The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The ''oval cell'' response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.

No abstract

S100 protein is a sensitive marker for melanomas and peripheral nerve sheath tumors. It is, however, expressed by other mesenchymal and epithelial tumors. Despite its low specificity, S100 protein is valuable for the diagnosis of desmoplastic melanomas and peripheral nerve sheath tumors, for which no specific marker is available. Sox10 is a neural crest transcription factor crucial for specification, maturation, and maintenance of Schwann cells and melanocytes. Anti-Sox10 antibody was applied to a variety of neural crest-derived tumors, mesenchymal and epithelial neoplasms, and normal tissues. Sox10 nuclear expression was found in 76 of 78 melanomas (97%) and 38 of 77 malignant peripheral nerve sheath tumors (49%) whereas S100 protein was expressed in 71 melanomas (91%) and 23 malignant peripheral nerve sheath tumors (30%). Sox10 was diffusely expressed in schwannomas and neurofibromas. Sox10 reaction was seen only in sustentacular cells of pheochromocytomas/paragangliomas, and occasionally carcinoid tumors from various organs, but it was not seen in the tumor cells. In normal tissues, Sox10 was expressed in Schwann cells, melanocytes, and myoepithelial cells of salivary, bronchial, and mammary glands. Sox10 reaction was not identified in any other mesenchymal and epithelial tumors except for myoepitheliomas and diffuse astrocytomas. Sox10 was expressed by metastatic melanomas and nodal capsular nevus in sentinel lymph nodes, but not by other lymph node components such as dendritic cells. Our results indicate that Sox10 will serve as a more sensitive and specific marker for the diagnosis of melanocytic and schwannian tumors than S100 protein.

BACKGROUND New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker. METHODS We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay. RESULTS Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). Fibulin-3 preferentially stained tumor cells in 26 of 26 samples. In an overall comparison of patients with and those without mesothelioma, the receiver-operating-characteristic curve for plasma fibulin-3 levels had a sensitivity of 96.7% and a specificity of 95.5% at a cutoff value of 52.8 ng of fibulin-3 per milliliter. In a comparison of patients with early-stage mesothelioma with asbestos-exposed persons, the sensitivity was 100% and the specificity was 94.1% at a cutoff value of 46.0 ng of fibulin-3 per milliliter. Blinded validation revealed an area under the curve of 0.87 for plasma specimens from 96 asbestos-exposed persons as compared with 48 patients with mesothelioma. CONCLUSIONS Plasma fibulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with effusion fibulin-3 levels, plasma fibulin-3 levels can further differentiate mesothelioma effusions from other malignant and benign effusions. (Funded by the Early Detection Research Network, National Institutes of Health, and others.)

IntroductionT-cell development is an ordered process thought to take place exclusively in the thymus where CD4 ϩ CD8 ϩ T cells develop into CD4 ϩ and CD8 ϩ T cells with mutually exclusive expression of these 2 receptors. Mature CD4 ϩ and CD8 ϩ T cells then leave the thymus and enter secondary lymphoid organs where they recognize their cognate antigen in the context of major histocompatibility complex (MHC) class II and class I molecules on antigenpresenting cells. Antigenic stimulation induces proliferation and differentiation into armed effector cells with the ability to home to infected organs and to participate in immune responses against intracellular pathogens.Contrary to this conventional dichotomy, circulating CD4 ϩ CD8 ϩ T cells have sporadically been reported in humans as well as in animals such as mice, chicken, swine, and monkeys. [1][2][3][4][5][6] The existence of this unconventional and rare (Ͻ 5%) lymphocyte population in the peripheral blood was explained as a premature release of CD4 ϩ CD8 ϩ T cells from the thymus into the periphery, 7,8 where their maturation into functionally competent single-positive cells continues. 7 There is, however, considerable evidence of an increased frequency of peripheral CD4 ϩ CD8 ϩ T cells during viral infections. [9][10][11][12] In human immunodeficiency virus (HIV) or EpsteinBarr virus (EBV) infections, for example, the percentage of CD4 ϩ CD8 ϩ T cells can increase to 20% of all circulating lymphocytes. 9,10 Yet, in humans, very little is known about their phenotype, antigen specificity, and function. This is particularly striking since in animal models CD4 ϩ CD8 ϩ T cells have been described as antigen-specific memory cells that can be induced by vaccination. 3,4,6 Thus, it is crucial to determine the role and biologic significance of peripheral CD4 ϩ CD8 ϩ T cells in humans, as they could potentially contribute to the adaptive immune response against pathogens.In this study, we addressed this issue with an extensive analysis of peripheral CD4 ϩ CD8 ϩ T cells in both healthy individuals and patients with past or present viral infections. We determined the frequency and ex vivo phenotype of CD4 ϩ CD8 ϩ T cells in regards to memory, activation, homing, differentiation, and maturation markers. Furthermore, we provide data on antigen specificity, proliferation, cytokine production, and cytotoxic potential of CD4 ϩ CD8 ϩ T cells in response to tetanus toxoid and to a large variety of antigens (lysates of infected cells, overlapping viral peptides, minimal optimal viral epitopes) from past (influenza A virus [IV] Materials and methods Blood samplesPeripheral blood mononuclear cells (PBMCs) were isolated from anticoagulant citrate dextrose (ACD)-anticoagulated blood of 10 healthy blood donors and 15 HCV-infected patients as described. 13 Viral lysates, peptides, proteins, and antibodiesLysates from CMV-or EBV-infected cells and the corresponding uninfected cells were purchased from Virion (Marristown, NJ). Lysates from HSV-1-, MV-, and VZV-infected cells and the c...

No abstract

Hepatitis C virus (HCV) establishes a chronic infection in the majority of exposed individuals and can cause cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. Here, we demonstrate that three broadly nAbs, AR3A, AR3B and AR4A, delivered with adeno-associated viral (AAV) vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a novel therapeutic approach to interfere with HCV infection exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes in order to sustain chronicity.

Chemokines, chemotactic cytokines, may promote hepatic inflammation in chronic hepatitis C virus (HCV) infection through the recruitment of lymphocytes to the liver parenchyma. We evaluated the association between inflammation and fibrosis and CXCR3-associated chemokines, interferon-␥ (IFN-␥)-inducible protein 10 (IP-10/CXCL10), monokine induced by IFN-␥ (Mig/CXCL9), and interferon-inducible T cell ␣ chemoattractant (I-TAC/ CXCL11), in HCV infection. Intrahepatic mRNA expression of these chemokines was analyzed in 106 chronic HCV-infected patients by real-time PCR. The intrahepatic localization of chemokine producer cells and CXCR3 ؉ lymphocytes was determined in selected patients by immunohistochemistry. We found elevated intrahepatic mRNA expression of all three chemokines, most markedly CXCL10, in chronic HCV-infected patients with higher necroinflammation and fibrosis. By multivariable multivariate analysis, intrahepatic CXCL10 mRNA expression levels were significantly associated with lobular necroinflammatory grade and HCV genotype 1. In the lobular region, CXCL10-expressing and CXCL9-expressing hepatocytes predominated in areas with necroinflammation. Strong CXCL11 expression was observed in almost all portal tracts, whereas CXCL9 expression varied considerably among portal tracts in the same individual. Most intrahepatic lymphocytes express the CXCR3 receptor, and the number of CXCR3 ؉ lymphocytes was increased in patients with advanced necroinflammation. Conclusion: These findings suggest that the CXCR3-associated chemokines, particularly CXCL10, may play an important role in the development of necroinflammation and fibrosis in the liver parenchyma in chronic HCV infection.