Biomedical subjects

William B Guggino

Publications and source records attributed to William B Guggino.

At least 19 recordsLinked to original sources

PubMed2004

Gene expression profile analysis of 4-phenylbutyrate treatment of IB3-1 bronchial epithelial cell line demonstrates a major influence on heat-shock proteins.

Most individuals with cystic fibrosis (CF) carry one or two mutations that result in a maturation defect of the full-length CFTR protein. The DeltaF508 mutation results in a mutant protein that is degraded by the proteosome instead of progressing to the apical membrane where it functions as a cAMP-regulated chloride channel. 4-Phenylbutyrate (PBA) modulates heat-shock protein expression and promotes trafficking of DeltaF508, thus permitting maturation and membrane insertion. The goal of this study was to gain insight into the genetic mechanism of PBA action through a large-scale analysis of gene expression. The Affymetrix genome-spanning U133 microarray set was used to compare mRNA expression levels in untreated IB3-1 cell line cultures with cultures treated with 1 mM PBA for 12 and 24 h. The most notable changes in mRNA levels were transient elevations in heat-shock proteins. The majority of genes downregulated throughout the application period were functionally associated with control of gene expression. Another set of genes increased in expression starting at 24 h, suggesting these are downstream effects of altered gene expression initiated by PBA. More than one-third of the genes in this late expressing set were identified as having potential significance in understanding the pathology of CF. Our results demonstrate the usefulness of gene expression profile analysis in understanding the consequences of PBA treatment and provide insights in how this drug exerts its effect on the trafficking of CFTR.

Animals↗

PubMed2004

A phase I trial of intranasal Moli1901 for cystic fibrosis.

BACKGROUND: The peptide drug Moli1901 activates an alternative chloride channel that is present in cystic fibrosis (CF) nasal and airway epithelia. Doing so bypasses the dysfunctional CF transmembrane regulator. STUDY OBJECTIVE: To determine whether intranasal Moli1901 is safe, tolerable, and will induce chloride transport in healthy volunteers and CF subjects. DESIGN: A single-blind (to the participant), randomized, placebo-controlled, dose-escalation study of intranasal Moli1901 was performed in four healthy non-CF participants and four participants with CF. Drug or placebo was administered by intranasal superfusion, and nasal potential difference responses were continuously monitored during sequential dose escalations at 1-min intervals from 0.01 through 10 micro mol/L. RESULTS: Neither Moli1901 nor placebo were associated with visible changes such as edema, erythema, drainage, secretions, or ulcer formation. No elevations in lactate dehydrogenase, albumin, or cell counts were observed in nasal lavage fluid after administration. No clinically significant changes in FEV(1) or other toxicity parameters occurred. Changes in the nasal potential difference (NPD) induced by chloride-free, amiloride-containing Ringers solution and by subsequent superfusion with the same solution plus 10 micro mol/L isoproterenol were consistent with both an acute and a sustained change in chloride transport in response to Moli1901. A similar analysis of NPD in the four CF participants demonstrated an acute response that resolved more quickly. A dose-response relationship to Moli1901 was observed in non-CF participants, but a greater range of variability within the CF participants contributed to the lack of a clear dose-response relationship in this group. CONCLUSION: Moli1901 stimulates chloride transport in normal and CF nasal epithelia in vivo, but may have a shorter duration of action in CF participants.

Administration, Intranasal↗

PubMed2003

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Adenosine Diphosphate↗

PubMed2003

Modulation of mature cystic fibrosis transmembrane regulator protein by the PDZ domain protein CAL.

We have previously identified the cystic fibrosis transmembrane regulator (CFTR)-interacting protein CAL and demonstrated that CAL modulates CFTR plasma membrane expression by retaining CFTR within the cell. Here, we report that in addition to regulating membrane expression, CAL also regulates the expression of mature CFTR. The co-expression of hemagglutinin-tagged or Myc-tagged CAL with green fluorescent protein (GFP)-CFTR in COS-7 cells causes a dose-dependent reduction in mature GFP-CFTR, independent of its tags. Bafilomycin A1, a lysosomal proton pump inhibitor, increases mature GFP-CFTR, confirming previous reports of lysosomal degradation of mature CFTR. Importantly, bafilomycin A1 reverses CAL-mediated CFTR degradation. The proteasome inhibitor, MG132, on the other hand, does not reverse the effect of CAL. CAL has no effect on CFTR maturation, suggesting that it exerts its effects on mature CFTR. Co-expression of CAL enhances the degradation of CFTR. We showed previously that CAL reduces the half-life of CFTR at the cell surface. Here we show that expression of dominant-negative dynamin 2 K44A, a large GTPase inhibitor that is known to inhibit clathrin-mediated endocytosis and vesicle formation in the Golgi, increases cell surface CFTR as measured by surface biotinylation. More importantly, dynamin 2 K44A also restores cell surface CFTR in CAL-overexpressing cells and partially blocks the CAL-mediated degradation of mature CFTR. These data suggest a model in which CAL retains CFTR in the cell and targets CFTR for degradation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines.

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

Actin Depolymerizing Factors↗

PubMed2003

Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study.

Recombinant adeno-associated serotype 2-based vectors (rAAV2) possess a number of theoretical advantages for cystic fibrosis (CF) gene therapy because they elicit little or no inflammatory response and generally result in stable expression. rAAV2 vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have previously been shown to mediate stable correction of the CF defect in CF bronchial epithelial cells and stable expression of CFTR in rabbit and nonhuman primate models. Here we report the results of the first trial initiated with rAAV in humans, a phase I study in 25 adult and adolescent CF patients with mild to moderate lung disease. Doses of the rAAV-CFTR vector (tgAAVCF) ranging from 3 x 10(1) to 1 x 10(9) replication units (RU), which is equivalent to approximately 6 x 10(4) to 2 x 10(12) DNase resistant particles (DRP), were administered to one side of the nose and to the superior segment of the lower lobe of the right lung. Several adverse events were noted prior to and/or after vector delivery, but most of them appeared to be related to the endogenous CF lung disease or a result of the bronchoscopic procedures. Only one of the serious events was judged to be possibly vector-related (based on temporal association), and this event was a pulmonary exacerbation very similar to several others experienced by the same subject in the three months preceding vector delivery. Vector shedding was minimal throughout the study, and serum-neutralizing antibodies were detected after vector delivery to subjects in the highest dosage cohorts. Gene transfer as measured by DNA polymerase chain reaction (PCR) was not observed until cohort 10 in nasal and bronchial epithelia. Sporadic low-level copy numbers suggested gene transfer of anywhere from 0.002 copies per cell up to 0.5 copies per cell was possible; however, DNA PCR was positive in lungs prior to direct dosing suggesting aspiration from the nasal dosing. These data indicate the need for continued evaluation of rAAV-CFTR vectors in additional clinical trials.

Administration, Intranasal↗

PubMed2003

Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.

Loss of the renal endosome-associated chloride channel, ClC-5, in Dent's disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected 125I-beta 2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

Absorption↗

PubMed2003

Estrogen modulates ClC-2 chloride channel gene expression in rat kidney.

ClC-2 is a CLC family member of chloride channels sensitive to changes in cell volume, pH and voltage. The ClC-2 is widely distributed along the nephron although in the kidney its role still not well understood. Aldosterone studies suggest that ClC-2 expression in the kidney may be hormonally regulated. To explore the possibility that estrogen control ClC-2 expression, we investigated whether its expression changed in the kidney of female Wistar rats subjected to ovariectomy with or without near-physiological or high doses of 17beta-estradiol benzoate treatment for 10 days. Total RNA isolated from rat kidney and dissected nephron segments was analyzed by ribonuclease protection assay and/or a semi-quantitative RT-PCR. The renal ClC-2 protein expression was analyzed by Western blot. The decreased renal expression of ClC-2 mRNA and protein observed in ovariectomized rats was restored to control levels after treatment with low doses of estradiol. Higher dose estradiol lead to an even greater increase in ClC-2 mRNA and protein expression. This change in overall expression was shown to be caused by the modulation of ClC-2 mRNA expression in the proximal tubule. These results suggest that ClC-2 may be involved in estrogen-induced Cl(-) transport in rat kidney.

Animals↗

PubMed2003

Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF.

Pancreatic duct cells express a Ca2+-activated Cl- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 microM) caused a fourfold increase in short-circuit current (Isc), a hyperpolarization of transepithelial potential difference (from -4.9 +/- 0.73 to -8.5 +/- 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 microM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K+ channel activity was necessary to maintain the ATP-stimulated Isc. Using a pharmacological approach, we found evidence for two types of K+ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 microM) and sensitive to clotrimazole (30 microM), as well as one blocked by clofilium (100 microM) but not chromanol 293B (5 microM). RT-PCR indicated expression of the Ca2+-activated K+ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K+ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells.

Adenosine Triphosphate↗

PubMed2003

Effect of adeno-associated virus-specific immunoglobulin G in human amniotic fluid on gene transfer.

Intra-amniotic administration of adeno-associated virus (AAV) vector may be an effective way to deliver gene therapy for treatment of congenital pulmonary and intestinal disorders. In an effort to understand potential barriers to intra-amniotic gene therapy better, we determined whether human amniotic fluid (AF) could act as an inhibitor of AAV2-mediated gene transfer. AF samples were obtained from 21 different human pregnancies during routine amniocentesis at 16-20 weeks of gestation. An immortalized fetal human tracheal epithelial cell line (FHTE) was infected with AAV2 containing a luciferase reporter gene driven by the SV40 promoter in the presence and absence of each AF sample. Inhibition of transgene expression was observed in 8 (38%) of the AF samples (inhibitory AF) and resulted in luciferase levels of only 1.4% +/- 0.6% of those obtained with infection in normal media. Infections in 13 samples (62%) resulted in transgene expression comparable or in excess of infection in media alone (noninhibitory AF). Removal of immunoglobulin G (IgG) from inhibitory AF samples with Protein A returned luciferase expression to control levels (119% +/- 37% of control), suggesting the possible presence of inhibiting antibody. Eleven of the AF samples were evaluated by enzyme-linked immunosorbent assay (ELISA) for specific anti-AAV antibodies. All noninhibitory AF samples were negative (titers of < 1:20; n = 3), and 6 of the 8 inhibitory samples contained specific anti-AAV antibodies at titers ranging from 1:40 to 1:160. These studies demonstrate that AF from some individuals contains AAV-specific IgG that can inhibit gene transfer.

Amniotic Fluid↗

PubMed2003

Successful transgene expression with serial doses of aerosolized rAAV2 vectors in rhesus macaques.

Bronchoscopic microspraying of recombinant adeno-associated viral (rAAV) vectors targets high doses of vector directly to pulmonary epithelium. Single-dose endobronchial gene therapy trials have been accomplished in cystic fibrosis patients; however, repeated dosing strategies are likely essential for lifetime correction. These studies address whether serial redosing with rAAV2 vectors results in an antiserotypic response and, furthermore, whether it triggers an inflammatory response prohibitive to transgene expression. Serial redosing of 9 x 10(11) infectious units of aerosolized rAAV2 vectors to rhesus macaques resulted in successful gene transfer by quantitative PCR (1.43 x 10(9) copies/g tissue) and transgene expression. Additionally, confocal microscopy and immunohistochemical analysis demonstrated in situ expression localized to the pulmonary epithelium. Although serial redosing did induce a heightened anti-neutralizing antibody response in sera, gene transfer prevailed with resultant expression. This study is the first to demonstrate successful gene transfer subsequent to repeated aerosolized doses of rAAV2 in immunocompetent nonhuman primates without associated inflammatory responses prohibitive to transgene expression.

Aerosols↗

PubMed2003

The ClC-5 knockout mouse model of Dent's disease has renal hypercalciuria and increased bone turnover.

Dent's disease is a nephrolithiasis disorder associated with hypercalciuria and low molecular weight proteinuria that is caused by mutations in the voltage-gated chloride channel ClC-5. Because the exact cause of hypercalciuria in this disease is unknown and could come from a renal, intestinal, or bone origin, we have investigated overall calcium handling in the ClC-5 knockout mouse (ClC-5 KO). On a high calcium diet, ClC-5 KO mice had elevated serum 1alpha,25-dihydroxyvitamin D3 (1alpha,25D3), alkaline phosphatase (AP), osteocalcin (OC), and urinary deoxypyridinoline (DPD), but serum parathyroid hormone (PTH), calcium, and intestinal calcium uptake was similar to that of wild-type (WT) mice. A 30-fold decrease in dietary calcium intake caused elevation of serum PTH and urinary cyclic adenosine monophosphate in ClC-5 KO mice and decreased the renal calcium excretion, which still remained 2-fold above that of WT mice. On this low calcium diet, both groups of mice had the same serum 1alpha,25D3, with similar increments in intestinal calcium absorption, serum AP, OC, and urinary DPD. These data indicate that the hypercalciuria in the ClC-5 KO mice on low and high calcium diets is of bone and renal origin and is not caused by increased intestinal calcium absorption, despite an elevated serum 1alpha,25D3. These mice data suggest that young patients with this disease may have a propensity for altered bone homeostasis that should be monitored clinically.

Animals↗

PubMed2002

The cytoplasmic tail of large conductance, voltage- and Ca2+-activated K+ (MaxiK) channel is necessary for its cell surface expression.

The large conductance, voltage- and Ca(2+)-activated K(+) channel (MaxiK) is expressed in several renal segments and functions in cell volume regulation and flow-mediated K(+) secretion. Previously, we cloned two MaxiK channel isoforms, named rbslo1 and rbslo2, from rabbit renal cells. rbslo1 has a 58-amino acid insertion after the S8 hydrophobic domain, whereas rbslo2 is truncated and cannot be activated. Here we use the sequence differences between the two variants to examine their plasma membrane processing. Plasma membrane localization of rbslo1 and 2 expressed in HEK293 cells was assayed by electrophysiology, immunocytochemistry, and biochemistry studies. Consistent with its functional silence, rbslo2 localized primarily within the cytoplasm, presumably in the endoplasmic reticulum and Golgi region. Coexpression with MaxiK beta subunits did not alter the cellular localization of either rbslo1 or rbslo2. When rbslo1 and 2 are cotransfected in non-polarized cells, they colocalized primarily within the cell with only rbslo1 detected at the plasma membrane. When transfected into polarized, medullary-thick ascending limb (mTAL) cells, rbslo1 is expressed at the apical membrane whereas the majority of rbslo2 localized throughout the cytoplasm. Given the high degree of similarity between the two isoforms, we conclude that the cytoplasmic tail of rbslo1 is important for the cell surface expression of MaxiK channels.

Animals↗

PubMed2002

Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63.

Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg2+ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH-induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl- revealed high nitrate permeability even at pH 7.4; P(NO3)/P(Cl) > 9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO3-/Cl- selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO3-/Cl- mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.

Amino Acid Sequence↗

PubMed2002

PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator.

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.

Amino Acid Motifs↗

PubMed2002

Basolateral chloride transporters in autosomal dominant polycystic kidney disease.

Transepithelial Cl(-) secretion mediated by apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels plays a key role in cyst fluid accumulation in autosomal dominant polycystic kidney disease (ADPKD). The molecular identity of the basolateral transporter(s) responsible for Cl(-) entry in ADPKD cells is unknown, although pharmacological studies suggest that a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC/BSC) is involved. We investigated the expression of NKCC1, CFTR and anion exchanger type I (AE1) in ADPKD kidneys and cultured ADPKD cells. Immunoblotting and immunoprecipitation detected NKCC1 at ~170 kDa in ADPKD cells and kidney extracts. Immunostaining located NKCC1 in one-third of ADPKD cysts, with a pattern of basolateral reactivity. Staining of serial sections showed that cysts positive for NKCC1 also stained for CFTR. Additional studies demonstrated that AE1 is expressed in ADPKD kidneys, and is located at the basolateral pole of CFTR-positive ADPKD cysts that do not express NKCC1. RT-PCR and sequence analyses confirmed the selective expression of NKCC1 or AEI in cultured ADPKD cells that also express CFTR. The fact that most CFTR-positive ADPKD cysts also express NKCC1 suggests that transepithelial Cl(-) secretion in ADPKD involves molecular mechanisms similar to secretory epithelia. AE1 might be an alternative basolateral pathway for Cl(-) in a minority of cysts.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2002

A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies.

tgAAVCF, an adeno-associated cystic fibrosis transmembrane conductance regulator (CFTR) viral vector/gene construct, was administered to 23 patients in a Phase II, double-blind, randomized, placebo-controlled clinical trial. For each patient, a dose of 100,000 replication units of tgAAVCF was administered to one maxillary sinus, while the contralateral maxillary sinus received a placebo treatment, thereby establishing an inpatient control. Neither the primary efficacy endpoint, defined as the rate of relapse of clinically defined, endoscopically diagnosed recurrent sinusitis, nor several secondary endpoints (sinus transepithelial potential difference [TEPD], histopathology, sinus fluid interleukin [IL]-8 measurements) achieved statistical significance when comparing treated to control sinuses within patients. One secondary endpoint, measurements of the anti-inflammatory cytokine IL-10 in sinus fluid, was significantly (p < 0.03) increased in the tgAAVCF-treated sinus relative to the placebo-treated sinus at day 90 after vector instillation. The tgAAVCF administration was well tolerated, without adverse respiratory events, and there was no evidence of enhanced inflammation in sinus histopathology or alterations in serum-neutralizing antibody titer to adeno-associated virus (AAV) capsid protein after vector administration. In summary, this Phase II trial confirms the safety of tgAAVCF but provides little support of its efficacy in the within-patient controlled sinus study. Various potentially confounding factors are discussed.

Administration, Intranasal↗

PubMed2002

Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.

Animals↗