Standardization and validation of a novel and simple method to assess lumbar dural sac size.

AIM: To develop and validate a simple, reproducible method to assess dural sac size using standard imaging technology. MATERIALS AND METHODS: This study was institutional review board-approved. Two readers, blinded to the diagnoses, measured anterior-posterior (AP) and transverse (TR) dural sac diameter (DSD), and AP vertebral body diameter (VBD) of the lumbar vertebrae using MRI images from 53 control patients with pre-existing MRI examinations, 19 prospectively MRI-imaged healthy controls, and 24 patients with Marfan syndrome with prior MRI or CT lumbar spine imaging. Statistical analysis utilized linear and logistic regression, Pearson correlation, and receiver operating characteristic (ROC) curves. RESULTS: AP-DSD and TR-DSD measurements were reproducible between two readers (r = 0.91 and 0.87, respectively). DSD (L1-L5) was not different between male and female controls in the AP or TR plane (p = 0.43; p = 0.40, respectively), and did not vary by age (p = 0.62; p = 0.25) or height (p = 0.64; p = 0.32). AP-VBD was greater in males versus females (p = 1.5 × 10(-8)), resulting in a smaller dural sac ratio (DSR) (DSD/VBD) in males (p = 5.8 × 10(-6)). Marfan patients had larger AP-DSDs and TR-DSDs than controls (p = 5.9 × 10(-9); p = 6.5 × 10(-9), respectively). Compared to DSR, AP-DSD and TR-DSD better discriminate Marfan from control subjects based on area under the curve (AUC) values from unadjusted ROCs (AP-DSD p < 0.01; TR-DSD p = 0.04). CONCLUSION: Individual vertebrae and L1-L5 (average) AP-DSD and TR-DSD measurements are simple, reliable, and reproducible for quantitating dural sac size without needing to control for gender, age, or height.

Understanding the population structure of North American patients with cystic fibrosis.

It is generally presumed that the cystic fibrosis (CF) population is relatively homogeneous, and predominantly of European origin. The complex ethnic make-up observed in the CF patients collected by the North American CF Modifier Gene Consortium has brought this assumption into question, and suggested the potential for population substructure in the three CF study samples collected from North America. It is well appreciated that population substructure can result in spurious genetic associations. To understand the ethnic composition of the North American CF population, and to assess the need for population structure adjustment in genetic association studies with North American CF patients, genome-wide single-nucleotide polymorphisms on 3076 unrelated North American CF patients were used to perform population structure analyses. We compared self-reported ethnicity to genotype-inferred ancestry, and also examined whether geographic distribution and cystic fibrosis transmembrane regulator (CFTR) mutation type could explain the population structure observed. Although largely Caucasian, our analyses identified a considerable number of CF patients with admixed African-Caucasian, Mexican-Caucasian and Indian-Caucasian ancestries. Population substructure was present and comparable across the three studies of the consortium. Neither geographic distribution nor CFTR mutation type explained the population structure. Given the ethnic diversity of the North American CF population, it is essential to carefully detect, estimate and adjust for population substructure to guard against potential spurious findings in CF genetic association studies. Other Mendelian diseases that are presumed to predominantly affect single ethnic groups may also benefit from careful analysis of population structure.

A susceptibility gene for type 2 diabetes confers substantial risk for diabetes complicating cystic fibrosis.

AIMS/HYPOTHESIS: Insulin-requiring diabetes affects 25-50% of young adults with cystic fibrosis (CF). Although the cause of diabetes in CF is unknown, recent heritability studies in CF twins and siblings indicate that genetic modifiers play a substantial role. We sought to assess whether genes conferring risk for diabetes in the general population may play a risk modifying role in CF. METHODS: We tested whether a family history of type 2 diabetes affected diabetes risk in CF patients in 539 families in the CF Twin and Sibling family-based study. A type 2 diabetes susceptibility gene (transcription factor 7-like 2, or TCF7L2) was evaluated for association with diabetes in CF using 998 patients from the family-based study and 802 unrelated CF patients in an independent case-control study. RESULTS: Family history of type 2 diabetes increased the risk of diabetes in CF (OR 3.1; p = 0.0009). A variant in TCF7L2 associated with type 2 diabetes (the T allele at rs7903146) was associated with diabetes in CF in the family study (p = 0.004) and in the case-control study (p = 0.02; combined p = 0.0002). In the family-based study, variation in TCF7L2 increased the risk of diabetes about three-fold (HR 1.75 per allele, 95% CI 1.3-2.4; p = 0.0006), and decreased the mean age at diabetes diagnosis by 7 years. In CF patients not treated with systemic glucocorticoids, the effect of TCF7L2 was even greater (HR 2.9 per allele, 95% CI 1.7-4.9, p = 0.00011). CONCLUSIONS/INTERPRETATION: A genetic variant conferring risk for type 2 diabetes in the general population is a modifier of risk for diabetes in CF.

Abnormal ion permeation through cystic fibrosis respiratory epithelium.

The epithelium of nasal tissue excised from subjects with cystic fibrosis exhibited higher voltage and lower conductance than tissue from control subjects. Basal sodium ion absorption by cystic fibrosis and normal nasal epithelia equaled the short-circuit current and was amiloride-sensitive. Amiloride induced chloride ion secretion in normal but not cystic fibrosis tissue and consequently was more effective in inhibiting the short-circuit current in cystic fibrosis epithelia. Chloride ion-free solution induced a smaller hyperpolarization of cystic fibrosis tissue. The increased voltage and amiloride efficacy in cystic fibrosis reflect absorption of sodium ions across an epithelium that is relatively impermeable to chloride ions.

Modulation of cystic fibrosis lung disease by variants in interleukin-8.

Cystic fibrosis pulmonary disease is characterized by excessive and prolonged inflammation. CF Pulmonary disease severity exhibits considerable variation that, to some extent, appears to be due to the presence of modifier genes. Several components of the inflammatory response are known to have altered regulation in the CF lung. Genetic variants in 52 inflammatory genes were tested for associations with lung disease indices in a CF patient population (n=737) homozygous for the DeltaF508 cystic fibrosis transmembrane conductance regulator mutation. Variants in three inflammatory genes showed significant genotypic associations with CF lung disease severity, including IL8 and previously reported TGFbeta1 (P< or =0.05). When analyzed by gender, it was apparent that IL8 variant associations were predominantly due to males. The IL8 variants were tested in an additional CF population (n=385) and the association in males verified (P< or =0.01). The IL8 variants were in strong linkage disequilibrium with each other (R2> or =0.82), while variants in neighboring genes CXCL6, RASSF6 and PF4V1 did not associate (P> or =0.26) and were in weaker LD with each other and with the IL8 variants (0.01< or =R2< or =0.49). Studies revealed differential expression between the IL8 promoter variant alleles (P<0.001). These results suggest that IL8 variants modify CF lung disease severity and have functional consequences.

Abnormal apical cell membrane in cystic fibrosis respiratory epithelium. An in vitro electrophysiologic analysis.

The transepithelial chloride permeability of airway and sweat ductal epithelium has been reported to be decreased in patients with cystic fibrosis (CF). In the present study, we investigated whether the airway epithelial defect was in the cell path by characterizing the relative ion permeabilities of the apical membrane of respiratory epithelial cells from CF and normal subjects. Membrane electric potential difference (PD) and the responses to luminal Cl- replacement, isoproterenol, and amiloride were measured with intracellular microelectrodes. The PD across the apical barrier was smaller for CF (-11 mV) than normal (-29 mV) epithelia whereas the PD across the basolateral barrier was similar, (-26 and -34 mV respectively). In contrast to normal nasal epithelium, the apical membrane in CF epithelia was not Cl- permselective and was not responsive to isoproterenol. Amiloride, a selective Na+ channel blocker, induced a larger apical membrane hyperpolarization and a greater increase in transepithelial resistance in CF epithelia. Both reduced apical cell membrane Cl- conductance and increased Na+ conductance appear to contribute to the abnormal function of respiratory epithelia of CF patients.

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell.

Chloride secretory response of cystic fibrosis human airway epithelia. Preservation of calcium but not protein kinase C- and A-dependent mechanisms.

Because the defect in Cl- secretion exhibited by cystic fibrosis (CF) epithelia reflects regulatory rather than conductive abnormalities of an apical membrane Cl- channel, we investigated the role of different regulatory pathways in the activation of Cl- secretion in freshly excised normal and CF nasal epithelia mounted in Ussing chambers. A beta agonist (isoproterenol [ISO]), a Ca2+ ionophore (A23187), and a phorbol ester (PMA) were all effective Cl- secretagogues in normal human nasal epithelia. Agonist addition studies indicated that ISO and PMA but not A23187 may share a common regulatory pathway. In contrast, only A23187 induced Cl- secretion in CF epithelia. Bradykinin raised cytosolic Ca2+ and induced Cl- secretion in both normal and CF tissues, indicating that receptor gated Ca2+ dependent Cl- secretory mechanisms were preserved in CF. The defective Cl- secretory response in CF epithelia to ISO and PMA did not reflect abnormalities in cAMP-dependent (A) and phospholipid Ca2+-dependent (C) kinase activities. We conclude that (a) a Ca2+-sensitive mechanism for regulating Cl- secretion is maintained in CF airway epithelia, and (b) a regulatory pathway shared by two distinct protein kinases is defective in CF, indicating that the CF genetic lesion is not tightly coupled to a single (e.g., cAMP dependent) regulatory mechanism.

Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.

To test whether a major contribution of airways epithelial ion transport to lung defense reflects the regulation of airway surface liquid (ASL) ionic composition, we measured ASL composition using the filter paper technique. On nasal surfaces, the Cl- concentration (approximately 125 meq/liter) was similar to plasma, but the Na+ concentration (approximately 110 meq/liter) was below plasma, and K+ concentration (approximately 30 meq/liter) above plasma. The resting ASL osmolarity [2(Na+ + K+); 277 meq/liter] approximated isotonicity. There were no detectable differences between cystic fibrosis (CF) and normal subjects. In the lower airways, the Na+ concentrations were 80-85 meq/liter, K+ levels approximately 15 meq/liter, and Cl- concentrations 75-80 meq/liter. Measurements of Na+ activity with Na(+)-selective electrodes and osmolality with freezing point depression yielded values consistent with the monovalent cation measurements. Like the nasal surfaces, no differences in cations were detected between CF, normal, or chronic bronchitis subjects. The tracheobronchial ASL hypotonicity was hypothesized to reflect collection-induced gland secretion, a speculation consistent with observations in which induction of nasal gland secretion produced hypotonic secretions. We conclude that there are no significant differences in ASL ion concentrations between CF, normal, and chronic bronchitis subjects and, because ASL ion concentrations exceed values consistent with defensin activity, the failure of CF lung defense may reflect predominantly factors other than salt-dependent defensins.

In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis.

Cystic fibrosis (CF) is a monogenetic disease that is associated with chronic airways disease and early death. The pulmonary disease reflects mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and associated abnormal epithelial ion transport, including defective cAMP-mediated (CFTR) Cl- secretion and an accelerated rate of basal Na+ transport. With the development of vectors for gene therapy, the airway epithelium of CF patients has been targeted for studies of gene transfer. The biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia can be assessed by in vivo measurements of the transepithelial potential difference (PD), a parameter of ion transport that reflects the expression and function of CFTR. This paper describes techniques that can be used to discriminate in vivo between the ion transport phenotype of normal subjects and patients with cystic fibrosis. Protocols are outlined to allow assessment of individual components of the electrolyte transport phenotype, i.e., the magnitude of the basal and cAMP-mediated (CFTR) Cl- secretion, and the rate of Na+ transport. The physiologic basis of the protocols and important technical features of these measurements are defined. If performed properly, the in vivo nasal PD technique clearly discriminates between normal subjects and cystic fibrosis patients, and can yield estimates of the biological efficacy of gene transfer to achieve correction of the electrolyte transport defects in CF patients.

Gene therapy for cystic fibrosis using E1-deleted adenovirus: a phase I trial in the nasal cavity. The University of North Carolina at Chapel Hill.

Cystic fibrosis (CF) is an autosomal recessive disease that reflects mutations in the CFTR gene. Multiple mutations in this gene have been detected that lead to a protein (CFTR) that is abnormally metabolized, dysfunction, or both. The full spectrum of the activities of the gene product have not been defined, but it is clear that CFTR can act as a cAMP-regulated Cl- channel. This type of defect is consistent with the physiologic characterization of CF epithelia, which has revealed abnormalities in salt and water transport. In the lung, abnormalities in epithelial salt and water metabolism lead to abnormal mucociliary clearance. This defect in clerance represents a major failure of lung defense and leads ultimately to infection of the lung with Staphylococcus aureus, Pseudomonas aeruginosa, and other bacterial organisms. The chronic inflammatory response to this persistent intraluminal bacterial infection leads to protease-induced destruction of airway walls and finally, lung failure. More than 95% of CF patients die of lung disease. The clinical therapy of CF lung disease is limited to agents designed to promote clearance of secretions from the lung and antibiotics to treat the chronic bacterial infection. Recent laboratory demonstrations that introduction of the normal CFTR cDNA into CF cells corrects the ion transport defects of these cells has led to the hypothesis that gene therapy in the lung can be an effective, novel mode of therapy for this lung disease. The classic gene transfer vectors, e.g., retroviruses, appear to be not well suited for therapy of lung disease because of the low proliferation rate of airway epithelia in vivo. Recently, adenoviruses, which have a natural tropism for airway epithelia, have been genetically modified (E1-deleted) in an attempt to reduce potential toxicity of this virus and provide space for the CFTR cDNA. A series of in vitro studies have shown that this vector is highly efficient for transferring CFTR into airway epithelial cells in culture and correcting the CF defect. Further, studies in whole animals appear to indicate that this mode of gene transfer is associated with a low degree of toxicity. The present study is a dose-effect study designed to test for the safety and efficacy of E1-deleted recombinant adenovirus containing the CFTR cDNA under a CMV-beta-actin promoter in CF nasal epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)

A double-blind, placebo controlled, dose ranging study to evaluate the safety and biological efficacy of the lipid-DNA complex GR213487B in the nasal epithelium of adult patients with cystic fibrosis.

UNLABELLED: GTAB1001: A Double-Blind, Placebo Controlled, Dose Ranging Study to Evaluate the Safety and Biological Efficacy of the Lipid-DNA Complex GR213487B in the Nasal Epithelium of Adult Patients with Cystic Fibrosis. OBJECTIVES: To evaluate the effectiveness of various dosages of the lipid-DNA complex GR213487B (0.4375mg and either 4.0mg or 0.0625mg) for producing CFTR gene transfer and correcting the chloride ion transport defect in the nasal epithelium of patients with cystic fibrosis. To assess the safety and tolerability of the lipid-DNA complex GR213487B when applied to the nasal epithelium of patients with cystic fibrosis. DESIGN: Single-center, double-blind, placebo controlled, dose ranging study. DURATION: Pre-treatment evaluations will be performed during two outpatient study visits (ie. between Day -7 to -3 and at Day -2). Patients will be admitted to the Clinical Research Unit (CRU) at the University of North Carolina at Chapel Hill on Day -1 for additional pre-treatment evaluations performed the day prior to administration of double-blind treatment (ie. gene transfer) on Treatment Day 0. Patients will remain in the CRU for 7 days (Day -1 to Day 6) and will be discharged on Day 6. Patients will subsequently be followed on an outpatient basis but will return for another assessment between Days 9-11, and may also return to the CRU for two optional study visits on Days 14 and 21. All patients will return to the CRU on an out-patient basis for follow-up evaluations on Day 28 +/- 3. SETTING: Patients will receive in-patient treatment in the CRU at the University of North Carolina at Chapel Hill and will remain in the CRU for 7 days. PATIENTS: A target enrollment of 12 evaluable patients is planned. STUDY TREATMENTS: Patients who meet all entry criteria will complete pre-treatment assessments, which will take place between Day -7 to Day -1, and will serve as a baseline for specific evaluations and to ensure clinical stability. Patients will return on Day -1 for admission to the CRU the day prior to gene transfer. Each nostril of the patients will be randomly assigned in a double blind manner to receive either GR213487B liquid nasal spray or the lipid alone (ie. control administered as liposome), by topical application directed at the inferior turbinate. The first four patients will receive an initial dosage of GR213487B containing 0.4375 mg of DNA. The decision to proceed to administer a higher dose (ie. 4.0mg DNA) or a lower dose (ie. 0.0625mg DNA) in the subsequent eight patients will be determined by the Principal Investigator in association with an FDA officer serving as an independent Clinical Ombudsman, according to the study plan (see Section 5.5 and Appendix 3-Dosing Flow Chart). MEASUREMENTS: Efficacy Evaluations The primary variables to determine the efficacy of transgene expression will be: * Evidence of vector derived CFTR (cystic fibrosis transmembrane conductance regulator) mRNA, as measured by reverse transcriptase polymerase chain reaction (RT-PCR) in nasal epithelial cells obtained from nasal scrapes on Day 3 and, nasal biopsies on Day 5, if sufficient tissue is available. * Correction of chloride ion transport across the nasal epithelium as measured by the transepithelial electrical potential difference (TEPD). The baseline TEPD will initially be measured, and again subsequently following perfusion of: --zero chloride perfusion containing amiloride (to induce chloride secretion) --zero chloride perfusion containing amiloride and isoproterenol (to increase cAMP-mediated chloride secretion) Secondary measures to determine the efficacy of gene transfer will be: * Evidence of delivery of plasmid DNA in the nasal lavage (Day 1-5, Day 9-11 and Day 28) * Evidence of vector derived CFTR mRNA from nasal scrapes performed after the nasal biopsy (ie. Day 9-11 and/or Day 28) * Percentage of cells from nasal biopsies expressing vector derived CFTR mRNA as measured by in situ hybridization * Evidence of vector derived CFTR

The effects of substrates, products and other ligands on the susceptibility of inositol monophosphatase to proteolysis by endoprotease lys-C.

Inositol monophosphatase is cleaved by endoprotease lys-C at a single site (Lys36-Ser37). The rate of proteolysis is greatly reduced in the presence of substrate (D,L-Ins(1)P) and Mg2+, and less so in the presence of Pi and Mg2+, consistent with protection of the susceptible bond in the E-P or E-Pi states of the enzyme. Potentiation by Li+ of the protection afforded by a substrate analogue, 1S-phosphoryloxy-2R,4S-dihydroxycyclohexane, and Mg2+ supports the idea that Li+ binds to the E-P state.

Characterisation of a chimeric hD3/D2 dopamine receptor expressed in CHO cells.

The D2 dopamine receptor is known to be functionally coupled when expressed in CHO cells, whereas the effector systems for the D3 dopamine receptor remain unclear. A chimeric, human D3/D2 receptor (hD3/D2) was constructed containing the third intracellular loop region of the D2 receptor. CHO cells stably expressing the D2, D3, or hD3/D2 receptors were created and the pharmacology of the receptors was examined. The chimeric hD3/D2 receptor retained D3-like affinities for dopaminergic ligands. However, in contrast to the D2 receptor neither the D3 receptor nor the hD3/D2 receptor could functionally couple to the adenylate cyclase or arachidonic acid release mechanisms.

Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells.

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, Bmax = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp-dependent shifts in [3H]N-0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1-10 microM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.

Depression of high-threshold calcium currents by activation of human D2 (short) dopamine receptors expressed in differentiated NG108-15 cells.

1. This study examined the regulation of calcium currents in differentiated NG108-15 cells that had been stably transfected with cDNA encoding the short isoform of the human D2 dopamine receptor. Whole cell calcium currents were recorded by nystatin-perforated patch clamp recording. 2. Transient low-threshold calcium currents elicited by depolarizations from -100 mV to -20 mV were reversibly depressed by NiCl2 (84 +/- 8% at 30 microM; n = 3) and by omega-agatoxin IVA (15 +/- 5%; 100 nM, n = 7). These currents were unaffected by hD2 receptor activation. 3. High-threshold calcium currents elicited by depolarizations from -80 mV to 0 mV were partly blocked by omega-conotoxin GVIA (67 +/- 6% at 100 nM, n = 4) and by the subsequent addition of the dihydropyridine, nisoldipine (94 +/- 3% at 1 microM). Consistent with the presence of at least two distinct types of high-threshold calcium channels, nisoldipine alone (38 +/- 15% at 1 microM, n = 6) did not preclude the inhibition caused by omega-conotoxin GVIA (69 +/- 13% at 100 nM, n = 4). The residual current was completely blocked by 100 microM CdCl2 (98.8 +/- 0.4%, n = 7). 4. In hD2-transfected cells, but not untransfected cells, high-threshold currents were depressed by quinpirole (30 +/- 4% at 100 nM; n = 15) with a pEC50 of 8.61 +/- 0.22 (n = 5), as well as by (-)-noradrenaline (28 +/- 5% at 1 microM, n = 9). Responses to both agonists were selectively antagonized by S-(-)sulpiride (100 nM) but not by the alpha-adrenoceptor antagonist, phentolamine (1O microM). The depression caused by (-)-noradrenaline was positively correlated with that of quinpirole for each cell(r2 = 0.91, slope = 0.99).5. hD2-receptor-mediated inhibition of high-threshold calcium currents was abolished by pretreatment of cells with omega-conotoxin GVIA (100 nM; n = 4). However, a component of the high-threshold current was reversibly depressed by omega-conotoxin GVIA (67% to 45% depression after 10 min wash). This current was also depressed by hD2 receptor activation (59 +/- 9% depression in 100 nM quinpirole, n = 3),and was completely blocked by nisoldipine (95 +/- 2% at 1 MicroM).6. These data demonstrate that activation of hD2(short) dopamine receptors can regulate both wconotoxinGVIA, and dihydropyridine-sensitive high-threshold calcium currents in neuroblastoma cells.Morever, the ability of human D2 dopamine receptors to regulate more than one type of calcium current supports the notion that these receptors have a diverse functional role in the central nervous system.

Discordant organ laterality in monozygotic twins with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by abnormal ciliary structure and function, impaired mucociliary clearance, and chronic middle ear, sinus, and lung disease. PCD is associated with situs inversus in approximately 50% of the patients. One proposed explanation for this relationship is that normal ciliary function plays a role in normal organ orientation, whereas organ orientation in PCD is a random event because of dysfunctional cilia in early embryonic development. Another hypothesis for the association between PCD and situs inversus is that mutated genes in PCD not only cause defective cilia, but are also linked to the control of organ laterality, such that abnormalities in this molecular pathway result in random left-right asymmetry. We report on a set of monozygotic twin women with PCD. In both patients, deficiency of the inner dynein arms was noted on ciliary ultrastructural analysis, associated with a clinical syndrome of bronchiectasis, chronic sinusitis, and middle ear disease. One of the twins has situs solitus, the other has situs inversus totalis. DNA analysis confirmed that the twins are monozygotic. This is consistent with the hypothesis that situs inversus occurring in patients with primary ciliary dyskinesia is a random but "complete" event in the fetal development of patients with PCD.

Ultrastructure of airway epithelial cell membranes among patients with cystic fibrosis.

Alterations in transepithelial ion fluxes are a primary pathophysiologic feature in cystic fibrosis (CF). Chronic respiratory infections and host responses are secondary aggravating pulmonary complications of this disease. In the present study, the application of the freeze-fracture technique to samples of nasal and tracheal epithelium from patients with CF has provided a perspective of large areas of cell membrane for the evaluation of possible structural correlates to the pathophysiology of this disease. A variety of aberrant configurations in stranding pattern and disorganization of the epithelial tight junctional complexes in CF airway epithelium are described. Additionally, examination of ciliary membranes revealed the presence of compound cilia and dysmorphology of ciliary necklace configuration. These features are thought to represent acquired structural lesions possibly derived from chronic infection and/or host responses which may further exacerbate abnormal ion transport properties and decrements of ciliary function that appear to be associated with the airway epithelium of individuals with CF.

Pharmacologic treatment of abnormal ion transport in the airway epithelium in cystic fibrosis.

Cystic fibrosis (CF) is a recessive genetic disease reflecting mutations in the gene coding for the CF transmembrane regulator (CFTR) protein, which normally functions as a cyclic adenosine monophosphate (cAMP)-regulated chloride (Cl-) channel. Functional abnormalities include thick airway secretions resulting from defective cAMP-mediated Cl- (liquid) secretion and a related defect, excessive sodium (Na+) (liquid) absorption. Novel pharmacologic agents are being tested as therapy for these ion transport defects. Aerosolized amiloride inhibits excessive Na+ absorption, and pilot studies in adult patients with CF show slowing of the disease-associated decline in lung function. Clinical trials of amiloride are currently underway in adults and adolescents, and short-term safety studies have been initiated in children. Aerosolized uridine triphosphate (UTP) induces Cl- (and liquid) secretion in CF airway epithelia via non-CFTR Cl- channels. Short-term aerosolized UTP is well tolerated by normal subjects and patients with CF, and pilot studies in normal subjects show that aerosolized UTP is an effective stimulator of mucociliary clearance. Pharmacotherapy that modifies airway epithelial ion transport may provide new opportunities for treatment of CF lung disease.

Nontuberculous mycobacteria in adult patients with cystic fibrosis.

Because patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual microorganisms, we screened the sputum of adult CF patients for mycobacterial organisms. Acid-fast bacilli (AFB) smears and mycobacterial culture were performed on 297 sputum specimens from 87 patients. Cultures for mycobacteria were frequently overgrown with other bacteria; 22.6 percent of cultures were contaminated. Despite this limitation of mycobacterial culture, 17 patients had at least one positive culture for a Mycobacterium other than tuberculosis (MOTT). Eleven patients were positive for Mycobacterium avium-intracellulare (MAI), two for MAI and M chelonei, three for M chelonei, and one for M fortuitum. None was positive for M tuberculosis. Patients with CF with MOTT were similar to patients with CF without MOTT; only a slightly different (older) age distribution was recognized. The clinical significance of MOTT was difficult to determine in any individual patient, but patients with positive AFB smears appeared more likely to suffer pathogenic effects. We conclude that MOTT is frequently recovered from adult CF patients in the southeastern United States. A specific risk factor for colonization and/or pathogenic infection in this patient group was not evident. The general prevalence and clinical pathogenesis in CF patients in the United States remains to be determined.