Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury.

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.

Smooth muscle cells can express cytokeratins of "simple" epithelium. Immunocytochemical and biochemical studies in vitro and in vivo.

Cytokeratins are a set of 19 proteins that together constitute the class of intermediate filament protein expressed by epithelial cells and tumors. Using a panel of 9 different monoclonal anti-cytokeratin antibodies, the authors have performed immunocytochemistry on methanol-fixed, frozen sections and methacarn-fixed, paraffin-embedded tissue of human myometrial specimens. Anomalous cytokeratin expression (ACE) by smooth muscle cells was found in all specimens. Immunoblots of this tissue confirmed the presence of cytokeratin 19, and possibly 8. In addition, immunocytochemical studies demonstrated ACE in human fetal tissues within the intestinal muscularis and the heart, especially in the region of the aortic outflow tract, and in 8 of 19 cases of leiomyosarcoma from adults. Indirect immunofluorescence studies were also performed on cells explanted from myometrial tissue; the overwhelming majority of cells derived from these cultures were smooth muscle cells as verified by expression of muscle actins, and a subpopulation of these cells was found to be cytokeratin-positive. ACE was confirmed in vitro by double labeling experiments demonstrating simultaneous expression of muscle actins and cytokeratins within the same cell. The significance of this smooth muscle cell ACE is unknown, but it may be a phenotypic marker of smooth muscle in a proliferative state. ACE could be a source of confusion in the immunocytochemical analysis of poorly differentiated malignancies if a complete panel of antibodies is not employed.

HHF35, a muscle-actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization.

A monoclonal antibody to muscle cell actin isotypes was produced and characterized. Immunocytochemical analysis of methanol-Carnoy's-fixed, paraffin-embedded human tissue revealed that this antibody, termed HHF35, reacts with skeletal muscle cells, cardiac muscle cells, smooth muscle cells, pericytes, and myoepithelial cells, but is nonreactive with endothelial, epithelial, neural, or connective tissue cells. When assayed by indirect immunofluorescence, HHF35 reacts with microfilament bundles from various cultured mammalian smooth muscle cells, but does not react with cultured human dermal fibroblasts or various epithelial tumor cell lines. In one-dimensional gel electrophoresis immunoblot experiments this antibody detects a 42-kd polypeptide from tissue extracts of uterus, ileum, aorta, diaphragm, and heart and extract from smooth muscle cells. The antibody also reacts with a comigrating 42-kd band of highly purified rabbit skeletal muscle actin. HHF35 is nonreactive on immunoblots of extracts from all tested nonmuscle cell extracts. Immunoelectrophoresis followed by immunoblotting performed in the presence of urea and reducing agents reveals recognition of the alpha isoelectrophoretic variant of actin from skeletal, cardiac, and smooth muscle sources and of the gamma variant from smooth muscle sources. Because HHF35 reacts with virtually all muscle cells, it will be useful as a marker for muscle and muscle-derived cells.

Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures.

We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.