Submucosal glands are the predominant site of CFTR expression in the human bronchus.

We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

On the occurrence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in rat brain.

The difference spectra of the carbon monoxide-complex of dithionite-reduced rat brain microsomes, compared with both reduced microsomes, alone, and the carbon monoxide-complex of oxidized microsomes, indicate the presence of small amounts of cytochrome P-450 in brain. As in liver, cytochrome P-450 in brain is degraded in vitro to its inactive form, cytochrome P-420 by methylmercury chloride. Aryl hydrocarbon hydroxylase activity is also present in rat brain microsomes and, at lower specific activity, in brain homogenates. This carcinogen metabolizing activity is increased four-fold in rats pretreated with 3-methylcholanthrene.

Calmodulin-stimulated protein kinase activity from rat pancreas.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

The effects of donor and recipient practices on transplant center finances.

Over the past several years we have noted a marked decrease in this profitability of our kidney transplant program. Our hypothesis is that this reduction in kidney transplant institutional profitability is related to aggressive donor and recipient practices. The study population included all adults with Medicare insurance who received a kidney transplant at our center between 1999 and 2005. Adopting the hospital perspective, multi-variate linear regression models to determine the independent effects of donor and recipient characteristics and era effects on total reimbursements and total hospital margin. We note statistically significant decreased medical center incremental margins in cases with ECDs (-$5887) and in cases of DGF (-4937). We also note an annual change in the medical center margin is independently associated with year and changes at a rate of -$5278 per year, related to both increasing costs and decreasing Medicare reimbursements. The financial loss associated with patient DGF and the use of ECD kidneys may resonate with other centers, and could hinder efforts to expand kidney transplantation within the United States. The Centers for Medicare and Medicaid Services (CMS) should consider risk-adjusted reimbursement for kidney transplantation.

Regulation of membrane chloride currents in rat bile duct epithelial cells.

This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using 36Cl and 125I efflux. Separate Ca(2+)- and cAMP-dependent Cl- currents were identified. Ca(2+)-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. Ionomycin (2 microM) increased this current from 357 +/- 72 pA to 1,192 +/- 414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of 125I > 36Cl in 15:15 studies. Ionomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) (150 microM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 microM) or cpt-cAMP (500 microM) increased this current from 189 +/- 50 pA to 784 +/- 196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of 36Cl > 125I in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca(2+)- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFTR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.

Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.

Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.

Expression of the cystic fibrosis gene in adult human lung.

Critical to an understanding of the pulmonary disease in cystic fibrosis (CF) and the development of effective gene therapies is a definition of the distribution and regulation of CF gene expression in adult human lung. Previous studies have detected the product of the CF gene, the CF transmembrane conductance regulator (CFTR), in submucosal glands of human bronchi. In this report, we have characterized the distribution of CFTR RNA and protein in the distal airway and alveoli of human lungs. Samples from eight human lungs were analyzed for CFTR expression by in situ hybridization and immunocytochemistry. CFTR was detected in a subpopulation of epithelial cells at every level of the distal lung, including proximal, terminal, and respiratory bronchioles, and the alveoli. However, there was substantial variation in the level of CFTR expression between samples. In bronchioles, CFTR protein localized to the apical plasma membrane and was found primarily in a subpopulation of nonciliated cells. CFTR was expressed in the same distribution as the Clara cell marker CC10 in proximal bronchioles, however, expression was discordant in the more distal bronchioles and alveoli where CC10 was not detected. These studies suggest that epithelial cells of the distal lung may play a primary role in the pathogenesis of CF as well as expand the spectrum of target cells that should be considered in the development of gene therapies.

Decision Making in Fuzzy Discrete Event Systems1.

The primary goal of the study presented in this paper is to develop a novel and comprehensive approach to decision making using fuzzy discrete event systems (FDES) and to apply such an approach to real-world problems. At the theoretical front, we develop a new control architecture of FDES as a way of decision making, which includes a FDES decision model, a fuzzy objective generator for generating optimal control objectives, and a control scheme using both disablement and enforcement. We develop an online approach to dealing with the optimal control problem efficiently. As an application, we apply the approach to HIV/AIDS treatment planning, a technical challenge since AIDS is one of the most complex diseases to treat. We build a FDES decision model for HIV/AIDS treatment based on expert's knowledge, treatment guidelines, clinic trials, patient database statistics, and other available information. Our preliminary retrospective evaluation shows that the approach is capable of generating optimal control objectives for real patients in our AIDS clinic database and is able to apply our online approach to deciding an optimal treatment regimen for each patient. In the process, we have developed methods to resolve the following two new theoretical issues that have not been addressed in the literature: (1) the optimal control problem has state dependent performance index and hence it is not monotonic, (2) the state space of a FDES is infinite.

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas.

A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function.

Evaluation of robotic-assisted laparoscopic and open pyeloplasty in children: single-surgeon experience.

INTRODUCTION: Robotic-assisted laparoscopic pyeloplasty (RALP), the most commonly undertaken paediatric robotic urologic surgery, has not been compared against open pyeloplasty (OPN) by a single surgeon. Here, we describe our experience and outcomes. METHODS: Children undergoing RALP or OPN from 2007 to 2013 were reviewed. Clinical success was defined as resolution of presenting symptoms and improved/stable hydronephrosis on ultrasound. RESULTS: RALP and OPN cohorts comprised 52 and 40 patients, respectively. RALP patients were significantly older (6.8 vs 1.2 years, p<0.01) and heavier (28.4 vs 8.4 kg, p<0.01). Operative times for RALP were longer (203.3 vs 135.0 min, p<0.01), but decreased significantly with increasing experience (r(2)=0.42, p<0.01). Seven type-IIIb Clavien-Dindo complications occurred in RALP patients compared with two in OPN cases. There were no differences in postoperative narcotic administration (p=0.92) or duration of stay in hospital (DOSH) (p=0.93). A total of 11/40 (28%) OPN patients required epidural analgesia but none were placed in the RALP cohort. A total of 49/52 (94%) RALP patients and 40/40 OPN cases had successful outcomes. Three RALP patients required revision RALP. CONCLUSIONS: These data show that outcomes for RALP and OPN were comparable. An initial learning curve with RALP is to be expected, but operative times for RALP approached those for OPN. Previously reported benefits of RALP (reduced analgesic requirements, DOSH) were not observed. This difference may have been due to comparison of a heterogeneous cohort. Close evaluation of complications allowed for improved placement of stents in RALP.

Evaluation of the policy of empiric treatment of suspected Toxoplasma encephalitis in patients with the acquired immunodeficiency syndrome.

PURPOSE: This study was designed to measure response rates and survival in patients with acquired immunodeficiency syndrome (AIDS) and suspected Toxoplasma encephalitis treated empirically and in AIDS patients treated for biopsy-proven toxoplasmosis. PATIENTS AND METHODS: AIDS patients identified at Bellevue Hospital between August 1985 and May 1986, who had abnormal computed tomographic scans of the brain and who received empiric treatment for toxoplasmosis, constitute the empirically treated cohort. A cohort with biopsy-proven toxoplasmosis was identified from Bellevue Hospital neuropathology records spanning 1981 through 1986. Patient records were reviewed with a standardized data form, and tomograms were evaluated by neuroradiologists unaware of the identity of the scans. Survival analysis was performed by the product limit method. RESULTS: Of 38 empirically treated patients, 26 responded clinically and radiographically within four weeks of initiation of therapy. Four of nine patients who underwent biopsy responded to treatment. There was no difference in these response rates (68% versus 44%, p = 0.24). The median survival of the empirically treated responders, from first diagnosis of AIDS to last follow-up, was 422 days. Among the 30 responders, five patients discontinued therapy and four of them had relapses. No relapses occurred in the 25 patients who continued full-dose therapy indefinitely (p = 0.0004). Sixteen of 30 patients (53%) receiving continuous therapy developed toxicity, which required a change in medication. There was no difference in the survival of patients who continued to receive sulfadiazine and pyrimethamine compared with those in whom clindamycin was substituted for sulfadiazine (median, 311 days versus 422 days, p = 0.25). CONCLUSION: A policy of empiric treatment of suspected Toxoplasma encephalitis is satisfactory, and patients who respond to such therapy and continue to take full therapeutic doses of anti-Toxoplasma drugs have relatively long survivals.

Stimulation of secretion by the T84 colonic epithelial cell line with dietary flavonols.

Flavonols are dietary compounds widely distributed in plants and characterized by a 2-phenyl-benzo(alpha)pyrane nucleus possessing hydroxyl and ketone groups at positions 3 and 4, respectively. Kaempferol, quercetin, and myricetin are flavonols that are further mono-, di-, or trihydroxylated on the phenyl ring, respectively. To test whether these ingested flavonols might exert a direct secretory effect on intestinal epithelial cells, monolayers of the T84 colonocyte cell line were mounted in Ussing chambers and examined for ion transport response. Twenty minutes after addition of 100 microM quercetin to either the serosal or mucosal side, the short-circuit current change was maximal at 16.6 microA/cm2. Kaempferol was less potent than quercetin, while myricetin and glycosylated quercetin (rutin) did not induce secretion. The secretion induced by quercetin did not seem to be mediated by the reactive oxygen species generated by quercetin through auto-oxidation and/or redox cycling (superoxide, hydrogen peroxide, and the hydroxyl radical) because it was neither enhanced by iron, nor inhibited by desferroxamine B or catalase (alone or in combination with superoxide dismutase). Like vasoactive intestinal peptide, quercetin induced a secretory response that was inhibited by barium chloride and bumetanide, and which exhibited synergism with carbachol. Quercetin also stimulated a modest increase in intracellular cAMP levels and the phosphorylation of endogenous protein substrates for cAMP-dependent protein kinase. Thus, quercetin is a potent stimulus of colonocyte secretion that resembles secretagogues which act via a cAMP-mediated signaling pathway.

Cystic fibrosis mutations and genetic predisposition to idiopathic chronic pancreatitis.

Idiopathic chronic pancreatitis is a leading cause of chronic pancreatitis. Work from this and other groups has shown that idiopathic chronic pancreatitis is associated with mutations of the cystic fibrosis gene (CFTR). Many idiopathic pancreatitis patients have compound heterozygote genotypes in which both copies of the CFTR gene are abnormal. In these patients, the pancreatic disease can be viewed as a mild variant of cystic fibrosis, in which there is sufficient residual CFTR function to prevent lung disease. This article summarizes the evidence associating these abnormal CFTR genotypes with idiopathic chronic pancreatitis and reviews the implications of this association for the pathogenesis, classification, and prevention of pancreatitis.

Are mutations in the cystic fibrosis gene important in chronic pancreatitis?

The leading causes of chronic pancreatitis are alcohol and idiopathic pancreatitis. The importance of genetic factors in chronic pancreatitis has been uncertain. Recently, however, it was learned that many patients with idiopathic chronic pancreatitis have mutations of the cystic fibrosis gene. This article reviews the evidence that links mutations of this gene to unexplained pancreatitis, and discusses the implications of this for the evaluation, pathogenesis, classification, and possible prevention of pancreatitis.