Journal of Histochemistry and Cytochemistry current issue

Expression of Aquaporin 9 in Rat Liver and Efferent Ducts of the Male Reproductive System After Neonatal Diethylstilbestrol Exposure

Wellejus, A., Jensen, H. E., Loft, S., Jonassen, T. E. — 2008-04-18

Aquaporins (AQP) have important solute transport functions in many tissues including the epididymal efferent ducts (ED) and in the liver. We investigated the effect of neonatal exposure to diethylstilbestrol (DES) on AQP9 expressions in the ED and in the liver of rats. DES was administered from day 2 to day 20 postnatally at a dose of 4,8 µg/day, and AQP9 protein and mRNA were measured by immunoblotting and real-time PCR, respectively, along with immunohistochemistry. DES caused hepatic downregulation of AQP9 at both the protein and mRNA level; however, decreased AQP9 labeling was only observed in the periportal zone. In the ED, AQP9 protein expression was increased in the DES-treated animals by 300% that could be ascribed to a widening of the ED lumen, whereas no difference was observed in AQP9 mRNA expression. Immunohistochemical findings revealed that AQP9 expression was confined to the epithelial cells of the ED. In conclusion, neonatal DES exposure appears to upregulate AQP9 channels in the ED in male rats, whereas a downregulation in the hepatic expression was observed, particularly in the periacinous area.(J Histochem Cytochem 56:425–432, 2008)

Cellular Expression Patterns of Genes Upregulated in Murine and Human Colonic Neoplasms

Chen, X., Ehrhardt, W. M., Halberg, R. B., Aronow, B. J., Dove, W. F. — 2008-04-18

Markers overexpressed in colonic tumors of the multiple intestinal neoplasia (Min) mouse have been recently identified by cDNA subtractive hybridization and by microarray analysis. The significance of such a marker depends on its expression in tumor vs stromal lineages and on its expression pattern in normal tissue. From 34 differentially expressed markers, 14 were found to be expressed from supporting lineages. The markers expressed in the tumor lineage were grouped into three classes on the basis of ISH in mouse models and IHC in human adenomas. The first class includes markers expressed both in neoplastic cells and in the proliferating cells residing at the bottom of normal colonic crypts. The second class of markers shows elevated expression in neoplastic cells and also in the postmitotic Paneth cells of the small intestine. Finally, the third class of marker shows detectable intestinal expression only within tumors but not in the normal intestinal epithelium. Is such a tumor-associated marker uniquely essential for tumor growth? Deficiency for the tumor-associated glycoprotein clusterin does not affect the multiplicity or growth rate of intestinal tumors in Min mice. Thus, clusterin is a candidate secreted colon cancer marker but not a single target for chemoprevention or therapy. (J Histochem Cytochem 56:433–441, 2008)

Voltage-gated Potassium Channel (Kv) Subunits Expressed in the Rat Cochlear Nucleus

2008-04-18

Because the neuronal membrane properties and firing characteristics are crucially affected by the depolarization-activated K⁺ channel (Kv) subunits, data about the Kv distribution may provide useful information regarding the functionality of the neurons situated in the cochlear nucleus (CN). Using immunohistochemistry in free-floating slices, the distribution of seven Kv subunits was described in the rat CN. Positive labeling was observed for Kv1.1, 1.2, 1.6, 3.1, 3.4, 4.2, and 4.3 subunits. Giant and octopus neurons showed particularly strong immunopositivity for Kv3.1; octopus neurons showed intense Kv1.1- and 1.2-specific reactions also. In the latter case, an age-dependent change of the expression pattern was also documented; although both young and older animals produced definite labeling for Kv1.2, the intensity of the reaction increased in older animals and was accompanied with the translocation of the Kv1.2 subunits to the cell surface membrane. The granule cell layer exhibited strong Kv4.2-specific immunopositivity, and markedly Kv4.2-positive glomerular synapses were also seen. It was found that neither giant nor pyramidal cells were uniform in terms of their Kv expression patterns. Our data provide new information about the Kv expression of the CN and also suggest potential functional heterogeneity of the giant and pyramidal cells. (J Histochem Cytochem 56:443–465, 2008)

Colloidal-gold Immunocytochemical Localization of Osteopontin in Avian Eggshell Gland and Eggshell

Hincke, M. T., Chien, Y.-C., Gerstenfeld, L. C., McKee, M. D. — 2008-04-18

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix–mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix–mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite—its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance. (J Histochem Cytochem 56:467–476, 2008)

Glycodelin Protein and mRNA Is Downregulated in Human First Trimester Abortion and Partially Upregulated in Mole Pregnancy

2008-04-18

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy. (J Histochem Cytochem 56:477–485, 2008)

Molecular Definition of High-resolution Multicolor Banding Probes: First Within the Human DNA Sequence Anchored FISH Banding Probe Set

2008-04-18

Fluorescence in situ hybridization (FISH) banding approaches are standard for the exact characterization of simple, complex, and even cryptic chromosomal aberrations within the human genome. The most frequently applied FISH banding technique is the multicolor banding approach, also abbreviated as m-band, MCB, or in its whole genomic variant multitude MCB (mMCB). MCB allows the differentiation of chromosome region–specific areas at the GTG band and sub-band level and is based on region-specific microdissection libraries, producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) mapped, comprehensive, genome-wide human MCB probe set. All 169 region-specific microdissection libraries were characterized in detail for their size and the regions of overlap. In summary, the unique possibilities of the MCB technique to characterize chromosomal breakpoints in one FISH experiment are now complemented by the feature of being anchored within the human DNA sequence at the BAC level. (J Histochem Cytochem 56:487–493, 2008)

Murine mCLCA6 Is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co-localizes With the Cystic Fibrosis Transmembrane Conductance Regulator

Bothe, M. K., Braun, J., Mundhenk, L., Gruber, A. D. — 2008-04-18

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine. (J Histochem Cytochem 56:495–509, 2008)

Cannabinoid CB1 Receptors Are Expressed by Parietal Cells of the Human Gastric Mucosa

2008-04-18

Experimental data suggest that the endogenous cannabinoid system is involved in gastric function in different animal species. In most of them, CB₁ receptors have been localized on vagal terminals innervating the external wall of the stomach. We aimed at studying the putative presence and distribution of these receptors in the human gastric mucosa. To this end, we first performed Western blotting, RT-PCR, in situ hybridization, and immunohistochemical analysis of CB₁ protein distribution in biopsy samples of healthy individuals. To determine the precise cell populations expressing CB₁ receptors, we performed double immunofluorescence plus confocal microscopy analysis of the same samples. Our results show that CB₁ receptors are present in the gastric epithelium of the mucosa. Specifically, they are expressed by a subpopulation of mucosal cells, the acid-secreting parietal cells, as shown by double immunohistochemical staining and by their differential abundance in subregions of the gastric mucosa. These results reinforce the notion of a prominent role for the endocannabinoid system in the gastric function in humans and postulate the use of cannabinoid CB₁ receptors in parietal cells as new therapeutic targets for the regulation of gastric acid production. (J Histochem Cytochem 56:511–516, 2008)

Pathogenic Role of NF-{kappa}B Activation in Tubulointerstitial Inflammatory Lesions in Human Lupus Nephritis

Zheng, L., Sinniah, R., Hsu, S. I-H. — 2008-04-18

In vitro and in vivo experimental studies suggest that the transcription factor NF-B plays a role in tubulointerstitial injury. We investigated possible cellular and molecular mechanisms involving NF-B activation in the progression of tubulointerstitial lesions in human lupus nephritis (LN). Paraffin-embedded renal biopsies from 50 patients with LN and six control patients with minimal change disease (MCD) were examined by Southwestern histochemistry for in situ detection of active NF-B and AP-1. Immunohistochemistry was performed to examine the expression of NF-B, AP-1, and NF-B regulatory proteins (IB-, p-IB-, and IKK- proteins), as well as NF-B and AP-1 downstream target proinflammatory molecules (ICAM-1, TNF-, IL-1β, IL-6, and GM-CSF) and NF-B upstream signaling molecules (CD40 and CD40L). We observed extensive upregulation of activated NF-B in renal tubular cells and interstitial cells, in parallel with overactivation of transcription factor AP-1 in LN, as compared with normal controls and MCD. Tubular expression of activated NF-B correlated well with the degree of tubulointerstitial histopathological indices and/or renal function. Tubulointerstitial IKK- expression was specifically upregulated in LN. IB- and p-IB- were detected only in interstitial cells in LN. Tubulointerstitial expression levels of NF-B and AP-1 downstream inflammatory molecules and NF-B upstream signaling molecules CD40 and CD40L were markedly enhanced in LN as compared with MCD or normal controls and were associated with tubulointerstitial histopathological indices and/or renal function. The results suggest that altered IKK- expression and NF-B activation along with AP-1 overexpression may play a pathogenic role in tubulointerstitial injury in human LN mediated through a network of downstream proinflammatory molecules. (J Histochem Cytochem 56:517–529, 2008)