Xenon tissue/blood partition coefficient for pig urinary bladder.

In four landrace pigs the tissue/blood partition coefficient (lambda) for xenon (Xe) for the urinary bladder was calculated after chemical analysis for lipid, water and protein content and determination of the haematocrit. The coefficients varied from bladder to bladder owing to small differences in both the haematocrit and tissue composition. In Xe washout studies of the blood flow of the urinary bladder, we recommend calculating the lambda for Xe from the actual haematocrit and from the median value of tissue composition found in the present study.

Concentrations of cefixime in bronchial mucosa and sputum after three oral multiple dose regimens.

In a study of 58 patients the concentrations of cefixime, a new oral cephem antibiotic, in bronchial mucosa were 35-40% of the concentrations found in simultaneously collected serum samples. The antibiotic was often undetectable in sputum despite a highly sensitive assay.

Esophageal carcinoma-associated proteins detected by two-dimensional polyacrylamide gel electrophoresis.

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.

Isolation of a potent cholesterol nucleation-promoting activity from human gallbladder bile: role in the pathogenesis of gallstone disease.

Gallbladder bile contains nucleation-promoting activity that binds to concanavalin A. The activity was found in gallbladder bile from cholesterol gallstone patients but also in gallbladder bile from patients without stones and patients with pigment stones. Bile from patients with multiple cholesterol gallstones contained high concanavalin A-binding nucleation-promoting activity. The activity was much lower in bile samples from pigment stone patients, patients without stones and patients with a solitary cholesterol stone. Serum contained very little activity and no concanavalin A-binding nucleation-promoting activity could be demonstrated in gallbladder mucosa. This suggests that concanavalin A-binding nucleation promoter is produced in the liver or bile duct epithelium. The activity was fully resistant to digestion with pronase but was heat labile and could be destroyed by prolonged incubation with a mixed glycosidase preparation indicating that sugar residues are important for this activity. On a Superose 12 gel permeation column, promoting activity eluted in two major peaks at apparent molecular weights of 150 +/- 30 kD (n = 5) and less than 5 kD respectively. The mobility on the column was not influenced by pronase digestion. The factor with the higher molecular weight could be isolated further by polyacrylamide gel electrophoresis under nondenaturing conditions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of the glycoprotein was 130 kD. In conclusion, gallbladder bile contains nucleation-promoting activity that binds to concanavalin A. The activity is increased in bile from patients with multiple cholesterol gallstones and could therefore play an important role in the pathogenesis of gallstone disease.

Receptors on airway gland cells.

Airway submucosal glands are by volume the most important source of macromolecules in airway secretions. These secretions, containing gel-forming mucins, antibacterial proteins, and antiproteases, comprise the major defensive barrier protecting the host against airborne pathogens. The identification of the mechanisms regulating secretion from the submucosal glands is key to understanding the genesis of this barrier and how it is altered by disease processes. Using a variety of methods, we and others have identified on the gland cells of several species receptors specific for ACh, norepinephrine, substance P, VIP, PGE1, PGE2, PGA1, PGD2, histamine and bradykinin. These receptors all participate in modulating the secretory activity of the airway submucosal glands. Studies of homogeneous cultures of bovine airway serous cells have yielded detailed information regarding the beta-adrenergic receptor on these cells. Using radioligand binding techniques, we found evidence for the presence of a single high affinity beta receptor of beta-2 subtype. Occupancy of this receptor by isoproterenol causes an elevation in the concentration of intracellular cAMP, which in turn stimulates the phosphorylation of a subset of cytoplasmic and membrane proteins. Based on the kinetics and pharmacology of these effects, it is likely that cAMP functions as a second messenger in the serous cell secretory pathway, probably acting through protein kinases. Current efforts are directed at identification of those phosphoproteins whose phosphorylation and dephosphorylation times are consistent with their possible roles in secretion.

On the use of absorbing discs to sample mucosal surface liquids.

A common technique to sample airway mucosal 'surface' liquids is with absorbing discs of filter paper. The present study examined the efficacy of this technique by analysing tracheal liquids of control and capsaicin (0.1 nmol)-exposed guinea-pig airways. Mucosal fluids, obtained by topically applied discs or by a specific lavage procedure, and tracheal tissue were sampled. The animals had received FITC-dextran (MW 70 kDa) intravenously and this specific plasma tracer was analysed in the sampled material. Under control conditions significantly more FITC-dextran was found in the discs than in the tracheal lavage fluids (P less than 0.001) despite the fact that the lavaged mucosal surface was much larger than that covered by the discs. Capsaicin significantly increased the content of FITC-dextran in all fluids sampled as well as in the airway tissue. In all cases concentrations of FITC-dextran in the disc fluids did not differ much from that in the tissue samples. These data suggest that absorbing discs severely disturb the epithelial-barrier function and sample subepithelial fluid and solutes including macromolecules. As demonstrated in this study by the elevated content of a plasma tracer molecule an inflammatory process may, nevertheless, be traced in the mixture of surface and tissue fluids that is sampled by the discs.

Expression of a multidrug resistance gene in esophageal adenocarcinoma. Correlation with response to chemotherapy and comparison with gastric adenocarcinoma.

The resistance of malignant tumors to chemotherapy is often associated with overexpression of the multidrug resistance gene MDR. Its gene product, P-glycoprotein, acts as a drug efflux pump for chemotherapeutic agents. The authors studied MDR expression in 28 adenocarcinomas arising in Barrett's esophagus (EAs) using a monoclonal antibody directed against this gene product. The results were compared with MDR expression in 27 gastric adenocarcinomas (GAs). P-glycoprotein was detected in both tumor and normal mucosa in 7 of 27 GAs and in 6 of 10 EAs that were resected without prior chemotherapy. Chemotherapy was given before surgical resection in 18 of the EAs studied. Five patients had a partial response to chemotherapy, and one had a complete eradication of his carcinoma; all of these tumors were negative for P-glycoprotein. Of 12 patients without chemotherapy response, 6 had tumors that expressed P-glycoprotein. The authors conclude that P-glycoprotein is present in EAs and GAs before exposure to chemotherapy. The presence of P-glycoprotein in tumors usually correlates with its presence in the adjacent mucosa. Its presence in tumor cells may be an indicator of lack of sensitivity to chemotherapy.

Use of lectins for detection of glycoconjugate changes in mucous epithelium of the chicken proventriculus.

The lectin binding pattern in the mucous epithelium of the chicken proventriculus was studied by using a peroxidase labeling method and correlated procedure. The nature of glycoconjugates in the mucous epithelium was found to change during the upward cell migration from deep to superficial parts. The superficial mucous epithelium (plica) was found to contain predominantly neutral glycoconjugates with N-acetyl-D-glucosamine residues and terminal galactose-N-acetylgalactosamine disaccharides whereas the deep epithelium (sulcus) contained acidic sulfated glycoconjugates with terminal sialic acid-galactose dimer. A continuous intraluminal mucous layer over the surface epithelium was found to contain a mixture of glycoconjugates secreted from both the plica and sulcus mucous cells. The data suggests that the changes in glycoconjugates of epithelial cells were closely correlated with cellular maturation.

Comparative flow cytometric deoxyribonucleic acid studies on exophytic tumor and random mucosal biopsies in untreated carcinoma of the bladder.

In 290 patients with untreated carcinoma of the bladder the deoxyribonucleic acid properties, as measured by flow cytometry, of 3 random mucosal biopsies were studied and compared to those of the exophytic tumors. Mucosal aneuploidy was found with few exceptions in aneuploid tumors only, and in a significantly lower frequency in aneuploid tumors of grade 2 than grade 3. The individual specificity of bladder tumors is emphasized by the observation that the level of ploidy was mostly the same in aneuploid mucosal biopsies as in the exophytic tumor. This is underlined further by the occurrence of cell populations of the same ploidy in different parts of the bladder mucosa. However, S-phase values of the concomitant intraurothelial lesions were significantly lower than those of the exophytic tumors. Therefore, we concluded that the process of evolution from malignantly transformed lesions, confined to the urothelium, to an exophytic or invasive tumor is dependent on a further elevated proliferation of the urothelial lesions.

Immunoreactivity of granular cell lesions of skin, mucosa, and jaw.

Granular cell lesions from many different sites share similar light and electron microscopic features. Immunologically, however, these lesions do not appear to be a homogenous group. This study determines the extent of immunologic heterogeneity of granular cell lesions from a wide variety of sites in skin, mucosa, and jaw. Thirty-one granular cell lesions (26 granular cell tumors [GCT] and five other granular cell lesions) from 18 different sites were evaluated immunohistochemically for keratins, vimentin, desmin, muscle actin, ACT, HLA-DR, and S-100 protein. Paraffin-embedded sections were utilized with an avidin-biotin complex immunoperoxidase technique. Except for ameloblastomas, all lesions were negative for keratin and positive for vimentin. All lesions were negative for desmin and actin. Positive ACT reactivity was found in one of seven GCT of tongue, a colonic lesion, a nose lesion, and a granular cell ameloblastic fibroma. All lesions were positive for HLA-DR except a few in which fixation appeared inadequate. S-100 immunoreactivity was found in all lesions except the congenital epulis, a GCT of the skin of the nose, a colonic lesion, and the odontogenic tumors. The antigenic profile of GCT of skin and mucosa is consistent with Schwann cell origin. However, some GCT and other granular cell lesions appear to be derived from macrophages, epithelial cells, or other cells. The expression of HLA-DR by granular cells is believed to be unrelated to cellular origin but rather to some common immunologic function.

Identification of a 185-kDa band 3-related polypeptide in oxyntic cells.

Polyclonal antibodies to the purified mouse erythrocyte anion exchange protein (band 3) and to a conserved COOH-terminal peptide of mouse band 3 (alpha-Ct) recognized a single major 185-kDa polypeptide in immunoblots of a membrane fraction prepared from rabbit gastric glands. Competition studies revealed that the epitopes shared between the rabbit gastric 185-kDa antigen and the approximately 100-kDa mouse erythrocyte band 3 protein are restricted to the COOH-terminal domain of band 3, which is known to contain the catalytic site for anion exchange activity. Immunofluorescence microscopy was used to demonstrate that this band 3-related polypeptide is associated with the plasma membrane in a subpopulation of gastric gland cells composed exclusively of oxyntic cells, as judged by the coincidence of immunofluorescence with alpha-Ct and with a monoclonal antibody to the gastric H+-K+-ATPase. This alpha-Ct-reactive antigen was further localized to the cytoplasmic face of the basolateral membrane of oxyntic cells, which correlates well with the physiologically determined site of anion exchange activity. These data demonstrate the presence in gastric oxyntic cells of a novel member of the family of proteins related to the erythrocyte anion exchanger. The possibility that the 185-kDa polypeptide is an anion exchanger is discussed.

An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates.

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.

The value of mucin histochemistry, autoradiography and analysis of cellular DNA content for cancer surveillance in patients with ulcerative colitis.

Although only a small proportion of patients with ulcerative colitis (UC) will develop cancer, colorectal carcinoma is still an important complication of UC. Traditionally, histopathological dysplasia has been used as a marker for colorectal carcinogenesis in patients with UC, however, wide within- and between-observer disagreements regarding the grading of dysplasia have become evident of late. Recently, mucin histochemistry, autoradiography and flow cytometric or static cytophotometric DNA analysis have been used for monitoring the development of colorectal carcinoma in patients with UC. A brief review of the recent literatures, however, has disclosed that the value of these modern techniques in the follow up surveillance of patients with UC of long standing is rather limited and that none of these measures should be used in isolation for the early detection of colorectal carcinoma arising in UC, or for selecting candidates for colectomy. Rather, the possible role of these modern techniques appears to be as a tool for elucidating the mechanism of colorectal carcinogenesis in patients with UC.

Mast cells in conjunctiva affected by cicatricial pemphigoid.

Ocular cicatricial pemphigoid (OCP) is characterized by progressive conjunctival subepithelial fibrosis often leading ultimately to corneal blindness. Mast cells have been shown to play a role in several fibrotic disorders, but the role of mast cells in OCP is unknown. The authors compared the mast cell population in conjunctival biopsy specimens from 14 OCP patients and from six controls by using specific histochemical stains for mast cell subsets. The total mast cell number and the ratio of connective tissue mast cells to mucosal mast cells (MMCs) were significantly higher in OCP than in normal conjunctiva (P less than 0.05). This report is the first analysis of mast cell subsets in human ocular tissue. The results suggest that connective tissue mast cells (CTMCs) may play an important role in OCP and that therapy directed toward mast cells and their mediators may be an appropriate avenue for further exploration.

Use of an antiserum against deglycosylated human mucins for cellular localization of their peptide precursors: antigenic similarities between bronchial and intestinal mucins.

Highly glycosylated regions of mucins, or glycopeptides, were obtained by proteolysis of human bronchial mucins. They were deglycosylated by treatment with a trifluoromethane sulfonic acid/anisole mixture and subsequent solvolysis with anhydrous liquid hydrogen fluoride. The resulting peptides were then used to raise an immune serum in rabbit. This immune serum was used to localize the peptide precursors of human respiratory mucins within bronchial cells, using an immunohistochemical method. Two main patterns of labeling were observed in the goblet cells: the entire cytoplasm of some goblet cells was immunoreactive, whereas in other cells the labeling was concentrated around the nucleus. In the respiratory mucous glands, the labeling was localized around or below the nucleus. The serous cells were not stained. Similar labeling was observed in human colon goblet cells. This immune serum seems to be specific for mucin-secreting cells and has a strong affinity for the perinuclear region of these cells.

Neutrophils and asthma.

The importance of inflammation in asthma has been recognized for a long time and recently proved in man and animal models. All inflammatory cells are probably involved in exacerbations of asthma. Neutrophils in particular are present in the airways during and after the spontaneous asthma attacks in man and during asthmatic reactions and airway hyperresponsiveness induced experimentally in man and animals. Depletion of neutrophils prevents these effects and repletion with neutrophils reconstitutes them. Moreover, the supernatant from stimulated human neutrophils causes transient hyperresponsiveness. However, neutrophils are not increased in stable asthmatics with hyperreactive airways and are not involved in airway hyperresponsiveness induced experimentally in some animals (e.g. guinea-pigs). The studies reviewed suggest that neutrophils may be involved in the transient increases of airway responsiveness associated with exacerbations of asthma, but not in the long-lasting hyperresponsiveness of stable asthmatics.

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin.

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.

A carbohydrate histochemical study on surface mucous cells, mucous neck cells and chief cells in the gastric mucosa of developing mice.

The ontogenesis of the mouse gastric mucosa was studied by carbohydrate histochemistry and 3H-thymidine autoradiography. Surface mucous cells and glandular cells were identified from day 16 of gestation. Sugar residues in the mucin of surface mucous cells seem to undergo no major changes throughout the period under study, since secretory granules of the cells were positive in periodic acid-Schiff (PAS) and galactose oxidase-Schiff (GOS) reactions and consistently bound certain lectins. Chief cells and mucous neck cells are not separated until the third postnatal week, though primitive chief cells are present during earlier developmental stages. Secretory granules of primitive chief cells shared positive PAS and GOS reactions with mucous neck cells and bound similar lectins, but the intensity was generally weaker. The granules of primitive chief cells were also stained by Bowie's solution which exclusively stained zymogen granules in chief cells in adults. These results suggest that secretory granules of primitive chief cells contain a complex carbohydrate similar to mucin in mucous neck cells, but with a lower sugar/protein ratio. It is concluded by studies using 3H-thymidine autoradiography combined with carbohydrate histochemistry that, though immature surface mucous cells, primitive chief cells and mucous neck cells actively proliferate, chief cells rarely undergo mitosis.