The psychological impact of diagnostic food challenges to confirm the resolution of peanut or tree nut allergy.

BACKGROUND: Twenty percent of children outgrow peanut allergy and 10% outgrow tree nut allergy. Resolution can be confirmed by a food challenge. Little is known about the psychosocial impact of the challenge. We aimed to investigate effects of a food challenge on anxiety, stress and quality of life (QoL) in children and their mothers on the day of a food challenge to peanuts or nuts, and in the months following the challenge. METHODS: One hundred and three families participated. Forty children undergoing food challenges to access resolution of allergy, and their mothers, completed validated questionnaires to measure generic and food specific quality of life, stress and anxiety prior to challenge, on the day of investigation and 3-6 months later. Sixty-three children with no clinical indication to challenge (i.e. in the opinion of the allergist had persistent allergy) acted as comparison group completing questionnaires 3-6 months apart. RESULTS: Mothers reported raised anxiety on the day of challenge (P = 0.007), but children were less anxious. The children (P = 0.01) and mothers (P = 0.01) had improved food-related, but not general, QoL 3-6 months following challenge. Children reported lower anxiety levels following the challenge (P = 0.02), but anxiety remained unchanged in mothers. The improvements in maternal and children's QoL and anxiety levels were irrespective of the challenge outcome and despite co-existing food allergies in 50% of children. CONCLUSIONS: Mothers experienced increased anxiety on the day of food challenge, unlike the children, perhaps reflecting the differences in their perceived risks. Food challenges are associated with improved food-related QoL in the following months even in those with a positive challenge.

Relationship between visual sensitivity and target localization in older adults.

Older adults commonly report problems in visual search tasks and experience a higher incidence of mobility problems (e.g. falls and vehicle crashes), which involve visual skills. We examined whether target localization problems in the elderly can be adequately explained by impairments in peripheral visual sensitivity, or whether deficits in higher order visual processing are also contributory. Fifty-nine older adults (59-88 yr) who exhibited varying degrees of visual field loss (none to severe) were asked to localize briefly-presented, high-contrast targets (3 x 5 deg) in the central 60 deg (diameter) of the visual field, while simultaneously performing a visual discrimination task at fixation. Visual sensitivity accounted for only 36% of the variance in localization performance across subjects, and this relationship grew weaker (13%) when the target was embedded in distracting stimuli, suggesting that impaired attentional skills also underlie older adults' localization problems. Not surprisingly, older adults with severe visual field loss were also poor at localizing targets. However, about half of those older patients with normal or near-normal visual fields also had severe localization problems. These results indicate that despite having good visual field sensitivity, many older adults have serious difficulty locating objects of interest in the environment. This study illustrates that clinical tests for identifying visual performance problems in the elderly must embody stimulus and task features which better reflect the visual demands of everyday life.

Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers.

To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.

Use of bronchoalveolar lavage semiquantitative cultures in cystic fibrosis.

To assess bronchoalveolar lavage (BAL) in adult CF patients with respiratory symptoms, we studied BAL fluid (BALF) culture results from 28 bronchoscopies in 11 patients. Patients were asked to provide sputum for culture. All but two patients were receiving antibiotics at the time of bronchoscopy, with 13 bronchoscopies done on patients who had been receiving antibiotics for more than 10 d. Gram stain of the BALF was positive in 18 cases. In all but one BALF, > 10,000 colony-forming units per milliliter (cfu/ml) BALF of one or more pathogens was identified. The final case grew Burkholderia cepacia, which was not grown in the sputum. In only six cases (21%) were the sputum and BALF culture results the same. Prior to 11 bronchoscopies, the sputum was not adequate. The remaining 11 cases either had different pathogens in the BAL (six cases), or had some but not all of the BALF pathogens in the sputum. BALF cultures changed therapy in 13 (48%) of cases. Semiquantitative culture of BALF was a useful diagnostic tool in CF in patients in whom empiric therapy failed.

Phosphorylation and activation of a bovine tracheal anion channel by Ca2+/calmodulin-dependent protein kinase II.

Secretion of Cl- by epithelial cells is fundamental to the processes of fluid and electrolyte transport by epithelia such as those of the airways, sweat-ducts, and gastrointestinal tract. In the present study, we show that a novel Cl- channel protein, immunoaffinity purified from bovine tracheal apical membrane vesicles, is sensitive to phosphorylation by Ca2+/calmodulin protein kinase II (CaMK II). The channel protein, which migrates with an M(r) of 140,000 under nonreducing conditions, is phosphorylated in vitro by CaMK II in a Ca(2+)- and calmodulin-dependent manner. When reconstituted into planar lipid bilayers, the protein behaves as an anion-selective, 4,4'-diisothiocyanostilbene- and dithiothreitol-sensitive channel. The open probability of this channel is significantly increased by Ca2+ alone but only at levels of Ca2+ (5-10 microM) that lie outside the physiological range. Addition of CaMK II to the presumptive cytoplasmic side of the bilayer in the presence of ATP and calmodulin dramatically increased the sensitivity of the channel to free Ca2+, shifting the dose-response curve for Ca(2+)-dependent channel activation to lower [Ca2+]i, the maximum increase in channel Po occurring between 0.6 and 1 microM. The addition of kinase in the absence of ATP or calmodulin or the addition of ATP or calmodulin in the absence of kinase was without effect on channel Po. Increasing [Ca2+] above 1 microM decreased channel mean current, causing a flickery block that was maximal at 2 microM. Increasing [Ca2+] as high as 10 microM in the presence of kinase did not further alter channel behavior. In contrast to CaMK II, the addition of the catalytic subunit of protein kinase A either alone or together with ATP had no effect on channel Po. These observations suggest that a novel Ca(2+)-sensitive anion channel isolated from bovine airway epithelium is regulated by CaMK II phosphorylation.

Changes in the inflammatory response of the lung during acute respiratory distress syndrome: prognostic indicators.

We studied paired bronchoalveolar lavage (BAL) in patients with sepsis-associated acute respiratory distress syndrome (ARDS). Patients were evaluated at one institution and underwent bronchoscopy with BAL within 48 h of the onset of ARDS. Patients were restudied with bronchoscopy and BAL after 4 d of treatment. Fifty-eight patients were initially studied, with 44 patients having follow-up bronchoscopy after 4 d. The overall 30-d survival for the ARDS group was 60%. In the initial lavage, there was no difference in the neutrophils between the survivors and nonsurvivors (survivors: 59 [0-98]%; Median [Range]; nonsurvivors: 55 [0-92]%). The follow-up lavage demonstrated a significant drop in the neutrophils for the survivors (36 [4-89]%, p < 0.002) which was not seen for the nonsurvivors (70 [26-95]%). Initial IL-8 concentrations in the BAL fluid were not significantly different between the two groups. In the follow-up lavage, there was a significant fall for the IL-8 concentrations for the survivors but not the nonsurvivors. We conclude that neutrophil influx in ARDS may rapidly resolve within a week of the onset of ARDS. The resolution of neutrophils was associated with a good prognosis.

Antibodies against purified epithelial sodium channel protein from bovine renal papilla.

Monoclonal and polyclonal antibodies against amiloride-sensitive sodium channel protein purified from bovine renal papilla have been produced. These antibodies show considerable specificity in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays and can immunoprecipitate radiolabeled channel protein. The polyclonal antibodies bind two sodium channel subunits on Western blots, namely, the 300- and 110-kDa polypeptides, and cross react with channel protein isolated from the amphibian A6 renal epithelial cell line. They can be used to immunoaffinity purify the channel in relatively high yield from a crude, detergent-solubilized bovine kidney homogenate. These antibodies should be useful in isolating the sodium channel gene, in studying the channel protein structure, in studying immunocytochemical localization, and in allowing purification of the channel from other tissue sources.

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface.

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride-sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)-channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.

G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes.

Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100 ng/ml) + NAD (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (Po) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 +/- 3 picosiemens (pS) in the +/- 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the Po of the linear 80-pS channels from 0.66 +/- 0.05 to >0.9, and revealed the presence of a small (16 +/- 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the Po of the larger conductance channel and restored PKA-sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 mum) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.

Jaundice in severe bacterial infection.

Thirty patients are described who developed jaundice during the course of severe bacterial infection. Although the infecting organism was variable, as was the site of infection, the patients were generally ill and pyrexial. The group had a very high mortality rate (43%). A positive blood culture was obtained in 11 patients. Biochemical abnormalities noted were those of an increased concentration of conjugated bilirubin in the serum with only a modest increase in alkaline phosphatase and transaminase levels. Serum cholesterol was found to be normal. The mean serum urea level was significantly elevated, as were creatine phosphokinase and lactic dehydrogenase. Most patients exhibited a neutrophil leukocytosis and an elevated sedimentation rate, and the mean hemoglobin level was low. Liver histology was studied in 13 patients. There was evidence of mild bile stasis in 5 and moderate bile stasis in 2. Findings were otherwise nonspecific and were characterized by fatty change and/or inflammatory cells in the portal areas. There was no correlation between degree or duration of juandice and prognosis, although all patients who died remained jaundiced until death. It is suggested that this syndrome is not one of true cholestasis in that all biliary substances were not shown to be elevated in the serum, but that it is rather a selective defect in the excretion of conjugated bilirubin.

Malignant human gliomas express an amiloride-sensitive Na+ conductance.

Human astrocytoma cells were studied using whole cell patch-clamp recording. An inward, amiloride-sensitive Na+ current was identified in four continuous cell lines originally derived from human glioblastoma cells (CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures of cells obtained from glioblastoma multiforme tumors (up to 4 passages). In addition, cells freshly isolated from a resected medulloblastoma tumor displayed this same characteristic inward current. In contrast, amiloride-sensitive currents were not observed in normal human astrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas. The only amiloride-sensitive Na+ channels thus far molecularly identified in brain are the brain Na+ channels (BNaCs). RT-PCR analyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 in these tumors and in normal astrocytes. These results indicate that the functional expression of amiloride-sensitive Na+ currents is a characteristic feature of malignant brain tumor cells and that this pathway may be a potentially useful target for therapeutic intervention.

Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system.

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.

Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers.

Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.