Nuclei Of Epithelial Cells Research Articles

Immunohistochemical expression of estrogen receptor α, Bcl-2 and NF-κB P65 in the polyps of patients with and without endometriosis.

To compare the expression of estrogen receptor α (ERα), Bcl-2 and NF-κB P65 in endometrial polyps from patients with and without endometriosis. Immunohistochemistry was used to characterize the expression of ERα, Bcl-2 and NF-κB P65 in proliferative phase endometrial polyps from patients with and without endometriosis. H-Scores indicating the staining intensity for ERα, Bcl-2 and NF-κB P65 in the glandular epithelium and stroma were measured separately. Apoptotic cells were detected with the use of the dUTP nick-end labeling (TUNEL) assay. Bcl-2 was expressed in the cytoplasm of glandular epithelial cells, whereas ERα was expressed in the nuclei of both glandular epithelial and stromal cells. NF-κB P65 was observed in the cytoplasm of both glandular epithelial and stromal cells. The H-scores for Bcl-2 in the endometrial glands significantly higher in the endometriosis polyp group than in the non-endometriosis polyp group. The H-scores for ERα in both stromal and glandular epithelial cells were significantly higher in the endometriosis polyp group than in the non-endometriosis polyp group. The H-scores for Bcl-2 and ERα were positively correlated in all of the women examined. Apoptotic cells in the endometriosis polyp group were significantly less than that of the non-endometriosis polyp group. There expression levels of Bcl-2 and ERα, both of which were significantly increased in the polyps of endometriosis patients compared to those of patients without endometriosis, were positively correlated. These results suggested that imbalanced apoptosis secondary to abnormally high ERα expression was responsible for the high prevalence of polyps in endometriosis patients.

Carnivore Protoparvovirus-1 Associated With an Outbreak of Hemorrhagic Gastroenteritis in Small Indian Civets.

Carnivore protoparvovirus-1 (CPPV-1) infection has been reported frequently in both domestic and wildlife species including wild carnivores. Fifty-five captive small Indian civets (Viverricula indica), farmed for perfume production in Eastern Thailand, showed clinical signs of acute bloody diarrhea, anorexia, vomiting, circling, and seizures. The disease spread within the farm and resulted in the death of 38 of the 55 civets (69% mortality) within a month. Fecal swabs were collected from the 17 surviving civets, and necropsy was performed on 7 of the dead civets. Pathologic findings were severe hemorrhagic gastroenteritis with generalized lymphadenopathy. CPPV-1 was identified in both fecal swabs and postmortem samples by species-specific polymerase chain reaction. Further whole-gene sequencing and restriction fragment length polymorphism analysis suggested feline panleukopenia virus (FPV) as the causative agent. The viral tropism and tissue distribution were confirmed by immunohistochemistry, with immunolabeling in the cytoplasm and nucleus of small intestinal crypt epithelial cells, villous enterocytes, histiocytes in lymphoid tissues, myenteric nerve plexuses, and cerebral and cerebellar neurons. Phylogenetic analysis of civet-derived CPPV-1 indicated a genetic similarity close to the FPV HH-1/86 strain detected in a jaguar (Panthera onca) in China. To our knowledge, this mass die-off of civets is the first evidence of disease associated with CPPV-1 infection in the subfamily Viverrinae. These findings support the multi-host range of parvovirus infection and raises awareness for CPPV-1 disease outbreaks in wildlife species.

P0013MAP KINASES-REGULATED PROTEINS IN SIGNALING PATHWAYS OF VASOPRESSIN IN KIDNEY COLLECTING DUCT

Abstract Background and Aims Activation of G protein-coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R. However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2 expression and phosphorylation, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R. Tripartite motif-containing protein 28 (TRIM28) was identified as a substrate of ERK1 as well as a vasopressin-responsive protein via bioinformatic tools. We further evaluated whether TRIM28 plays a role in vasopressin-mediated regulation of AQP2 in the kidney CD. Method To identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on high-throughput profiles of transcriptome and proteome (http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); and 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate protein (TRIM28) was examined by immunohistochemistry. The role of TRIM28 in vasopressin-mediated AQP2 regulation was examined by quantitative real-time PCR (qRT-PCR) and semiquantitative immunoblotting after RNA interference of TRIM28 in mpkCCDc11 cells. Results Immunoblotting of mpkCCDc11 cells revealed that both p-ERK1/2 and pS261-AQP2 expression was decreased in response to dDAVP (10-9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase-substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed TRIM28, as the substrate of ERK1, has the only one phosphorylation site which was down-regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin-protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP-induced AQP2 mRNA and protein up-regulation was significantly attenuated in mpkCCDc11 cells with siRNA-mediated knockdown of TRIM28. Conclusion We identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.

Effect of Nuclear Stiffness on Cell Mechanics and Migration of Human Breast Cancer Cells.

The migration and invasion of cancer cells through 3D confined extracellular matrices is coupled to cell mechanics and the mechanics of the extracellular matrix. Cell mechanics is mainly determined by both the mechanics of the largest organelle in the cell, the nucleus, and the cytoskeletal architecture of the cell. Hence, cytoskeletal and nuclear mechanics are the major contributors to cell mechanics. Among other factors, steric hindrances of the extracellular matrix confinement are supposed to affect nuclear mechanics and thus also influence cell mechanics. Therefore, we propose that the percentage of invasive cells and their invasion depths into loose and dense 3D extracellular matrices is regulated by both nuclear and cytoskeletal mechanics. In order to investigate the effect of both nuclear and cytoskeletal mechanics on the overall cell mechanics, we firstly altered nuclear mechanics by the chromatin de-condensing reagent Trichostatin A (TSA) and secondly altered cytoskeletal mechanics by addition of actin polymerization inhibitor Latrunculin A and the myosin inhibitor Blebbistatin. In fact, we found that TSA-treated MDA-MB-231 human breast cancer cells increased their invasion depth in dense 3D extracellular matrices, whereas the invasion depths in loose matrices were decreased. Similarly, the invasion depths of TSA-treated MCF-7 human breast cancer cells in dense matrices were significantly increased compared to loose matrices, where the invasion depths were decreased. These results are also valid in the presence of a matrix-metalloproteinase inhibitor GM6001. Using atomic force microscopy (AFM), we found that the nuclear stiffnesses of both MDA-MB-231 and MCF-7 breast cancer cells were pronouncedly higher than their cytoskeletal stiffness, whereas the stiffness of the nucleus of human mammary epithelial cells was decreased compared to their cytoskeleton. TSA treatment reduced cytoskeletal and nuclear stiffness of MCF-7 cells, as expected. However, a softening of the nucleus by TSA treatment may induce a stiffening of the cytoskeleton of MDA-MB-231 cells and subsequently an apparent stiffening of the nucleus. Inhibiting actin polymerization using Latrunculin A revealed a softer nucleus of MDA-MB-231 cells under TSA treatment. This indicates that the actin-dependent cytoskeletal stiffness seems to be influenced by the TSA-induced nuclear stiffness changes. Finally, the combined treatment with TSA and Latrunculin A further justifies the hypothesis of apparent nuclear stiffening, indicating that cytoskeletal mechanics seem to be regulated by nuclear mechanics.

Seasonal expressions of SPAG11A and androgen receptor in the epididymis of the wild ground squirrels (Citellus dauricus Brandt).

Sperm-associated antigen 11A (SPAG11A), a protein expressed exclusively in the epididymis, plays a vital and special role in regulating mammalian sperm maturation. The aim of this study was to investigate the seasonal expressions of SPAG11A and androgen receptor (AR) in the epididymis of the wild ground squirrels (Spermophilus dauricus Brandt). Morphologically, the results showed that epididymis length and weight in the breeding season were significantly higher than those of the non-breeding season. Histologically, the results revealed that enlarged lumen diameters, thickened epithelium and abundant sperm in the breeding season while reduced lumen diameters and epithelium with no sperm in the non-breeding season. SPAG11A was intensely expressed in cytoplasm and nucleus of epithelial cells and smooth muscle cells in the breeding season, and weaker staining in the non-breeding season. In the lumen of epididymis, SPAG11A immunostaining in the sperm of the epididymal corpus and cauda was higher than those in the caput during the breeding season. The immunostaining of AR was only presented in nucleus of smooth muscle cells and epithelial cells in the epididymis from the breeding season rather than the non-breeding season. The results of real-time quantitative PCR also showed that the mRNA levels of SPAG11A and AR in the epididymis during the breeding season were significantly higher than those of the non-breeding season. In addition, the circulating testosterone, follicle- stimulating hormone and luteinizing hormone levels in the squirrels were higher in the breeding season compared with those in the non-breeding season. Taken together, these results implied that SPAG11A might be involved in regulating seasonal changes in epididymal functions of the wild ground squirrels.

Oestrogen receptors and hypoxia inducible factor 1 alpha expression in abdominal wall endometriosis

What are the protein levels and localization of oestrogen receptors (including ERa, ERb and G protein-coupled oestrogen receptor [GPER]) and hypoxia-inducible factor-1alpha (HIF-1a) in normal control endometrium (COEM) and ectopic endometrium from abdominal wall endometriosis (AWE). AWE (n = 20) were obtained during surgery; COEM (n = 40) were collected by curettage. All tissues were obtained during the proliferative or secretory phase. Formalin-fixed paraffin-embedded tissues were used for immunohistochemical study for oestrogen receptors and HIF-1a proteins. The expression of oestrogen receptors and HIF-1a in AWE differed from that in the corresponding menstrual cycle phase of COEM. Compared with COEM, ERa and HIF-1a were decreased whereas ERb and GPER were increased in AWE. The greatest difference was in GPER, with increased protein expression in both the cytoplasm and nucleus of endometrial epithelial and stromal cells, as well as a distinct change in localization from cytoplasmic expression to nuclear and cytoplasmic expression, compared with COEM. Our data suggest that the expression changes of oestrogen receptors and HIF-1a, especially GPER, are associated with AWE, which may provide new clues to understanding the cause of endometriosis.

Follicle Stimulating Hormone Receptor Expression In Histologically Normal And Neoplastic Canine Mammary Gland Tissues

The roles of Follicle Stimulating Hormone (FSH) in reproductive physiology have been recognized for a long time. The hormone achieves its functions by binding to the Follicle Stimulating Hormone Receptor (FSHRc) that is strongly expressed in both male and female gonads. Over the last 30 years FSH and its receptor have been discovered also in normal and neoplastic extragonadal tissues including: placenta, uterus, and most recently mammary glands. In neoplasias they contribute to cancer development, cancer cell migration, invasion and neovascularization. Canine mammary gland tumors (MGTs) are the most frequent tumors in intact bitches. Though frequently studied, their pathogenesis is still not completely understood and the presence of FSHRc has not been investigated. The purpose of this study was to evaluate histologically normal and neoplastic mammary glands for the presence of FSHRc using immunohistochemistry (IHC) and in situ hybridization (ISH). Positive immunohistochemical cytoplasmic staining is noticed in epithelial and the underlying myoepithelial cells of normal and neoplastic mammary glands and also in the cytoplasm of chondrocytes in mixed MGTs. In situ hybridization detected the expression of FSHRc in the nucleus and perinuclear cytoplasm of epithelial and myoepithelial cells of normal and neoplastic mammary glands. The signal was observed in the cytoplasm and, to a lesser degree, in the nucleus of chondrocytes in mixed MGTs. Further studies are necessary to elucidate the physiological and pathophysiological roles of FSH and FSHRc in histologically and neoplastic mammary glands and to investigate their potential use as targets for highly specific chemo‐ or immunotherapies of MGTs.

Epithelial Repair in the Rabbit Cornea Following Aspects of Epithelial Injury and Repair after Mustard Exposure

Sulfur mustard (SM; bis (2‐chloroethyl) sulfide) is a potent vesicating agent that has been used in chemical warfare. Exposure of the eyes to SM can cause corneal erosions, epithelial defects, neovascularization, microblistering, and/or dry eye disease, all injuries that can lead to corneal epithelium sloughing and ultimately blindness. In the present studies, the effects of ocular exposure to SM vapor (0.420 mg/L for 8 min) on the corneas of male New Zealand white rabbits was characterized at 1, 3, 7, 14, 21 and 28 days post‐exposure. During this time, corneal thickness increased from ~400 microns to 600–900 microns. Unexposed control corneas were clear throughout the time course. However, after SM exposure increased corneal thickness was associated with decreased corneal opacity and neovascularization. Histologically, SM exposure induced epithelial‐stromal separation at 3 days, by which time 16% of the epithelia had separated from the stroma, and by 28 days detachment had decreased to 9%. Also observed was post‐SM epithelial thinning, increased stromal disorganization, and edema. Sporadic keratocytes were also evident in the edematous stroma by day 14. At 28 days post‐SM, an inflammatory cell infiltrate was observed in the stroma. In control corneas, keratin 17, a marker of wound repair, was constitutively expressed in the limbus and bulbar conjunctiva. By 14 days post‐SM, keratin 17 was expressed in the peripheral corneal epithelium, and by 28 days it was expressed in the central cornea. PCNA, a proliferation marker, was constitutively expressed in the nuclei of control corneal epithelial cells. Three days post‐SM, PCNA expression was decreased along the damaged basal layer. Twenty eight days post‐SM, PCNA was upregulated in the nuclei of hyperplastic cells of the central cornea and cells of the limbus, indicating wound repair. These data suggest that limbal and /or conjunctival epithelial cells play a role in the repair of SM‐induced corneal injury. A better understanding of the mechanisms of SM‐induced corneal damage will lead to the development of new therapeutics for the treatment of these debilitating ocular injuries. Supported by NIH grant AR055073.Support or Funding InformationSupported by NIH grant AR055073.

MAP kinases‐Regulated Proteins in Downstream Signaling Pathways of Vasopressin V2 Receptor in Kidney Collecting Duct

BackgroundActivation of G protein‐coupled vasopressin V2 receptor (V2R) is critical in water and electrolyte transport in the kidney collecting duct (CD) cells. Stimulation of V2R affects several downstream pathways, including PKA, PI3K/AKT, Wnt, and Ca2+/calmodulin. Previous studies have shown that MAP kinases are also involved as an apparent downstream signaling pathway of V2R, which affects AQP2 phosphorylation (Ser261). However, the role of MAP kinases and their substrate proteins in the vasopressin signaling, including the regulation of AQP2, are unclear. In the present study, substrates of MAP kinases were identified using bioinformatic analyses, and they were mapped on the downstream signaling pathways of V2R.MethodsTo identify comprehensive substrates of MAP kinases in the kidney CD, we investigated 1) the expression of MAP kinases in CD cells by use of databases based on highthroughput profiles of transcriptome and proteome ( http://hpcwebapps.cit.nih.gov/ESBL/Database/index.html); 2) MAP kinases substrates expressed in the CD cells by use of protein phosphorylation databases (PhosphoNetworks and RegPhos 2.0). The identified substrates were mapped on the downstream signaling of V2R. Cellular and subcellular localization of selected substrate proteins was examined by immunohistochemistry. Substrate protein‐mediated AQP2 regulation was examined quantitative real‐time PCR (qRT‐PCR) and semiquantitative immunoblotting.ResultsImmunoblotting of mpkCCDc11 cells revealed that both p‐ERK1/2 and pS261‐AQP2 expression was decreased in response to dDAVP (10−9 M) stimulation. In silico analyses demonstrated that five MAP kinases (ERK1, ERK2, ERK3, JNK2, and MAPK p38 alpha) were identified as the MAP kinases expressed in kidney CD cells. Based on several protein kinase‐substrates databases, 189 proteins were identified as the substrates of the five MAP kinases. In particular, sequential data mining revealed that tripartite motif‐containing 28 (TRIM28), as the substrate of ERK1, has the only one phosphorylation site which was down‐regulated by vasopressin stimulation. Since TRIM28 is a transcription cofactor and also a ubiquitin‐protein E3 ligase, we examined whether TRIM28 is involved in the regulation of AQP2 expression as a mediator of MAP kinases action. Immunofluorescence labeling of mouse and rat kidneys revealed that TRIM28 was exclusively expressed in the nuclei of the tubular epithelial cells, including CD cells. dDAVP‐induced AQP2 mRNA and protein up‐regulation was significantly attenuated in mpkCCDc11 cells with siRNA‐mediated knockdown of TRIM28.ConclusionsWe identify MAP kinase substrates in the kidney CD, which are mapped on the downstream signaling pathways of V2R. TRIM28 is identified as a substrate of MAP kinases that involves in vasopressin signaling pathways. TRIM28 is likely to play a role in the regulation of AQP2 expression, particularly in the AQP2 transcription.Support or Funding InformationThis work was supported by the National Research Foundation of Korea funded by the Ministry of Science, Information and Communication Technology, Korea (2019R1A2C1007753).

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis.

To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). Murine models of A. fumigatus keratitis were established by scraping the central epithelium of mouse cornea, daubing A. fumigatus on the cornea and covering with a contact lens. The mice were randomly divided into the control group and the A. fumigatus-infected (A.F.) group for 1, 3 and 5d respectively, which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection. In this study, immunofluorescence staining was used to detect the expression and localization of AhR in mouse corneas, and the mRNA and protein of AhR were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. In addition, mouse peritoneal macrophages were stimulated by A. fumigatus with or without the pretreatment of AhR antagonist CH223191 and AhR agonist FICZ, and the tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and Arg-1 mRNA were detected by RT-PCR. According to the results of the slit light photography, it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3rd day after the infection of A. fumigatus. Contrasted with the control group, the expression of AhR in the corneal epithelial cells infected with A. fumigatus was significantly increased detected by immunofluorescence staining. AhR mainly expressed in the nucleus and cytoplasm of corneal epithelial cells. Consistent with the transcriptional level of AhR mRNA, the expression level of AhR protein reached the peak on the 3rd day after infection which was detected by Western blot. Furthermore, RT-PCR showed that CH223191 up-regulated the expression of TNF-α and iNOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages; inversely, FICZ reduced the expression of TNF-α and iNOS while elevated the expression of IL-10 and Arg-1. AhR is involved in the pathogenesis of A. fumigatus keratitis and induced immune protection in anti-A. fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

INFLUENCE OF SPELEOCLIMATOTHERAPY ON THE PSYCHO-EMOTIONAL STATE, GENETIC STABILITY AND ACTIVITY OF а-AMYLASE IN THE ORAL CAVITY OF THE STUDENTS

The goal of investigation was to evaluate the effect of speleoclimate on the psycho-emotional state, the frequency of occurrence of buccal epithelial cells with core aberrations and the level of а-amylase activity in the oral cavity of students. Second-year students of VSMU named N. N. Burdenko were selected as subjects. The collection of psychological parameters, buccal epithelium, to assess genetic stability, and oral fluid, for evaluation of the activity of а-amylase was carried out in the first, fourth, eighth, eleventh day visit speleochamber and four days after a course of speleoclimate. The psycho-emotional state of the subjects was assessed using the SAN questionnaire and the scale of C.D. Spielberger and Y.L. Khanin. The state of genetic homeostasis of students was evaluated using a micronucleus test in the buccal epithelium. The level of activity of а-amylase was carried out by the method of solid-phase enzyme immunoassay. Comparison of the studied indicators was carried out using the nonparametric Van-der-Warden criterion. The relationship between the dynamics of а-amylase activity and cytological parameters was revealed using the Spearman correlation coefficient (rs). The results obtained indicate a positive effect of speleoclimate on the psychological and genetic stability of a person. Subjects with low а-amylase activity showed a higher level of cells with abnormal nuclei. A negative correlation was found between the dynamics of the frequency of abnormal nuclei in buccal epithelial cells and the activity of а-amylase in the group with initially low activity (rs= - 0,8; P < 0,05). Speleoclimatotherapy can be recommended as a method of non-medicamental correction of the effects of stress influence on health of students.

Морфогенез ателектазов легких при экспериментальном радиоиндуцированном воздействии

Introduction. Pulmonary atelectasis that occur in the chest organs exposed to radiation is often accompanied by the development of radiation alveolitis, fibrosis, and respiratory failure. Different forms of alveolitis cause severe disturbance of the structure of alveolocytes. However, the effect of radiation on the vascular compartment of pulmonary tissue, bronchioles and alveolocytes, and intercellular structures in the formation of atelectasis was not studied in detail. The importance of quantitative and qualitative changes in pulmonary surfactant (PS) in the development of atelectasis was understudied. The aim of the work was to explore the development of pulmonary atelectasis in rats after radiation exposure. Materials and methods. The study was carried out on 60 male Wistar rats aged 8-9 weeks, body weight 200-220 g. Single local irradiation of animals under Nembutal anesthesia (40 mg / kg weight) was carried out by the RUM-17 apparatus (voltage 200 kV, current 15 mA, filters 0.5 mm Cu + 1.0 mm Al, skin-focal length 25 cm) at a dose of 12 Gy. Phospholipids (PL) in pulmonary surfactant were quantitatively determined in lavage fluid extracted from the rat lung. The material was examined histologically (hematoxylin and eosin, alcian blue, picrofuchsin according to Van Gieson) and under transmission electron microscope. The thickness of the epithelial lining of bronchioles, the average diameter of the nuclei of epithelial cells of bronchioles and the thickness of the interalveolar partitions were calculated morphometrically on control samples at different time periods after exposure to ionizing radiation. Results. Atelectasis occurred at the early stages of the experiment is surfactant-dependent, which is confirmed by the decreased amount of PS and PL in lavage fluid. The formation of atelectasis in later periods is caused by the obturation, narrowing of the lumen of the bronchi and bronchioles due to thickening of the epithelial lining and pronounced bronchial bronchoconstriction. Conclusion. Understanding the mechanisms of development of atelectasis is necessary in order to prevent the pathology described above and reduce the risk of complications. Keywords: atelectasis, distelectasis, morphometry, radiation fibrosis, radiation alveolitis

A Study on Immunohistochemical Expression of Progesterone Receptor and Cell Proliferation by AgNOR in Canine Mammary Tumors (CMT)

Mammary gland tumors are the most commonly diagnosed tumors in domestic dogs. Although immunohistochemical methods provide valuable information such as the location and semi-quantitative data of the interested antigens in particular tumors, conventional methods like histopathological diagnosis remain useful and necessary for identification and classification of tumors. In the present study, we combined histopathology with immunohistochemical staining for progesterone receptor (PR) and special staining argyrophilic nucleolar organizer regions (AgNOR) for cell proliferation in canine mammary gland tumors. Twenty-nine dogs with primary mammary gland neoplasms (11 benign and 18 malignant) were included in this study. All 29 tumors were stained with AgNOR but only 15 cases expressed positive PR signals in the nuclei of neoplastic alveolar and ductal epithelial cells. A weak positive correlation (r = 0.2) was observed between PR and AgNOR index of benign tumors and a very weak negative correlation (r = -0.1833) between PR and AgNOR index of malignant tumors suggesting that higher cellular proliferation is associated with low expression of progesterone receptors.

Histological Classification of Thymoma

Background: Many thymoma classifications have been followed and have been updated by newer or alternative schemes. Many classifications were based on the morphology and histogenesis of normal thymus as the backbone, while other classifications have followed a more simplified scheme, whereby thymomas were grouped based on biological behavior. The WHO classification is currently the advocated one, which is based on “organotypical” features (i.e. histological characteristics mimicking those observed in the normal thymus) including cytoarchitecture (encapsulation and a “lobular architecture”) and the cellular composition, mostly the nuclear morphology is generally appreciated.
 Objectives: This study aims to re-classify thymomas by establishing certain morphometric parameters to evaluate the epithelial cells nuclei. An appraisal of thymoma classification as cortical/ lymphocytic/ type B1 and B2 or medullary/ spindle cells/ type A will be attempted as objective re-evaluation of thymoma.
 Patients: This study is a retrospective evaluation of 50 cases of thymoma, 20 cases of thymic hyperplasia and 10 cases of normal thymus (control group). Using a 5 µm formalin-fixed paraffin embeded tissue sections, stained with hematoxylin-eosin stain, these cases were previously classified histologically into lymphocytic (6 cases), lymphoepithelial (mixed) (28 cases) and epithelial (16 cases), including 2 cases of spindle cell thymoma.
 Methods: Computer-assisted morphometry was performed for 80 cases. This involves digitization of the histopathological features and application of morphometric analysis through software. The morphometric parameters used are nuclear area, maximum nuclear diameter and Form factor of epithelial cells nuclei. Ten normal thymus glands from the archived cases were also evaluated as a control groups.
 Results: The results showed that epithelial thymomas possess significantly different nuclear areas from that of a normal thymus, the maximum nuclear diameter (D-Max) follows the same pattern and adds no further outcomes. The numerical morphometric analysis showed no significant differences between lymphocytic predominant thymoma and those classified as cortical thymoma (Type B2). Thus it does not support such a re-classification. Form factor is an indication of pleomorphism, but it should be cautiously used when spindle cells are present since it may give a false indication of pleomorphism.
 Conclusion: Computer-Assisted morphometric analysis provides an objective, reproducible and comparable results for thymoma histological classification.
 Keywords: Thymoma, Histological classification, Grading, Morphometry, Computer-assistedmorphometry.

Characterisation of transcription factor profiles in polycystic kidney disease (PKD): identification and validation of STAT3 and RUNX1 in the injury/repair response and PKD progression

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, caused in the majority of the cases by a mutation in either the PKD1 or the PKD2 gene. ADPKD is characterised by a progressive increase in the number and size of cysts, together with fibrosis and distortion of the renal architecture, over the years. This is accompanied by alterations in a complex network of signalling pathways. However, the underlying molecular mechanisms are not well characterised. Previously, we defined the PKD Signature, a set of genes typically dysregulated in PKD across different disease models from a meta-analysis of expression profiles. Given the importance of transcription factors (TFs) in modulating disease, we focused in this paper on characterising TFs from the PKD Signature. Our results revealed that out of the 1515 genes in the PKD Signature, 92 were TFs with altered expression in PKD, and 32 of those were also implicated in tissue injury/repair mechanisms. Validating the dysregulation of these TFs by qPCR in independent PKD and injury models largely confirmed these findings. STAT3 and RUNX1 displayed the strongest activation in cystic kidneys, as demonstrated by chromatin immunoprecipitation (ChIP) followed by qPCR. Using immunohistochemistry, we showed a dramatic increase of expression after renal injury in mice and cystic renal tissue of mice and humans. Our results suggest a role for STAT3 and RUNX1 and their downstream targets in the aetiology of ADPKD and indicate that the meta-analysis approach is a viable strategy for new target discovery in PKD.Key messagesWe identified a list of transcription factors (TFs) commonly dysregulated in ADPKD.Out of the 92 TFs identified in the PKD Signature, 35% are also involved in injury/repair processes.STAT3 and RUNX1 are the most significantly dysregulated TFs after injury and during PKD progression.STAT3 and RUNX1 activity is increased in cystic compared to non-cystic mouse kidneys.Increased expression of STAT3 and RUNX1 is observed in the nuclei of renal epithelial cells, also in human ADPKD samples.

Effects of high progesterone in in-vitro fertilization cycle on DNA methylation and gene expression of adhesion molecules on endometrium during implantation window.

High progesterone is associated with low implantation rate. Our previous study demonstrated that DNA methylation in endometrium was increased in women with high progesterone in IVF cycles. However, the DNA methylation status is still not yet confirmed, and how it affects endometrial receptivity in high progesterone is still unknown. Current study investigated the effects of high progesterone on DNA methylation and gene expression of adhesion molecules on endometrium during implantation window. A cohort study included 20 women with high progesterone (HP) and 20 with normal progesterone (NP) on the day of human chorionic gonadotropin (hCG) administration after controlled ovarian hyperstimulation in IVF cycle. Endometrial tissues were collected on the 7th day after hCG administration. Immunohistochemical staining of DNA methyltransferases (DNMT1 and DNMT3B) and adhesion molecules (MUC1, CDH1 and CTNNB1) were performed. Methylation of MUC1, CDH1, and CTNNB1 promoter regions was detected by Sequenom MassARRAY or bisulfite sequencing PCR. RT-qPCR was used to quantify mRNA expression levels, and correlation of methylation and gene expression level of the adhesion molecules were determined. DNMT3B, but not DNMT1, in nucleus of luminal and glandular epithelial cells in HP group was significantly higher than that in NP group. Promoter regions of CDH1 and CTNNB1, but not MUC1, in endometrium of HP group were hypermethylated. Protein and mRNA expression of MUC1, CDH1, and CTNNB1 in endometrium of HP group was significantly lower than that in NP group. Level of DNA methylation was negatively correlated with the gene expression of CDH1 and CTNNB1, but not MUC1. DNA hypermethylation and low expression of adhesion molecules on endometrium were associated with high progesterone during implantation window, which may contribute to the underlying epigenetic mechanism in the failure of IVF treatment.

Using high-throughput sequencing to explore the anti-inflammatory effects of \u03b1-mangostin

Lipopolysaccharide (LPS) causes an inflammatory response, and α-mangostin (α-MG) is an ingredient of a Chinese herbal medicine with anti-inflammatory effects. We investigated the mechanism by which α-MG reduces LPS-stimulated IEC-6 cells inflammation. A genome-wide examination of control, LPS-stimulated, and α-MG-pretreated cells was performed with the Illumina Hiseq sequencing platform, and gene expression was verified with quantitative real-time PCR (qPCR). Among the 37,199 genes profiled, 2014 genes were regulated in the LPS group, and 475 genes were regulated in the α-MG group. GO enrichment and KEGG pathway analyses of the differentially expressed genes (DEGs) showed that they were mainly related to inflammation and oxidative stress. Based on the transcriptomic results, we constructed a rat model of inflammatory bowel disease (IBD) with LPS and investigated the effects of α-MG on NLRP3 inflammasomes. After LPS stimulation, the rat intestinal villi were significantly detached, with congestion and hemorrhage; the intestinal epithelial cell nuclei were deformed; and the mitochondria were swollen. However, after pretreatment with α-MG, the intestinal villus congestion and hemorrhage were reduced, the epithelial nuclei were rounded, and the mitochondrial morphology was intact. qPCR and western blotting were used to detect NLRP3, caspase 1, interleukin (IL)-18, and IL-1β expression at the gene and protein levels. Their expression increased at both the transcript and protein levels after LPS stimulation, whereas it decreased after pretreatment with α-MG. This study provides new methods and ideas for the treatment of inflammation. α-MG may have utility as a drug for intestinal inflammation.

Long-read sequencing identified repeat expansions in the 5′UTR of the NOTCH2NLC gene from Chinese patients with neuronal intranuclear inclusion disease

BackgroundNeuronal intranuclear inclusion disease (NIID) is a heterogenous neurodegenerative disorder named after its pathological features. It has long been considered a disease of genetic origin. Recently, the GGC repeated expansion...

CDX2 Protein Expression in Colorectal Cancer and ItsCorrelation with Clinical and Pathological Characteristics, Prognosis, and Survival Rate of Patients.

Caudal-type homeobox transcription factor 2 (CDX2) is expressed in the nucleus of the intestinal epithelial cells and is essential for embryonic formation and differentiation of the intestine, whose reduced expression can result in metastatic tumors. This study was to investigate the association of CDX2 expression level in colorectal cancer (CRC) with age, gender, microscopic histopathology, tumor staging, tumor grading, 3-year survival rate, and prognosis. After preparing paraffin tissue blocks, CDX2 protein expression was assayed by immunohistochemistry in 82 CRC patients. Hematoxylin and eosin staining was used to detect tumor histology, tumor grading, tumor staging, and blood-lymphatic, and neural invasion. The collected data includes age, gender, tumor site, and 3-year survival rate of patients after diagnosis. The CDX2 expression was significantly higher in men than in women, and it was significantly lower in right-sided tumors as in transverse colon and left-sided tumors. Also, the CDX2 expression was significantly higher in adenocarcinoma than in mucinous. In addition, a significant correlation was found between downregulated CDX2 and lymph node involvement. In tumor grading, there was a significant correlation between CDX2 downregulation and high-grade tumor. Moreover, there was a significant correlation between downregulated CDX2 expression and overall pathological staging. The downregulated CDX2 expression is associated with female gender, right-sided tumors, mucinous tumors, lymph node involvement, high-grade tumor, and advanced overall pathological staging and can be considered as a possible prognostic factor for patients follow-up. However, our study is a preliminary study and further studies with larger sample sizes in different ethnic groups are required.

Lactoferrin translocates to the nucleus of bovine rectal epithelial cells in the presence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC–MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a “delta” isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging.