Cryostat Sections Research Articles

Abstract 2935: Sex disparity in the response of the immune microenvironment to colon cancer liver metastases

Abstract Background Metastasis remains the most life-threatening aspect of malignant disease and a major clinical challenge. Myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes (Tregs), which are frequently found in the tumor microenvironment, are known to impair immune surveillance and promote a state of immune tolerance that leads to metastatic expansion. Previously, we have shown that the incidence of colon carcinoma liver metastases was reduced in TNF receptor 2 (TNFR2) deficient female mice, as a consequence of reduced recruitment of MDSCs and Tregs into the liver. Intriguingly however, no reduction in liver metastasis or MDSC/Treg accumulation was observed in TNFR2 deficient male mice, suggesting that their recruitment and survival were distinctly regulated. The objective of this study was, therefore, to elucidate the mechanisms underlying the sex disparity observed in the response of the immune microenvironment to liver metastases, in particular the role of sex hormones in immune response regulation in this context. Methods Hormone-ablated female mice were generated using surgical ovariectomy (OVX). Estrogen-deprived mice, sham-operated controls and hormone reconstituted OVX mice were injected with syngeneic murine colon carcinoma MC-38 cells via the intrasplenic/portal route and experimental liver metastases enumerated 2 weeks later. MDSCs and Tregs recruited into the livers of tumor-bearing mice were analyzed 7-10 days post-tumor injection using flow cytometry, as well as IHC performed on liver cryostat sections. Results We found that the number of experimental liver metastases was markedly reduced in ovariectomized female mice, as compared to sham-operated controls but was restored in estradiol supplemented OVX mice. Reduced incidence of hepatic metastases in OVX mice correlated with reduced accumulations of CD11b+Gr-1+ myeloid cells (MDSCs) and CD4+Foxp3+ cells (Tregs) at metastatic sites. IHC also revealed an increased CD8+ : CD4+Foxp3+ (i.e. cytotoxic T cell : Treg) ratio around hepatic metastases in OVX mice as compared to sham-operated controls. Estradiol reconstitution in OVX mice restored MDSC and Treg cell accumulation to the levels observed in control mice. Conclusion Together, our data identify female sex hormones as major players in the regulation of the tumor immune microenvironment and a potential contributing factor in the patients’ response to immune-based cancer therapy. Supported by the Canadian Institute for Health Research, MDEIE and FRSQ. Citation Format: Simon Milette, Ni Wang, Stéphanie Perrino, Pnina Brodt. Sex disparity in the response of the immune microenvironment to colon cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2935. doi:10.1158/1538-7445.AM2017-2935

Expression of cd35 and cd57 in placenta of women with asthma

Background. Prevalence of asthma (BA) among pregnant women reaches 12%. Obstetric complications (preeclampsia, placental insufficiency, IUGR, etc.) mainly occur in the absence of control of asthma. Aspects of the placenta status in women with chronic allergic respiratory disease are not well understood.
 Purpuses and tasks — studying the expression of markers of immature dendritic cells (CD35) and natural killer (CD57) cells in the placenta of patients with asthma, depending on the of severity, control and the type of therapy.
 Materials and methods. The study involved 200 pregnant women, including 170 with asthma (44 patients with mild intermittent, 52 with mild persistent, 61 with moderate and 13 with severe asthma), and 30 patients without pulmonary and allergic diseases. We analized complications and pregnancy outcomes, conducted the immunohistochemical study of placental samples of 134 patients for the expression of CD35 (marker of immature dendritic cells) and CD57 (marker of NK cells) on cryostat sections, immunomorphological study of the placenta with determination of the fixation of pathogenic immune complexes (PIC), IL-6, IL- 4, IL-10 in the placenta.
 Results. CD35 expression in the placenta of women with asthma is increased, while in patients undergoing preeclampsia CD35 expression is lower, especially in cases of uncontrolled asthma in the 3rd trimester. In all cases with persistent and incompletely controlled asthma, the expression of the natural killer marker (CD57) in the placenta correlates with the presence of PIC, IL-6, IL-4, IL-10 and inflammatory changes in the placenta. Conclusions. The results of the study suggest that a decrease in CD35 expression in cases with the combination of uncontrolled persistent asthma and preeclampsia seems to deplete the mechanisms of the immune response. Increased NK cell marker expression (CD57) in the placenta of women with uncontrolled and persistent asthma indicates on the cytotoxic role of this subpopulation of cells, leading to morphological and immune changes in the placenta that adversely affect the fetus and newborn.

Multiple sclerosis: Serum anti-CNS autoantibodies

Background: It is uncertain whether there are autoantibodies detectable by indirect immunofluorescence in the serum of patients with multiple sclerosis (MS). Objective: To determine whether there are anti-central nervous system (CNS) autoantibodies detectable by indirect immunofluorescence in the serum of MS patients. Methods: Sera and in some cases cerebrospinal fluid from 106 patients with multiple sclerosis, 156 patients with other neurological diseases, and 70 healthy control subjects were examined by indirect immunofluorescence using cryostat sections of rat cerebrum fixed by perfusion with paraformaldehyde. Results: Autoantibodies were detected that recognized more than 30 neuronal, glial, and mesodermal structures in 28 of 106 MS cases. Most were also detected in patients with other related and unrelated neurological diseases and several were also found in healthy controls. Novel anti-CNS autoantibodies recognizing particular sets of interneurons were detected in both normal controls and in subjects with CNS diseases. Interpretation: Serum anti-CNS autoantibodies of diverse specificities are common in MS patients. The same anti-CNS autoantibodies are not uncommon in patients with other neurological diseases. The findings provide no support for the proposition that myelin breakdown in MS is caused by exposure of intact myelin sheaths or oligodendrocytes to a pathogenic serum anti-myelin or anti-oligodendrocyte autoantibody.

Matrix-assisted laser desorption/ionization mass spectrometric imaging for the rapid segmental analysis of methamphetamine in a single hair using umbelliferone as a matrix

Segmental hair analysis offers a longer period for retrospective drug detection than blood or urine. Hair is a keratinous fiber and is strongly hydrophobic. The embedding of drugs in hydrophobic hair at low concentrations makes it difficult for extraction and detection with matrix-assisted laser desorption/ionization (MALDI) coupled with mass spectrometric imaging (MSI). In this study, a single scalp hair was longitudinally cut with a cryostat section to a length of 4 mm and fixed onto a stainless steel MALDI plate. Umbelliferone was used as a new hydrophobic matrix to enrich and assist the ionization efficiency of methamphetamine in the hair sample. MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS profiling and imaging were performed for direct detection and mapping of methamphetamine on the longitudinal sections of the single hair sample in positive ion mode. Using MALDI-MSI, the distribution of methamphetamine was observed throughout five longitudinally sectioned hair samples from a drug abuser. The changes of methamphetamine were also semi-quantified by comparing the ratios of methamphetamine/internal standard (I.S). This method improves the detection sensitivity of target drugs embedded in a hair matrix for imaging with mass spectrometry. The method could provide a detection level of methamphetamine down to a nanogram per milligram incorporated into hair. The results were also compared with the conventional high performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) method. Changes in the imaging results over time by the MSI method showed good semi-quantitative correlation to the results from the HPLC-MS/MS method. This study provides a powerful tool for drug abuse control and forensic medicine analysis in a narrow time frame, and a reduction in the sample amount required.

Analysis of jejunal crypt morphology from diabetic mice fed genistein diet using two methods: traditional cryostat and novel optical clearing

Gastrointestinal dysfunctions are commonly associated with obesity and diabetes. We have previously shown a significant decrease intransepithelial short circuit current (Isc) in jejunal segments from ob/ob diabetic mice, compared to lean controls. Moreover, we recently demonstrated a significant increase/rescue in Isc in ob/ob mice following chronic consumption(4‐weeks) of a genistein‐containing diet, 600 mg genistein/kg food (600G). Therefore, the current study is evaluating whether or not morphological changes in jejunal crypts of ob/ob mice are responsible, at least in part, for the rescue of Isc. Male ob/ob mice were fed either a genistein‐free diet orgenistein‐containing diet (600G), and comparisons made to lean controls. Assessments of crypt morphology were performed via two methodologies:traditional cryostat sections in optimal cutting temperature compound (OCT), and a novel 3‐D optical clearing method. At completion of the 4‐week diet study, mice were given an injection of EdU (5‐ethynyl‐2′‐deoxyuridine) to tag for actively dividing DNA prior to euthanizing. Freshly isolated segments of jejunum were used. Cryostat sections of OCT embedded jejunum (8 μm) were stained with an EdU Click‐iT assay, imaged at a 20× objective with an inverted microscope (15 measures/mouse, n=3/group). Additional jejunum segments underwent the clearing process, fixed with 4% PFA, stained with an EdU Click‐iT assay and imaged at a 40× objective with a confocal microscope using 100–160 μm total thickness of jejunum, with reconstructions of 0.44 μm z‐stacks (12 measures/mouse, n=3/group). The following measures were taken per image: crypt depth and width and number of EdU+ cells. Interestingly, each method gave differing results. Using the cryostat, there was a significant decrease in EdU+ cells in the ob/ob mice fed genistein‐free diet (7.03±0.506, P<0.05) compared to leans (8.93±0.544) and genistein diet significantly reversed this (8.911±1.09, P<0.05). With cryostat technique, we noted no differences in crypt depth or width. In contrast, analysis of the optically cleared samples demonstrated a significant decrease in crypt depth (CD) and crypt width (CW) in ob/ob genistein‐free fed mice (CD=58.02±2.92μm and CW=31.28±2.30 μm, P<0.05) compared to leans (CD=71.75±3.08 μm and CW=41.30±3.08 μm) and genistein rescued this (CD=94.33±4.75 μm and CW=41.08±2.35 μm, P<0.05). In addition, we will determine measures of crypt volume utilizing the 3‐D optical clearing method. Additional measures with a further four mice/group will determine if our measures and statistical evidence are consistent with a larger sample size. The data suggest that caution should be used with cryostat methodology, whereby one ‘sees a snapshot’ of the jejunum versus the optical clearing methodology, which enables visualization of 3‐D crypts in their entirety, thus providing a virtual reconstruction of the intact tissue. The optical clearing methodology demonstrates a more thorough view of whole tissue samples, thereby generating a more accurate account of the tissue, and specific morphometrics under investigation. Thus, we conclude that given the data shown above, increased cellular proliferation within the jejunal crypts does not appear to account for the genistein‐mediated increase in Isc in ob/ob mice fed 600G.Support or Funding InformationMidwestern University Department of Biomedical Sciences (NS‐S and SR), DAREF (LA), Midwestern Intramural funds (LA and JK)

Morin Stain Detects Aluminum-Containing Macrophages in Macrophagic Myofasciitis and Vaccination Granuloma With High Sensitivity and Specificity.

Macrophagic myofasciitis (MMF) is an inflammatory condition associated with the intramuscular (i.m.) injection of aluminum adjuvant-containing vaccines. It is clinically characterized by myalgia, weakness, and chronic fatigue and histologically by aggregates of cohesive macrophages with abundant basophilic, periodic acid-Schiff (PAS)-positive, diastase-resistant granules that percolate through the peri- and endomysium without eliciting substantial myofiber damage. The definitive diagnosis of MMF requires demonstration of aluminum within these macrophages. We evaluated the Morin stain, a simple, 2-step histochemical stain for aluminum, as a confirmatory diagnostic tool for MMF. Among 2270 muscle biopsies processed at UTSW between 2010 and 2015, a total of 12 MMF cases and 1 subcutaneous vaccination granuloma case were identified (11 pediatric, 2 adults). With the Morin stain, all 13 cases showed strong granular reactivity within the cytoplasm of macrophages but not in myofibers or connective tissue. Three cases of inflammatory myopathy with abundant macrophages (IMAM), 8 cases of granulomatous inflammation and 23 other deltoid muscle biopsies used as controls were all negative. Morin stain could be used in both formalin-fixed paraffin-embedded and cryostat sections. Thus, Morin stain detects aluminum with high sensitivity and specificity in human muscle and soft tissue and may improve the diagnostic yield of MMF and vaccination granuloma.

An easy, fast and “low-tech”-equipment-requiring alternative method to optimize immunolabelling conditions for pre-embedding immunogold electron microscopy and to correlate light and electron microscopical immunogold labelling results

Correlating light microscopic immunolabelling results with electron microscopic data is of great interest in many fields of biomedical research but typically requires very specialized, expensive equipment and complex procedures which are not available in most labs. In this technical study, we describe an easy and “low-tech”-equipment-requiring pre-embedding immunolabelling approach that allows correlation of light microscopical immunolabelling results with electron microscopic (EM) data as demonstrated by the example of immunolabelled synaptic ribbons from retinal rod photoreceptor synapses. This pre-embedding approach does not require specialized embedding devices but only commonly available equipment. The cryostat section-based procedure allows optimization of the pre-embedding immunolabelling conditions at the less laborious and time-consuming light microscopic (LM) level before the ultrastructural analyses of the immunolabelled structures can be performed on the same sample after ultrathin sectioning without further modification. The same photoreceptor synapse that has been first studied at the light microscopic level can be subsequently analyzed with this approach at the electron microscopic level at individual ultrathin sections or serial ultrathin sections from individual, identical synapses. Higher resolution EM analyses of the immunolabelled synapses can be performed with only minor modifications of the combined LM/EM procedure. The detergent-free procedure is applicable even for weakly fixed cryostat sections which is a relevant aspect for many antibodies that do not work with more strongly fixed biological samples.

Beta-3 adrenergic receptor is expressed in acetylcholine-containing nerve fibers of the human urinary bladder: An immunohistochemical study.

To identify in the human bladder the structures which express the Beta-3 adrenoceptor (β3AR). Human bladders from cadaveric organ donors (equally balanced in sex and age) were collected. Bladders were immediately fixed in paraformaldehyde and further processed for cryostat sectioning. Single and double immunohistochemistry was performed using antibodies against β3AR C-terminal, β3AR N-terminal, a pan-neuronal marker (β3-Tubulin) and markers of cholinergic (Vesicular Acetylcholine Transporter), adrenergic (Tyrosine Hidroxylase), and peptidergic (Calcitonin Gene-Related Peptide) nerve fibers. Nerve fibers expressing immunoreactivity for β3AR were abundantly found in the mucosa and muscular layers of the human bladder. No β3AR-IR was detected on urothelial or smooth muscle cells. The presence of β3AR-IR in nerve fibers was confirmed by co-expression with β3-Tubulin. Nerve fibers expressing β3AR-IR were cholinergic, VAChT+ , and abundantly observed in the suburothelium. The cholinergic fibers were in close proximity and intermingled with adrenergic TH+ and peptidergic CGRP+ fibers. We demonstrated that β3AR is abundantly located in acetylcholine-containing nerve fibers. These findings have important consequences to understand the mechanism of action of β3AR agonists currently used for the treatment of OAB.

Morphological and histochemical investigation of the response of Olea europaea leaves to fungal attack by Spilocaea oleagina

This study investigated the response of Olea europaea (cv. Conservolea) leaves to attack by the fungal pathogen Spilocaea oleagina. Cryostat and semithin sections of healthy and S. oleagina‐infected olive leaves were analysed histochemically for polyphenol oxidase (PPO) activity and tested for programmed cell death (PCD) induction by means of terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL). At all stages of infection, the fungus remained localized between the external and internal layers of cuticle without crossing the pectocellulosic layer. No PCD phenomena could be detected in plant cells at any stage of the disease. However, extensive degeneration of palisade parenchyma cells was observed in advanced infections, with massive loss of cytoplasmic contents and disappearance of cell compartments. Polyphenol oxidases are enzymes that, in olive, oxidize o‐diphenols (principally oleuropein and rutin) to produce o‐diquinones and melanins, substances that are toxic to many pathogens. No significant increase in overall PPO activity was found in infected leaves; on the contrary, enzyme activity was gradually lost as infection progressed, most probably due to degradation of plastids within mesophyll cells, in which such enzymes are normally confined. Only a limited local PPO activation occurred in a few upper epidermal cells of the leaf, indicating a feeble induction of a plant response.

Vision Recovery and Connectivity by Fetal Retinal Sheet Transplantation in an Immunodeficient Retinal Degenerate Rat Model.

PurposeTo characterize a recently developed model, the retinal degenerate immunodeficient S334ter line-3 rat (SD-Foxn1 Tg(S334ter)3Lav) (RD nude rat), and to test whether transplanted rat fetal retinal sheets can elicit lost responses to light.MethodsNational Institutes of Health nude rats (SD-Foxn1 Tg) with normal retina were compared to RD nude rats with and without transplant for morphology and visual function. Retinal sheets from transgenic rats expressing human placental alkaline phosphatase (hPAP) were transplanted into the subretinal space of RD nude rats between postnatal day (P) 26 and P38. Transplant morphology was examined in vivo using optical coherence tomography (OCT). Visual function was assessed by optokinetic (OKN) testing, electroretinogram (ERG), and superior colliculus (SC) electrophysiology. Cryostat sections were analyzed for various retinal/synaptic markers and for the expression of donor hPAP.ResultsOptical coherence tomography scans showed the placement and laminar development of retinal sheet transplants in the subretinal space. Optokinetic testing demonstrated a deficit in visual acuity in RD nude rats that was improved after retinal sheet transplantation. No ERG responses were detected in the RD nude rats with or without transplantation. Superior colliculus responses were absent in age-matched control and sham surgery RD nude rats; however, robust light-evoked responses were observed in a specific location in the SC of transplanted RD nude rats. Responsive regions corresponded to the area of transplant placement in the eye. The quality of visual responses correlated with transplant organization and placement.ConclusionsThe data suggest that retinal sheet transplants integrate into the host retina of RD nude rats and recover significant visual function.

Recovery and amplification of ancient DNA from Herculaneum victims killed by the 79 AD Vesuvius hot surges

n thermally damaged archaeological bones the quantity, quality, and amplifiability of DNA are very much reliant on both the extent of heating and the environmental conditions of the burial context. In this study we tested the possibility of extracting and amplifying ancient DNA from human bone remains of Herculaneum victims of the 79 AD eruption of Vesuvius using a combination of histochemical and molecular methods. Long bone samples with variable degrees of chromatic and morphological alterations consistent with exposure to temperatures of about 300 °C were taken from four specimens. Using histochemical stains, bone cryostat sections from three individuals revealed DNA within osteocyte lacunae but only for one sample the DNA was suitable for PCR amplification obtained, namely from reactions with the primer pair for X and Y amelogenin (AMEL) loci. The relative sequence differed from the homologous trait of AMELX deposited in GenBank for six bases, probably due to degradation processes following death. Our data are indicative that archaeological bones exposed to high temperatures of about 300 °C should be considered for DNA analysis, given the favorable conditions of corpse burial and skeleton preservation, such as those that occurred for the 79 AD victims in Herculaneum and Pompeii.

Changes in Somatostatin-Like Immunoreactivity in the Sympathetic Neurons Projecting to the Prepyloric Area of the Porcine Stomach Induced by Selected Pathological Conditions.

The aim of the present study was to define changes in the expression of somatostatin (SOM) in the sympathetic perikarya innervating the porcine stomach prepyloric area during acetylsalicylic-acid-induced gastritis (ASA) and experimentally induced hyperacidity (HCL) and following partial stomach resection (RES). On day 1, the stomachs were injected with neuronal retrograde tracer Fast Blue (FB). Animals in the ASA group were given acetylsalicylic acid orally for 21 days. On the 22nd day after FB injection, partial stomach resection was performed in RES animals. On day 23, HCL animals were intragastrically given 5 ml/kg of body weight of a 0.25 M aqueous solution of hydrochloric acid. On day 28, all pigs were euthanized. Then, 14-μm thick cryostat sections of the coeliac-superior mesenteric ganglion (CSMG) complexes were processed for routine double-labelling immunofluorescence. All pathological conditions studied resulted in upregulation of SOM-like (SOM-LI) immunoreactivity (from 14.97 ± 1.57% in control group to 33.72 ± 4.39% in the ASA group, to 39.02 ± 3.65% in the RES group, and to 29.63 ± 0.85% in the HCL group). The present studies showed that altered expression of SOM occurs in sympathetic neurons supplying the prepyloric area of the porcine stomach during adaptation to various pathological insults.

Chronic pancreatites or pancreatic ductal adenоcarcinoma?

Based on a comprehensive histological, immunohistochemical and ultrastructural study, the authors have developed criteria of histologic differential diagnosis between chronic pancreatitis and pancreatic ductal adenocarcinoma useful in doubtful cases, especially in core- biopsies and cryostat sections, as well as for novice doctors pathologists.

Identification of IL-17F/frequent exacerbator endotype in asthma

Severe asthma might be associated with overexpression of Th17 cytokines, which induce neutrophil recruitment via neutrophil-mobilizing cytokines in airways. To study IL-17-related cytokines in nasal/bronchial biopsies from controls and mild asthmatics (MAs) to severe asthmatics (SAs) in relation to exacerbation rate. Inflammatory cells and IL-17A+, IL-17F+, IL-21+, IL-22+, and IL-23+ cells were examined by immunohistochemistry in cryostat sections of bronchial/nasal biopsies obtained from 33 SAs (21 frequent exacerbators [FEs]), 31MAs (3 FEs), and 14 controls. IL-17F protein was also measured by ELISA in bronchial/nasal lysates and by immunohistochemistry in bronchial tissue obtained from subjects who died because of fatal asthma. Immunofluorescence/confocal microscopy was used for IL-17F colocalization. Higher number (P<.05) of neutrophils, IL-17A+, IL-17F+, and IL-21+ cells in bronchial biopsies and higher numbers (P<.01) of IL-17F+ and IL-21+ cells in nasal biopsies were observed in SAs compared with MAs. Bronchial IL-17F+ cells correlated with bronchial neutrophils (r=0.54), exacerbation rate (r=0.41), and FEV1 (r=-0.46). Nasal IL-17F+ cells correlated with bronchial IL-17F (r=0.35), exacerbation rate (r=0.47), and FEV1 (r=-0.61). FEs showed increased number of bronchial neutrophils/eosinophils/CD4+/CD8+ cells and bronchial/nasal IL-17F+ cells. Receiver operating characteristic curve analysis evidenced predictive cutoff values of bronchial neutrophils and nasal/bronchial IL-17F for discriminating between asthmatics and controls, between MAs and SAs and between FEs and non-FEs. IL-17F protein increased in bronchial/nasal lysates of SAs and FEs andin bronchial tissue of fatal asthma. IL-17F colocalized in CD4+/CD8+ cells. IL-17-related cytokines expression was amplifiedin bronchial/nasal mucosa of neutrophilic asthma prone to exacerbation, suggesting a pathogenic role of IL-17F in FEs.

Distribution and sub-types of afferent fibre in the mouse urinary bladder

AimIncreased afferent fibre activity contributes to pathological conditions such as the overactive bladder syndrome. Nerve fibres running near the urothelium are considered to be afferent as no efferent system has yet been described. The aim of this study was to identify sub-types of afferent nerve fibres in the mouse bladder wall based on morphological criteria and analyse regional differences. Materials and methods27 bladders of six month old C57BL/6 mice were removed and tissues were processed for immunohistochemistry. Cryostat sections were cut and stained for Protein Gene Product 9.5 (PGP), calcitonin gene related polypeptide (CGRP), neurofilament (NF), vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS). ResultsIn the sub-urothelium, different types of afferent nerve fibre were found, i.e. immunoreactive (IR) to; CGRP, NF, VAChT, and/or nNOS. At the bladder base, the sub-urothelium was more densely innervated by CGRP-IR and VAChT-IR nerve fibres, then at the lateral wall. NF- and nNOS nerves were sparsely distributed in the sub-urothelium throughout the bladder. At the lateral wall the inner muscle is densely innervated by CGRP-IR nerve fibres. NF, VAChT and nNOS nerves were evenly distributed in the different muscle layers throughout the bladder. Nerve fibre terminals expressing CGRP and NF were found within the extra-mural ganglia at the bladder base. ConclusionsDifferent types of afferent nerve fibres were identified in the sub-urothelium of the mouse bladder. At the bladder base the sub-urothelium is more densely innervated than the lateral wall by CGRP-IR and VAChT-IR afferent nerve fibres. CGRP and NF afferent nerve fibres in the muscle layer probably relay afferent input to external ganglia located near the bladder base. The identification of different afferent nerves in the sub-urothelium suggests a functional heterogeneity of the afferent nerve fibres in the urinary bladder.

The nature of the white opaque substance within colorectal neoplastic epithelium as visualized by magnifying endoscopy with narrow-band imaging.

Background and study aims: We previously reported our discovery of a white opaque substance (WOS) that is opaque to endoscopic light inside the epithelium while using magnifying endoscopy (ME) to examine gastric epithelial neoplasia. Histopathologic analysis revealed that the WOS comprises minute lipid droplets (LDs) accumulated within the neoplastic epithelium. In addition, the WOS was found in colorectal epithelial neoplasia, although it was unclear whether this WOS corresponded to an accumulation of LDs, as in the stomach. Therefore, the aim of the current study was to elucidate whether the WOS observed in colorectal epithelial tumors comprises LDs. Patients and methods: A consecutive series of 40 WOS-positive and 40 WOS-negative colorectal epithelial tumors was analyzed. One biopsy specimen was taken from each neoplasm. Cryostat sections were stained with oil red O for LD, and sections after formalin-fixation for LD were immunostained with anti-adipophilin antibody. Results: The prevalence of LDs stained with oil red O in WOS-positive vs. WOS-negative lesions was 47.5 % (19/40) vs. 5 % (2/40), respectively (P < 0.001). Furthermore, the WOS coincided with the expression of adipophilin; the prevalence of LDs stained by anti-adipophilin antibody in WOS-positive vs. WOS-negative lesions was 100 % (40/40) vs. 62.5 % (25/40), respectively (P < 0.001). Conclusions: This study elucidated for the first time that endoscopically visualized WOS in colorectal epithelial neoplasia may be composed of LDs accumulated in the neoplastic epithelium.

РОЛЬ ЭСТРАДИОЛА В ПОДДЕРЖАНИИ ФЕТОПЛАЦЕНТАРНОЙ СИСТЕМЫ

The aim of the study is to define the role of abnormalities in the formation of estriol hormones in the development of hormonal dysfunction of fetoplacental system. There were examined 39 placentas from women with exacerbation of chronic cytomegalovirus infection at the third trimester of pregnancy and IgG antibody titer 1:1600 and 30 placentas from women with latent CMV infection and titer 1:400. The activity of androstenedione dehydrogenase was found out with histochemical method on cryostat sections of freshly frozen tissues of placentas by Lloyd’s method. The assessment of intensiveness of histochemical reaction was done with cytophotometric method by Scion program. Estradiol in homogenate of placenta was measured with immune-enzyme method. The quantity of nuclei of placenta syncytiotrophoblast in the state of apoptosis was done with cytophotometric method on paraffin sections stained by ISEL method. By the results of the study the exacerbation of CMV infection at the third trimester of pregnancy leads to the decrease of intensiveness of histochemical reaction in placentas to androstenedione dehydrogenase till 32.1±2.88 pixel/mcm2 (at the latent course it is 54.2±4.31 pixel/mcm2, р&amp;#60;0.001), and in homogenate of placenta to the decrease of estradiol till 18443.2±117.53 pmole/l (at the latent course of the disease it is 28977.7±158.13 pmole/l, p&amp;#60;0.001), on paraffin sections in syncytiotrophoblast by the increase of nuclei number in the state of apoptosis till 5.0±0.03% (at latent course of the disease it is 1.2±0.001%, р&amp;#60;0.001) and vacuole formation, which leads to destructive damages of syncytiotrophoblast cytoplasm. The obtained results prove that the exacerbation of CMV infection at the third trimester of pregnancy causes the decrease of estriol hormones formation, which is accompanied by the damage of morphostructure and exchange processes in cell elements of placenta.

Autofluorescence discrimination of metabolic fingerprint in nutritional and genetic fatty liver models

Liver tissue autofluorescence (AF) has been characterized in two models with a different potential to undergo disease progression to steatohepatitis: Wistar rats, administered with a methionine, choline deficient diet (MCD), and Zucker (fa/fa) rats, homozygous for a spontaneous mutation of leptin receptor. AF spectra were recorded from liver tissue cryostatic sections by microspectrofluorometry, under 366nm excitation. Curve fitting analysis was used to estimate the contribution of different endogenous fluorophores (EFs) to the overall AF emission: i) fluorescing fatty acids, a fraction of liver lipids up to now poorly considered and complicated to detect by conventional procedures; ii) lipofuscin-like lipopigments, biomarkers of oxidizing events; iii) NAD(P)H and flavins, biomarkers of energy metabolism and tissue redox state. AF data and biochemical correlates of hepatocellular injury resulted to depend more on rat strain than on intratissue bulk lipid or ROS levels, reflecting a different metabolic ability of the two models to counteract potentially harmful agents. AF analysis can thus be proposed for extensive applications ranging from experimental hepatology to the clinics. AF based diagnostic procedures are expected to help both the prediction of the risk of fatty liver disease progression and the prescreening of marginal organs to be recruited as donors for transplantation. A support is also foreseen in the advancement and personalization of strategies to ameliorate the donor organ preservation outcome and the follow up of therapeutic interventions.

Transcriptional Analysis by Nascent RNA FISH of &lt;em&gt;In Vivo&lt;/em&gt; Trophoblast Giant Cells or &lt;em&gt;In Vitro&lt;/em&gt; Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.