Urea-based amino sugar agent clears murine liver and preserves protein fluorescence and lipophilic dyes.

Five established clearing protocols were compared with a modified and simplified method to determine an optimal clearing reagent for three-dimensionally visualizing fluorophores in the murine liver, a challenging organ to clear. We report successful clearing of whole liver lobes by modification of an established protocol (UbasM) using only Ub-1, a urea-based amino sugar reagent, in a simpler protocol that requires only a 24-h processing time. With Ub-1 alone, we observed sufficiently preserved liver tissue structure in three dimensions along with excellent preservation of fluorophore emissions from endogenous protein reporters and lipophilic tracer dyes. This streamlined technique can be used for 3D cell lineage tracing and fluoroprobe-based reporter gene expression to compare various experimental conditions.

Prominin-1-expressing hepatic progenitor cells induce fibrogenesis in murine cholestatic liver injury.

Cholestatic liver injury is associated with intrahepatic biliary fibrosis, which can progress to cirrhosis. Resident hepatic progenitor cells (HPCs) expressing Prominin-1 (Prom1 or CD133) become activated and participate in the expansion of cholangiocytes known as the ductular reaction. Previously, we demonstrated that in biliary atresia, Prom1(+) HPCs are present within developing fibrosis and that null mutation of Prom1 significantly abrogates fibrogenesis. Here, we hypothesized that these activated Prom1-expressing HPCs promote fibrogenesis in cholestatic liver injury. Using Prom1CreERT2-nLacZ/+ ;Rosa26Lsl-GFP/+ mice, we traced the fate of Prom1-expressing HPCs in the growth of the neonatal and adult livers and in biliary fibrosis induced by bile duct ligation (BDL). Prom1-expressing cell lineage labeling with Green Fluorescent Protein (GFP) on postnatal day 1 exhibited an expanded population as well as bipotent differentiation potential toward both hepatocytes and cholangiocytes at postnatal day 35. However, in the adult liver, they lost hepatocyte differentiation potential. Upon cholestatic liver injury, adult Prom1-expressing HPCs gave rise to both PROM1(+) and PROM1(-) cholangiocytes contributing to ductular reaction without hepatocyte or myofibroblast differentiation. RNA-sequencing analysis of GFP(+) Prom1-expressing HPC lineage revealed a persistent cholangiocyte phenotype and evidence of Transforming Growth Factor-ß pathway activation. When Prom1-expressing cells were ablated with induced Diphtheria toxin in Prom1CreERT-nLacZ/+ ;Rosa26DTA/+ mice, we observed a decrease in ductular reactions and biliary fibrosis typically present in BDL as well as decreased expression of numerous fibrogenic gene markers. Our data indicate that Prom1-expressing HPCs promote biliary fibrosis associated with activation of myofibroblasts in cholestatic liver injury.

Sebaceous Nevus of the Scalp.

Excisions of scalp nevus sebaceous (NS) presents a unique challenge due to limited soft tissue laxity, hair-bearing skin, and convex surfaces which often leave the surgeon and patient underwhelmed with the reconstructive outcome. In this study, the authors conducted an institutional review board-approved retrospective review of patients who underwent excision of pathologically proven scalp primary NS from 2003 to 2017 at our institution to better define the reconstructive outcomes and options for treatment of pediatric scalp NS. 92 patients were included in the study, 54 males (58.7%) and 38 females (41.3%). The average age at surgery was 7.24 years (0.5-16.0; SD 4.7). Local tissue undermining/galeal scoring with primary closure (LTUGS) was used for lesions with average surface area of 3.6 cm, rotational or transposition flaps (RF/TF) for lesions averaging 4.3 cm, completed serial excision for lesions averaging 13.9 cm, and tissue expansion (TE) for lesions averaging 21 cm (P <0.001). One or more poor outcomes were experienced by 35 patients (38%), with a significant difference between the surgical groups; LTUGS 37.2% (29/78), RF/TF 60% (3/5), serial excision 100% (3/3), TE 0% (0/6) (P = 0.022). Univariant binary regression analysis within the LTUGS and RF/TF groups showed that lesion size was a significant predictor of poor outcomes (P = 0.012). All specimens in this study were negative for carcinoma. Therefore, most pediatric nevus sebaceous of the scalp can be managed by a single-phase procedure though risk of poor outcomes increase with nevus size with high rates of poor outcomes even with small lesions.

Some Aspects of CD8+ T-Cell Exhaustion Are Associated With Altered T-Cell Mitochondrial Features and ROS Content in HIV Infection.

BACKGROUND: Reversing or preventing T-cell exhaustion is an important treatment goal in the context of HIV disease; however, the mechanisms that regulate HIV-specific CD8 T-cell exhaustion are incompletely understood. Since mitochondrial mass (MM), mitochondrial membrane potential (MMP), and cellular reactive oxygen species (ROS) content are altered in exhausted CD8 T cells in other settings, we hypothesized that similar lesions may arise in HIV infection. METHODS: We sampled cryopreserved peripheral blood mononuclear cells from HIV-uninfected (n = 10) and HIV-infected participants with varying levels and mechanisms of viral control: viremic (VL > 2000 copies/mL; n = 8) or aviremic (VL < 40 copies/mL) due to antiretroviral therapy (n = 11) or natural control (n = 9). We characterized the MM, MMP, and ROS content of bulk CD8 T cells and MHC class I tetramer+ HIV-specific CD8 T cells by flow cytometry. RESULTS: We observed higher MM, MMP, and ROS content across bulk effector-memory CD8 T-cell subsets in HIV-infected compared with HIV-uninfected participants. Among HIV-specific CD8 T cells, these features did not vary by the extent or mechanism of viral control but were significantly altered in cells displaying characteristics associated with exhaustion (eg, high PD-1 expression, low CD127 expression, and impaired proliferative capacity). CONCLUSIONS: While we did not find that control of HIV replication in vivo correlates with the CD8 T-cell MM, MMP, or ROS content, we did find that some features of CD8 T-cell exhaustion are associated with alterations in mitochondrial state. Our findings support further studies to probe the relationship between mitochondrial dynamics and CD8 T-cell functionality in HIV infection.