Journal of Histochemistry & Cytochemistry

Grönberg, Malin; Tsolakis, Apostolos V.; Magnusson, Linda; Janson, Eva T.; Saras, Jan

doi: 10.1369/jhc.2008.951145pmid: 18474938

Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immuno-reactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.

O'Donnell, Rebekah K.; Feldman, Michael; Mick, Rosemarie; Muschel, Ruth J.

doi: 10.1369/jhc.2008.950790pmid: 18505934

Tumor invasion into blood and/or lymphatic channels is an important component of cancer staging and prognosis. Standard pathological methods do not provide sufficient contrast to discriminate between invasion into each type of vessel and are complicated by tissue retraction artifacts. We evaluated the ability of a triple-stain immunohistochemical method, combining cytokeratin, CD34, and podoplanin stains in a single section, to distinguish blood from lymphatic vascular invasion in oral squamous cell carcinoma and confirmed its results using multispectral analysis. The triple-stain method was significantly more sensitive in detecting invasive events than the standard hematoxylin and eosin staining method and easily discriminated between blood and lymphatic vessel invasion. Invasive events were present in blood and/or lymphatic vessels in the majority of patients with and without presentation of lymph node metastasis, indicating that vessel invasion in this cancer model is common and is not a rate-limiting step for lymph node metastasis.

Mori, Jun; Ishihara, Yasunori; Matsuo, Kensuke; Nakajima, Hisakazu; Terada, Naoto; Kosaka, Kitaro; Kizaki, Zenro; Sugimoto, Tohru

doi: 10.1369/jhc.2008.951244pmid: 18505932

Recent studies have shown that cells from bone marrow (BM) can give rise to differentiated skeletal muscle fibers. However, the mechanisms and identities of the cell types involved remain unknown. We performed BM transplantation in acid α-glucosidase (GAA) knockout mice, a model of glycogen storage disease type II, and our observations suggested that the BM cells contribute to skeletal muscle fiber formation. Furthermore, we showed that most CD45+:Sca1+ cells have a donor character in regenerating muscle of recipient mice. Based on these findings, CD45+:Sca1+ cells were sorted from regenerating muscles. The cell number was increased with granulocyte colony-stimulating factor after cardiotoxin injury, and the cells were transplanted directly into the tibialis anterior (TA) muscles of GAA knockout mice. Sections of the TA muscles stained with anti-laminin-α2 antibody showed that the number of CD45+:Sca1+ cells contributing to muscle fiber formation and glycogen levels were decreased in transplanted muscles. Our results indicated that hematopoietic stem cells, such as CD45+:Sca1+ cells, are involved in skeletal muscle regeneration.

Haase, Michael; Gmach, Christine C.; Eke, Iris; Hehlgans, Stephanie; Baretton, Gustavo B.; Cordes, Nils

doi: 10.1369/jhc.2008.951095pmid: 18505933

Integrin-linked kinase (ILK), a mediator of β integrin signals, has emerged as a therapeutic target in malignant tumors. Because malignant transformation is accompanied by dedifferentiation, ILK expression was evaluated in diverse normal and tumor tissue samples with regard to tissue differentiation. In single sections and in a tissue microarray (323 tumor tissues, 181 normal tissues), immunohistochemistry was performed [ILK, Akt, phospho-Akt-S473, loricrin, transforming growth factor β2 (TGFβ2)], and staining intensities were semiquantitatively scored. Increased ILK expression was clearly associated with increased differentiation in normal gastrointestinal, neural, bone marrow, renal tissue, and in more differentiated areas of malignant tumors. ILK colocalized with its putative downstream target Akt and with loricrin or TGFβ2. Our findings clearly show that elevated levels of ILK are associated with cellular differentiation in high turnover tissues but not generally with a malignant phenotype. Our study indicates that ILK is not a general molecular target for cancer therapy but rather an indicator of differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Kirkpatrick, Lisa J.; Allouh, Mohammed Z.; Nightingale, Chantale N.; Devon, Heidi G.; Yablonka-Reuveni, Zipora; Rosser, Benjamin W.C.

doi: 10.1369/jhc.2008.951608pmid: 18541708

Intrafusal fibers within muscle spindles make up a small subpopulation of muscle fibers. These proprioceptive fibers differ from most extrafusal fibers because, even in maturity, their diameters remain small, and they retain expression of developmental myosins. Although both extrafusal and intrafusal fibers contain satellite cells (SCs), comparatively little is known about intrafusal SCs. Analyzing chicken fast-phasic posterior (PLD) and slow-tonic anterior (ALD) latissimus dorsi muscles, we show that SCs of both intrafusal and extrafusal fibers express Pax7. We further test the hypotheses that intrafusal fibers display parameters reflective of extrafusal immaturity. These hypotheses are that intrafusal fibers contain (a) higher SC frequencies (number of SC nuclei/all nuclei within basal lamina) and concentrations (closer together) and (b) smaller myonuclear domains than do adjacent extrafusal fibers. IHC techniques were applied to PLD and ALD muscles excised at 30 and 138 days posthatch. The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.

Tornehave, Ditte; Kristensen, Peter; Rømer, John; Knudsen, Lotte Bjerre; Heller, R. Scott

doi: 10.1369/jhc.2008.951319pmid: 18541709

We studied the intra-islet localization of the glucagon-like peptide 1 receptor (GLP-1R) by colocalization studies of the GLP-1R mRNA and protein with islet cell hormones in mice, rats, and humans. In contrast to previous reports, we show that the GLP-1R is selectively located on the β cells. The localization of GLP-1R in islets and ducts was studied using ISH and double and triple fluorescence microscopy. In normal pancreatic tissue from mice and rats, GLP-1R mRNA was only detectable in the β cells. Double and triple immunofluorescence using two different GLP-1R antisera and combinations of insulin, glucagon, pancreatic polypeptide, and somatostatin showed that GLP-1R protein is almost exclusively colocalized with insulin. The same pattern was observed in human pancreas, but the GLP-1R expression was more heterogeneous, with populations of insulin immunoreactive cells with high and low expression. This is the first time that the GLP-1R has been localized in human islets. Furthermore, GLP-1R immunoreactivity was found in the pancreatic ducts in mouse, rat, and human pancreas. As an important confirmation of the specificity of our methods, we found no signals for GLP-1R mRNA or protein in pancreatic tissue from gene-targeted GLP-1R—deficient mice. In conclusion, our data suggest that the GLP-1 receptor is restricted to the pancreatic β cells and the lack of receptor immunoreactivity on δ cells cannot be explained suitably to correspond with published in vivo and in vitro data. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Azzarello, Joseph; Fung, Kar-Ming; Lin, Hsueh-Kung

doi: 10.1369/jhc.2008.951384pmid: 18574251

Human aldo-keto reductase (AKR) 1C3 (type 2 3α-hydroxysteroid dehydrogenase/type 5 17β-hydroxysteroid dehydrogenase) catalyzes androgen, estrogen, and prostaglandin metabolism. AKR1C3 is therefore implicated in regulating ligand access to the androgen receptor, estrogen receptor, and peroxisome proliferator activating receptor γ in hormone target tissues. Recent reports on close relationships between ARK1C3 and various cancers including breast and prostate cancers implicate the involvement of AKR1C3 in cancer development or progression. We previously described the characterization of an isoform-specific monoclonal antibody against AKR1C3 that does not cross-react with related, >86% sequence identity, human AKR1C1, AKR1C2, or AKR1C4, human aldehyde reductase AKR1A1, or rat 3α-hydroxysteroid dehydrogenase (AKR1C9). In this study, a clone of murine monoclonal antibody raised against AKR1C3 was identified and characterized for its recognition of rat homolog. Tissue distribution of human AKR1C3 and its rat homolog in adult genitourinary systems including kidney, bladder, prostate, and testis was studied by IHC. A strong immunoreactivity was detected not only in classically hormone-associated tissues such as prostate and testis but also in non—hormone-associated tissues such as kidney and bladder in humans and rats. The distribution of these two enzymes was comparable but not identical between the two species. These features warrant future studies of AKR1C3 in both hormone- and non—hormone-associated tissues and identification of the rodent homolog for establishing animal models.

Ostojić, Jelena; Grozdanić, Siniś; Syed, Nasreen A.; Hargrove, Mark S.; Trent, James T.; Kuehn, Markus H.; Kardon, Randy H.; Kwon, Young H.; Sakaguchi, Donald S.

doi: 10.1369/jhc.2008.951392pmid: 18574250

This study provides a detailed description of immunolocalization of two oxygen-binding proteins, neuroglobin (Ngb) and cytoglobin (Cygb), in the anterior segment of healthy human and canine eyes. Specific antibodies against Ngb and Cygb were used to examine their distribution patterns in anterior segment structures including the cornea, iris, trabecular meshwork, canal of Schlemm, ciliary body, and lens. Patterns of immunoreactivity (IR) were imaged with confocal scanning laser and conventional microscopy. Analysis of sectioned human and canine eyes showed Ngb and Cygb IR in the corneal epithelium and endothelium. In the iris, Ngb and Cygb IR was localized to the anterior border and the stroma, iridal sphincter, and dilator muscle. In the iridocorneal angle, Ngb and Cygb were detected in endothelial cells of the trabecular meshwork and canal of Schlemm in human. In the ciliary body, Ngb and Cygb IR was localized to the non-pigmented ciliary epithelium of the pars plana and pars plicata and in ciliary body musculature. Ngb and Cygb distribution was similar and colocalized within the same structures of healthy human and canine anterior eye segments. Based on their immunolocalization and previously reported biochemical features, we hypothesize that Ngb and Cygb may function as scavengers of reactive oxygen species. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.