The mesothelial cell

Mesothelial cells form a monolayer of specialised pavement-like cells that line the body's serous cavities and internal organs. The primary function of this layer, termed the mesothelium, is to provide a slippery, non-adhesive and protective surface. However, mesothelial cells play other pivotal roles involving transport of fluid and cells across the serosal cavities, antigen presentation, inflammation and tissue repair, coagulation and fibrinolysis and tumour cell adhesion. Injury to the mesothelium triggers events leading to the migration of mesothelial cells from the edge of the lesion towards the wound centre and desquamation of cells into the serosal fluid which attach and incorporate into the regenerating mesothelium. If healing is impaired, fibrous serosal adhesions form between organs and the body wall which impede vital intrathoracic and abdominal movement. Neoplastic transformation of mesothelial cells gives rise to malignant mesothelioma, an aggressive tumour predominantly of the pleura. Although closely associated with exposure to asbestos, recent studies have implicated other factors including simian virus 40 (SV40) in its pathogenesis.     

A comparison of asbestos burden in non-urban patients with and without lung cancer

Asbestos is a recognized carcinogen which is widely available for environmental exposure. Since all members of our society are exposed to asbestos containing environments and, indeed, have asbestos fibres in their lungs, the concern exists as to its significance in contributing to the incidence of lung cancer in such populations. The asbestos burden was compared in lung tissue from control and lung cancer patients who had resided in a non-urban environment. There were no significant differences between the asbestos burdens in both age matched groups; however, the proportions of amphiboles to chrysotile were different from those reported in previous urban based studies. This difference was suggested to be attributable to chrysotile exposure in urban air. All patients had appreciable non-asbestos fibres within their lungs. The results indicate that when comparing any dust burden in lungs, it is necessary to have data from regional control populations before attempting to explore causal-disease relationships.     

Presence of simian virus 40 sequences in malignant mesotheliomas and mesothelial cell proliferations

Malignant mesotheliomas (MMs) are pleural‐, pericardial‐, or peritoneal‐based neoplasms usually associated with asbestos exposure. Mesothelial cells are biphasic and may give rise to epithelial and sarcomatous MMs. In addition, benign or atypical proliferations of mesothelial cells may occur in response to many stimuli. There have been recent reports of simian virus 40 (SV40) DNA large T antigen (Tag) sequences in pleural MMs. To further understand the relationship between SV40, MMs, and mesothelial proliferations, we studied 118 MMs from multiple sites in Germany and North America, including 93 epithelial pleural, 14 sarcomatous or mixed pleural MMs, and 11 peritoneal MMs. In 12 pleural MMs, adjacent noninvasive tumor foci were identified and studied separately. Information about asbestos exposure (detailed history and/or microscopic examination for asbestos bodies) was available from 43 German patients. In addition, 13 examples of reactive mesothelium and 20 lung cancers from the United States were tested. DNA was extracted from frozen tumor and adjacent nontumorous tissues or after microdissection of archival formalin‐fixed, paraffin‐embedded microslides. Two rounds of PCR were performed with primers SVFor 3 and SVRev, which amplify a 105 bp region specific for SV40 Tag. The specificity of the PCR product was confirmed in some cases by sequencing. Our major findings were: 1) Specific SV40 viral sequences were present in 57% of epithelial invasive MMs, of both pleural and peritoneal origin. No significant geographic differences were found, and frozen and paraffin‐embedded tissues were equally suitable for analysis. 2) There was no apparent relationship between the presence of SV40 sequences and asbestos exposure. 3) SV40 sequences were present in the surface (noninvasive) components of epithelial MMs. 4) SV40 sequences were not detected in MMs of sarcomatous or mixed histologies. 5) Viral sequences were present in two of 13 samples (15%) of reactive mesothelium. 6) Lung cancers lacked SV40 sequences, as did non‐malignant tissues adjacent to MMs. Our findings demonstrate the presence of SV40 sequences in epithelial MMs of pleural and peritoneal origin and their absence in tumors with a sarcomatous component. Viral sequences may be present in reactive and malignant mesothelial cells, but they are absent in adjacent tissues and lung cancers. J. Cell. Biochem. 76:181–188, 1999. © 1999 Wiley‐Liss, Inc.     

Mesotheliomas and asbestos type in asbestos textile workers: a study of lung contents.

The asbestos contents of the lungs of former employees of an asbestos textile factory were determined at necropsy using a transmission electron microscope. Those who had died of mesothelioma were compared with a matched sample of those who had died of other causes. The predominant fibre processed in the factory was chrysotile, but crocidolite had also been used. The lung content was consistent with the known exposure to chrysotile, but the crocidolite content was also high, being about 300 times that of the general population of the United Kingdom. The lungs of those with mesothelioma did not contain more of either chrysotile or crocidolite than the lungs of the controls, so no particular type of asbestos could be implicated in causing the mesotheliomas. The evidence of substantial exposure to crocidolite means that the mesotheliomas that occurred in this factory could not be attributed with any certainty to the exposure to chrysotile.     

Tissue reaction following a second exposure to amosite asbestos.

An inherently long latency period exists between the time of asbestos exposure and the development of asbestos related clinical signs in man. By this stage the events reflect the cumulative responses and offer little with regard to characterizing the acute inflammatory reactions. Similarly, studies of asbestos-induced diseases employing animal models have often emphasized the investigation of chronic events, particularly the development of fibrosis and/or cancer. However, short-lived neutrophils, which exhibit a substantial potential to produce tissue damage through the generation of superoxide radicals and elastase, have been shown to constitute a component of the acute response to asbestos in this animal model. Repetitious exposures to asbestos could logically simulate extensions of this acute response and thus be an important contributor to the development of fibrosis. In order to assess this concept, animals received two exposures to asbestos. The parenchyma exhibited both 'established' lesions consisting primarily of foci of closely-packed, fibre-laden macrophages within alveoli, and 'new' lesions consisting of a mixed cell inflammatory response (including neutrophils and macrophages) as well as considerable alveolar exudate. Repeated infiltration of neutrophils to the site of renewed lung injury following a second exposure to asbestos may correspond to events occurring in human lungs exposed similarly to repeated exposures of the dust.     

Cytology of experimental mesotheliomas induced with crocidolite asbestos

The cellular features of 12 pleural and 5 peritoneal effusions, derived from experimental mesotheliomas induced with crocidolite asbestos in white rats, are described. The fluids were obtained 11 to 18 months after the introduction of asbestos into the body cavity. The morphologic characteristics of the cytoplasm, nuclei and nucleoli in the neoplastic mesothelial cells were studied using the Pappenheim, periodic acid-Schiff and Smetana stains. Nearly all effusions examined contained numerous normal and abnormal mitoses. Cell configurations suggestive of amitotic divisions were also observed. The study of the morphologic features of mesothelial cells in effusions in experimental asbestos-induced mesotheliomas may contribute to the understanding of the neoplastic transformation of the mesothelium.     

Mitochondrial-derived free radicals mediate asbestos-induced alveolar epithelial cell apoptosis

Asbestos causes pulmonary toxicity by mechanisms that in part involve reactive oxygen species (ROS). However, the precise source of ROS is unclear. We showed that asbestos induces alveolar epithelial cell (AEC) apoptosis by a mitochondrial-regulated death pathway. To determine whether mitochondrial-derived ROS are necessary for causing asbestos-induced AEC apoptosis, we utilized A549-rho(omicron) cells that lack mitochondrial DNA and a functional electron transport. As expected, antimycin, which induces an oxidative stress by blocking mitochondrial electron transport at complex III, increased dichlorofluoroscein (DCF) fluorescence in A549 cells but not in A549-rho(omicron) cells. Compared with A549 cells, rho(omicron) cells have less asbestos-induced ROS production, as assessed by DCF fluorescence, and reductions in total glutathione levels as well as less caspase-9 activation and apoptosis, as assessed by TdT-mediated dUTP nick end labeling staining and DNA fragmentation. A mitochondrial anion channel inhibitor that prevents ROS release from the mitochondria to the cytoplasm also blocked asbestos-induced A549 cell caspase-9 activation and apoptosis. Finally, a role for nonmitochondrial-derived ROS with exposure to high levels of asbestos (50 microg/cm(2)) was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) provided additional protection against asbestos-induced caspase-9 activation and DNA fragmentation in rho(omicron) cells. We conclude that asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in mitochondrial-derived ROS production that in turn mediates AEC apoptosis. Nonmitochondrial-associated ROS may also contribute to AEC apoptosis, particularly with high levels of asbestos exposure.     

Asbestos bodies in bronchoalveolar lavage fluid. A study of 20 asbestos-exposed individuals and comparison to patients with other chronic interstitial lung diseases

We studied the asbestos body (AB) content of bronchoalveolar lavage fluid from 20 patients with a history of occupational asbestos exposure, 31 patients with sarcoidosis and 5 patients with idiopathic pulmonary fibrosis. The cellular lavage pellet was digested in sodium hypochlorite and filtered onto Nuclepore filters for AB quantification by light microscopy. ABs were found in 15 of 20 asbestos-exposed individuals, 9 of 31 sarcoidosis cases and 2 of 5 patients with idiopathic pulmonary fibrosis. There was a statistically significant difference in the number of ABs per million cells recovered or per milliliter of recovered lavage fluid in the asbestos-exposed group as compared to the other categories of chronic interstitial lung disease. The highest levels occurred in patients with asbestosis. Large numbers of asbestos bodies in the lavage fluid (greater than 1 AB/10(6) cells) were indicative of considerable occupational asbestos exposure, whereas occasional bodies were a nonspecific finding.     

Asbestos and cigarette smoke cause increased DNA strand breaks and necrosis in bronchiolar epithelial cells in vivo

Coexposures to asbestos and cigarette smoke cause increased risks of lung cancer in asbestos workers. Although these carcinogens cause DNA damage to epithelial cells in vitro via generation of reactive oxygen species (ROS), it is unclear whether they cause injury to bronchiolar epithelial cells (i.e., the target cells of lung cancers in vivo). We exposed rats to amosite asbestos, cigarette smoke, and the two agents in combination for 1, 2, and 14 d. Numbers of cells exhibiting DNA strand breaks in comparison to sham rats were then evaluated in lungs using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end labeling (TUNEL) method and by transmission electron microscopy (TEM). Increases in TUNEL-positive, necrotic epithelial cells occurred after exposure to asbestos alone and in an additive fashion after smoke and asbestos in combination. These results indicate that DNA strand breakage and necrosis are prominent mechanisms of injury by asbestos fibers and cigarette smoke in vivo to epithelial cells of the respiratory tract, thus validating in vitro observations from a number of laboratories.     

Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers

We investigated the role of glutathione and nitric oxide synthase (NOS) in fiber-induced cell and DNA toxicity using alkaline (pH 13) single-cell gel electrophoresis (the Comet assay). Transformed cultured human pleural mesothelial (MeT-5A) cells and alveolar epithelial cells (A549) were exposed to crocidolite asbestos fibers (1-10 microg/cm(2)) in the presence of buthionine sulfoximine (BSO) or L-arginine-methyl ester (L-NAME). BSO inhibits gamma-glutamylcysteine synthetase (gamma-GCS) and causes glutathione depletion, and L-NAME inhibits nitric oxide generation. Studies were also conducted to assess the expression of the heavy and light subunits of gamma-GCS in human pleural mesothelium and bronchial epithelium in vivo and the induction of inducible NOS (iNOS) by asbestos fibers. Asbestos fibers caused DNA single-strand breaks, and the process was significantly enhanced by BSO (69% compared to the non-treated cells). A549 cells had a 3.5-fold glutathione content compared to MeT-5A cells, which was consistent with the higher resistance of these cells against oxidants and fibers. Flow cytometry of iNOS showed no change of iNOS by the fibers in either cell type in vitro. L-NAME had no effects on the DNA single-strand breaks in the Comet assay, either. Studies on lung biopsies showed that the immunoreactivities of both gamma-GCS subunits were very low in healthy human mesothelium in vivo. We conclude that glutathione may play an essential role in protecting intact cells against fiber-induced oxidative DNA alterations, and low gamma-GCS reactivity in pleural mesothelium may be associated with the high sensitivity of mesothelial cells to fiber-induced toxicity.     

The Incidence of Asbestos Bodies in the Lungs at Random Necropsies in Montreal

The incidence of asbestos bodies in the lungs of adult patients selected at random, who died in four Montreal hospitals, was studied by examining fresh unstained smears of lungs obtained at necropsy. Two techniques were used for preparation of the smears and an arbitrary grading system was developed to estimate the degree of contamination of the lungs by asbestos bodies.Asbestos bodies were present in 48 out of 100 necropsies; they were found in 32 of 56 men (57%) and in 16 of 44 women (34%). Men were more heavily contaminated. The proportion of positive smears depended on the technique used and the amount of lung sampled. No particular association was noted between asbestos bodies in the lungs and the presence of cancer in the 33 patients in this series with malignant disease. The high incidence in this random series suggests that asbestos is a significant air contaminant in Montreal.     

Study of the Secular Trend in Asbestos Bodies in Lungs in London 1936-66

Thick sections (30μ unstained) cut from blocks of lung tissue from 100 consecutive necropsies for the years 1936, 1946, 1956, and 1966 at the Archway Hospital, London, have been searched for asbestos bodies. The incidence rose progressively—0, 3, 14, and 20% respectively. The rise was not explained by the increasing age of death in the later years or by the likely effects of changes in the areas within London in which the deaths occurred. There was no similar increase in the incidence of other bodies in the lungs which might be mistaken for asbestos bodies. The rising incidence is shown to fit reasonably with a model based on the hypothesis that the risk of inhaling asbestos increases in relation to the cumulative total of asbestos imported into the country from 1910 onwards. The rising incidence does not fit a model in which the risk depends simply on the current level of asbestos imports.     

In vivo bioassays of acute asbestosis and its correlation with ESR spectroscopy and imaging in redox status

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.     

The protein coating of asbestos bodies

1. Asbestos bodies were isolated from human lungs and the amino acid composition of the protein content was determined. 2. The hydroxyproline, glycine, leucine and phenylalanine values indicate that the protein in the coating cannot be principally collagen. 3. Albumin can be adsorbed on chrysotile asbestos as a monolayer but more than a monolayer is adsorbed if iron is also adsorbed. 4. Ferritin is adsorbed on chrysotile to give a thick layer. 5. The amino acid composition and adsorption studies are discussed in the light of the suggestions that the protein coating of asbestos is collagen (Beattie, 1961) or ferritin (Davis, 1964).     

Redistribution of pulmonary EC-SOD after exposure to asbestos.

Inhalation of asbestos fibers leads to interstitial lung disease (asbestosis) characterized by inflammation and fibrosis. The pathogenesis of asbestosis is not fully understood, but reactive oxygen species are thought to play a central role. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects the lung in a bleomycin-induced pulmonary fibrosis model, but its role has not been studied in asbestos-mediated disease. EC-SOD is found in high levels in the extracellular matrix of lung alveoli because of its positively charged heparin-binding domain. Proteolytic removal of this domain results in clearance of EC-SOD from the matrix of tissues. We treated wild-type C57BL/6 mice with 0.1 mg of crocidolite asbestos by intratracheal instillation and euthanized them 24 h later. Compared with saline- or titanium dioxide-treated control mice, bronchoalveolar lavage fluid (BALF) from asbestos-treated mice contained significantly higher total protein levels and increased numbers of inflammatory cells, predominantly neutrophils, indicating acute lung injury in response to asbestos. Decreased EC-SOD protein and activity were found in the lungs of asbestos-treated mice, whereas more EC-SOD was found in the BALF of these mice. The EC-SOD in the BALF was predominantly in the proteolyzed form, which lacks the heparin-binding domain. This redistribution of EC-SOD correlated with development of fibrosis 14 days after asbestos exposure. These data suggest that asbestos injury leads to enhanced proteolysis and clearance of EC-SOD from lung parenchyma into the air spaces. The depletion of EC-SOD from the extracellular matrix may increase susceptibility of the lung to oxidative stress during asbestos-mediated lung injury.     

Elemental Analysis of Histological Specimens: A Method to Unmask Nano Asbestos Fibers

There is an increasing amount of evidence that nanoparticles may enhance toxicological potential in comparison to the same material in the bulk form. The aim of this study was to develop a new method to unmask asbestos nanofibers from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. For the first time, in this study we applied Energy Dispersive X-ray (EDX) microanalysis through transmission electron microscopy to demonstrate the presence of asbestos nanofibers in histological specimens of patients with possible occupational exposure to asbestos. The diagnostic protocol was applied to 10 randomly selected lung cancer patients with no history of previous asbestos exposure. We detected asbestos nanofibers in close contact with lung cancer cells in two lung cancer patients with previous possible occupational exposure to asbestos. We were also able to identify the specific asbestos iso-type, which in one of the cases was the same rare variety used in the workplace of the affected patient. By contrast, asbestos nanofibers were not detected in lung cancer patients with no history of occupational asbestos exposure.The proposed technique can represent a potential useful tool for linking the disease to previous workplace exposure in uncertain cases. Furthermore, Formalin-Fixed Paraffin-Embedded (FFPE) tissues stored in the pathology departments might be re-evaluated for possible etiological attribution to asbestos in the case of plausible exposure. Since diseases acquired through occupational exposure to asbestos are generally covered by workers’ insurance in most countries, the application of the protocol used in this study may have also relevant social and economic implications.Key words: Asbestos fibers, nanofibers, EDX microanalysis, Transmission Electron Microscopy, lung cancer, occupational exposure     

Enhanced lipid peroxidation in lung lavage of rats after inhalation of asbestos.

Exposure of phagocytic cells to asbestos in vitro results in an augmented production of reactive oxygen metabolites and increased peroxidation of lipids. The aim of this investigation was to assess the extent of lipid peroxidation both in cells and fluid obtained from bronchoalveolar lavage (BAL), and in lungs of rats exposed to crocidolite asbestos or titanium dioxide (TiO2), a nonfibrous particulate control. In comparison to sham and TiO2-exposed rats, the BAL fluid and cells of crocidolite-exposed animals contained significantly elevated levels of malondialdehyde (MDA), a breakdown product of lipid peroxidation detected using high-pressure liquid chromatography (HPLC). In contrast, no significant differences in MDA were detected in lavaged lung tissue from these animals. Inhalation of crocidolite caused an early inflammatory response characterized by elevated numbers of polymorphonuclear leukocytes and lymphocytes, as well as enhanced total protein in BAL. Pulmonary fibrosis and increased lung hydroxyproline also were observed after 20 days of exposure. Exposure to TiO2 did not cause inflammation, pulmonary fibrosis, or elevated amounts of hydroxyproline in the lung. Our results show that exposure to the fibrogenic and inflammatory mineral, crocidolite, results in an enhanced lipid peroxidation in BAL cells and fluid not observed after inhalation of the particulate TiO2. These novel observations suggest that MDA in BAL may be useful as a biomarker of exposure to inhaled asbestos or other oxidants.     

Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica.

Several studies indicate that active oxygen species play an important role in the development of pulmonary disease (asbestosis and silicosis) after exposure to mineral dust. The present study was conducted to determine if inhaled fibrogenic minerals induced changes in gene expression and activities of antioxidant enzymes (AOE) in rat lung. Two different fibrogenic minerals were compared, crocidolite, an amphibole asbestos fiber, and cristobalite, a crystalline silicon dioxide particle. Steady-state mRNA levels, immunoreactive protein, and activities of selected AOE were measured in lungs 1-10 days after initiation of exposure and at 14 days after cessation of a 10-day exposure period. Exposure to asbestos resulted in significant increases in steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) at 3 and 9 days and of glutathione peroxidase at 6 and 9 days. An increase in steady-state mRNA levels of copper, zinc-containing superoxide dismutase (CuZnSOD), was observed at 6 days. Exposure to asbestos also resulted in overall increased enzyme activities of catalase, glutathione peroxidase and total superoxide dismutase in lung. In contrast, silica caused a dramatic increase in steady-state levels of MnSOD mRNA at all time periods and an increase in glutathione peroxidase mRNA levels at 9 days. Activities of AOE remained unchanged in silica-exposed lungs. In both models, increases in gene expression of MnSOD correlated with increased amounts of MnSOD immunoreactive protein in lung and the pattern and extent of inflammation. These data indicate that the profiles of AOE are dissimilar during the development of experimental asbestosis or silicosis and suggest different mechanisms of lung defense in response to these minerals.     

Asbestos bodies in rat lung following intratracheal instillation of chrysotile

Asbestos bodies in rat lung have rarely been reported, and just one previous record of their formation from chrysotile fibres in the rat is known. This paper illustrates the production of numerous true asbestos bodies in the lungs of Lister hooded rats after a single small intratracheal dose of lightly milled chrysotile. The demonstration of these bodies is particularly useful because uncoated chrysotile fibres in lung tissue cannot normally be visualized by light microscopy; the detection of asbestos bodies, and therefore of asbestos fibres, provides a means of directly relating asbestos exposure to observed tissue lesions. The asbestos bodies detected in the present study were nearly always associated with small pulmonary fibrotic lesions. The bodies ranged in length from 5 to 80 micron and were up to 5 micron in diameter. Small spheres, rods and bodies the shape of a comma were common; larger beaded structures were somewhat rarer. The bodies were visible in tissue sections stained routinely with modified Azan stain and with haematoxylin and eosin, but their detection and localization was enhanced by the use of Perls' Prussian blue stain in association with a pale eosin counterstain.     

ULTRASTRUCTURAL STUDIES ON THE PERMEABILITY OF THE MESOTHELIUM TO HORSERADISH PEROXIDASE

Peritoneal mesothelium was exposed for 2–60 min to solutions of horseradish peroxidase by incubation in vitro, or after intraperitoneal injection in vivo. Peroxidase was localized, with the electron microscope in the intercellular clefts of the mesothelium, often along their entire lengths, in vesicles adjoining or contiguous with the clefts, and along the peritoneal and basal surfaces of the cell, and also in intracytoplasmic vacuoles. The intercellular junctions of peroxidase-treated mesothelium did not differ from those of controls: open and closed junctions were present in both groups. Intercellular localization was also obtained when the mesothelium was exposed to peroxidase during or after fixation. Although intracellular absorption of peroxidase and its incorporation into larger vacuoles were observed, there was no clearcut evidence of vesicular transport across the mesothelium in these experiments. These findings are consistent with physiologic data which postulate that mesothelial transport can be accounted for, at least in part, by passive diffusion through a system of pores, and they suggest that these pores are located in the intercellular clefts.