CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

The Influence of Fat Grafting on Skin Quality in Cosmetic Foot Grafting: A Randomized, Cross-Over Clinical Trial.

BACKGROUND: Pedal fat grafting is a cosmetic procedure to treat the functional and aesthetic sequelae of pedal fat pad atrophy. Fat grafting has been found to mitigate these symptoms, but the exact mechanism is unknown. OBJECTIVES: The authors hypothesized that pedal fat grafting may improve skin quality, accounting for prolonged symptomatic improvement despite loss of grafted fat. METHODS: Patients with pedal atrophy were enrolled in a randomized crossover clinical trial. Group 1 underwent fat grafting upon enrollment with 2-year follow-up. Group 2 was managed conservatively for 1 year then placed into the fat grafting group with 1-year follow-up. Patients underwent pedal ultrasounds to determine thicknesses of the fat pad and dermis, and photographs were taken to assess skin quality. RESULTS: Three men and 20 women with an average age of 63 ± 6 years and an average BMI of 26.0 ± 4.6 kg/m2 were enrolled in the study. Twenty-six feet were injected in Group 1 and 17 were injected in Group 2. Group 1 dermal thickness increased at 6 months post-injection (P < 0.05). This increase persisted through 24 months. Group 2 dermal thickness decreased prior to injection (P < 0.05) but returned to baseline after injection and through 12-month follow-up (P < 0.05). Fat pad thickness returned to baseline by study completion in both groups (P < 0.05). CONCLUSIONS: Pedal fat grafting yielded a significant, sustained increase in dermal thickness, though grafted fat was not retained. Fat grafting may improve skin quality, which could contribute to improved clinical outcomes despite loss of grafted fat.

Metabolic plasticity of human T cells: Preserved cytokine production under glucose deprivation or mitochondrial restriction, but 2-deoxy-glucose affects effector functions.

The strong link between T-cell metabolism and effector functions is well characterized in the murine system but hardly investigated in human T cells. Therefore, we analyzed glycolytic and mitochondrial activity in correlation to function in activated human CD4 and CD8 T cells. Glycolysis was barely detectable upon stimulation but accelerated beyond 24 h, whereas mitochondrial activity was elevated immediately in both T-cell populations. Glucose deprivation or mitochondrial restriction reduced proliferation, had only a transient impact on "on-blast formation" and no impact on viability, IFN-γ, IL-2, IL-4, and IL-10 production, whereas TNF was reduced. Similar results were obtained in bulk T cells and T-cell subsets. Elevated respiration under glucose restriction demonstrated metabolic flexibility. Administration of the glycolytic inhibitor 2-deoxy-glucose suppressed both glycolysis and respiration and exerted a strong impact on cytokine production that persisted for IFN-γ after removal of 2-deoxy-glucose. Taken together, glycolytic or mitochondrial restriction alone compromised proliferation of human T cells, but barely affected their effector functions. In contrast, effector functions were severely affected by 2-deoxy-glucose treatment.

Social policy for child custody: a multidiciplinary framework.

The need for collaboration between mental health professionals and those in the legal system in the development of a preventively-oriented social policy for children involved in custody disputes is discussed. Prior efforts toward this end are examined, and systemic influences that may impede the process are highlighted. Suggestions are offered for improving such efforts, and directions for needed empirical work are elaborated.