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.

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.

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.

Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro.

Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes. We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands. Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures. Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase. Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences.

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

Association between virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection.

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.

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.

Detection of phencyclidine in urine using a polarization fluoroimmunoassay.

A polarization fluoroimmunoassay was developed for the detection of phencyclidine in urine and the reagents were adapted for use on the Abbott TDx analyser. The assay was used to look for evidence of phencyclidine abuse, over a 6 month period, amongst patients attending drug abuse clinics in the East End of London. Although over 2000 patients' samples tested negative, the assay successfully detected phencyclidine in an external quality control scheme.

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.

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 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.

Experience with cost-effective in-house reagents for the assay of carbamazepine in serum, using the Abbott TDx.

The development of inexpensive in-house reagents for the assay of carbamazepine in serum, using a commercially available derivative and antiserum is described. They were adapted for use on the Abbott TDx as direct replacements for commercial reagents. Comparison with a high-performance liquid chromatography method is discussed together with an evaluation of their performance in an external quality control scheme. They proved robust and reliable and reagent costs were reduced to around 3p per test.