Journal of Histochemistry & Cytochemistry

Saarikoski, Sirkku T.; Wikman, Harriet A.-L.; Smith, Gillian; J. Wolff, C. Henrik; Husgafvel-Pursiainen, Kirsti

doi: 10.1369/jhc.4C6576.2005pmid: 15872048

CYP2S1 is a recently discovered dioxin-inducible member of the cytochrome P450 superfamily. It has been shown to be involved in the metabolism of some aromatic hydrocarbons as well as retinoic acid, suggesting a role in biotransformation of both exogenous and endogenous compounds. In this study, we used mRNA in situ hybridization and immunohistochemistry to investigate the cellular localization of CYP2S1 in various human tissues using tissue microarrays. High expression levels were observed mainly in epithelial cell types, especially in the epithelia frequently exposed to xenobiotics. In the respiratory tract, the expression was strong in nasal cavity, bronchi, and bronchioli, whereas it was low in the alveolar lining cells. Similarly, CYP2S1 was highly expressed in the epithelial cells throughout the gastrointestinal tract. Strong epithelial expression was also observed in uterine cervix, urinary bladder, and skin. In many exocrine glands (e.g., adrenal gland and pancreas), secretory epithelial cells showed moderate to strong expression levels. In the liver, the expression was low. CYP2S1 was highly expressed in epithelial cells that are major targets for carcinogen exposure and common progenitor cells to tumor development. Indeed, we found strong CYP2S1 expression in many tumors of epithelial origin.

Schevzov, Galina; Vrhovski, Bernadette; Bryce, Nicole S.; Elmir, Sarah; Qiu, Min Ru; O'Neill, Geraldine M.; Yang, Nan; Verrills, Nicole M.; Kavallaris, Maria; Gunning, Peter W.

doi: 10.1369/jhc.4A6505.2005pmid: 15872049

Puolakkainen, Pauli A.; Bradshaw, Amy D.; Brekken, Rolf A.; Reed, May J.; Kyriakides, Themis; Funk, Sarah E.; Gooden, Michel D.; Vernon, Robert B.; Wight, Thomas N.; Bornstein, Paul; Sage, E. Helene

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Wakefield, Larissa; Cornish, Valerie; Broackes-Carter, Fiona; Sim, Edith

doi: 10.1369/jhc.4A6496.2005pmid: 15872051

Murine arylamine N-acetyltransferase 2 (NAT2) is expressed in the developing heart and in the neural tube at the time of closure. Classically described as a xenobiotic metabolizing enzyme, there is increasing evidence for a distinct biological role for murine NAT2. We have characterized the expression of arylamine N-acetyltransferase 2 during cardiogenesis, mapping its expression in vivo, using a lacZ insertion deletion, and also in vitro, by measuring NAT2 enzyme activity. These findings show that cardiac Nat2 expression is both temporally and spatially regulated during development. In neonatal mice, cardiac Nat2 expression is most extensive in the central fibrous body and is evident in the atrioventricular valves and the valves of the great vessels. Whereas Nat2 expression is not detected in ventricular myocardial cells, Nat2 is strongly expressed in scattered cells in the region of the sinus node, the epicardium of the right atrial appendage, and in the pulmonary artery. Expression of active NAT2 protein is maximal when the developing heart attains the adult circulation pattern and moves from metabolizing glucose to fatty acids. NAT2 acetylating activity in cardiac tissue from Nat2−/- and Nat2+/- mice indicates a lack of compensating acetylating activity either from other acetylating enzymes or by NAT2 encoded by the wild-type Nat2 allele in Nat2+/- heterozygotes. The temporal and spatial control of murine Nat2 expression points to an endogenous role distinct from xenobiotic metabolism and indicates that Nat2 expression may be useful as a marker in cardiac development.

Jiang, Xi; Kalajzic, Zana; Maye, Peter; Braut, Alen; Bellizzi, Justin; Mina, Mina; Rowe, David W.

doi: 10.1369/jhc.4A6401.2005pmid: 15872052

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a β-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.

Clough, Maria H.; Schneider, David J.; Sobel, Burton E.; White, Morris F.; Wadsworth, Marilyn P.; Taatjes, Douglas J.

doi: 10.1369/jhc.4A6590.2005pmid: 15872053

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE−/-) and in ApoE−/- mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2+/- ApoE−/- mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2+/- ApoE−/- mice. The extent of the aortic intima occupied by lesion was increased in the IRS2+/- ApoE−/- compared with ApoE−/- mice (79 ± 3% compared with 68 ± 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 ± 6% to 30 ± 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 ± 1% to 19 ± 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

Takano, Ken-ichi; Kojima, Takashi; Go, Mitsuru; Murata, Masaki; Ichimiya, Shingo; Himi, Tetsuo; Sawada, Norimasa

doi: 10.1369/jhc.4A6539.2005pmid: 15872054

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, −4, −7, −8, −12, −13, and −14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, −4, and −7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

Al-Mulla, Fahd; Abdulrahman, Mahera; Varadharaj, Govindarajulu; Akhter, Nadeem; Anim, Jehoram T.

doi: 10.1369/jhc.4A6544.2005pmid: 15872055

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard.

Maher, Diane M.; Zhang, Zhi-Qiang; Schacker, Timothy W.; Southern, Peter J.

doi: 10.1369/jhc.4A6534.2005pmid: 15872056

The majority of newly acquired HIV infections are believed to occur following transmission of virus infectivity across mucosal surfaces, although many mechanistic details still remain unresolved. We have used human ex vivo organ cultures and primary cell populations to analyze the cellular and molecular basis for mucosal HIV transmission. By using human palatine tonsil from routine tonsillectomies and semen from HIV-positive donors, we have created an experimental equivalent to oral HIV transmission. HIV infection was readily transferred into tonsillar lymphocytes, but this transmission into lymphocytes was dramatically reduced when the exposed lymphocyte populations were protected by intact mucosal surfaces. In this study, we consider the impact that leukocyte activation and morphological aberrations in surface structure may have on susceptibility to primary HIV infection and introduce novel time-lapse confocal microscopy procedures that begin to reveal the dynamic complexity associated with cell-mediated HIV transmission. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Nishikawa, Sumio

doi: 10.1369/jhc.4A6533.2005pmid: 15872057

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin ∗∗∗L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.