A Highly Sensitive and Robust Method for Hepatitis B Virus Covalently Closed Circular DNA Detection in Single Cells and Serum.

Despite implications of persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the development of hepatocellular carcinoma (HCC), little is known about serum cccDNA in HBV-infected diseases. We developed a cccDNA-selective droplet digital PCR (ddPCR) to assess cccDNA content and dynamics across different stages of HCC development. One hundred forty-seven serum samples and 35 formalin-fixed, paraffin-embedded tumor tissues were derived from patients with HCC or HBV hepatitis/cirrhosis. After specific amplification and selective digestion, probe-based ddPCR was used to quantify cccDNA copy numbers in single cells and clinical samples. The cccDNA in single HepG2.2.15 cells ranged from 0 to 10.8 copies/cell. Compared with non-HCC patients, HCC patients showed a higher cccDNA-positive rate (89.9% versus 53.2%; P = 4.22 × 10-6) and increased serum cccDNA contents (P = 0.002 and P = 0.041 for hepatitis and cirrhosis patients, respectively). Serum cccDNA ranged from 84 to 1.07 × 105 copies/mL. Quantification of serum cccDNA and HBV-DNA was an effective way to discriminate HCC patients from non-HCC patients, with areas under the curve of receiver operating characteristic of 0.847 (95% CI, 0.759-0.935; sensitivity, 74.5%; specificity, 93.7%). cccDNA-selective ddPCR is sensitive to detect cccDNA in single cells and different clinical samples. Combined analysis of serum cccDNA and HBV-DNA may be a promising strategy for HBV-induced HCC surveillance and antiviral therapy evaluation.

Targeted In Situ Biosynthetic Transcriptional Activation in Native Surface-Level Human Articular Chondrocytes during Lesion Stabilization.

OBJECTIVE: Safe articular cartilage lesion stabilization is an important early surgical intervention advance toward mitigating articular cartilage disease burden. While short-term chondrocyte viability and chondrosupportive matrix modification have been demonstrated within tissue contiguous to targeted removal of damaged articular cartilage, longer term tissue responses require evaluation to further clarify treatment efficacy. The purpose of this study was to examine surface chondrocyte responses within contiguous tissue after lesion stabilization. METHODS: Nonablation radiofrequency lesion stabilization of human cartilage explants obtained during knee replacement was performed for surface fibrillation. Time-dependent chondrocyte viability, nuclear morphology and cell distribution, and temporal response kinetics of matrix and chaperone gene transcription indicative of differentiated chondrocyte function were evaluated in samples at intervals to 96 hours after treatment. RESULTS: Subadjacent surface articular cartilage chondrocytes demonstrated continued viability for 96 hours after treatment, a lack of increased nuclear fragmentation or condensation, persistent nucleic acid production during incubation reflecting cellular assembly behavior, and transcriptional up-regulation of matrix and chaperone genes indicative of retained biosynthetic differentiated cell function. CONCLUSIONS: The results of this study provide further evidence of treatment efficacy and suggest the possibility to manipulate or induce cellular function, thereby recruiting local chondrocytes to aid lesion recovery. Early surgical intervention may be viewed as a tissue rescue, allowing articular cartilage to continue displaying biological responses appropriate to its function rather than converting to a tissue ultimately governed by the degenerative material property responses of matrix failure. Early intervention may positively impact the late changes and reduce disease burden of damaged articular cartilage.

Histopomorphic evaluation of radiofrequency mediated débridement chondroplasty.

The use of radiofrequency devices has become widespread for surgical ablation procedures. When ablation devices have been deployed in treatment settings requiring tissue preservation like débridement chondroplasty, adoption has been limited due to the collateral damage caused by these devices in healthy tissue surrounding the treatment site. Ex vivo radiofrequency mediated débridement chondroplasty was performed on osteochondral specimens demonstrating surface fibrillation obtained from patients undergoing knee total joint replacement. Three radiofrequency systems designed to perform débridement chondroplasty were tested each demonstrating different energy delivery methods: monopolar ablation, bipolar ablation, and non-ablation energy. Treatment outcomes were compared with control specimens as to clinical endpoint and histopomorphic characteristics. Fibrillated cartilage was removed in all specimens; however, the residual tissue remaining at the treatment site displayed significantly different characteristics attributable to radiofrequency energy delivery method. Systems that delivered ablation-based energies caused tissue necrosis and collateral damage at the treatment site including corruption of cartilage Superficial and Transitional Zones; whereas, the non-ablation system created a smooth articular surface with Superficial Zone maintenance and without chondrocyte death or tissue necrosis. The mechanism of radiofrequency energy deposition upon tissues is particularly important in treatment settings requiring tissue preservation. Ablation-based device systems can cause a worsened state of articular cartilage from that of pre-treatment. Non-ablation energy can be successful in modifying/preconditioning tissue during débridement chondroplasty without causing collateral damage. Utilizing a non-ablation radiofrequency system provides the ability to perform successful débridement chondroplasty without causing additional articular cartilage tissue damage and may allow for other cartilage intervention success.

Native Chondrocyte Viability during Cartilage Lesion Progression: Normal to Surface Fibrillation.

OBJECTIVE: Early surgical intervention for articular cartilage disease is desirable before full-thickness lesions develop. As early intervention treatments are designed, native chondrocyte viability at the treatment site before intervention becomes an important parameter to consider. The purpose of this study is to evaluate native chondrocyte viability in a series of specimens demonstrating the progression of articular cartilage lesions to determine if the chondrocyte viability profile changes during the evolution of articular cartilage disease to the level of surface fibrillation. DESIGN: Osteochondral specimens demonstrating various degrees of articular cartilage damage were obtained from patients undergoing knee total joint replacement. Three groups were created within a patient harvest based on visual and tactile cues commonly encountered during surgical intervention: group 1, visually and tactilely intact surfaces; group 2, visually intact, tactilely soft surfaces; and group 3, surface fibrillation. Confocal laser microscopy was performed following live/dead cell viability staining. RESULTS: Groups 1 to 3 demonstrated viable chondrocytes in all specimens, even within the fibrillated portions of articular cartilage, with little to no evidence of dead chondrocytes. Chondrocyte viability profile in articular cartilage does not appear to change as disease lesion progresses from normal to surface fibrillation. CONCLUSIONS: Fibrillated partial-thickness articular cartilage lesions are a good therapeutic target for early intervention. These lesions retain a high profile of viable chondrocytes and are readily diagnosed by visual and tactile cues during surgery. Early intervention should be based on matrix failure rather than on more aggressive procedures that further corrupt the matrix and contribute to chondrocyte necrosis of contiguous untargeted cartilage.