Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( HCO3- ), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with HCO3- secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

Native small airways secrete bicarbonate.

Since the discovery of Cl(-) impermeability in cystic fibrosis (CF) and the cloning of the responsible channel, CF pathology has been widely attributed to a defect in epithelial Cl(-) transport. However, loss of bicarbonate (HCO3(-)) transport also plays a major, possibly more critical role in CF pathogenesis. Even though HCO3(-) transport is severely affected in the native pancreas, liver, and intestines in CF, we know very little about HCO3(-) secretion in small airways, the principle site of morbidity in CF. We used a novel, mini-Ussing chamber system to investigate the properties of HCO3(-) transport in native porcine small airways (∼ 1 mm φ). We assayed HCO3(-) transport across small airway epithelia as reflected by the transepithelial voltage, conductance, and equivalent short-circuit current with bilateral 25-mM HCO3(-) plus 125-mM NaGlu Ringer's solution in the presence of luminal amiloride (10 µM). Under these conditions, because no major transportable anions other than HCO3(-) were present, we took the equivalent short-circuit current to be a direct measure of active HCO3(-) secretion. Applying selective agonists and inhibitors, we show constitutive HCO3(-) secretion in small airways, which can be stimulated significantly by ß-adrenergic- (cAMP) and purinergic (Ca(2+)) -mediated agonists, independently. These results indicate that two separate components for HCO3(-) secretion, likely via CFTR- and calcium-activated chloride channel-dependent processes, are physiologically regulated for likely roles in mucus clearance and antimicrobial innate defenses of small airways.

A young child with bilateral diaphragmatic palsy after bilateral bidirectional Glenn shunt.

A 13-months old boy was admitted in National Heart Foundation Hospital and Research Institute on 3 August 2011 with the diagnosis of Dextrocardia, A-V discordance, DORV, large perimembranous VSD, severe infundibular and valvular PS, bilateral SVC. He was operated on 10 August 2011. Bilateral bidirectional Glenn shunt was done off pump along with interruption of PDA. Antegrade pulmonary blood flow was minimized by tight PA banding. Baby was extubated 3 hours after surgery but had to reintubate immediately due to intense respiratory distress. Subsequent three trials of extubation failed. Chest x-ray revealed elevation of both the hemidiaphragm. Ultrasonogram of abdomen and Bronchogram along with fluoroscopy done and bilateral diaphragmatic palsy was diagnosed. Tracheostomy was done on 25th August 2011. Plication of left hemidiaphragm was done on 27th August and right hemidiaphragm plication was done on 10th September 2011. Though it took long period of time we managed to take him out of ventilator on 57th postoperative day. He was oxygen dependent for a period of time and finally he managed to take his own breath without tracheostomy tube from 67th postoperative day. After a long eventful postoperative hospital stay he was discharged home on 78th postoperative day. Discharge Chest x-ray revealed well expanded lung with flattened diaphragm. Echo revealed well functioning bilateral Glenn shunt. Tracheostomy wound healed nicely and there was no evidence of tracheal stenosis.

ß-adrenergic sweat secretion as a diagnostic test for cystic fibrosis.

RATIONALE: ß-Adrenergically induced sweat secretion offers an expedient method to assess native cystic fibrosis transmembrane conductance regulator (CFTR) secretory function in vivo. OBJECTIVES: To evaluate the sensitivity, specificity, and reliability of a test based on the activity and secretory function of CFTR in the sweat gland. METHODS: Primary and validation trials with prospectively ascertained healthy control subjects, obligate heterozygotes, and patients with a CFTR-related disorder and CF (pancreatic sufficient and insufficient). MEASUREMENTS AND MAIN RESULTS: Diagnostic accuracy and reliability of ß-adrenergic sweat secretory rates using an evaporimeter was assessed and compared with sweat chloride concentrations. The cholinergically stimulated mean sweat rate did not differ among groups. The mean maximal ß-adrenergically stimulated sweat rate in heterozygotes was about half the rate of healthy control subjects, and completely absent in pancreatic-insufficient patients with CF and pancreatic-sufficient patients with CF (P < 0.0001). Subjects with a CFTR-related disorder showed reduced or absent ß-adrenergic sweat secretion. The ß-adrenergic secretory response demonstrated high diagnostic accuracy (area under a characteristic receiver-operator curve = 0.99; 95% confidence interval, 0.97-1.00) and reliability (intraclass correlation, 0.90; 95% confidence interval, 0.81-0.95). The diagnostic cutoff level for CF, derived from the primary trial, correctly identified all control subjects, heterozygotes, and patients with CF in the validation cohort, whereas concurrent sweat chloride measurements misclassified one heterozygote and five subjects with CF. The cholinergic and ß-adrenergic sweat secretion rates were lower in women compared with men (P < 0.001). CONCLUSIONS: ß-Adrenergic sweat secretion rate determined by evaporimetry is an accurate and reliable technique to assess different levels of CFTR function and to identify patients with CF.

Surface fluid absorption and secretion in small airways.

Native small airways must remain wet enough to be pliable and support ciliary clearance, but dry enough to remain patent for gas flow. The airway epithelial lining must both absorb and secrete ions to maintain a critical level of fluid on its surface. Despite frequent involvement in lung diseases, the minuscule size has limited studies of peripheral airways. To meet this challenge, we used a capillary to construct an Ussing chamber (area <1 mm(2)) to measure electrolyte transport across small native airways (∼1 mm ø) from pig lung. Transepithelial potentials (V(t)) were recorded in open circuit conditions while applying constant current pulses across the luminal surface of dissected airways to calculate transepithelial electrical conductance (G(t)) and equivalent short circuit current (I(eq)(sc)) in the presence and absence of selected Na(+) and Cl(-) transport inhibitors (amiloride, GlyH-101, Niflumic acid) and agonists (Forskolin + IBMX, UTP). Considered together the responses suggest an organ composed of both secreting and absorbing epithelia that constitutively and concurrently transport fluids into and out of the airway, i.e. in opposite directions. Since the epithelial lining of small airways is arranged in long, accordion-like rows of pleats and folds that run axially down the lumen, we surmise that cells within the pleats are mainly secretory while the cells of the folds are principally absorptive. This structural arrangement could provide local fluid transport from within the pleats toward the luminal folds that may autonomously regulate the local surface fluid volume for homeostasis while permitting acute responses to maintain clearance.

Enhanced heat loss responses induced by short-term endurance training in exercising women.

We investigated the effects of short-term endurance training and detraining on sweating and cutaneous vasodilatation during exercise in young women, taking into account changes in maximal oxygen uptake (VO2max) and the phase of the menstrual cycle. Eleven untrained women participated in endurance training; cycle exercise at approximately 60% VO2max for 60 min day(-1), 4-5 days week(-1) (30 degrees C, 45% relative humidity) for three complete menstrual cycles. The standard exercise test consisted of exercise at 50% VO2max for 30 min (25 degrees C, 45% relative humidity), and was conducted before training (Pre), during training sessions (T1, T2 and T3) and after cessation of training (D1 and D2). Values of VO2max increased significantly from 32.7 +/- 1.2 to 37.8 +/- 1.2 ml min(-1) kg(-1) at the end of the training. Local sweat rate in the chest and thigh, but not in the back and forearm, were significantly greater during T1 and T2 only in women who started training from the midfollicular phase. Cutaneous blood flow did not change with training. The threshold oesophageal temperatures for heat loss responses were significantly decreased during T1 versus Pre (averaged values for each body site: sweating, 37.49 +/- 0.08 versus 37.22 +/- 0.12 degrees C; and cutaneous vasodilatation, 37.40 +/- 0.07 versus 37.17 +/- 0.10 degrees C) and maintained through T3; the sensitivities of heat loss responses were not altered. These changes returned to the Pre level by D1. Our data indicate that physical training improves heat loss responses by decreasing the threshold temperatures and that these effects occur within a month of training and disappear within a month after cessation of training. The degree of increase in sweating with training differs among body sites and might be affected by the phase of the menstrual cycle.

Colon epithelium. I. Light microscopic, histochemical, and ultrastructural features of normal colon epithelium of male Fischer 344 rats.

Although the colon of the inbred F344 rat is not distinctly demarcated into ascending, transverse, and descending segments as in the human colon, it can roughly be divided into ascending and descending portions that show distinct light microscopic, histochemical, and ultrastructural features. The ascending colon is characterized by a "herringbone" pattern of mucosal folds and test-tube-shaped uniform crypts that contain mucous cells (MC) with abundant mucin (acidic mucopolysaccharide--mostly sialomucin) in the lower one-third of the crypt, whereas the upper one-third contains two putative cell populations: 1) MC containing large globules of neutral mucopolysaccharide or sulfomucin and 2) columnar cells (CC), the full capabilities of which are unknown. The descending colon has longitudinal folds and contains sparse MC with small mucous granules at the lower one-third of the crypt, whereas the upper one-third contains numerous goblet cells. Neutral mucopolysaccharide is sparse and the acidic mucin is exclusively sulfated. Histochemically, the descending segment of the rat colon resembles the human descending colon in that the predominant type of mucus is sulfomucin. Ultrastructurally, the cell types observed in both the ascending colon and the descending colon are: a) MC, b) CC, c) endocrine cells, and d) undifferentiated cells.

Colon epithelium. III. In vitro studies of colon carcinogenesis in Fischer 344 rats. N-methyl-N'-nitro-N-nitrosoguanidine-induced changes in colon epithelium in explant culture.

Colon explants from the inbred F344 rat descending colon pretreated in vivo with azoxymethane and maintained in explant culture were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). One week after the MNNG treatment, the colon crypts showed marked crowding, hypercellularity, and stratification of cells. Nine weeks after the treatment, the explants showed epithelial papillary projections on the surface epithelium and within the crypts, in addition to hypercellularity and stratification. The control untreated explants maintained a single layer of epithelium during the entire culture period. Ultrastructurally, the treated cells showed an unusual concentration of free polysomes and thin and thick filaments, multiple and bizarre nucleoli, nuclear indentations and pseudoinclusions, and intracellular lumina. Sulfomucin was the predominant component in the control untreated explants as well as in the normal descending colons of rats and humans. One week after treatment the crypts of the carcinogen-treated explants showed an increase in sialomucin, and by 9 weeks after treatment, they showed mostly sialomucin. These features, compared and correlated with those of the parallel in vivo animal model as well as with human material, lend additional support to de novo histogenesis of colon carcinoma.

Colon epithelium. IV. Human colon carcinogenesis. Changes in human colon mucosa adjacent to and remote from carcinomas of the colon.

To verify the popular belief that the mucosa of the colon remote from a carcinoma is normal, in a retrospective study colon mucosae from 30 patients were studied; 15 patients had colon carcinoma and the other 15 patients without any obvious tumor in their colons served as controls. All the patients having colon carcinoma showed definite abnormalities in the mucosa remote from the tumor. None of the 15 control patients showed such morphologic changes in the mucosal sections sampled at random. The mucous changes observed were: dilatation and distortion of the crypts with flattening of the lining cells, overcrowding of crypts with mucous cells and basophilic cells, lining of the crypt with eosinophilic surface epithelial cells, and focal cellular stratification in the crypts. These abnormalities were also consistently observed in the transitional mucosa adjacent to the tumor in all of the 15 patients with colon cancer. Histochemical studies for the detection of epithelial acidic mucosubstances showed that sialomucin predominated in the colon mucosa harboring a carcinoma irrespective of the location of the tumor, whereas colon mucosa from otherwise normal individuals and patients with noncarcinomatous diseases showed a predominance of sulfomucin. Therefore, mucosa of the colon harboring a carcinoma was conclusively demonstrated to be morphologically and histochemically abnormal. The significance of these abnormalities and their possible role in the de novo histogenesis of colon carcinoma are discussed.

Detection of benzo(a)pyrene:DNA adducts in human white blood cells.

Metabolic activation of benzo(a)pyrene (BP) to its ultimate carcinogenic form, 7 beta, 8 alpha-diol-9 alpha, 10 alpha-benzo(a)pyrene epoxide (BPDE), and the binding of BPDE to DNA are important steps in BP carcinogenicity in experimental animals. Since people of certain occupations are exposed to high concentrations of BP, we have used enzyme-linked immunosorbent assay and ultrasensitive enzymatic radioimmunoassay to measure BPDE:DNA adducts in white blood cells from 2 of these occupational groups. Seven of 28 samples from roofers and 7 of 20 samples from foundry workers were positive for BPDE:DNA adducts (range, 2 to 120 fmol BPDE/50 micrograms DNA). In a group of nine volunteers without these industrial exposures to BP, the two positive DNA samples were from cigarette smokers. Control DNA obtained from human lymphocyte cell line RPMI 4265 was negative. These results indicate that the metabolic activation of BP and formation of BPDE:DNA adducts occurs in humans.

Malignant conversion and metastasis of mouse skin tumors: a comparison of SENCAR and CD-1 mice.

The progression of papillomas to squamous cell carcinomas (malignant conversion) was studied in the skin of SENCAR and Charles River CD-1 mice, using a three-stage treatment protocol. After initiation with 7,12-dimethylbenz(a)anthracene (DMBA) (stage 1) and limited promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) (stage II), papilloma-bearing mice were treated (stage III) with either tumor initiators, such as urethane, N-methyl-N'nitro-N-nitrosoguanidine (MNNG) or 4-nitroquinoline-n-oxide (R-NQO), the promoter TPA, or solvent (acetone). Similar final carcinoma yields were found in the mice treated in stage III with TPA or acetone, although carcinomas developed earlier in the TPA-treated mice. In contrast, treatment with tumor initiators in stage III increased both the rate of appearance and the final yield of carcinomas. Similar results were obtained in both SENCAR and CD-1 mice. A papilloma stage appears to be necessary for carcinoma development since elimination of TPA treatment in stage II greatly reduced the incidence of both papillomas and carcinomas in both stocks of mice. The heterogeneity of papillomas with regard to progression to carcinomas is demonstrated by the low rate of conversion of TPA-dependent papillomas and the high rate of conversion of persistent papillomas in CD-1 mice. The carcinomas that develop using the three-stage regimen vary in metastatic potential. In CD-1 mice, the frequency of metastases to lymph nodes were similar in groups treated in stage III with MNNG, urethane, 4-NQO, TPA, or acetone, but treatment with urethane substantially increased metastases to the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of benzo(a)pyrene-DNA adducts in human tissue.

Specimens of human lung, uterine cervix, ovary, and placenta were studied for the presence of benzo(a)pyrene 7,8-diol 9,10 epoxide (BPDE)-DNA adducts by using rabbit anti-BPDE-DNA antibody and light microscopic immunocytochemistry. BPDE-DNA antigenicity was detected in the bronchial epithelial cells, cervical epithelium, oocytes, luteal cells, corpora albicans, and hyalinized media of arteries within the ovaries and trophoblastic cells of the placental villi. In conjunction with immunoassay detection of BPDE-DNA adducts in human peripheral blood lymphocytes, this study demonstrates that a variety of human tissues can metabolize and bind the ubiquitous carcinogen benzo(a)pyrene. The identification and localization of this carcinogen-DNA antigenicity in various tissues and cells may not only help in monitoring exposed persons but also give insight to organ site carcinogenesis, transplacental carcinogenesis, and teratogenesis.

Ultrastructural pathology in Hashimoto's thyroiditis.

Ultrastructural studies of the thyroid tissue in two cases of Hashimoto's thyroiditis demonstrated that the inflammatory cells do not pass through the follicular cells. Indeed these cells travelled between the epithelial cells in a manner similar to the neutrophil emigration (diapedesis) through the vessel wall in acute inflammation. Inflammatory infiltrates were composed of lymphocytes, plasma cells, or transformed lymphocytes that showed features intermediate between those of lymphocytes and plasma cells. These inflammatory cells were observed to travel from the stroma to the follicular lumen in a vectorial manner - similar to neutrophilic chemotaxis in acute inflammation. The basement membrane around the thyroid follicles remained intact around some follicles whereas it was reduplicated or focally increased in thickness around others. The basement membrane material seemed to have been secreted by the follicular cells, and strands of early collagen fiber formation were seen within the excess basement membrane material. The follicular cells showed evidence fo sublethal injury characterized by prominent defects of the rough endoplasmic reticulum or the mitochondria. Cells from areas that appeared as foci of squamous metaplasia by light microscopy showed an increased number of cytoplasmic filaments (120 to 160 A), bundles of tonofilaments, large desmosomes, an increased number of desmosomes, and intracellular desmosomes. The colloid content of the follicles was diminished, and it seemed that instead of secreting the protein colloid, the follicular cells in Hashimoto's thyroiditis were producing either excessive proteinaceous material similar to colloid or other types of proteins such as basement membrane material or keratin.

Primary leiomyosarcoma of bone.

A primary tumor of bone, the light microscopic features of which were suggestive of malignant fibrous histiocytoma, proved to be a primary leiomyosarcoma upon electron microscopic examination. Ultrastructurally the tumor cells were smooth muscle cells having all the characteristic features, such as cytoplasmic filaments, cytoplasmic and sarcolemmal dense bodies, and pinocytotic vesicles, with a basal lamina surrounding the cells. This example emphasizes the importance of electron microscopy in diagnostic pathology. This is the second ultrastructural report of a primary leiomyosarcoma of bone.

Barr bodies in testis with Klinefelter syndrome.

Barr bodies are described in the interstitial cells of testes of a chromatin-positive patient with Klinefelter syndrome. Ultrastructural studies confirm the origin of the Barr body from the nuclear chromatin. Ultrastructurally the interstitial cells showed diminished, smooth endoplasmic reticulum and lipid droplets probably indicating impaired function.

Carcinoma in-situ and "micro invasive" adenocarcinoma of colon.

A case of in-situ and "micro invasive" carcinoma of the colon is reported in a patient who was neither a member of familial polyposis nor had suffered from ulcerative colitis. The in-situ and "micro-invasive" areas were seen in the flat otherwise non-raised mucosa of the colon remote from a large fungating carcinoma. Similar foci were also observed in random sections distal to the large mass. This is the first report of an in-situ and "micro-invasive" carcinoma of colon in a "non-ulcerative colitis" individual.

Long-term organ culture of adult rat colon.

Colon explants from adult rats were maintained in culture for over 3 months in our laboratories with good epithelial preservation and cellular differentiation. The light and transmission electron microscopic features of rat colon mucosa during the culture period are described. In all the explants that remained viable, there was an initial phase of degeneration of the surface and crypt cells, later these areas were repopulated in one week, showing well-formed crypts, goblet cells, and ultrastructural features such as extensive lateral interdigitations, microvilli and glycocalyx--typical of colon. The effect of in vivo carcinogen pretreatment was also studied. The explant culture from control untreated animals showed good epithelial differentiation with crypts until 6 weeks. In contrast, the explants from animals pretreated with 4 weekly doses of azoxymethane consistently showed epithelial differentiation with well-formed crypts up to 13 weeks.

Rectal mucosa. Malignant and premalignant changes after radiation therapy.

A spectrum of changes that range from crypt basophilia through varying degrees of dysplasia and carcinoma in situ have been observed in the flat, nonraised mucosa of the rectum in patient who received pelvic irradiation for carcinoma of the cervix. This case demonstrates (1) the morphological evidence of the relationship between radiation and large-bowel carcinoma, (2) that large-bowel carcinoma may arise directly from the flat mucosa without having to go through a benign polyp-cancer sequence, (3) that early carcinoma arising from the flat mucosa may clinically resemble radiation proctocolitis, and therefore, (4) that increased vigilance in needed for the follow-up of patients who undergo pelvic irradiation.

Preneoplastic and neoplastic changes in colonic mucosa in Crohn's disease.

In order to further substantiate the associated risk of colonic adenocarcinoma in cases of Crohn's disease, colonic mucosa from 12 patients with documented Crohn's disease was studied. All of the cases exhibited a wide spectrum of mucosal changes. These changes included the following: dilation and distortion of the crypts, cellular basophilia with decreased mucus, Paneth's cell metaplasia, variable degrees of atypia, and carcinoma in situ. These findings are consistent with the increased incidence of carcinoma of the colon in cases of Crohn's disease.

Experimental Staphylococcus epidermidis endocarditis in rabbit model.

To study the natural course of catheter-induced endocarditis secondarily infected with Staphylococcus epidermidis, 29 rabbits had catheters introduced surgically through the carotid artery to the aortic valve. Forty-eight hours later the catheters were removed from five rabbits. The rabbits were inoculated intravenously with 10(8) colony-forming units of S epidermidis. Autopsies done at various intervals showed all rabbits with indwelling catheters had noticeable aortic valve vegetations with positive cultures for S epidermidis. In the group with catheters removed after 48 hours, less severe valvular lesions were noted. Metastatic seeding to kidneys, spleen, and liver were noted in both groups. Because of low cure rate in the treatment of S epidermidis endocarditis, this rabbit model could be used to study antibiotic regimens for valvular endocarditis.