Journal of Histochemistry & Cytochemistry

Lyck, Lise; Dalmau, Ishar; Chemnitz, John; Finsen, Bente; Schrøder, Henrik Daa

doi: 10.1369/jhc.7A7187.2007pmid: 17998570

Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage–specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2′, 3′ cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25α-antigen, and S100β as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.

Nosaka, Mizuho; Ishida, Yuko; Tanaka, Aki; Hayashi, Takahito; Miyashita, Tomoko; Kaminaka, Chikako; Eisenmenger, Wolfgang; Furukawa, Fukumi; Kimura, Akihiko

doi: 10.1369/jhc.7A7290.2007pmid: 17998569

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1–4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.

Verstege, Marleen I.; Kate, Fiebo J. W. ten; Reinartz, Susanne M.; Drunen, Cornelis M. van; Slors, Frederik J. M.; Bemelman, Willem A.; Vyth-Dreese, Florry A.; Velde, Anje A. te

doi: 10.1369/jhc.7A7308.2007pmid: 18040077

Dendritic cells (DCs) are key cells in innate and adaptive immune responses that determine the pathophysiology of Crohn's disease. Intestinal DCs migrate from the mucosa into mesenteric lymph nodes (MLNs). A number of different markers are described to define the DC populations. In this study we have identified the phenotype and localization of intestinal and MLN DCs in patients with Crohn's disease and non-IBD patients based on these markers. We used immunohistochemistry to demonstrate that all markers (S-100, CD83, DC-SIGN, BDCA1-4, and CD1a) showed a different staining pattern varying from localization in T-cell areas of lymph follicles around blood vessels or single cells in the lamina propria and in the MLN in the medullary cords and in the subcapsular sinuses around blood vessels and in the T-cell areas. In conclusion, all different DC markers give variable staining patterns so there is no marker for the DC.

Motomura, Wataru; Yoshizaki, Takayuki; Ohtani, Katsuki; Okumura, Toshikatsu; Fukuda, Mituko; Fukuzawa, Jun; Mori, Kenichiro; Jang, Seong-Jae; Nomura, Naoki; Yoshida, Itsuro; Suzuki, Yasuhiko; Kohgo, Yutaka; Wakamiya, Nobutaka

Oyasu, Miho; Fujimiya, Mineko; Kashiwagi, Kaori; Ohmori, Shiho; Imaeda, Hirotsugu; Saito, Naoaki

doi: 10.1369/jhc.7A7291.2007pmid: 18040079

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation.

Frolova, Lenka; Drastich, Pavel; Rossmann, Pavel; Klimesova, Klara; Tlaskalova-Hogenova, Helena

doi: 10.1369/jhc.7A7303.2007pmid: 18040078

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 inbiopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis.

Bowen, Kara B.; Reimers, Aaron P.; Luman, Sarah; Kronz, Joseph D.; Fyffe, William E.; Oxford, Julia Thom

doi: 10.1369/jhc.7A7310.2007pmid: 18040076

In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three a chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and α1(XI) and α2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon.

Williams, Diana L.; Schwartz, Michael W.; Bastian, L. Scot; Blevins, James E.; Baskin, Denis G.

doi: 10.1369/jhc.7A7331.2007pmid: 18040081

Current evidence suggests that leptin reduces food intake in part by enhancing the hindbrain neuronal response to meal-related gastrointestinal signals, including cholecystokinin (CCK), but the phenotypes of the relevant cells are not known. To identify neurons that participate in this interaction in the rat nucleus of the solitary tract (NTS), we induced c-Fos gene expression in NTS neurons with leptin and CCK. We focused on NTS catecholamine neurons because these cells have been implicated in the feeding response to CCK. Hindbrain sections from rats that received CCK with or without leptin pretreatment were immunostained for c-Fos and tyrosine hydroxylase (TH) by a double immunofluorescence procedure. Leptin pretreatment increased the number of NTS cells expressing c-Fos-like immunoreactivity (cFLI) 3-fold relative to CCK alone, but the number of TH-positive cells with cFLI was increased 6-fold. Next, cells detected by immunofluorescence for TH were collected by laser capture microdissection and pooled for real-time quantitative PCR of c-Fos mRNA. Here, neither leptin nor CCK alone affected the relative amount of mRNA in the TH cell–enriched samples, but leptin plus CCK substantially increased c-Fos mRNA content. These histochemical findings identify hindbrain catecholamine cells as potential mediators of the interaction between leptin and CCK.

Kadoya, Kuniko; Fukushi, Jun-ichi; Matsumoto, Yoshihiro; Yamaguchi, Yu; Stallcup, William B.

doi: 10.1369/jhc.7A7349.2007pmid: 18040080

In early postnatal mouse skin, the NG2 proteoglycan is expressed in the subcutis, the dermis, the outer root sheath of hair follicles, and the basal keratinocyte layer of the epidermis. With further development, NG2 is most prominently expressed by stem cells in the hair follicle bulge region, as also observed in adult human skin. During telogen and anagen phases of the adult hair cycle, NG2 is also found in stem cell populations that reside in dermal papillae and the outer root sheaths of hair follicles. Ablation of NG2 produces alterations in both the epidermis and subcutis layers of neonatal skin. Compared with wild type, the NG2 null epidermis does not achieve its full thickness due to reduced proliferation of basal keratinocytes that serve as the stem cell population in this layer. Thickening of the subcutis is also delayed in NG2 null skin due to deficiencies in the adipocyte population.

Shanmugam, Chandrakumar; Katkoori, Venkat R.; Jhala, Nirag C.; Grizzle, William E.; Siegal, Gene P.; Manne, Upender

doi: 10.1369/jhc.7A7362.2007pmid: 18071066

For subsets of colorectal adenocarcinoma (CRC) patients, nuclear accumulation of p53 (p53nac) and Bcl-2 expression are prognostic indicators. To understand their role in the progression of CRC we evaluated 90 CRCs and their contiguous adenomatous components (CAdCs) for immunohistochemical expression of these markers. In general, p53nac and Bcl-2 expression was significantly increased when comparing normal colonic epithelia to CAdCs and CRCs. Thirteen (14%) CAdCs that demonstrated p53nac continued to express p53nac in their contiguous CRCs. A similar trend was observed in Bcl-2 expression in that the majority of CAdCs expressing Bcl-2 continued to express it in their matching CRCs (39/44). Patients whose CAdCs and their contiguous CRCs demonstrate p53nac had shorter median survival (35.9 months) than those patients whose CAdCs and CRCs did not (80.56 months). However, patients whose CAdCs had p53nac and lacked Bcl-2 expression had the lowest median survival (15.74 months) when compared with patients whose CAdCs did not demonstrate p53nac but had increased expression of Bcl-2 (71.77 months). These findings suggest that in those adenomas that demonstrate p53nac but lack Bcl-2 expression, their contiguous CRCs are more likely to be aggressive as they progress.