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1.  Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies 
Histochemistry and cell biology  2015;144(6):613-621.
The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages, and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.
doi:10.1007/s00418-015-1364-9
PMCID: PMC4758119  PMID: 26403093
Biotinylated; IHC; macrophage; rat; Ki-67; double stain
2.  A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: Comparison of fibroblasts and diverse cancer cell lines 
Histochemistry and cell biology  2015;144(6):533-542.
Differing stimuli affect cell-stiffness while cancer metastasis further relates to cell-stiffness. Cell-stiffness determined by atomic Force Microscopy (AFM) has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. 90 μm square fields were recorded from 10 sites of cultured Human Dermal Fibroblasts (HDF), and 3 sites each for melanoma (MM39, WM175, MeIRMu), osteosarcoma (SAOS-2, U2OS), and ovarian carcinoma (COLO316, PEO4) cell lines, each site providing 1,024 measurements as 32x32 square grids. Stiffness recorded below 0.8 μm height was occasionally influenced by substratum, so only stiffness recorded above 0.8 μm was analyzed, but all sites were included for height and volume analysis. COLO316 had the lowest cell height and volume, followed by HDF (p<0.0001), and then PEO4, SAOS-2, MeIRMu, WM175, U2OS, and MM39. HDF were more stiff than all other cells (p < 0.0001), while in descending order of stiffness were PEO4, COLO316, WM175, SAOS-2, U2OS, MM39, and MeIRMu (p < 0.02). Stiffness-fingerprints comprised scattergrams of stiffness values plotted against the height at which each stiffness value was recorded, and appeared unique for each cell type studied, although in most cases the overall form of fingerprints was similar, with maximum stiffness at low height measurements and a second lower peak occurring at high height levels. We suggest our stiffness-fingerprint analytical method provides a more nuanced description than previously reported, and will facilitate study of the stiffness response to cell stimulation.
doi:10.1007/s00418-015-1363-x
PMCID: PMC4874187  PMID: 26357955
Cell-stiffness; Atomic Force Microscopy; Scanning Strategy; Cancer Cells; Fibroblasts
3.  Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input 
Histochemistry and cell biology  2010;134(2):145-157.
Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-β-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and α-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.
doi:10.1007/s00418-010-0723-9
PMCID: PMC5082709  PMID: 20607273
Myopia; Blood flow; Immunohistochemistry; Nitric oxide
4.  An Immunohistochemical Analysis of SERT in the Blood-Brain Barrier of the Male Rat Brain 
Histochemistry and cell biology  2015;144(4):321-329.
5-hydroxytryptamine (5-HT) was originally discovered as a vasoconstrictor. 5-HT lowers blood pressure when administered peripherally to both normotensive and hypertensive male rats. Because the serotonin transporter (SERT) can function bidirectionally, we must consider whether 5-HT can be transported from the bloodstream to the central nervous system (CNS) in facilitating the fall in blood pressure. The blood-brain barrier (BBB) is a highly selective barrier that restricts movement of substances from the bloodstream to the CNS and vice-versa, but the rat BBB has not been investigated in terms of SERT expression. This requires us to determine whether the BBB of the rat, the species in which we first observed a fall in blood pressure to infused 5-HT, expresses SERT. We hypothesized that SERT is present in the BBB of the male rat. To test this hypothesis, over 500 blood vessels were sampled from coronal slices of six male rat brains. Immunofluorescence of these coronal slices was used to determine if SERT and RecA-1 (an endothelial cell marker) colocalized to the BBB. Blood vessels were considered to be capillaries if they were between 1.5 and 23 μm (intraluminal diameter). SERT was identified in the largest pial vessels of the BBB (mean ± SEM= 228.70 ± 18.71 μm, N=9) and the smallest capillaries (mean ± SEM= 2.75 ± 0.12 μm, N=369). SERT was not identified in the endothelium of blood vessels ranging from 20 to 135 μm (N=45). The expression of SERT in the rat BBB means that 5-HT entry into the CNS must be considered a potential mechanism when investigating 5-HT-induced fall in blood pressure.
doi:10.1007/s00418-015-1343-1
PMCID: PMC4575874  PMID: 26223876
5-HT; blood brain barrier; rat; serotonin transporter
5.  The trophoblast plug during early pregnancy: a deeper insight 
Histochemistry and Cell Biology  2016;146(6):749-756.
During the first trimester of pregnancy, foetal endovascular trophoblasts invade into maternal spiral arteries, accumulate and form plugs in the lumen of the vessels. These plugs only allow blood plasma to seep through. Hence, during the first trimester of pregnancy, a first flow of fluids through the placental intervillous space is established, resulting in a physiological oxygen gradient between mother and foetus. The trophoblast plugs block spiral arteries until the beginning of the second trimester (11–14 weeks). In parallel, uterine glands are invaded and opened by endoglandular trophoblasts towards the intervillous space of the placenta, without showing the formation of plugs (Moser et al. in Hum Reprod 25:1127–1136, 2010, Hum Reprod Oxf Engl 30:2747–2757, 2015). This enables histiotrophic nutrition of the embryo prior to onset of maternal blood flow into the placenta. Failure of these endovascular and endoglandular invasion processes may lead to miscarriage or pregnancy disorders such as intrauterine growth restriction (IUGR). After dissolution of the plugs, the onset of maternal blood flow allows maternal blood cells to enter the intervillous space and oxygen concentrations rise up. In this study, we demonstrate for the first time serial cross sections through a trophoblast plug in a first trimester placental bed specimen. Invaded and plugged arteries as well as invaded uterine glands in week 11 of gestation are visualized with specific immunohistochemical double staining techniques. We show that spiral artery plugs appear throughout the placental invasion zone and illustrate erythrocytes stowed due to trophoblast plugs. In addition, we give evidence of the presence of MMP-1 in plugs of invaded spiral arteries. The results reveal a better understanding and a closer insight into the morphological appearance of trophoblast plugs and the consequences for placental and uterine blood flow.
doi:10.1007/s00418-016-1474-z
PMCID: PMC5101277  PMID: 27510415
Placenta; Spiral artery; Trophoblast plug; Endovascular trophoblast; Endoglandular trophoblast; Uterine gland
7.  Colocalization coefficients evaluating the distribution of molecular targets in microscopy methods based on pointed patterns 
Histochemistry and Cell Biology  2016;146(4):391-406.
In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders’ colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders’ colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.
doi:10.1007/s00418-016-1467-y
PMCID: PMC5037163  PMID: 27460592
Colocalization; Quantitative analysis; Pointed patterns; Transmission electron microscopy; Manders’ coefficients; Immunohistochemistry
8.  The high correlation between counts and area fractions of lipofuscin granules, a biomarker of oxidative stress in muscular dystrophies 
Histochemistry and Cell Biology  2016;146(5):627-634.
Images of cryostat unstained sections of two skeletal muscles, diaphragm and extensor digitorum longus (EDL), from wild-type normal and dystrophic mdx mice were captured with a fluorescence microscope, binarised and analysed by an automated procedure using ImageJ free software. The numbers, Feret diameters and areas of autofluorescent lipofuscin (LF)-like granules in the sections were determined from the binary images. The mean numbers of counted LF granules per mm3 muscle tissue correlated highly (r ≥ 0.9) with the area fractions of the granules in sections of both normal and mdx muscles. The similar distribution patterns of granule sizes in sections of diaphragm and EDL muscles are consistent with the high correlations.
doi:10.1007/s00418-016-1462-3
PMCID: PMC5073145  PMID: 27394427
Lipofuscin; Quantification; Image analysis; Skeletal muscle; Mdx mouse; Muscular dystrophy
9.  A role for GPR55 in human placental venous endothelial cells 
Histochemistry and cell biology  2015;144(1):49-58.
Endocannabinoids and their G protein-coupled receptors have been suggested to play a key role in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labour. G protein-coupled receptor 55 (GPR55) was previously postulated to be another cannabinoid receptor, since specific cannabinoids were shown to act independently of the classical cannabinoid receptors CB1 or CB2. Current knowledge about GPR55 expression and function in human placenta is very limited and motivated us to evaluate human placental GPR55 expression in relation to other human peripheral tissues and to analyze spatiotemporal GPR55 expression in human placenta. Gene expression analysis revealed low GPR55 levels in human placenta, when compared to spleen and lung, the organs showing highest GPR55 expression. Moreover, expression analysis showed 5.8 fold increased placental GPR55 expression at term compared to first trimester. Immunohistochemistry located GPR55 solely at the fetal endothelium of first trimester and term placenta. qPCR and immunocytochemistry consistently confirmed GPR55 expression in isolated primary placental arterial and venous endothelial cells. Incubation with L-α-lysophosphatidylinositol (LPI), the specific and functional ligand for GPR55, at a concentration of 1 μM, significantly enhanced migration of venous, but not arterial endothelial cells. LPI enhanced migration was inhibited by the GPR55 antagonist O-1918, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function.
doi:10.1007/s00418-015-1321-7
PMCID: PMC4487816  PMID: 25869640
Human placenta; Primary placental endothelial cells; Cannabinoid receptors; G protein-coupled receptor 55
10.  Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons 
Histochemistry and Cell Biology  2016;146(4):489-512.
Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47 kDa) in Drosophila. Sap47 null mutant larvae show reduced short-term synaptic plasticity and a defect in associative behavioral plasticity. In cultured adipocytes, Syap1 functions as part of a complex that phosphorylates protein kinase Bα/Akt1 (Akt1) at Ser473 and promotes differentiation. The role of Syap1 in the vertebrate nervous system is unknown. Here, we generated a Syap1 knock-out mouse and show that lack of Syap1 is compatible with viability and fertility. Adult knock-out mice show no overt defects in brain morphology. In wild-type brain, Syap1 is found widely distributed in synaptic neuropil, notably in regions rich in glutamatergic synapses, but also in perinuclear structures associated with the Golgi apparatus of specific groups of neuronal cell bodies. In cultured motoneurons, Syap1 is located in axons and growth cones and is enriched in a perinuclear region partially overlapping with Golgi markers. We studied in detail the influence of Syap1 knockdown and knockout on structure and development of these cells. Importantly, Syap1 knockout does not affect motoneuron survival or axon growth. Unexpectedly, neither knockdown nor knockout of Syap1 in cultured motoneurons is associated with reduced Ser473 or Thr308 phosphorylation of Akt. Our findings demonstrate a widespread expression of Syap1 in the mouse central nervous system with regionally specific distribution patterns as illustrated in particular for olfactory bulb, hippocampus, and cerebellum.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-016-1457-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-016-1457-0
PMCID: PMC5037158  PMID: 27344443
Brain; BSTA; Viability; Syap1 localization; Glutamatergic synapses; PKB/Akt phosphorylation
11.  Dynamic changes of nuclear RNA foci in proliferating DM1 cells 
Histochemistry and cell biology  2015;143(6):557-564.
Introduction
Nuclear RNA foci are molecular hallmarks of myotonic dystrophy type 1 (DM1). However, no designated study has investigated their formation and changes in proliferating cells. Proliferating cells, as stem cells, consist of an important cellular pool in the human body. The revelation of foci changes in these cells might shed light on the effects of the mutation on these specific cells and tissues. In this study, we used human DM1 iPS-cell derived neural stem cells (NSCs) as cellular models to investigate the formation and dynamic changes of RNA foci in proliferating cells.
Materials and Methods
Human DM1 NSCs derived from human DM1 iPS cells were cultured under proliferation conditions and non-proliferation conditions following Mitomycin C treatment. The dynamic changes of foci during the cell cycle were investigated by fluorescence in situ hybridization (FISH).
Results
We found RNA foci formed and dissociated during the cell cycle. Nuclear RNA foci were most prominent in number and size just prior to entering mitosis (early prophase). During mitosis, most foci disappeared. After entering interphase, RNA foci accumulated again in the nuclei. After stopping cell dividing by treatment of Mitomycin C, the number of nuclear RNA foci increased significantly.
Conclusion
DM1 NSC nuclear RNA foci undergo dynamic changes during cell cycle and mitosis is a mechanism to decrease foci load in the nuclei, which may explain why dividing cells are less affected by the mutation. The dynamic changes need to be considered when using foci as a marker to monitor the effects of therapeutic drugs.
doi:10.1007/s00418-015-1315-5
PMCID: PMC4439307  PMID: 25715678
Muscular Dystrophy; Foci; Molecular Marker; Neural Stem Cell
12.  Placental fractalkine mediates adhesion of THP-1 monocytes to villous trophoblast 
Histochemistry and cell biology  2015;143(6):565-574.
The chemokine fractalkine (CX3CL1) recently attracted increasing attention in the field of placenta research due to its dual nature, acting both as membrane-bound and soluble form. While the membrane-bound form mediates flow resistant adhesion of leukocytes to endothelial and epithelial cells via its corresponding receptor CX3CR1, the soluble form arises from metalloprotease dependent shedding and bears chemoattractive activity for monocytes, natural killer cells and T-cells. In human placenta, fractalkine is expressed at the apical microvillous plasma membrane of the syncytiotrophoblast, which may enable close physical contact with circulating maternal leukocytes. Based on these observations we tested the hypothesis that fractalkine mediates adhesion of monocytes to the villous trophoblast. Forskolin-induced differentiation and syncytialization of the trophoblast cell line BeWo was accompanied with a substantial upregulation in fractalkine expression and led to increased adhesion of the monocyte cell line THP-1, which preferentially bound to syncytia. Blocking as well as silencing of the fractalkine receptor CX3CR1 proved involvement of the fractalkine/CX3CR1 system in adherence of THP-1 monocytes to villous trophoblast. Pre-incubation of THP-1 monocytes with human recombinant fractalkine as well as silencing of CX3CR1 expression in THP-1 monocytes significantly impaired their adherence to BeWo cells and primary term trophoblasts. The present study suggests fractalkine as another candidate amongst the panel of adhesion molecules enabling stable interaction between leukocytes and the syncytiotrophoblast.
doi:10.1007/s00418-014-1304-0
PMCID: PMC4487366  PMID: 25566740
placental fractalkine; chemokine; trophoblast; monocyte; adhesion
13.  Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage 
Histochemistry and Cell Biology  2016;146(4):445-455.
Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt’s base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus–surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt–villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-016-1453-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-016-1453-4
PMCID: PMC5037145  PMID: 27246004
Cyclic guanosine monophosphate; Enteroendocrine cells; Guanylyl cyclase C; Intestinal fluid transport
14.  Immunolabeling of Gamma-glutamyl transferase 5 in Normal Human Tissues Reveals Expression and Localization Differs from Gamma-glutamyl transferase 1 
Histochemistry and cell biology  2014;143(5):505-515.
Gamma-glutamyl transferase (GGT5) was discovered due to its ability to convert leukotriene C4 (LTC4, a glutathione S-conjugate) to LTD4 and may have an important role in the immune system. However, it was not known which cells express the enzyme in humans. We have developed a sensitive and specific antibody that can be used to detect human GGT5 on western blots and in fixed tissue sections. We localized GGT5 expression in normal human tissues. We observed GGT5 expressed by macrophages present in many tissues, including tissue-fixed macrophages such as Kupffer cells in the liver and dust cells in the lung. GGT5 was expressed in some of the same tissues that have been shown to express gamma-glutamyl transferase (GGT1), the only other enzymatically active protein in this family. But, the two enzymes were often expressed by different cell types within the tissue. For example, GGT5 was expressed by the interstitial cells of the kidney; whereas, GGT1 is expressed on the apical surface of the renal proximal tubules. Other tissues with GGT5-positive cells included: adrenal gland, salivary gland, pituitary, thymus, spleen, liver, bone marrow, small intestine, stomach, testis, prostate and placenta. GGT5 and GGT1 are cell surface enzymes. The different pattern of expression results in their access to different extracellular fluids and therefore different substrates. GGT5 has access to substrates in blood and intercellular fluids, while GGT1 has access primarily to fluids in ducts and glands throughout the body. These data provide new insights into the different functions of these two related enzymes.
doi:10.1007/s00418-014-1295-x
PMCID: PMC4393757  PMID: 25377544
Gamma-glutamyl transferase; gamma-glutamyl transpeptidase; Leukotriene C4; glutathione; glycoproteins
15.  Altered expression of P2X3 in vagal and spinal afferents following esophagitis in rats 
Histochemistry and cell biology  2009;132(6):585-597.
Purinergic P2X3 receptors are predominantly expressed in small diameter primary afferent neurons and activation of these receptors by adenosine triphosphate is reported to play an important role in nociceptive signaling. The objective of this study was to investigate the expression of P2X3 receptors in spinal and vagal sensory neurons and esophageal tissues following esophagitis in rats. Two groups of rats were used including 7 days fundus-ligated (7D-ligated) esophagitis and sham-operated controls. Esophagitis was produced by ligating the fundus and partial obstruction of pylorus that initiated reflux of gastric contents. The sham-operated rats underwent midline incision without surgical manipulation of the stomach. Expressions of P2X3 receptors in thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophageal tissues were evaluated by RT–PCR, western blot and immunohistochemistry. Esophageal neurons were identified by retrograde transport of Fast Blue from the esophagus. There were no significant differences in P2X3 mRNA expressions in DRGs (T1–T3) and NGs between 7D-ligated and sham-operated rats. However, there was an upregulation of P2X3 mRNA in DRGs (T6–T12) and in the esophageal muscle. At protein level, P2X3 exhibited significant upregulation both in DRGs and in NGs of rats having chronic esophagitis. Immunohistochemical analysis exhibited a significant increase in P2X3 and TRPV1 co-expression in DRGs and NGs in 7D-ligated rats compared to sham-operated rats. The present findings suggest that chronic esophagitis results in upregulation of P2X3 and its co-localization with TRPV1 receptor in vagal and spinal afferents. Changes in P2X3 expression in vagal and spinal sensory neurons may contribute to esophageal hypersensitivity following acid reflux-induced esophagitis.
doi:10.1007/s00418-009-0639-4
PMCID: PMC4820021  PMID: 19784665
P2X3 receptor; Dorsal root ganglia; Vagus; Acid reflux; Esophagitis
16.  Expression of area-specific M2-macrophage phenotype by recruited rat monocytes in duct-ligation pancreatitis 
Acute pancreatitis remains a disease of uncertain pathogenesis and no established specific therapy. Previously, we found a predominant increase and active proliferation of macrophages in the inflamed tissues of a rat duct-ligation pancreatitis model. To analyze the origin and possible role of these macrophages, we investigated their in situ cellular kinetics in a rat model of duct-ligation pancreatitis using a recently established method of multicolor immunostaining for macrophage markers and for proliferating cells with ethynyl deoxyuridine. To detect monocyte-derived macrophages, green fluorescent protein-transgenic (GFP+) leukocytes were transferred to monocyte-depleted recipients. In the inflamed pancreas, infiltrating macrophages were mainly two phenotypes, CD68+CD163− round cells and CD68+CD163+ large polygonal cells, both of which showed active proliferation. In the interlobular area, the proportions of CD68+CD163low and CD68+CD163high cells increased over time. Most expressed the M2-macrophage markers CD206 and arginase 1. In contrast, in the interacinar area, CD68+ cells did not upregulate CD163 and CD206, but ~30 % of them expressed the M1 marker nitric oxide synthase 2 on day 4. GFP+-recruited cells were primarily CD68+CD163− monocytes on day 1 and showed phenotypic changes similar to those of the monocyte non-depleted groups. In conclusion, infiltrating macrophages mostly formed two distinct subpopulations in different areas: monocyte-derived macrophages with the M2 phenotype in the interlobular area or non-M2 phenotype in the interacinar area. Involvement of resident macrophages might be minor in this model. These results are the first demonstration of an upregulated M2 phenotype in rat inflammatory monocytes, which may promote tissue repair.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-016-1406-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-016-1406-y
PMCID: PMC4848343  PMID: 26860866
Pancreatitis; Multicolor immunostaining; Cellular kinetics; Proliferation; Monocyte recruitment; M2 macrophage
17.  Molecular insights into the premature aging disease progeria 
Hutchinson–Gilford progeria syndrome (HGPS) is an extremely rare premature aging disease presenting many features resembling the normal aging process. HGPS patients die before the age of 20 years due to cardiovascular problems and heart failure. HGPS is linked to mutations in the LMNA gene encoding the intermediate filament protein lamin A. Lamin A is a major component of the nuclear lamina, a scaffold structure at the nuclear envelope that defines mechanochemical properties of the nucleus and is involved in chromatin organization and epigenetic regulation. Lamin A is also present in the nuclear interior where it fulfills lamina-independent functions in cell signaling and gene regulation. The most common LMNA mutation linked to HGPS leads to mis-splicing of the LMNA mRNA and produces a mutant lamin A protein called progerin that tightly associates with the inner nuclear membrane and affects the dynamic properties of lamins. Progerin expression impairs many important cellular processes providing insight into potential disease mechanisms. These include changes in mechanosignaling, altered chromatin organization and impaired genome stability, and changes in signaling pathways, leading to impaired regulation of adult stem cells, defective extracellular matrix production and premature cell senescence. In this review, we discuss these pathways and their potential contribution to the disease pathologies as well as therapeutic approaches used in preclinical and clinical tests.
doi:10.1007/s00418-016-1411-1
PMCID: PMC4796323  PMID: 26847180
Lamins; Premature aging; Progeria; Nucleoplasmic lamins; Chromatin; Signaling; Adult stem cells; Senescence
18.  Morphology of nuclear transcription 
Gene expression control is a fundamental determinant of cellular life with transcription being the most important step. The spatial nuclear arrangement of the transcription process driven by RNA polymerases II and III is nonrandomly organized in foci, which is believed to add another regulatory layer on gene expression control. RNA polymerase I transcription takes place within a specialized organelle, the nucleolus. Transcription of ribosomal RNA directly responds to metabolic requirements, which in turn is reflected in the architecture of nucleoli. It differs from that of the other polymerases with respect to the gene template organization, transcription rate, and epigenetic expression control, whereas other features are shared like the formation of DNA loops bringing genes and components of the transcription machinery in close proximity. In recent years, significant advances have been made in the understanding of the structural prerequisites of nuclear transcription, of the arrangement in the nuclear volume, and of the dynamics of these entities. Here, we compare ribosomal RNA and mRNA transcription side by side and review the current understanding focusing on structural aspects of transcription foci, of their constituents, and of the dynamical behavior of these components with respect to foci formation, disassembly, and cell cycle.
doi:10.1007/s00418-016-1412-0
PMCID: PMC4796325  PMID: 26847177
Nuclear architecture; Nucleoli; Transcription factory; Chromatin; Loop formation; Epigenetic gene regulation
19.  Integration of CD45-positive leukocytes into newly forming lymphatics of adult mice 
The embryonic origin of lymphatic endothelial cells (LECs) has been a matter of controversy since more than a century. However, recent studies in mice have supported the concept that embryonic lymphangiogenesis is a complex process consisting of growth of lymphatics from specific venous segments as well as the integration of lymphangioblasts into the lymphatic networks. Similarly, the mechanisms of adult lymphangiogenesis are poorly understood and have rarely been studied. We have recently shown that endothelial progenitor cells isolated from the lung of adult mice have the capacity to form both blood vessels and lymphatics when grafted with Matrigel plugs into the skin of syngeneic mice. Here, we followed up on these experiments and studied the behavior of host leukocytes during lymphangiogenesis in the Matrigel plugs. We observed a striking co-localization of CD45+ leukocytes with the developing lymphatics. Numerous CD45+ cells expressed the LEC marker podoplanin and were obviously integrated into the lining of lymphatic capillaries. This indicates that, similar to inflammation-induced lymphangiogenesis in man, circulating CD45+ cells of adult mice are capable of initiating lymphangiogenesis and of adopting a lymphvasculogenic cellular differentiation program. The data are discussed in the context of embryonic and inflammation-induced lymphangiogenesis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-015-1399-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-015-1399-y
PMCID: PMC4848334  PMID: 26748643
Lymphatic endothelial cell; Lymphangioblast; Lymphangiogenesis; Lymphvasculogenesis; Endothelial progenitor cell; Mesenchymal stem cell
20.  Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth 
Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.
doi:10.1007/s00418-015-1390-7
PMCID: PMC4735258  PMID: 26671787
Calcium signaling; Cell differentiation; Electron microscopy; Neuritogenesis; Protein–protein interaction
21.  Elevated DNA damage response in pancreatic cancer 
Histochemistry and cell biology  2014;142(6):713-720.
Pancreatic cancer is one of the most aggressive and intractable human malignant tumors and a leading cause of cancer-related deaths across the world, with incidence equaling mortality. Because of the extremely high malignance, this disease is usually diagnosed at its advanced stage and recurs even after surgical excision. Pancreatic adenocarcinoma is generally thought to arise from pathological changes of pancreatic duct, and the pancreatic ductal adenocarcinoma (PDA) accounts for more than 90% of malignant neoplasms of the pancreas. To date, scientists have revealed several risk factors for pancreatic cancer, including smoking, family history, and ageing. However, the underlying molecular mechanism remains unclear. Meanwhile, more mutations of DNA damage response factors have been identified in familial pancreatic cancers, implying a potential link between DNA damage and pancreatic cancer. DNA damage is a recurring phenomenon in our bodies which could be induced by exogenous agents and endogenous metabolism. Accumulated DNA lesions cause genomic instability which eventually results in tumorigenesis. In this study, we showed obvious DNA damages existed in human pancreatic cancer, which activated DNA damage response and the DNA repair pathway including ATM, DNA-PK, CHK1 and CHK2. The persistent DNA damage in pancreatic tissue may be the source for its tumorigenesis.
doi:10.1007/s00418-014-1245-7
PMCID: PMC4242800  PMID: 25002126
DNA damage response; pancreatic cancer; checkpoint
22.  Galanin regulates blood glucose level in the zebrafish: a morphological and functional study 
The present study has demonstrated the galaninergic innervation of the endocrine pancreas including sources of the galaninergic nerve fibers, and the influence of galanin receptor agonists on blood glucose level in the zebrafish. For the first time, a very abundant galaninergic innervation of the endocrine pancreas during development is shown, from the second day post-fertilization to adulthood. The fibers originated from ganglia consisting of galanin-IR, non-adrenergic (non-sensory) neurons located rostrally to the pancreatic tissue. The ganglia were found on the dorsal side of the initial part of the anterior intestinal segment, close to the intestinal branch of the vagus nerve. The galanin-IR neurons did not show immunoreactivity for applied antibodies against tyrosine hydroxylase, choline acetyltransferase, and vesicular acetylcholine transporter. Intraperitoneal injections of galanin analog NAX 5055 resulted in a statistically significant increase in the blood glucose level. Injections of another galanin receptor agonist, galnon, also caused a rise in blood glucose level; however, it was not statistically significant. The present findings suggest that, like in mammals, in the zebrafish galanin is involved in the regulation of blood glucose level. However, further studies are needed to elucidate the exact mechanism of the galanin action.
doi:10.1007/s00418-015-1376-5
PMCID: PMC4710661  PMID: 26496922
Galanin; Blood glucose; Insulin; Somatostatin; Parasympathetic ganglia; Zebrafish
23.  A novel interaction between ATOH8 and PPP3CB 
ATOH8 is a bHLH transcription factor playing roles in a variety of developmental processes such as neurogenesis, differentiation of pancreatic precursor cells, development of kidney and muscle, and differentiation of endothelial cells. PPP3CB belongs to the catalytic subunit of the serine/threonine phosphatase, calcineurin, which can dephosphorylate its substrate proteins to regulate their physiological activities. In our study, we demonstrated that ATOH8 interacts with PPP3CB in vitro with different approaches. We show that the conserved catalytic domain of PPP3CB interacts with both the N-terminus and the bHLH domain of ATOH8. Although the interaction domain of PPP3CB is conserved among all isoforms of calcineurin A, ATOH8 selectively interacts with PPP3CB instead of PPP3CA, probably due to the unique proline-rich region present in the N-terminus of PPP3CB, which controls the specificity of its interaction partners. Furthermore, we show that inhibition of the interaction with calcineurin inhibitor, cyclosporin A (CsA), leads to the retention of ATOH8 to the cytoplasm, suggesting that the interaction renders nuclear localization of ATOH8 which may be critical to control its activity as transcription factor.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-015-1368-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-015-1368-5
PMCID: PMC4710663  PMID: 26496921
ATOH8; PPP3CB; Calcineurin; Cyclosporin A
24.  β- and γ-Actins in the nucleus of human melanoma A375 cells 
Histochemistry and Cell Biology  2015;144(5):417-428.
Actin is a highly conserved protein that is expressed in all eukaryotic cells and has essential functions in the cytoplasm and the nucleus. Nuclear actin is involved in transcription by all three RNA polymerases, chromatin remodelling, RNA processing, intranuclear transport, nuclear export and in maintenance of the nuclear architecture. The nuclear actin level and polymerization state are important factors regulating nuclear processes such as transcription. Our study shows that, in contrast to the cytoplasm, the majority of endogenous nuclear actin is unpolymerized in human melanoma A375 cells. Most mammalian cells express the two non-muscle β- and γ-actin isoforms that differ in only four amino acids. Despite their sequence similarity, studies analysing the cytoplasmic functions of these isoforms demonstrated that β- and γ-actins show differences in localization and function. However, little is known about the involvement of the individual actin isoforms in nuclear processes. Here, we used the human melanoma A375 cell line to analyse actin isoforms in regard to their nuclear localization. We show that both β- and γ-non-muscle actin isoforms are present in nuclei of these cells. Immunolocalization studies demonstrate that both isoforms co-localize with RNA polymerase II and hnRNP U. However, we observe differences in the ratio of cytoplasmic to nuclear actin distribution between the isoforms. We show that β-actin has a significantly higher nucleus-to-cytoplasm ratio than γ-actin.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-015-1349-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-015-1349-8
PMCID: PMC4628621  PMID: 26239425
Actin; Nucleus; Nuclear actin; Actin isoform; Actin polymerization state
25.  Macrophage expression of tartrate-resistant acid phosphatase as a prognostic indicator in colon cancer 
Histochemistry and cell biology  2014;142(2):195-204.
Recent research has indicated that separate populations of macrophages are associated with differing outcomes in cancer survival. In our study, we examine macrophage expression of tartrate resistant acid phosphatase (TRAP) and its effect on survival in colon cancer. Immunohistochemical analysis on colorectal adenocarcinomas confirmed macrophage expression of TRAP. Co-localization of TRAP with CD68, a pan-macrophage marker, revealed that TRAP is present in some but not all subpopulations of macrophages. Further co-localization of TRAP with CD163, an M2 marker, revealed that TRAP is expressed by both M2 and non-M2 macrophages. TRAP expression was then measured using the AQUA method of quantitative immunofluorescence in a tissue microarray consisting of 233 colorectal cancer patients seen at Yale-New Haven Hospital. Survival analysis revealed that patients with high TRAP expression have a 22% increase in 5-year survival (uncorrected log rank p=0.025) and a 47% risk reduction for disease specific death (p=0.02). This finding was validated in a second cohort of older cases consisting of 505 colorectal cancer patients. Patients with high TRAP expression in the validation set had a 19% increase in 5-year survival (log rank p=0.0041) and a 52% risk reduction of death (p=0.0019). These results provide evidence that macrophage expression of TRAP is associated with improved outcome, and implicates TRAP as a potential biomarker in colon cancer.
doi:10.1007/s00418-014-1181-6
PMCID: PMC4101067  PMID: 24429833
Tartrate-resistant acid phosphatase; M1; M2; macrophage; colon cancer; biomarker

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