Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma

Wnt inhibitory factor-1 (WIF-1) is a secreted protein that antagonizes Wnt signaling. We recently demonstrated the importance of aberrant activation of the Wnt signaling pathway in various cancers including malignant pleural mesothelioma. In this study, we revealed downregulated WIF-1 expression in cell lines and primary tissue when compared to normal mesothelial cell lines and adjacent pleura, respectively. We observed hypermethylation in four of four mesothelioma cell lines, but not in two normal mesothelial cell lines. In primary tissue samples, we observed methylation in three paired tumor specimens compared to their adjacent normal pleura and methylation in eight of nine unpaired tumor tissue samples. Taken together, our studies suggest that WIF-1 silencing due to its promoter hypermethylation is an important mechanism underlying the constitutively activated Wnt signaling in mesothelioma. New therapies toward inhibition of the Wnt pathway through WIF-1 might be promising for the future treatment of malignant mesothelioma.     

Hydraulic conductivity of canine parietal pleura in vivo

The hydraulic conductivity (Lp) of the parietal pleura was measured in vivo in spontaneously breathing anesthetized dogs in either the supine (n = 8) or the prone (n = 7) position and in an excised portion of the chest wall in which the pleura and its adjacent tissue were intact (n = 3). A capsule was glued to the exposed parietal pleura after the intercostal muscles were removed. The capsule was filled with either autologous plasma or isotonic saline. Transpleural fluid flow (V) was measured at several transpleural hydrostatic pressures (delta P) from the rate of meniscus movement within a graduated pipette connected to the capsule. Delta P was defined as the measured difference between capsule and pleural liquid pressures. The Lp of the parietal pleura was calculated from the slope of the line relating V to delta P by use of linear regression analysis. Lp in vivo averaged 1.36 X 10(-3) +/- 0.45 X 10(-3) (SD) ml.h-1.cmH2O-1.cm-2, regardless of whether the capsule was filled with plasma or saline and irrespective of body position. This value was not significantly different from that measured in the excised chest wall preparation (1.43 X 10(-3) +/- 1.1 X 10(-3) ml.h-1.cmH2O-1.cm-2). The parietal pleura offers little resistance to transpleural protein movement, because there was no observed difference between plasma and saline. We conclude that because the Lp for intact parietal pleura and extrapleural interstitium is approximately 100 times smaller than that previously measured in isolated stripped pleural preparations, removal of parietal pleural results in a damaged preparation.     

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Effects of SNP, ouabain, and amiloride on electrical potential profile of isolated sheep pleura.

The fluid and solute transport properties of pleural tissue were studied by using specimens of intact visceral and parietal pleura from adult sheep lungs. The samples were transferred to the laboratory in a Krebs-Ringer solution at 4 degrees C within 1 h from the death of the animal. The pleura was then mounted as a planar sheet in a Ussing-type chamber. The results that are presented in this study are the means of six different experiments. The spontaneous potential difference and the inhibitory effects of sodium nitroprusside (SNP), ouabain, and amiloride on transepithelial electrical resistance (R(TE)) were measured. The spontaneous potential difference across parietal pleura was 0.5 +/- 0.1 mV, whereas that across visceral pleura was 0.4 +/- 0.1 mV. R(TE) of both pleura was very low: 22.02 +/- 4.1 Omega. cm2 for visceral pleura and 22.02 +/- 3.5 Omega. cm2 for parietal pleura. There was an increase in the R(TE) when SNP was added to the serosal bathing solution of parietal pleura and to the serosal or mucosal bathing solution in visceral pleura. The same was observed when ouabain was added to the mucosal surface of visceral pleura and to either the mucosal or serosal surface of parietal pleura. Furthermore, there was an increase in R(TE) when amiloride was added to the serosal bathing solution of parietal pleura. Consequently, the sheep pleura appears to play a role in the fluid and solute transport between the pleural capillaries and the pleural space. There results suggest that there is a Na+ and K+ transport across both the visceral and parietal pleura.     

Isolation of multipotent progenitor cells from pleura and pericardium for tracheal tissue engineering purposes

Tissue engineering (TE) of long tracheal segments is conceptually appealing for patients with inoperable tracheal pathology. In tracheal TE, stem cells isolated from bone marrow or adipose tissue have been employed, but the ideal cell source has yet to be determined. When considering the origin of stem cells, cells isolated from a source embryonically related to the trachea may be more similar. In this study, we investigated the feasibility of isolating progenitor cells from pleura and pericard as an alternative cells source for tracheal tissue engineering. Porcine progenitor cells were isolated from pleura, pericard, trachea and adipose tissue and expanded in culture. Isolated cells were characterized by PCR, RNA sequencing, differentiation assays and cell survival assays and were compared to trachea and adipose-derived progenitor cells. Progenitor-like cells were successfully isolated and expanded from pericard and pleura as indicated by gene expression and functional analyses. Gene expression analysis and RNA sequencing showed a stem cell signature indicating multipotency, albeit that subtle differences between different cell sources were visible. Functional analysis revealed that these cells were able to differentiate towards chondrogenic, osteogenic and adipogenic lineages. Isolation of progenitor cells from pericard and pleura with stem cell features is feasible. Although functional differences with adipose-derived stem cells were limited, based on their gene expression, pericard- and pleura-derived stem cells may represent a superior autologous cell source for cell seeding in tracheal tissue engineering.     

Karyometric marker features in tissue adjacent to in situ cervical carcinomas

Subtle changes in nuclear chromatin structure have been documented in the histologically normal mucosa adjacent to neoplastic lesions. To evaluate the expression of such "marker features" in tissue adjacent to squamous carcinomas in situ (CIS) of the uterine cervix, normal-appearing ectocervical tissues from five cases of CIS were compared with ectocervical tissues from control patients with squamous metaplasia. Formalin-fixed, paraffin-embedded tissue sections were Feulgen stained and analyzed with the microTICAS video microphotometer. Discriminant analysis revealed seven features that helped to distinguish nuclei from ectocervical tissue adjacent to CIS from those of control tissue. These features reflected changes in nuclear shape and chromatin distribution that were not detected by routine histopathologic analysis. The findings may reflect a subtle premalignant change in the apparently normal mucosa adjacent to a cervical neoplasm; they may also reflect the influence of the neoplasm on the adjacent mucosa.     

Ultrasonic diagnosis of pleurisy

Ultrasound investigation of changes in the pleural cavity was conducted in 146 patients with infectious and infectious-allergic pleuritides, beginning with the first clinical manifestations to the outcome of the disease. The following stages were noted: thickening of the pleural layer in a limited area, sometimes of the endothoracic fascia and connective tissue layer between it and the parietal pleura; the appearance of fibrinous effusion; fluid accumulation; encapsulation and multilocular pleurisy; exudate resorption, fibrinolysis; fibrin involvement by connective tissue and formation of adhesions. Besides, an ultrasound picture of the normal chest wall and pleural cavity is presented.     

Histological events leading to somatic embryo formation in cultured petioles of alfalfa

Summary An optimum 10-day exposure of petioles of alfalfa [Medicago sativa ssp.falcata (L.) Arcangeli] to 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid results in the semisynchronous production of somatic embryos starting about 4 days after transfer to a non-auxin-containing medium. The timing of cell division induction in the petiole explants was found to vary depending on the petiole tissue type. Cells adjacent to the vascular bundles divide first at about 48 h after exposure to auxin, closely followed by those of the inner parenchyma, whereas most of the cells of the subepidermal and epidermal layers start to divide later, between 72 and 120 h. Two different sources of callus were also evident. Cells adjacent to the vascular bundles and the inner parenchyma cells were the primary source of callus when a short, 2-day (non-embryo-producing) exposure to auxin was used. In contrast, the subepidermal and epidermal cells were the primary source of callus tissue when a longer, 10-day (embryo producing) exposure was used. It is concluded that the source of somatic embryos is primarily the daughter cells of the subepidermal or epidermal tissue or both.     

Genome-Wide Profile of Pleural Mesothelioma versus Parietal and Visceral Pleura: The Emerging Gene Portrait of the Mesothelioma Phenotype

Background

Malignant pleural mesothelioma is considered an almost incurable tumour with increasing incidence worldwide. It usually develops in the parietal pleura, from mesothelial lining or submesothelial cells, subsequently invading the visceral pleura. Chromosomal and genomic aberrations of mesothelioma are diverse and heterogenous. Genome-wide profiling of mesothelioma versus parietal and visceral normal pleural tissue could thus reveal novel genes and pathways explaining its aggressive phenotype.

Methodology and Principal Findings

Well-characterised tissue from five mesothelioma patients and normal parietal and visceral pleural samples from six non-cancer patients were profiled by Affymetrix oligoarray of 38 500 genes. The lists of differentially expressed genes tested for overrepresentation in KEGG PATHWAYS (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) terms revealed large differences of expression between visceral and parietal pleura, and both tissues differed from mesothelioma. Cell growth and intrinsic resistance in tumour versus parietal pleura was reflected in highly overexpressed cell cycle, mitosis, replication, DNA repair and anti-apoptosis genes. Several genes of the “salvage pathway” that recycle nucleobases were overexpressed, among them TYMS, encoding thymidylate synthase, the main target of the antifolate drug pemetrexed that is active in mesothelioma. Circadian rhythm genes were expressed in favour of tumour growth. The local invasive, non-metastatic phenotype of mesothelioma, could partly be due to overexpression of the known metastasis suppressors NME1 and NME2. Down-regulation of several tumour suppressor genes could contribute to mesothelioma progression. Genes involved in cell communication were down-regulated, indicating that mesothelioma may shield itself from the immune system. Similarly, in non-cancer parietal versus visceral pleura signal transduction, soluble transporter and adhesion genes were down-regulated. This could represent a genetical platform of the parietal pleura propensity to develop mesothelioma.

Conclusions

Genome-wide microarray approach using complex human tissue samples revealed novel expression patterns, reflecting some important features of mesothelioma biology that should be further explored.     

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration.     

Regional blood flow to canine parietal pleura and internal intercostal muscle

Transcapillary Starling forces in the parietal pleura and the underlying interstitium may potentially contribute to the exchange of fluid across this barrier. However, the extent of blood flow to the parietal pleura has not been measured. Thus, using standard microsphere techniques, we compared blood flow to the parietal pleura, including the subpleural interstitium, with blood flow to the adjacent internal intercostal muscle, as well as with flows to other serous tissues, including mediastinal pleura, pericardium, and parietal peritoneum, in anesthetized dogs that were either breathing spontaneously (n = 9) or ventilated to control arterial PCO2 (n = 5). Blood flow (ml.min-1.g-1) was measured after 20 min of equilibration in four successive body positions: right lateral decubitus, supine, left lateral decubitus, and prone. Overall, flow to parietal pleura was not different in spontaneous [1.07 +/- 0.14 (SE)] and mechanically ventilated animals (0.74 +/- 0.11). Flow to the internal intercostal muscle was significantly less than pleural blood flow, averaging 0.24 +/- 0.03 and 0.16 +/- 0.03 in the same groups, although again there was no effect of ventilation mode. Blood flow to other serous tissues in the thoracic cavity, specifically the mediastinal pleura (0.67 +/- 0.14) and pericardium (0.88 +/- 0.22), was similar to parietal pleural flow, whereas that to the parietal peritoneum was an order of magnitude lower (0.09 +/- 0.02, P less than 0.05). Changing body position had no effect on blood flow to any of the sampled tissues. Blood flow to the dorsal aspect of the chest wall muscle in spontaneously breathing animals tended to be greater than that to lateral or ventral portions of the chest wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological, histochemical and immunohistochemical differences between tumorous and adjacent tissues in chemically induced colon cancer in rats.

Methods of morphological, histochemical and immunohistochemical analyses were used to further characterize differences between tumourous and adjacent grossly normal tissues in chemically-induced colon cancer in rats. Colon tumors were induced by the treatment of rats with 1,2-dimethylhydrazine or with N-methyl-N'-nitro-N-nitrosoguanidine alone or with subsequent treatment with deoxycholic bile acid. Tissues were studied morphologically (for the presence of goblet cells in the colon crypts, and the extent of infiltration of lymphocytes into the crypts and between them), histochemically (for the presence of positive reaction to neutral and acid mucopolysaccharides) and immunohistochemically (for the presence of tissue polypeptide antigen). All data were evaluated quantitatively, and index of tissue damage was calculated for both tumorous and non-tumorous tissues. Significant morphological differences were found between tumorous and adjacent apparently normal tissue. Histochemically and immunohistochemically, both types of tissue reacted very similarly to exposure to the carcinogens. Index of damage was significantly different from normal untreated colon in both kinds of tissue. It was suggested that precancerous state in tissue adjacent-to-tumor could be detected using the combination of these methods.     

Remodeling in myocardium adjacent to an infarction in the pig left ventricle

Changes in the structure of the "normal" ventricular wall adjacent to an infarcted area involve all components of the myocardium (myocytes, fibroblasts and the extracellular matrix, and the coronary vasculature) and their three-dimensional structural relationship. Assessing changes in these components requires tracking material markers in the remodeling tissue over long periods of time with a three-dimensional approach as well as a detailed histological evaluation of the remodeled structure. The purpose of the present study was to examine the hypotheses that changes in the tissue adjacent to an infarct are related to myocyte elongation, myofiber rearrangement, and changes in the laminar architecture of the adjacent tissue. Three weeks after myocardial infarction, noninfarcted tissue adjacent to the infarct remodeled by expansion along the direction of the fibers and in the cross fiber direction. These changes are consistent with myocyte elongation and myofiber rearrangement (slippage), as well as a change in cell shape to a more elliptical cross section with the major axis in the epicardial tangent plane, and indicate that reorientation of fibers either via "cell slippage" or changes in orientation of the laminar structure of the ventricular wall are quantitatively important aspects of the remodeling of the normally perfused myocardium.     

Respiratory and circulatory effects of parietal pleural afferent stimulation in rabbits.

Respiratory symptoms accompanying pleural diseases combine dyspnea, tachypnea, rapid shallow breathing, and sometimes hypotension. There are no experimental data on the changes in respiratory and circulatory functions elicited by the activation of pleural afferents. After removal of all muscles covering the 5th to 10th intercostal spaces, we investigated in paralyzed, vagotomized rabbits the changes in phrenic discharge, transpulmonary pressure, and systemic arterial pressure in response to an outwardly directed force exerted on the parietal pleura or the local application of solutions containing lactic acid or inflammatory mediators. Mechanical stimulation of the pleura induced an immediate decrease in both integrated phrenic discharge and arterial blood pressure, the responses being positively correlated with the magnitude of force applied on the pleura. No accompanying changes in ventilatory timing, transpulmonary pressure, or heart rate were measured. Lactic acid solution also elicited an inhibition of phrenic activity and a fall in blood pressure. Section of the internal intercostal nerves supplying the stimulated intercostal spaces totally abolished the responses to mechanical stimulation or lactic acid. An inflammatory mixture elicited only modest respiratory and circulatory effects. We concluded that an acute mechanical distension of the parietal pleura as well as its chemical stimulation by lactic acid elicit a marked inhibition of phrenic motoneurons combined to a reduction of the sympathetic outflow to the circulatory system.     

Effect of chronic morphine exposure on response properties of rat barrel cortex neurons.

Chronic exposure to morphine can impair performance in tasks which need sensory processing. Using single unit recordings we investigate the effect of chronic morphine exposure on the firing properties of neurons in layers IV and V of the whisker-related area of rat primary somatosensory cortex. In urethane-anesthetized animals, neuronal activity was recorded in response to principal and adjacent whisker deflections either stimulated independently or in a conditioning test paradigm. A condition test ratio (CTR) was calculated for assessing the inhibitory receptive field. In layer IV, chronic morphine treatment did not change the spontaneous discharge activity. On responses to principal and adjacent whisker deflections did not show any significant changes following chronic morphine exposure. The magnitude Off responses to adjacent whisker deflection decreased while its response latency increased. In addition, there was a significant increase in the latency of Off responses to principal whisker deflection. CTR did not change significantly following morphine exposure. Layer V neurons, on the other hand, did not show any significant changes in their spontaneous activity or their evoked responses following morphine exposure. Our results suggest that chronic morphine exposure has a subtle modulatory effect on response properties of neurons in barrel cortex.     

Pneumothorax after mediastinal emphysema. The site and mechanism of pleural rupture

By rupture of the mediastinal pleura a mediastinal emphysema may lead to a pneumothorax. An experiment imitating this process is able to point out two spots where the pleura is most likely to tear: An area as large as a thumb tip above the root of the left lung; there mediastinal pleura covers a space the width of which changes most in respirating. The border of a fatty fold based on the pericardium and covered by mediastinal pleura; it is there that maxima of tension occur by emphysematic inflation. In general, air from the mediastinum far more often enters the left pleural cavity than the right one.     

Stereological analysis of cotyledon tissue inVicia faba L. var. aquadulce during germination

Summary Stereological analysis was carried out on cotyledon tissue at three different stages of germination. The tissue was selected by reference to adjacent sections which were assayed histochemically for protease activity. Day six tissue had no activity while eight and 14-day tissue did. Results show that during germination both tissue and cellular components undergo changes. The cells increase in size, the intercellular spaces increase, the cytoplasm increases in volume, the protein bodies swell and fuse, and small starch grains appear while the large starch grains do not undergo any major changes.     

Argon laser irradiation of rabbits' eyes-changes in prostaglandin E2 levels

Laser irradiation of the eye is a widely used therapeutic measure in various ocular disorders. We investigated in laser-treated rabbits' eyes the changes in prostaglandin E2 (PGE2) levels of the tissue affected by the laser (the retina/choroid) and of its adjacent vitreous over a two-week period. The parameters studied were; PGE2 in vitro production by the retina/choroid, as well as PGE2 and protein levels in the vitreous, the latter indicative of a break in the blood retinal barrier (BRB). The effect of noncoherent light exposure used for illumination, and that of the mechanical manipulation involved (sham exposure) were also studied. Following laser exposure vitreal PGE2 levels were increased two-fold above baseline (days three and 14), whereas light exposure resulted in a single peak. PGE2 in vitro production by the retina/choroid in the laser-exposed group was elevated throughout the observation period, peaking twice (days 3 and 14), in the light-exposed group the enhanced production was evident during a shorter period, whereas in the sham group it remained unchanged from baseline. An elevation in vitreal protein levels to above baseline levels occurred in both the laser- and, to a lesser degree, in the noncoherent light-exposed groups, but not in the sham group. Our study demonstrated an enhanced PGE2 in vitro production by retina/choroid of laser-exposed eyes, which might be attributable to the additive effect of the laser induced trauma, and the noncoherent light photochemical changes; the clinical significance of the recurrent increase in vitreal PGE2 levels in laser-treated eyes might be related to its anti-inflammatory properties.     

Mesothelioma in Scotland

In a retrospective study of the incidence of mesothelioma in Scotland for 1950-67 80 cases were traced from pathological reports and biopsy material of malignant tumours invading the pleura and peritoneum. These cases were matched with two sets of controls. Detailed histories of residence, occupation, and degree of exposure to asbestos confirmed that the incidence of mesothelioma in Scotland is similar to that in other parts of Britain.     

Alterations in cation levels and Na-K ATPase activity in rat cerebral cortex during the development of cobalt-induced epilepsy

Abstract— This study was carried out to ascertain what biochemical changes might be present in cobalt-induced epilepsy in the rat. Sodium, potassium, calcium, magnesium, Na-K ATPase activity, water content, protein content and the ability of the tissue to utilize oxygen were measured in (1) the area of the cerebral cortex in which the cobalt was implanted; (2) in an area adjacent to but not including the area of the lesion; and (3) in the homotopic area of the contralateral cerebral cortex. The greatest changes were observed in the area of the lesion itself, with marked increases in calcium, magnesium and sodium contents and decreases in potassium content, Na-K ATPase activity, protein content and the ability of the tissue to utilize oxygen. The only significant findings in the area adjacent to the lesion and in the contralateral cortex were a modest elevation of sodium and a modest decrease in potassium at different time periods after implantation of the cobalt. We feel that the changes observed at the site of cobalt implantation may reflect tissue destruction which is unrelated to the epileptic process.