Biomedical subjects
Publications and source records attributed to W E Finkbeiner.
Digoxigenin-labeled riboprobes of six groups of human mucins were evaluated for sensitivity in archival tissue, using protease XXIV or proteinase K during in situ hybridization. (J Histochem Cytochem 49:923-924, 2001)
We have compared the distribution, numbers and volume of mucous glands in the tracheas of 11 mammalian species. No glands were present in the rabbit. The mouse only contained glands at the border between the trachea and larynx. In the rat, glands were commonest in the cephalad third of the trachea, but on average were much scarcer than in the larger species. Between species, there was a significant correlation between airway diameter and gland volume per unit surface area, suggesting that the rate of deposition of inhaled particles may increase in large airways. In the ventral portion of the trachea of about half the species, the glands were concentrated between the cartilaginous rings; in others they were evenly distributed over and between the rings. In most species in which the trachealis muscle attached to the internal surface of the cartilaginous rings, the glands were external to the muscle. In all species in which the muscle attached to the external surface of the cartilaginous rings, the glands were internal to the muscle. In the ox, goat, dog and sheep, the volume of glands per unit tracheal surface area was markedly greater in the ventral than the dorsal aspect of the trachea. The reverse was true of the pig. In humans, gland density in the 2 regions was similar. The frequency of gland openings was determined in the ox, goat, pig, dog and sheep tracheas, and ranged from 0.3 per mm2 in the dorsal portion of the sheep trachea to 1.5 per mm2 in the ventral portion of the ox trachea. For these 5 species, the volume of gland acini per unit luminal surface area varied linearly with the numbers of gland openings, with the volume of individual glands being constant at approximately 120 nl.
The tracheobronchial glands, composed of mucous and serous secretory cells, provide a mucin-rich, antimicrobial-rich secretion for the conducting airways. The secretory processes of these cells are under complex neurohumoral control. Several diseases demonstrate considerable increases in the volume of secretory glands, the amount of glandular secretions or the character of the secretory product. The role of the tracheobronchial glands in the pathophysiology of chronic bronchitis, asthma and cystic fibrosis is discussed.
BACKGROUND: Bronchiolitis obliterans occurs in 30% to 80% of lung-transplant recipients and is a direct cause of death in more than 40% of patients with this complication. This study assessed the potential utility of measuring fibroblast-proliferative activity in bronchoalveolar lavage fluid from lung-transplant recipients to better understand the pathogenesis of this process. METHODS: The capacity of bronchoalveolar lavage fluid obtained from transplant recipients, during routine surveillance bronchoscopy, to stimulate the proliferation of human lung fibroblasts in vitro was assessed retrospectively and compared to that of control subjects. For each recipient, a correlation was made between the fibroblast-proliferative activity in serial lavage samples over time and the other modalities employed for detecting post-transplant complications including spirometry, transbronchial lung biopsy, and high-resolution computed tomography. RESULTS: There was a significant difference in fibroblast-proliferative activity between volunteer and transplant recipient groups (p = 0.002). Further, for each transplant recipient, the decline in the forced expired flow rate between 25% and 75% of expired volume (FEF(25%-75%)) was correlated with the mean fibroblast-proliferative activity during the period of this study (r = 0.83; p = 0.04). CONCLUSIONS: A sustained increase in fibroblast-proliferative activity in lavage supernatant precedes both histologic and physiologic evidence of bronchiolitis obliterans. Relative to an increase in fibroblast-proliferative activity or abnormalities in FEF25%-75%, a decrease in forced expiratory volume in 1 second is a late finding.
Reperfusion lung injury is an important cause of morbidity and mortality after orthotopic lung transplantation. The purpose of this study was to investigate the function of the alveolar epithelium in the setting of reperfusion lung injury. Simultaneous samples of pulmonary edema fluid and plasma were collected from eight patients with severe post-transplantation reperfusion edema. The edema fluid to plasma protein ratio was measured, an indicator of alveolar-capillary barrier permeability. The initial edema fluid to plasma protein ratio was > 0.75 in six of eight patients, confirming the presence of increased permeability of the alveolar-capillary barrier. Graft ischemic time was positively correlated with the degree of permeability (r = 0.77, p < 0.05). In four of six patients with serial samples, there was a high rate of alveolar fluid clearance (19 +/- 9%/h, mean +/- SD). Alveolar fluid clearance was calculated from serial samples in six patients. Intact alveolar fluid clearance correlated with less histologic injury, rapid resolution of hypoxemia, and more rapid resolution of radiographic infiltrates. The two patients with no net alveolar fluid clearance had persistent hypoxemia and more severe histologic injury. This study provides the first direct evidence that increased permeability to protein is the usual cause of reperfusion edema after lung transplantation, with longer ischemic times associated with greater permeability to protein in the transplanted lung. The high rates of alveolar fluid clearance indicate that the fluid transport capacity of the alveolar epithelium may be well preserved in the allograft despite reperfusion lung injury. The ability to reabsorb fluid from the alveolar space was a marker of less severe reperfusion injury, whereas the degree of alveolar-capillary barrier permeability to protein was not. Measurement of alveolar fluid clearance may be useful to assess the severity of reperfusion lung injury and to predict outcome when pulmonary edema develops after lung transplantation.
In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV >> E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.
Centrins are small calcium-binding proteins found in a variety of cell types, often in association with microtubule-organizing centers. Here we present results regarding the expression of centrins during the in vitro differentiation of human tracheal epithelial cells. When grown at an air-liquid interface, these cells differentiate into mucus-secreting cells or undergo ciliogenesis. In immunofluorescence and immunoelectron microscopy experiments, an anti-centrin antibody stained exclusively the basal bodies of the ciliated cells. There was no staining over the axonemes or the striated rootlets. Northern blots and RT-PCR analysis of the three known human centrin genes showed that these genes have distinct patterns of expression during the growth and differentiation of human tracheal epithelial cells. Centrin-1 is never transcribed. Centrin-2 mRNA is present at all times, and its concentration increases when ciliogenesis occurs. Centrin-3 mRNA is found at a constant level throughout the entire process. This differential regulation suggests that centrins are not interchangeable but instead have unique functions.
1. There is evidence that defective submucosal gland secretion contributes to the airway pathology of cystic fibrosis (CF). Using a capacitance probe technique, we have compared fluid transport across submucosal gland cultures from individuals with and without CF. 2. Under baseline conditions, approximately 60% of non-CF cultures secreted fluid; the rest absorbed. In secreting tissues, amiloride increased secretion, whereas in absorbing tissues it reduced or reversed absorption. 5-Nitro-2(3-phenylpropylamino)-benzoate (NPPB) a blocker of the CF transmembrane conductance regulator (CFTR), converted secretion to absorption. Thus, the direction and magnitude of baseline fluid movement depended on a balance between active absorption of Na+ and cAMP-dependent secretion of Cl-. 3. 8-(4-Chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), methacholine and luminal uridine 5'-triphosphate (UTP) all induced or increased fluid secretion across non-CF cultures. Results with NPPB and with 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), a blocker of Ca(2+)-activated Cl- channels, suggested that fluid secretion induced by CPT-cAMP was mediated primarily by CFTR; UTP acted entirely via Ca(2+)-activated Cl- channels, and methacholine activated both pathways. 4. All CF cultures showed baseline fluid absorption, which was abolished by amiloride. 5. CF cultures showed a normal secretory response to UTP, a reduced response to methacholine, and no response to CPT-cAMP. 6. Thus, the absorptive processes of airway glands are retained in CF, but the cAMP-dependent secretory process is lost. This would markedly reduce the water content of gland secretions. The resulting change in viscosity would contribute to the accumulation of airway mucus which is characteristic of this disease.
We describe the pathological examination of 11 cases of fetal congenital cystic adenomatoid malformation of the lung. All patients were treated in utero between 21 and 27 weeks of gestation with either lobectomy or placement of a thoracoamniotic shunt. Ten cases involved a single lobe, and one case involved two lobes. The lesions contained both solid and cystic areas. On the basis of microscopic appearances, we separated the malformations into two distinct types. The first type consisted of seven cases showing scattered bronchiole-like dilated spaces lined by pseudostratified ciliated epithelium with intervening tightly packed small tubules lined by columnar cells with subnuclear vacuoles. This pattern superficially resembled the pseudoglandular period of lung development. The remaining four cases were of the other histologic type. They contained scattered bronchiole-like structures with intervening irregularly branching glands lined by cuboidal epithelium within loose mesenchymal stroma. This pattern superficially resembled the canalicular period of lung development. We observed that the malformations contained cysts of various sizes and that cyst size varied widely within a single lesion. Moreover, predominantly cystic and predominantly solid lesions could not be separated histologically. Thus, we identify two patterns of fetal congenital cystic adenomatoid malformations, pseudoglandular and canalicular, the clinical significance of which is yet to be determined.
This study delineates the clinical spectrum of 15 patients with polymorphic ventricular tachycardia and normal QT intervals in the absence of apparent structural heart disease, adverse drug effects, or electrolyte disturbances. Patients presented with either palpitations (n = 2), presyncope (n = 5), syncope (n = 4), no symptoms (n = 1), or aborted sudden death (n = 3). Mean age was 41 years (range 20 to 64), and mean follow-up 38 months (range 4 to 109). Left ventricular function was normal as determined by either echocardiogram (n = 9) or left ventriculography (n = 9). Episodes of polymorphic ventricular tachycardia (VT) were analyzed in terms of the preceding interval, and the relation of the initiating coupling interval to the QT interval (coupling interval/QT interval = polymorphic VT index). The mean QT for the group as a whole was 0.41 +/- 0.02 second. Patients could be separated into 3 distinct groups. Four patients had polymorphic VT reproducibly induced by exercise and initiated by late-coupled beats (mean polymorphic VT index 1.27 +/- 0.21). Isoproterenol induced polymorphic VT in 3 of 4 patients, and all 4 responded to chronic beta blockade. Two patients had polymorphic VT during episodes of coronary artery spasm, and both responded to calcium channel blockade. Polymorphic VT unrelated to exertion or coronary vasospasm occurred in 9 patients. Tachycardia onset was initiated by closely coupled beats (mean polymorphic VT index 0.95 +/- 0.16), and was preceded by a pause in 4 patients, and no pause in 5 patients. Sudden death occurred in 5 of 9 patients with the shortest polymorphic VT indexes.(ABSTRACT TRUNCATED AT 250 WORDS)
BACKGROUND: Distinguishing chronic major vessel thromboembolic pulmonary hypertension from primary pulmonary hypertension is critical because the treatment options differ markedly. Surgical thromboendarterectomy is potentially curative in the former condition, whereas oxygen, vasodilators, perhaps anticoagulation, and lung transplantation are the options for the latter. The development of large thrombi in the main, right, or left pulmonary arteries has not been previously described in patients with primary pulmonary hypertension. METHODS AND RESULTS: Three pulmonary hypertensive patients with massive thrombi in the central pulmonary arteries are described. The data indicate that the large central thrombi in these three patients were not hemodynamically significant. In none did perfusion lung scans demonstrate segmental or larger defects. CONCLUSIONS: Large central thrombi can develop in patients with primary pulmonary hypertension. Perfusion lung scans that do not demonstrate segmental or larger defects should alert physicians to this possibility. Chest computed tomography and other studies identifying such thrombi are not adequate in distinguishing such a development from operable chronic major vessel thromboembolic hypertension. Careful review of lobar and segmental artery findings and the pulmonary angiogram, angioscopy, and cardiac catheterization data demonstrating the hemodynamic significance (or lack thereof) of these thrombi are essential in making this important distinction. Furthermore, these observations may constitute an additional indication for anticoagulant therapy in primary pulmonary hypertension.
We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.
The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. Recent advances in culturing primary epithelial cells and the development of transformed airway epithelial cell lines have been particularly important in enhancing our understanding of the pathology associated with cystic fibrosis and lung cancer. The establishment of conditions that enhance the proliferative capacity of airway epithelial cells in primary culture was the first technical hurdle overcome in the development of in vitro culture systems. Research is now being geared toward the development of cell culture conditions that facilitate the expression in culture of the differentiated characteristics found in the native epithelium. Aside from the advances that have been made in defining the growth media and extracellular matrixes that enhance the expression of differentiated features, the use of an air-liquid interface has been a significant advance in the culture of airway epithelial cells. The implementation of the in vitro cell culture systems that have now been established and the research into optimizing the conditions for the growth of airway epithelial cells have been and will continue to be essential in the development of therapies for airway disease.
We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.
OBJECTIVES: The purpose of this study was to better understand the effects of long-term right ventricular pacing on left ventricular perfusion, innervation, function and histology. BACKGROUND: Long-term right ventricular apical pacing is associated with increased congestive heart failure and mortality compared with atrial pacing. The exact mechanism for these changes is unknown. In this study, left ventricular perfusion, sympathetic innervation, function and histologic appearance after long-term pacing were studied in dogs in an attempt to see whether basic changes might be present that might ultimately be associated with the adverse clinical outcome. METHODS: A total of 24 dogs were studied. Sixteen underwent radiofrequency ablation of the atrioventricular (AV) junction to produce complete AV block. Seven of these underwent long-term pacing from the right ventricular apex (ventricular paced group), and nine had atrial and right ventricular apical pacing with AV synchrony (dual-chamber paced group). A control group of eight dogs had sham ablations with normal AV conduction. These dogs had atrial pacing only. Regional perfusion and sympathetic innervation were studied in all dogs by imaging with thallium-201 and [I123]metaiodobenzylguanidine, respectively. The degree of innervation was also determined by assay of tissue norepinephrine levels. Left ventricular function was assessed by radionuclide ventriculography. Cardiac histology was studied with both light and electron microscopy. RESULTS: Mismatching of perfusion and innervation in the ventricular paced group was noted, with perfusion abnormalities of both the septum and free wall. Regional [I123]metaiodobenzylguanidine distribution was homogeneous. Tissue norepinephrine levels were elevated in both the ventricular and dual-chamber paced groups compared with the control group. No light or electron microscopic findings were noted in any groups. In the dual-chamber paced group, diastolic dysfunction was noted, with normal systolic function. CONCLUSIONS: Ventricular pacing resulted in regional changes in tissue perfusion and heterogeneity between perfusion and sympathetic innervation. Both ventricular and dual-chamber pacing were associated with an increase in tissue catecholamine activity. The abnormal activation of the ventricles via right ventricular apical pacing may result in multiple abnormalities of cardiac function, which may ultimately affect clinical outcome.
This study is an extension of in situ hybridization experiments showing expression of mRNA encoding CHIP28 in selected epithelial or endothelia in spleen, colon, lung, and eye (H. Hasegawa, R. Zhang, A. Dohrman, and A. S. Verkman. Am. J. Physiol. 264 (Cell Physiol. 33): C237-C245, 1993). Additional tissues from rat were screened by in situ hybridization, and tissues from rat and humans were stained with a polyclonal anti-CHIP28 antibody. Northern blot showed the 2.8-kilobase mRNA encoding CHIP28 in kidney, lung, and heart. In situ hybridization showed strong hybridization in epithelial cells in choroid plexus, iris, ciliary body, and lens and in epithelial and subepithelial layers of trachea. Except for colonic crypts, specific hybridization was not observed in the gastrointestinal tract, liver, thyroid gland, and muscle. Immunoblot of tissues from exsanguinated rats showed immunoreactive CHIP28 protein in kidney, lung, trachea, and heart. In fixed frozen rat and/or human tissues, the anti-CHIP28 antibody stained epithelial cells in kidney proximal tubule and thin limb of Henle, lung alveolus, bronchial mucosa and glands, choroid plexus, ciliary body, iris, lens surface, colonic crypt, sweat gland, pancreatic acini, gallbladder epithelium, and placental syncytial trophoblast cells. Endothelial cells were stained in many tissues. These studies indicate a wide and selective CHIP28 tissue distribution, suggesting an important role for CHIP28 in fluid transport. The absence of CHIP28 in many nonrenal membranes believed to be water permeable suggests the existence of non-CHIP28 water transporters.
Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.
Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.