Differential activation of airway eosinophils induces IL-13-mediated allergic Th2 pulmonary responses in mice.

BACKGROUND: Eosinophils are hallmark cells of allergic Th2 respiratory inflammation. However, the relative importance of eosinophil activation and the induction of effector functions such as the expression of IL-13 to allergic Th2 pulmonary disease remain to be defined. METHODS: Wild-type or cytokine-deficient (IL-13(-/-) or IL-4(-/-) ) eosinophils treated with cytokines (GM-CSF, IL-4, IL-33) were adoptively transferred into eosinophil-deficient recipient mice subjected to allergen provocation using established models of respiratory inflammation. Allergen-induced pulmonary changes were assessed. RESULTS: In contrast to the transfer of untreated blood eosinophils to the lungs of recipient eosinophil deficient mice, which induced no immune/inflammatory changes either in the lung or in the lung draining lymph nodes (LDLN), pretreatment of blood eosinophils with GM-CSF prior to transfer elicited trafficking of these eosinophils to LDLN. In turn, these LDLN eosinophils elicited the accumulation of dendritic cells and CD4(+) T cells to these same LDLNs without inducing pulmonary inflammation. However, exposure of eosinophils to GM-CSF, IL-4, and IL-33 prior to transfer induced not only immune events in the LDLN, but also allergen-mediated increases in airway Th2 cytokine/chemokine levels, the subsequent accumulation of CD4(+) T cells as well as alternatively activated (M2) macrophages, and the induction of pulmonary histopathologies. Significantly, this allergic respiratory inflammation was dependent on eosinophil-derived IL-13, whereas IL-4 expression by eosinophils had no significant role. CONCLUSION: The data demonstrate the differential activation of eosinophils as a function of cytokine exposure and suggest that eosinophil-specific IL-13 expression by activated cells is a necessary component of the subsequent allergic Th2 pulmonary pathologies.

Eosinophil activities modulate the immune/inflammatory character of allergic respiratory responses in mice.

BACKGROUND: The importance and specific role(s) of eosinophils in modulating the immune/inflammatory phenotype of allergic pulmonary disease remain to be defined. Established animal models assessing the role(s) of eosinophils as contributors and/or causative agents of disease have relied on congenitally deficient mice where the developmental consequences of eosinophil depletion are unknown. METHODS: We developed a novel conditional eosinophil-deficient strain of mice (iPHIL) through a gene knock-in strategy inserting the human diphtheria toxin (DT) receptor (DTR) into the endogenous eosinophil peroxidase genomic locus. RESULTS: Expression of DTR rendered resistant mouse eosinophil progenitors sensitive to DT without affecting any other cell types. The presence of eosinophils was shown to be unnecessary during the sensitization phase of either ovalbumin (OVA) or house dust mite (HDM) acute asthma models. However, eosinophil ablation during airway challenge led to a predominantly neutrophilic phenotype (>15% neutrophils) accompanied by allergen-induced histopathologies and airway hyper-responsiveness in response to methacholine indistinguishable from eosinophilic wild-type mice. Moreover, the iPHIL neutrophilic airway phenotype was shown to be a steroid-resistant allergic respiratory variant that was reversible upon the restoration of peripheral eosinophils. CONCLUSIONS: Eosinophil contributions to allergic immune/inflammatory responses appear to be limited to the airway challenge and not to the sensitization phase of allergen provocation models. The reversible steroid-resistant character of the iPHIL neutrophilic airway variant suggests underappreciated mechanisms by which eosinophils shape the character of allergic respiratory responses.

Unraveling nanotubes: field emission from an atomic wire.

Field emission of electrons from individually mounted carbon nanotubes has been found to be dramatically enhanced when the nanotube tips are opened by laser evaporation or oxidative etching. Emission currents of 0.1 to 1 microampere were readily obtained at room temperature with bias voltages of less than 80 volts. The emitting structures are concluded to be linear chains of carbon atoms, Cn, (n = 10 to 100), pulled out from the open edges of the graphene wall layers of the nanotube by the force of the electric field, in a process that resembles unraveling the sleeve of a sweater.

Growth and sintering of fullerene nanotubes.

Carbon nanotubes produced in arcs have been found to have the form of multiwalled fullerenes, at least over short lengths. Sintering of the tubes to each other is the predominant source of defects that limit the utility of these otherwise perfect fullerene structures. The use of a water-cooled copper cathode minimized such defects, permitting nanotubes longer than 40 micrometers to be attached to macroscopic electrodes and extracted from the bulk deposit. A detailed mechanism that features the high electric field at (and field-emission from) open nanotube tips exposed to the arc plasma, and consequent positive feedback effects from the neutral gas and plasma, is proposed for tube growth in such arcs.

Changes in the translational activity of polyadenylated messenger RNA of HL60 promyelocytic leukemia cells associated with myeloid or macrophage differentiation.

In order to characterize the events which commit the HL60 human promyelocytic leukemia cell line to differentiate into macrophages or mature myeloid cells, we have analyzed the in vitro [35S]methionine-labeled translational products obtained from polyadenylated messenger RNA of the HL60 cells before and after exposure to: (a) dimethylformamide (DMF), an inducer of myeloid differentiation; (b) 12-O-tetradecanylphorbol-13-acetate (TPA), an inducer of macrophage differentiation; or (c) a combination of the two inducers. Exposure of the HL60 cells to either TPA or DMF results in decreases in the relative abundancy of translational products with molecular weights of 20,000, 17,000, and 15,000. Exposure of the HL60 cells so as to generate macrophage differentiation results in elevations of translational products with molecular weights of 60,000, 47,000, 42,000, 32,000, 27,000, 14,000, and 12,300, while DMF-induced myeloid differentiation is associated with increases in the abundancy of translational products with molecular weights of 60,000, 42,000, 35,000, 32,000, 27,000, 13,000 and 12,300. The addition of the macrophage inducer TPA to HL60 cells previously exposed to the myeloid inducer DMF results in changes in the relative abundance of several translational products, yielding a pattern which differs quantitatively from that obtained from cells treated with DMF or TPA alone. These changes in the relative abundancies of the HL60 translational products suggest that the steady state levels of several different populations of mRNA or the ability of these mRNAs to be translated are being modified during the induction of myeloid or macrophage differentiation in the HL60 promyelocytic leukemia cell line.

Transcription of the non-repetitive genome in liver hypertrophy and the homology between nuclear RNA of normal and 12 H-regenerating liver.

The percentage of the non-repetitive genome transcribed and the complexity of nuclear RNA were estimated in normal and 12 h-regenerating rat liver. Nuclear RNA from normal or 12 h-regenerating liver hybridizes with approximately 6.1% of non-repetitive DNA (12.2% of the single-copy genome, assuming assymetric transcription). The estimated complexity of either of these nuclear RNA populations is 7.6 . 10(10) daltons, which is approximately 7 times higher than that calculated for polysomal mRNA. Cross hybridization experiments did not show differences between the nuclear RNA populations of normal and 12 h-regenerating liver. The results indicate that liver hypertropy (without hyperplasia) may be brought about without a large increase in the proportion of the non-repetitive genome transcribed.

The effect of fluorescein labels on the affinity of antisera to small haptens.

Small haptens such as methylamphetamine (MW 149) cannot, on their own, induce an immune response. It is also unlikely that they fill the binding site of any antibody that recognises them. Under such circumstances any attached label might be expected to enter the area of the binding site and exert an influence on overall binding. To investigate the possible influence of the label on binding, a range of fluorescein-labelled derivatives, differing in bridge length, were prepared. Antiserum binding of these labelled derivatives was then compared to that of the unlabelled drug. Evidence is presented which suggests that, with small haptens, the closeness of the fluorescein molecule can markedly influence antibody binding. Significant differences were found in titre, sensitivity, and assay kinetics. These overall effects appear to be brought about by the change in affinity of the antibody for the labelled hapten.

A randomized single-blind controlled study of cultured epidermal allografts in the treatment of split-thickness skin graft donor sites.

BACKGROUND AND DESIGN: In uncontrolled studies, cultured keratinocytes derived from donor tissue (allografts) appear to accelerate healing in a variety of acute and chronic skin wounds ranging from burns to leg ulcers. A randomized clinical trial was undertaken to compare the healing time of split-thickness skin graft donor sites in elderly patients using cultured epidermal allografts vs nonadherent dressings. Fresh-cultured epidermal grafts were used in 10 split-thickness skin graft donor sites in nine patients ranging in age from 63 to 87 years. In each patient, half the donor site was allografted and the other half treated with nonadherent dressings. To provide information about allograft survival, biopsy specimens were taken from allografted areas in three patients 2 months after the grafting procedure, for multilocus DNA analysis. RESULTS: The mean time to complete healing was 8.4 days in allografted sites compared with 15.3 days in control sites. There was no evidence of survival of cultured allogeneic cells in allografted areas. CONCLUSION: Cultured allografts can accelerate healing in split-thickness skin graft donor sites in elderly patients compared with nonadherent dressings. Cultured allografts do not survive permanently on the wound bed.

Acute yellow oleander (Thevetia peruviana) poisoning: cardiac arrhythmias, electrolyte disturbances, and serum cardiac glycoside concentrations on presentation to hospital.

OBJECTIVE: To describe the cardiac arrhythmias, electrolyte disturbances, and serum cardiac glycoside levels seen in patients presenting to hospital with acute yellow oleander (Thevetia peruviana) poisoning and to compare these with published reports of digitalis poisoning. DESIGN: Case series. SETTING: Medical wards of Anuradhapura District General Hospital, Sri Lanka, and coronary care unit of the Institute of Cardiology, National Hospital of Sri Lanka, Colombo, the national tertiary referral centre for cardiology. PATIENTS: 351 patients with a history of oleander ingestion. MEASUREMENTS: ECG and blood sample analysis on admission. RESULTS: Most symptomatic patients had conduction defects affecting the sinus node, the atrioventricular (AV) node, or both. Patients showing cardiac arrhythmias that required transfer for specialised management had significantly higher mean serum cardiac glycoside and potassium but not magnesium concentrations. Although there was considerable overlap between groups, those with conduction defects affecting both sinus and AV nodes had significantly higher mean serum cardiac glycoside levels. CONCLUSIONS: Most of these young previously healthy patients had conduction defects affecting the sinus or AV nodes. Relatively few had the atrial or ventricular tachyarrhythmias or ventricular ectopic beats that are typical of digoxin poisoning. Serious yellow oleander induced arrhythmias were associated with higher serum cardiac glycoside concentrations and hyperkalaemia but not with disturbances of magnesium.

Development of fluoroimmunoassays for the specific detection of morphine in urine.

Two fluoroimmunoassays for the specific detection of morphine in urine are described based on the use of ovine antibodies and fluorescein-labelled normorphine. The first, a polarisation fluoroimmunoassay, is performed by adding 10 microliter of urine to 1.5 ml of a single-reagent, comprising premixed antiserum and tracer, incubation for a few minutes at ambient temperature and measurement of fluorescence polarisation. The assay gives results which compare well with those by thin-layer chromatography, EMIT d.a.u., and the Boehringer opiate drug test. Although adequate for routine screening for drug abuse, the technique is not as sensitive as some radioimmunoassays. Therefore, a second fluoroimmunoassay was developed based on the use of the same antibodies covalently coupled to magnetisable particles to facilitate both the separation of the bound and free fractions and the removal of non-specific interfering substances. Thus, larger sample volumes could be employed and greater sensitivity achieved.

A broad spectrum immunoassay using fluorescence polarization for the detection of amphetamines in urine.

Rapid immunoassays are widely used to screen for amphetamine abuse. Broad spectrum immunoassays are the most useful for this purpose followed by physicochemical techniques for verification and identification of particular drugs. We describe the production of an antiserum with a broad specificity for the amphetamine group of drugs. The antiserum was produced in a sheep using an immunogen linking amphetamine to keyhole limpet haemocyanin via an N-aminobutyl bridge. This antiserum was used to develop a fluorescence polarization immunoassay for application to urine samples. A limited investigation into the use of saliva as an alternative sample was also performed. The effects of chemical modifications to the basic amphetamine structure on antibody binding are discussed.

Single-reagent polarisation fluoroimmunoassay for the cocaine metabolite, benzoylecgonine, in urine.

A polarisation fluoroimmunoassay is described for the detection in urine of benzoylecgonine, the major metabolite of cocaine. The method uses a single reagent comprising sheep anti-benzoylecgonine serum pre-mixed with a fluorescein-labelled benzoylecgonine derivative as tracer. Under the assay conditions the antiserum has equal cross-reactivity with cocaine and the metabolite. The test is performed by the addition of 10 microL of urine to an aliquot of the single reagent, incubation for a few minutes and measurement of fluorescence polarisation. The assay has a positive/negative cut-off level of 1 mg/L benzoylecgonine and results compare favourably with the EMIT-d.a.u.TM (Syva) system.

Reducing the cost of phenytoin assays with in-house TDx reagents.

The Abbott TDx is frequently used for therapeutic drug monitoring of phenytoin concentrations, but reagent costs are high. In an attempt to reduce these costs, we investigated the preparation of in-house reagents. A commercial antiserum was available at reasonable cost and the fluorescent tracer was easily prepared. We describe the preparation of these reagents and their application to the TDx system. Substantially reduced costs were obtained using the in-house reagents.

A reliable non-separation fluorescence quenching assay for total glycated serum protein: a simple alternative to nitroblue tetrazolium reduction.

A simple non-separation assay for the measurement of total glycated serum protein is described. It was found that the fluorescence intensity of a solution of a fluorescein-boronic acid derivative was quenched in proportion to the amount of serum added. This led to the development of an assay in which 10 microL of serum is added to 4 mL of a solution of the fluorescein-boronic acid derivative and the fluorescence intensity is measured after 15 min. The results, as measured by drop in fluorescence intensity, calibrated by a single standard, were compared with the results for nitroblue tetrazolium (NBT) reduction of fructosamine and showed good correlation (r=0.936, n=114). The intra-assay precision (seven samples each measured 10 times) was less than 2.1% (concentration range 190-660 micromol/L); inter-assay precision for seven samples in 10 assays was less than 2.5% (over the same concentration range). Dilution of serum that had a high concentration of total glycated protein showed the assay to be linear. Serum samples (with low, medium and high total glycated protein concentrations) showed less than 2.1% difference from base results with added glucose (up to 60 mmol/L), less than 9.7% difference with added bilirubin (up to 250 micromol/L) and less than 6.9% with added triglycerides (up to 50 mmol/L). Addition of haemoglobin (up to 0.9 g/dL) with high glycation (11.7% HbA1c) to plasma (298 micromol/L total glycated protein) showed less than 10% difference from the base result. Assays performed over a range of temperatures (12-34 degrees C) showed no significant differences in the results. The assay gives similar results to the currently used NTB method but with significantly less susceptibility to interferences. As such the method should be a useful aid in the management of diabetes.

Drug abuse screening with immunoassays: unexpected cross-reactivities and other pitfalls.

Immunoassays are the only practical means of coping with the increasing demand for drug abuse screening because of their simplicity and ease of automation. However, it is essential that the analyst understands the strengths and weaknesses of such assays to enable correct interpretation of results. This is especially important with the increasing popularity of near-patient testing where assays are liable to be carried out by inexperienced and untrained staff. False-positive and false-negative results, unexpected cross-reactivities and other pitfalls are discussed, together with an attempt to explain the reasons for these problems.

Mortality after hip fracture and assessment of some contributory factors.

Of 881 patients with hip fracture operated on in Galway between 1968 and 1973 only 77 per cent were alive after six months from the time of injury, 72 per cent survived 1 year and 63 per cent survived two years. A control sample, sex and age matched, showed survival rates of 96 per cent, 93 per cent and 84 per cent for corresponding periods. After six months the hip fracture appears to have little bearing on survival.Factors having an adverse effect on the outcome were the following in their order of importance: atheroma, bedsores on admission, male sex, concomitant illness and sustaining the injury in the first quarter of the year. A better than average outcome was associated with the absence of these factors or their logical converse. Factors which appear to play an insignificant role in relation to mortality are the type of operation, the length of operation or the accident circumstances except road traffic accidents, which were often in young healthy patients and so gave a better than expected survival. In assessing the influence of these factors on mortality each group was again compared with matched samples from the general population.

A nonclinical model for predicting scaling and root planing case difficulty.

As students progress through their clinical education, the integration of patient types challenging the development of scaling and root planing skills is necessary. A clinical screening to determine case type is ideal; however, some programs do not have the time or resources for a clinical screening, and some patients do not want to take the time for the additional screening appointment. The intent of the screening process is to determine case type and/or treatment needs. In some cases, this information is used to match student educational requirements with patient needs. Even though an institution or program may have a clinical screening process, patient assignment can still be haphazard, resulting in an inequitable distribution of patient case types to students. It seems the problem is due, in part, to the absence of a mechanism for early identification and distribution of case types. The purpose of this study was to develop a model for initially predicting patient scaling and root planing case difficulty from nonclinical, patient variables obtained during a telephone interview. Dental hygiene student evaluation forms (n = 1,356) at the University of North Carolina at Chapel Hill School of Dentistry were gathered and categorized by patient case difficulty. A sample of charts was selected from each scaling classification. The scaling classifications were then subdivided into two samples for developing and testing the model. Variables selected from the patient charts as possible predictors were smoking status, race, age, gender, date of last prophylaxis, periodontal classification, and oral hygiene habits (brushing and flossing).(ABSTRACT TRUNCATED AT 250 WORDS)