A single slide multiplex assay for the evaluation of classical Hodgkin lymphoma.

Classical Hodgkin lymphoma can be diagnosed with confidence in the majority of cases, but there is a significant subset that remains a diagnostic challenge. The authors have investigated the utility of a novel hyperplexing technology, MultiOmyx™, which may be applied to stain with >60 antibodies on single tissue sections from formalin-fixed paraffin-embedded tissue as an aid to the diagnosis of classical Hodgkin lymphoma. The multiplexing protocol included CD30, CD15, PAX-5, CD20, CD79a, CD45, BOB.1, OCT-2, and CD3 antibodies. The technology showed a high degree of sensitivity, specificity, and precision. Comparison studies with routine hematoxylin and eosin and immunohistochemical assessment of hematopathology cases in which classical Hodgkin lymphoma was included in the differential diagnosis showed concordance in 54 of 56 cases, with the 2 discordant cases illustrating the potential of this multiplexed immunofluorescence technology to improve on traditional immunohistochemistry for classical Hodgkin lymphoma diagnosis. This technology is practical for routine diagnosis and may be particularly useful in cases in which the sample size is limited, few Hodgkin-like cells are present, or in CD30-positive lymphoma cases with difficult morphology. MultiOmyx may potentially benefit other areas of research and diagnostic pathology.

Notch signaling is necessary to maintain quiescence in adult muscle stem cells.

Satellite cells (SCs) are myogenic stem cells found in skeletal muscle that function to repair tissue damaged by injury or disease. SCs are quiescent at rest, although the signaling pathways required to maintain quiescence are unknown. Using a transgenic Notch reporter mouse and quantitative reverse-transcription polymerase chain reaction analysis of Notch target genes, we determined that Notch signaling is active in quiescent SCs. SC-specific deletion of recombining binding protein-Jκ (RBP-Jκ), a nuclear factor required for Notch signaling, resulted in the depletion of the SC pool and muscles that lacked any ability to regenerate in response to injury. SC depletion was not due to apoptosis. Rather, RBP-Jκ-deficient SCs spontaneously activate, fail to self-renew, and undergo terminal differentiation. Intriguingly, most of the cells differentiate without first dividing. They then fuse with adjacent myofibers, leading to the gradual disappearance of SCs from the muscle. These results demonstrate the requirement of Notch signaling for the maintenance of the quiescent state and for muscle stem cell homeostasis by the regulation of self-renewal and differentiation, processes that are all critical for normal postnatal myogenesis.

Essence of keratin in lips and associated structures of a freshwater fish Puntius sophore in relation to its feeding ecology: histochemistry and scanning electron microscope investigation.

Morphological specializations in the lips and associated structures of Puntius sophore were examined by scanning electron microscopy and histochemically. The upper lip (UL), in P. sophore, is associated with the horny upper jaw sheath (HUJS) on its ventral side and with the rostral cap (RC) on its dorsal side through a thin and extensive fold of skin (FSUR). The lower lip (LL) is greatly enlarged, conspicuous and associated with horny lower jaw sheath (HLJS) on the dorsal side and ventrally continues with ventral head skin (VHS). On the lateral sides there is a thin and extensive fold of skin (FSLS) between the lower lip and VHS. In contrast to the mucogenic epithelia of the UL, LL, the RC and fold of skins, the horny jaw sheaths are keratinized in nature and surface epithelial cells are characteristically modified into unculi. The UL and the LL are equipped with epithelial cells (EC), mucous cells (MC) and taste buds (TB) while in addition to these cells club cells (CC) are also present in the RC. Keratin found in unculi is an extremely strong protein which is tough and insoluble, they form the hard but un-mineralized structures. Keratin in unculi could be regarded as an adaptation for browsing or scraping food materials from the substrate as the fish grubs about the bottom. The elaboration of mucus is considered to lubricate the surface and protect the epithelia from abrasions. Taste buds are associated to locate and select palatable food and to trigger a 'pick-up' reflex.

A comparison of anion-exchange and steric-exclusion HPLC assays of mouse plasma lipoproteins.

We compared two HPLC methods (anion exchange [AE] and steric exclusion [SE]) for analysis of mouse lipoprotein profiles by determining coefficients of variability (CVs) under varying conditions. CVs for AE and SE were comparable on fresh samples. There was an inverse relationship between subfraction curve area and CV [r = -0.65 (AE) and -0.50 (SE)], consistent with the interpretation that as curve area decreased, error variance increased and signal-to-noise ratio decreased. Sample storage did not affect SE. In contrast, with AE, alterations in measured lipoproteins were apparent after storage, including a decrease in the HDL subfraction [66.8% (baseline) vs. 15.9% (1 week); P < 0.01] and an increase in areas under LDL and VLDL peaks. Concomitant with decreasing HDL area, reproducibility deteriorated with the duration of storage. Analysis of the effects of decreasing sample injectate volume to <25 microl on SE lipoprotein subfractions revealed that areas under LDL and VLDL peaks decreased and persisted as volume was decreased further. Areas under all lipoprotein subfractions measured with either AE or SE were linearly correlated with the amount of cholesterol [r = 0.69 (AE) and 0.87 (SE)]. Both AE and SE yield reproducible, accurate, and rapid measurements of lipoproteins from small amounts of serum. AE yields more sensitive, high-amplitude, well-defined peaks that can be easily distinguished and necessitates the use of smaller sample volumes compared with SE, but sample storage causes alterations in the chromatogram. SE appears better suited to serial analyses involving stored samples.

Increased glucocorticoid receptor and 11{beta}-hydroxysteroid dehydrogenase type 1 expression in hepatocytes may contribute to the phenotype of type 2 diabetes in db/db mice.

Excess tissue glucocorticoid action may contribute to the hyperglycemia and insulin resistance associated with type 2 diabetes, but the associated mechanisms are poorly understood. 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inactive 11-dehydrocorticosterone into active corticosterone, thus amplifying glucocorticoid receptor-mediated tissue glucocorticoid action, particularly in the liver. To examine the role of tissue glucocorticoid action in type 2 diabetes, we analyzed expression of glucocorticoid receptor and 11beta-HSD1 and their regulation by endogenous hormones in vivo and in vitro in hepatocytes from db/db mice (a model of type 2 diabetes). We observed positive relations between expression of both glucocorticoid receptor and 11beta-HSD1 in liver and insulin sensitivity and expression of PEPCK mRNA in db/db mice and db/+ controls. Increased expression of glucocorticoid receptor and 11beta-HSD1 in the liver of db/db mice was correlated with elevated circulating levels of corticosterone, insulin, and blood glu-cose. Treatment of db/db mice with glucocorticoid antagonist RU486 reversed the increases in the expression of glucocorticoid receptor and 11beta-HSD1 within the liver and attenuated the phenotype of type 2 diabetes. Addition of corticosterone to db/db mouse primary hepatocytes activated expression of glucocorticoid receptor, 11beta-HSD1, and PEPCK, and these effects were abolished by RU486. Incubation of primary hepatocytes with increasing concentrations of glucose caused dose-dependent increases in glucocorticoid receptor and 11beta-HSD1 expression, whereas insulin did not affect the expression of 11beta-HSD1 and glucocorticoid receptor in primary hepatocytes. These findings suggest that activation of glucocorticoid receptor and 11beta-HSD1 expression within the liver may contribute to the development of type 2 diabetes in db/db mice.

Increased expression of membrane type 3-matrix metalloproteinase in human atherosclerotic plaque: role of activated macrophages and inflammatory cytokines.

BACKGROUND: Matrix metalloproteinases (MMPs) are thought to play a prominent role in atherogenesis and destabilization of plaque. Pericellularly localized membrane-type (MT)-MMPs activate secreted MMPs. We investigated the hypothesis that MT3-MMP is expressed in human atherosclerotic plaques and is regulated by locally produced inflammatory cytokines and oxidized low-density lipoprotein (Ox-LDL). METHODS AND RESULTS: Expression and cellular localization of MT3-MMP in normal and atherosclerotic human coronary arteries were examined using specific antibodies. Abundant MT3-MMP expression was noted in medial smooth muscle cells (SMCs) of normal arteries. In atherosclerotic arteries, MT3-MMP expression was observed within complex plaques and colocalized with SMCs and macrophages (Mphi). Cultured human monocyte-derived Mphi constitutively expressed MT3-MMP mRNA and proteolytically active protein, as demonstrated by mRNA analyses, immunoblotting, and gelatin zymography, respectively. Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor caused dose- and time-dependent increases in steady-state levels of MT3-MMP mRNA in cultured Mphi. This correlated with a 2- to 4-fold increase in levels of MT3-MMP immunoreactive protein and enzymatic activity in Mphi membranes. Confocal microscopy and flow cytometry confirmed induction and spatial distribution of MT3-MMP protein from intracellular domains to the Mphi plasma membrane by Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor. CONCLUSIONS: MT3-MMP is expressed by SMCs and Mphi in human atherosclerotic plaques. Proinflammatory molecules cause a progressive increase in the expression of MT3-MMP in cultured Mphi. Our results suggest a mechanism by which inflammatory molecules could promote Mphi-mediated degradation of extracellular matrix and thereby contribute to plaque destabilization.

Increased expression of macrophage colony-stimulating factor after coronary artery balloon injury is inhibited by intracoronary brachytherapy.

BACKGROUND: The mechanisms underlying the reduced neointimal proliferation (NP) by intracoronary brachytherapy (ICBT) are unknown. We hypothesized that ICBT inhibits NP by reducing expression of macrophage colony-stimulating factor (M-CSF). METHODS AND RESULTS: Thirty coronary arteries from 10 pigs were divided into 3 groups of 10 each: control (C), balloon injury (BI), and BI followed by ICBT (16 Gy at 0.5-mm tissue depth with a (32)P balloon system). Pigs were killed at 24 hours (n=3) and at 7 (n=4) and 14 (n=3) days. Expression of M-CSF was assessed by Western blot, ELISA, and quantitative immunostaining. Persistently increased levels of M-CSF after BI (to 1.4+/-0.2 nmol/L [ELISA] and 29.4+/-4.9% of cross-sectional area stained [immunocytochemistry]; P< 0.001 versus control for both) were observed in the injured arteries. Treatment of BI arteries with ICBT reduced M-CSF expression compared with BI alone (to 0.7+/-0.1 nmol/L [ELISA] and 13.5+/-2.9% of cross-sectional area stained [immunocytochemistry]; P<0.001 versus BI and P=NS versus control for both) and remained similar to control M-CSF expression for the 14-day study period. Neointimal thickness increased after BI (to 4.8+/-2.9 mm(2); P<0.001 versus control), but this was reduced by ICBT (1.4+/-0.4 mm(2); P<0.001 versus BI). CONCLUSIONS: In porcine coronary arteries, BI is associated with increased expression of M-CSF and NP, but neither occurs after ICBT. The beneficial effects of ICBT on NP involve inhibition of M-CSF expression.

Rationale for the role of osteoclast-like cells in arterial calcification.

Atherosclerotic arteries frequently become calcified, and these calcium deposits are associated with a high risk of adverse clinical events. Descriptive studies suggest calcification is an organized and regulated process with many similarities to osteogenesis, yet the mechanism and its relationship to atherosclerosis remain largely unknown. In bone development and homeostasis, mineral deposition by osteoblasts and mineral resorption by osteoclasts are delicately balanced such that there is no overall gain or loss in bone mass. We hypothesize that there exists in arteries a mechanism that similarly balances mineral deposition with resorption. We propose that the cellular mediators of arterial mineral resorption are osteoclast-like cells (OLCs) derived from hematopoietic precursors of the mononuclear phagocytic lineage. In arterial microenvironments, mononuclear precursors are induced to differentiate toward OLCs by macrophage-colony stimulating factor and receptor activator of NF-kappaB ligand, both of which are necessary and sufficient for osteoclastogenesis and mineral resorption in bone. OLCs may participate in normal mineral homeostasis within the arterial wall or, alternatively, may be recruited to specific sites within developing plaque. Net calcium deposition occurs as a result of focal perturbation of the balance between the activity of osteoblast-like cells and OLCs. Our proposed mechanism thus views arterial mineral deposition not so much as an active pathological process, but as a localized failure of protective mechanisms that actively oppose mineral deposition within the disordered metabolic milieu of developing atherosclerotic plaque.