Phase II trial of ipilimumab in melanoma patients with preexisting humoural immune response to NY-ESO-1.

BACKGROUND: Immune checkpoint therapy has dramatically changed treatment options in patients with metastatic melanoma. However, a relevant part of patients still does not respond to treatment. Data regarding the prognostic or predictive significance of preexisting immune responses against tumour antigens are conflicting. Retrospective data suggested a higher clinical benefit of ipilimumab in melanoma patients with preexisting NY-ESO-1-specific immunity. PATIENTS AND METHODS: Twenty-five patients with previously untreated or treated metastatic melanoma and preexisting humoural immune response against NY-ESO-1 received ipilimumab at a dose of 10 mg/kg in week 1, 4, 7, 10 followed by 3-month maintenance treatment for a maximum of 48 weeks. Primary endpoint was the disease control rate (irCR, irPR or irSD) according to immune-related response criteria (irRC). Secondary endpoints included the disease control rate according to RECIST criteria, progression-free survival and overall survival (OS). Humoural and cellular immune responses against NY-ESO-1 were analysed from blood samples. RESULTS: Disease control rate according to irRC was 52%, irPR was observed in 36% of patients. Progression-free survival according to irRC was 7.8 months, according to RECIST criteria it was 2.9 months. Median OS was 22.7 months; the corresponding 1-year survival rate was 66.8%. Treatment-related grade 3 AEs occurred in 36% with no grade 4-5 AEs. No clear association was found between the presence of NY-ESO-1-specific cellular or humoural immune responses and clinical activity. CONCLUSION: Ipilimumab demonstrated clinically relevant activity within this biomarker-defined population. NY-ESO-1 positivity, as a surrogate for a preexisting immune response against tumour antigens, might help identifying patients with a superior outcome from immune checkpoint blockade. CLINICAL TRIAL INFORMATION: NCT01216696.

Evaluation of a novel point-of-care test for active matrix metalloproteinase-8: agreement between qualitative and quantitative measurements and relation to periodontal inflammation.

BACKGROUND AND OBJECTIVES: Severe periodontitis affects about 10% of the world population. In addition, associations between periodontitis and systemic diseases exist. Therefore, the diagnosis should be made quickly and at an early stage. Matrix metalloproteinase-8 (MMP-8) is the most prominent collagenase found in inflamed periodontal tissues. Its active form (aMMP-8) is increasingly used as a diagnostic biomarker. Aim of the present study is to evaluate the diagnostic accuracy of a novel aMMP-8 point-of-care (POC) test in comparison to the standard laboratory test to diagnose the disease rapidly and reliably. MATERIAL AND METHODS: In a prospective, mono-center, double-blinded, case-control study, participants with healthy gums (n = 35), gingivitis (n = 60) and periodontitis (n = 35) were investigated before and after therapy. Beside clinical variables for plaque and inflammation, aMMP-8 concentrations were determined in oral rinsing specimens by the enzyme-linked immunosorbent assay (ELISA) and by POC. Positive and negative percent agreements with their exact one-sided lower 95% confidence limits were calculated. RESULTS: Of 130 participants, 111 finished the study. Overall, positive percent agreements were 75.8% (57.7-88.9) before treatment and 73.7% (56.9-86.6) after treatment. Negative percent agreements were 92.8% (85.7-97.0) before and 93.3% (85.1-97.8) after treatment. Positive test results (POC and ELISA) ranged from 5.7% to 8.6% in healthy patients, 25.0-29.8% in patients with gingivitis and 40.0-48.1% in patients with periodontitis. Patients who had positive aMMP-8 test results (POC) showed higher scores for plaque and inflammation. CONCLUSIONS: The novel POC test to detect aMMP-8 has proved to agree strongly with the standard method, ELISA. The test can be recommended to screen patients at risk for periodontitis in dental offices, at the general practitioner and at specialists for associated diseases.

Physicians declining patient enrollment in a critical care trial: a case study in thromboprophylaxis.

PURPOSE: To analyze the frequency, rationale and determinants of attending physicians requesting that their eligible patients not be approached for participation in a thromboprophylaxis trial. METHODS: Research personnel in 67 centers prospectively documented eligible non-randomized patients due to physicians declining to allow their patients to be approached. RESULTS: In 67 centers, 3,764 patients were enrolled, but 1,460 eligible patients had no consent encounter. For 218 (14.9 %) of these, attending physicians requested that their patients not be approached. The most common reasons included a high risk of bleeding (31.2 %) related to fear of heparin bioaccumulation in renal failure, the presence of an epidural catheter, peri-operative status or other factors; specific preferences for thromboprophylaxis (12.4 %); morbid obesity (9.6 %); uncertain prognosis (6.4 %); general discomfort with research (3.7 %) and unclear reasons (17.0 %). Physicians were more likely to decline when approached by less experienced research personnel; considering those with[10 years of experience as the reference category, the odds ratios (OR) for physician refusals to personnel without trial experience was 10.47 [95 % confidence interval (CI) 2.19-50.02] and those with less than 10 years experience was 1.72 (95 % CI 0.61-4.84). Physicians in open rather than closed units were more likely to decline (OR 4.26; 95 % CI 1.27-14.34). Refusals decreased each year of enrollment compared to the pilot phase. CONCLUSIONS: Tracking, analyzing, interpreting and reporting the rates and reasons for physicians declining to allow their patients to be approached for enrollment provides insights into clinicians' concerns and attitudes to trials. This information can encourage physician communication and education, and potentially enhance efficient recruitment.

The efficiency of sputum cell counts in cystic fibrosis.

BACKGROUND: Technical factors relating to processing viscid sputum in cystic fibrosis (CF) and their influence on the reproducibility and validity of cell counts need to be evaluated. In addition, the methods need to be standardized so that they can be applied clinically and in research. OBJECTIVE: To examine the efficiency, reliability and validity of processing small volumes of spontaneously expectorated sputum from subjects with CF. METHODS: Sputum was collected from adults with CF (n=35) and compared with sputum from adults with infective bronchitis or bronchiectasis (IB/B) (n=16), or with asthma or chronic obstructive pulmonary disease (AS/COPD) (n=25). Selected sputum (100 mg to 200 mg) was processed with dithiothreitol (0.1%) and filtered. Total cell count (TCC) and viability were obtained in a counting chamber and cytospins were prepared and stained with Wright's for a differential cell count. Sputum and filter remnant were processed for TCC, viability and differential cell count, and the efficiency was determined by comparing the mean loss in cell yield to the filter. Two different portions from the same sputum sample were processed for cell counts to determine reproducibility. Results were compared with those from IB/B and AS/COPD groups. RESULTS: Efficiency of cell dispersal was excellent and similar to that in AS/COPD and IB/B groups. Reproducibility of cell counts from two portions of a sputum sample was high (>or=0.80). CF sputum demonstrated a raised TCC and neutrophilia similar to IB/B but significantly higher than AS/COPD. CONCLUSION: The selection method of evaluating cell counts in viscid CF sputum is efficient, reproducible and valid.

Predictors of pulmonary exacerbations in patients with cystic fibrosis infected with multi-resistant bacteria.

BACKGROUND: This study examined characteristics of adult and adolescent patients with cystic fibrosis (CF) to determine factors associated with an increased risk of pulmonary exacerbations. METHODS: 249 patients with CF infected with multidrug resistant bacteria were recruited and prospectively followed for up to 4.5 years until they experienced a pulmonary exacerbation severe enough to require intravenous antibiotics. Multivariable regression analyses were used to compare the characteristics of patients who experienced an exacerbation with those who did not. RESULTS: 124 of the 249 patients (50%) developed a pulmonary exacerbation during the first year and 154 (62%) experienced an exacerbation during the 4.5 year study period. Factors predictive of exacerbations in a multivariable survival model were younger age (OR 0.98, 95% CI 0.96 to 0.99), female sex (OR 1.45, 95% CI 1.07 to 1.95), lower forced expiratory volume in 1 second (FEV(1)) (OR 0.98, 95% CI 0.97 to 0.99), and a previous history of multiple pulmonary exacerbations (OR 3.16, 95% CI 1.93 to 5.17). Chronic use of inhaled corticosteroids was associated with an increased risk of exacerbation (OR 1.92, 95% CI 1.00 to 3.71) during the first study year. CONCLUSIONS: Patients who experience pulmonary exacerbations are more likely to be younger, female, using inhaled steroids, have a lower FEV(1), and a history of multiple previous exacerbations. It is hoped that knowledge of these risk factors will allow better identification and closer monitoring of patients who are at high risk of exacerbations.

Uptake of 18fluorodeoxyglucose in the cystic fibrosis lung: a measure of lung inflammation?

Positron emission tomography is a three-dimensional imaging technique that measures physiological effects, including metabolism. 18Fluorodeoxyglucose has been extensively used as a tracer of cellular energy metabolism in the brain and in tumour detection. As neutrophils utilise glucose as an energy source during their respiratory burst, it was hypothesised that 18fluorodeoxyglucose uptake, by these cells, could be interpreted as a measure of neutrophil activation in cystic fibrosis (CF). Ten adult CF patients were given a bolus intravenous injection of 18fluorodeoxyglucose, followed by a 90-min dynamic mid-lung acquisition scan. Right-lung 18fluorodeoxyglucose uptake was assessed using a Patlak plot and values were converted to glucose utilisation. Three clinically inactive pulmonary sarcoidosis patients served as controls. From the 10 CF patients with baseline sputum neutrophils of 14 x 10(6) cells x mL(-1) who were investigated, seven were found to have sputum at a normal or slightly depressed glucose utilisation rate (mean 1.33 micromol x g(-1) x h(-1)) compared with a mean of 2.82 micromol x g(-1) x h(-1) for the sarcoidosis patients. In eight patients, receiving inhaled tobramycin therapy, no change in lung glucose utilisation or sputum neutrophil counts were found. Despite high-sputum neutrophil levels, lung glucose utilisation was not elevated in patients with cystic fibrosis.

Adjudicating ventilator-associated pneumonia in a randomized trial of critically ill patients.

PURPOSE: The purpose of this study was to evaluate an adjudication strategy for diagnosing ventilator-associated pneumonia (VAP) in a randomized trial. MATERIALS AND METHODS: In a double-blind trial of sucralfate versus ranitidine, one of four pairs of adjudicators examined each case of clinically suspected VAP. Nurse and physician notes and all relevant laboratory data were allocated to each adjudication pair in groups of five patients. Each reader in the pair decided whether the patient had VAP; differences were resolved by consensus discussion. RESULTS: The overall unadjusted study odds ratio for VAP was 0.82 (P = .21) representing a trend toward less pneumonia with sucralfate compared with ranitidine. The odds ratio adjusted for adjudication pair was 0.85 (P = .27). The proportion of charts adjudicated as VAP positive among pairs ranged from 50% to 92%; crude agreement between readers in each pair varied from 50% to 82%. When adjudicators disagreed, the final consensus was split evenly between the two adjudicators' initial opinions in two pairs; in the other two pairs, the final decision reflected one dominant initial opinion. Personnel time to adjudicate all patients with a suspicion of VAP was 74 days. CONCLUSIONS: Though adjudication of outcomes such as VAP is time-consuming, consistent decision-making requires strict criteria, training, and calibration. Patients should be assigned to adjudication teams through random allocation.

Phosphodiesterase inhibitors suppress alpha2-adrenoceptor-mediated 5-hydroxytryptamine release from tracheae of newborn rabbits.

The outflow of 5-hydroxytryptamine (5-HT) from isolated tracheae of newborn rabbits was determined by high pressure liquid chromatography with electrochemical detection. This 5-HT outflow reflects release from neuroendocrine epithelial cells of the airway mucosa, as previously shown. Phenylephrine, via alpha2B-adrenoceptors, caused a transient increase in 5-HT outflow, maximally by about 250%, an effect mediated by liberation of intracellular Ca2+, as previously shown. The non-selective phosphodiesterase inhibitor 2-isobutyl-1-methylxanthine (IBMX) concentration-dependently inhibited phenylephrine-induced 5-HT release (completely at 100 microM, IC50: 1.3 microM). Likewise, benzafentrine (inhibitor of phosphodiesterase 3 and 4) and siguazodan (inhibitor of phosphodiesterase 3) also almost completely inhibited phenylephrine-induced 5-HT release with IC50 values of 1.7 and 4.2 microM, respectively. Rolipram (inhibitor of phosphodiesterase 4), in a concentration of 10 microM, which exceeds more than 10-fold the reported IC50 for phosphodiesterase 4, did not significantly affect phenylephrine-induced 5-HT release. 5-HT release induced by depolarizing concentrations of K+ (45 mM), which largely depends on extracellular Ca2+, was not affected by IBMX. In conclusion, phosphodiesterases, with characteristics of phosphodiesterase 3, appear to play an important role in the control of cyclic nucleotide mediated inhibition of 5-HT release from neuroendocrine epithelial cells.

The immune response in halo nevi.

The mechanism(s) responsible for halo nevus presents a provocative link with the immune response to melanoma. Although no direct demonstration of melanocyte killing has been observed by the immune effector cells found within the halo, the abundance of antigen-presenting cells in the regressing nevus and the presence of T lymphocytes at the site of depigmentation suggest that these cells participate in the halo phenomenon. Within the latter population of cells, evidence points to the involvement of CD8+ T cells as potential effectors in the destruction of nevomelanocytes. The break in tolerance that triggers migration and the presumed activation of these and other lymphocytes in the nevus in the apparent absence of disease remains unexplained. This brief overview reviews the evidence for the participation of the immune response in the genesis of the halo nevus.

Nitric oxide, via activation of guanylyl cyclase, suppresses alpha2-adrenoceptor-mediated 5-hydroxytryptamine release from neuroendocrine epithelial cells of rabbit tracheae.

Isolated tracheae of newborn rabbits were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by HPLC with electrochemical detection. Evidence has previously been provided that this 5-HT outflow derives from neuroendocrine epithelial (NEE) cells of the airway mucosa. Phenylephrine, at a maximally effective concentration of 10 microM, caused a transient increase in 5-HT outflow by about 250%, an effect mediated by alpha2B-adrenoceptors, as previously shown. The phenylephrine-induced 5-HT release remained unchanged in calcium-free medium, but was reduced by 75% when the tracheae were incubated in calcium-free medium which contained 0.5 mM EDTA, a treatment known to lower also intracellular calcium. The NO donor SNAP (S-nitroso-N-acetyl-penicillinamine, 10 microM) almost completely inhibited phenylephrine-induced 5-HT release. The inhibitory effect of SNAP was prevented by ODQ, (1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one), an inhibitor of soluble guanylyl cyclase. In contrast, 5-HT release induced by depolarizing concentrations of potassium (45 mM), which was reduced by 96% in calcium-free medium, was not affected by SNAP. In conclusion, NO, via activation of soluble guanylyl cyclase, inhibits 5-HT release from NEE cells in a stimulus-dependent manner. Alpha2-adrenoceptor-mediated 5-HT release, which appears to be triggered by liberation of calcium from intracellular stores, is suppressed by NO, whereas high potassium-evoked 5-HT release which is triggered by calcium influx through voltage regulated channels, is not affected.

Adrenoceptor- and cholinoceptor-mediated mechanisms in the regulation of 5-hydroxytryptamine release from isolated tracheae of newborn rabbits.

1. Isolated tracheae of newborn rabbits were incubated in vitro and the outflow of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. with electrochemical detection. Evidence has previously been provided that this 5-HT outflow derives from neuroendocrine epithelial (NEE) cells of the airway mucosa. 2. Phenylephrine (1, 10 and 30 microM) enhanced the outflow of 5-HT by 80, 290 and 205%, respectively. 5-HT outflow evoked by 10 microM phenylephrine was not affected by the presence of the neurotoxin tetrodotoxin (1 microM). 3. Rauwolscine, ARC 239 (an alpha(2B)-adrenoceptor preferring antagonist), yohimbine and prazosin antagonized the effect of 10 microM phenylephrine in a concentration-dependent manner with IC50 values of 150, 295, 300 and 1,700 nM, respectively. Comparison of the ratios (between all antagonists) of the present IC50 values with the corresponding ratios of Ki values obtained in binding studies for the alpha(2A)-, alpha(2B)-, alpha(2C)- and alpha(2D)-adrenoceptor subtypes strongly suggests the involvement of an alpha(2B)-receptor. 4. 5-HT outflow evoked by 10 microM phenylephrine was inhibited by 65% in the presence of 1 microM forskolin and abolished in the presence of 10 microM forskolin. 5. 5-HT outflow evoked by 10 microM phenylephrine was inhibited by about 45 and 70% in the presence of 0.1 and 1 microM isoprenaline, respectively. The inhibitory effect of 1 microM isoprenaline was only marginally antagonized by 1 microM, but blocked by 10 microM propranolol. 6. 5-HT outflow was not affected by the muscarine receptor agonist oxotremorine (10 microM), but was enhanced by 175% by 100 microM nicotine. The effect of nicotine was blocked by 100 microM hexamethonium and prevented by 1 microM tetrodotoxin or 1 microM yohimbine. 7. In conclusion, 5-HT release from NEE cells of the rabbit trachea is stimulated via alpha-adrenoceptors most likely of the alpha(2B)-subtype localized directly at the NEE cells. Activation of beta-adrenoceptors as well as direct activation of adenylyl cyclase by forskolin exert inhibitory effects on 5-HT release. Activation of nicotinic, but not of muscarinic receptors, also evokes the release of 5-HT. However, the effect of nicotine appears to be mediated indirectly via the release of noradrenaline.

Effects of bacterial lipopolysaccharides (LPS) and tumour necrosis factor-alpha (TNF alpha) on rat tracheal epithelial cells in culture: morphology, proliferation and induction of nitric oxide (NO) synthase.

Rat tracheal epithelial cells were cultured and the effects of LPS and TNF alpha on cell morphology, rate of proliferation and NO synthase activity were studied. NO synthase activity was determined by measuring the accumulation of 3H-L-citrulline during incubation of confluent monolayer with 3H-L-arginine. In untreated cells no significant 3H-L-citrulline formation was detected, and bradykinin and the calcium ionophore A 23187 failed to stimulate 3H-L-citrulline formation excluding a constitutively expressed, calcium-dependent NO synthase activity. After culturing the cells for 18 h in the presence of LPS (10 micrograms/ml) and TNF alpha (500 U/ml) a marked formation of 3H-L-citrulline could be detected, which was largely inhibited by N(G)-monomethyl-L-arginine (L-NMMA) indicating the induction of NO synthase activity which could be prevented by dexamethasone. Exposure of confluent monolayer to LPS and TNF alpha for up to 4 days resulted in a reduction in cell density by 20% within 1 to 2 days and in additional marked changes in cell morphology. The normal honeycomb-like structure of the culture was lost and a considerable number of cells developed "dendritic' outgrowths. These morphological changes as well as the reduction in cell density was attenuated by dexamethasone, but not by the NO synthase inhibitor L-NMMA. The rate of cell proliferation was determined in non-confluent cultures 24 h after passage by determination of the incorporation of tritium into DNA during 24 h of incubation with 3H-thymidine. 3H-thymidine incorporation was reduced by about 40-45% when LPS or TNF alpha was present during exposure to 3H-thymidine, and by about 65%, when LPS and TNF alpha were present in combination. Neither L-NMMA nor dexamethasone significantly affected the 3H-thymidine incorporation nor the inhibitory effects of LPS and TNF alpha. In conclusion, airway epithelial cells are markedly affected by LPS and TNF alpha and the various responses (changes in the cell morphology, inhibition of cell proliferation and induction of NO synthase activity) appear to be caused by different (dexamethasone-sensitive and -insensitive), cellular mechanisms. An enhanced formation of endogenous NO may not be responsible for the observed morphological changes or the inhibition of cell proliferation.

[ARDS caused by military zinc fumes exposure]. / ARDS durch militärische Zinknebelexposition.

A previously healthy 19-year-old male was exposed to concentrated hexite smoke (ZnCl2) for several minutes during military training. The initial symptoms (vomiting, cough, dyspnea) disappeared after a few hours. After 48 hours the patient developed acute respiratory distress syndrome requiring tracheal intubation and mechanical ventilation for 8 days. He left hospital 10 days after extubation. Spirometry at this time revealed a restrictive defect (vital capacity 50% predicted). Four months after the accident the patient had returned to work without symptoms of respiratory insufficiency. Lung function tests had normalized with the exception of a slight reduction in carbon monoxide diffusion. This case illustrates the typical course of inhalative intoxication with hexite. It underlines the importance of prolonged clinical surveillance in view of the latency between the initial symptoms of hexite intoxication and possible later development of ARDS.

Characterization of 5-hydroxytryptamine release from isolated rabbit and rat trachea: the role of neuroendocrine epithelia cells and mast cells.

Rabbit or rat isolated tracheae were incubated in vitro, and the release of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection. Release of 5-HT from rabbit tracheae could be evoked by the calcium ionophore A 23187 and, in a calcium-dependent manner, by depolarizing concentrations of potassium (45 mmol/l), but not by the mast cell degranulating drug compound 48/80. High potassium- and A 23187-evoked release of 5-HT was markedly higher from tracheae of newborn compared to adult rabbits. In rabbit tracheae, mechanical removal of the mucosa resulted in 80-90% reduction in tissue 5-HT and in a similar reduction in high potassium-evoked 5-HT release. 5-Hydroxytryptophan, but not tryptophan, caused a marked increase in the spontaneous outflow of 5-HT and 5-HIAA from tracheae of newborn rabbits, and the effect on 5-HT, but not that on 5-HIAA, required an intact mucosa. Furthermore, treatment with 5-hydroxytryptophan caused an increase in tissue 5-HT and 5-HIAA, and these effects required an intact mucosa. In tracheae of adult rabbits 5-hydroxytryptophan caused similar, although less profound, effects. Adrenaline (1 micromol/1) enhanced the release of 5-HT from newborn rabbit tracheae, and this effect was inhibited by 1 micro mol/l phentolamine or 1 micromol/1 prazosin, but not affected by 100 nmol/1 propranolol. In rat tracheae, compound 48/80 evoked a large release of 5-HT, whereas depolarizing concentrations of potassium (45 mmol/1) had only a very minor effect. In rat tracheae, 5-hydroxytryptophan had small effects on the outflow and tissue contents of 5-HT and 5-HIAA in comparison to the effects on rabbit tracheae; and removal of the mucosa resulted in only a minor reduction in tissue 5-HT. In conclusion, neuroendocrine epithelial (NEE) cells and mast cells are the major source of 5-HT in tracheae of the rabbit and rat, respectively. Isolated tracheae of newborn rabbits appear to be a useful model to study 5-HT secretion from NEE cells. 5-HT secretion from NEE cells is activated by a rise in intracellular calcium, and calcium influx through voltage-regulated channels appears to be one activating pathway. 5-HT secretion from NEE cells can be stimulated via alpha-adrenoceptors.

Effect of a platelet activating factor antagonist, WEB 2086, on allergen induced asthmatic responses.

BACKGROUND: Platelet activating factor (PAF) has been implicated in the pathogenesis of airway hyperresponsiveness in asthma. The purpose of this study was to evaluate the effects of a selective PAF antagonist (WEB 2086), given in doses known to antagonise the effects of inhaled PAF in human subjects, on allergen induced early and late asthmatic responses and on airway hyperresponsiveness. METHODS: Eight atopic, mildly asthmatic subjects were studied during a screening period and two treatment periods. During the screening period subjects inhaled an allergen to which they were known to be sensitised and the response was measured as the fall in the forced expired volume in one second (FEV1) to show the presence of early (0-1 h) and late (3-7 h) asthmatic responses. On another day the subjects inhaled allergen diluent. During the treatment periods subjects inhaled allergen after one week's pretreatment with WEB 2086 (100 mg three times a day) or placebo administered in a randomised, double blind, crossover fashion. Histamine airway responsiveness was measured 24 hours before and 24 hours after allergen and the results were expressed as the provocative concentration causing a 20% fall in FEV1 (PC20). RESULTS: The maximal early asthmatic response after allergen with placebo treatment was 18.4% (SE 4.4%) and with WEB 2086 18.9% (4.4%). The maximal late response with placebo treatment was 21.7% (5.3%) and with WEB 2086 21.2% (3.0%). The log difference (before and after allergen) in histamine PC20 was 0.35 (0.06) after placebo treatment and 0.30 (0.1) after WEB 2086. CONCLUSIONS: These results indicate that one week of treatment with an orally administered PAF antagonist (WEB 2086) does not attenuate allergen induced early or late responses or airway hyperresponsiveness.

Changes in the cellular profile of induced sputum after allergen-induced asthmatic responses.

Allergen inhalation causes airway inflammation and an increase in histamine airway responsiveness. We have used cell counts in sputum induced by hypertonic saline aerosol to assess airway inflammation before and 32 h after asthmatic responses to allergen. Twelve asthmatic subjects (mean age, 27.4 yr; range, 20-38 yr) had an inhalation test with D. farinae, ragweed pollen, or cat extract. All of them developed an early response with a fall in FEV1, of 24.8% (SD, 6.3%); nine of 12 had a definite late response (fall in FEV1 greater than or equal to 15%), and 10 of 12 had an increase in airway responsiveness to histamine at 32 h (PC20 reduced by greater than twofold). Sputum was induced by hypertonic saline after the histamine test, before and 32 h after the allergen challenge, at the same time of day. The quality of the sample was scored according to visual inspection and inverted microscopy and by salivary contamination. Plugs arising from the lower respiratory tract were selected for further evaluation. Differential cell counts of eosinophils (Eo) and metachromatic cells (MCC) (mast cell and basophils) were obtained from direct smears, blind to the clinical procedures. The mean fall in FEV1 after hypertonic saline was 6.4% (range, zero to 28%). The sputum samples were adequate in 79.5% of attempts. Eo and MCC increased significantly from 3.8 (4.4) to 18.2 (22.8)% (p = 0.01) and from 0.05 (0.17) to 0.25 (0.76)% (p = 0.04), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular distribution and differential gene expression of the three alpha subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.

We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.