Patients were followed-up for 6months. The primary study outcome was safety. Secondary outcomes included recurrence of tense ascites and pump performance. RESULTS: Surgical complications occurred early in the study and became less frequent. The pump system removed 90% of the ascites and significantly reduced the median number of large volume paracentesis per month [3.4 (range 1-6) vs. 0.2 (range 0-4); p <0.01]. Cirrhosis-related adverse events decreased along follow-up. CONCLUSIONS: The automated pump seems an efficacious tool to move out ascites from the peritoneal cavity to the bladder. Its safety is still moderate, but a broad use in different countries will improve the surgical technique as well as the medical surveillance. A prospective randomized clinical trial vs. large volume paracentesis is underway to confirm these preliminary results.

Intrathecal delivery of fluorescent labeled butyrylcholinesterase to the brains of butyrylcholinesterase knock-out mice: visualization and quantification of enzyme distribution in the brain.

Exogenously delivered butyrylcholinesterase (BChE) has proven to be an efficient bioscavenger against highly toxic organophosphorus poisons and nerve agents. The scavenger properties of BChE when delivered via intramuscular, intravenous, subcutaneous, or intraperitoneal routes are limited to the body's peripheral sites because the 340 kDa enzyme does not cross the blood-brain barrier (BBB). Overcoming the BBB is an important step toward evaluating the neuroprotective properties of BChE within the central nervous system (CNS). This study examines the feasibility of delivering BChE to the brain and spinal cord by intrathecal (IT) injection. Mice completely devoid of BChE were injected intrathecally with either BChE (80 units) that was labeled with near-infrared fluorescent dye (BChE/IRDye) or a molar equivalent amount of carboxylate dye. The BChE/IRDye and carboxylate dye were tracked using an in vivo imaging system demonstrating the real-time distribution of BChE in the brain and the residence time in the brain and spinal cord through 25 h post-dosing. BChE/IRdye levels in the brain peaked at 6h post-dosing. BChE enzyme activity was quantified in plasma and brain sections by BChE activity assays of plasma and of perfused tissues. Average BChE activity levels were 0.6 units/g in the brains of mice treated with BChE/IRDye at 4h post-dosing. Intense fluorescent signal in the cortex, dentate gyrus and ventricles of the brain at 25 h post-dosing was visualized by confocal microscopy and the presence of BChE was confirmed with activity assays of frozen sections. This procedure proved to be an efficient, safe and rapid method to deliver BChE to the CNS of mice, providing a research tool for determining neural protection by BChE following OP exposure.

Heart xenograft survival with chimeric pig donors and modest immune suppression.

OBJECTIVE: To assess the use of donor pigs with cellular chimerism for prevention of acute rejection with modest immune suppression. The clinical use of pig organ xenografts is currently precluded by severe acute rejection, which resists standard immune suppression. SUMMARY BACKGROUND DATA: For long-term survival of pig organ xenografts, immune suppression significantly greater than used with allografts would currently be necessary, leaving the recipient immune deficient and at increased risk for infections. Induction of immune tolerance and tissue accommodation could enhance xenograft survival but would lead to complications and frequent graft failure. Induction of cellular chimerism within the donor pigs, however, could accomplish these goals before transplantation, significantly reducing the risk. METHODS: Marrow cells from sheep were infused into fetal pigs. Heart xenografts from chimeric or nonchimeric pigs were transplanted heterotopically into recipient sheep, simultaneous with infusion of splenocytes. Posttransplant suppression consisted of cyclosporine and tapered corticosteroids, comparable with allotransplants. RESULTS: All of the control grafts (n = 12) were rejected by acute vascular rejection in 4 to 8 days. In contrast, only one episode of vascular rejection was observed in the experimental group (n = 13). Four experimental recipients had an episode of moderate diffuse cellular rejection (grade 3) and one had moderate focal cellular rejection (grade 2). Each episode responded to pulse steroids. Seven grafts showed no significant rejection. There was little evidence of immune deficiency, infection, or toxicity. CONCLUSIONS: Acute vascular rejection was prevented in a large animal model without the need for severe immune suppression.