Determination of Ole e 1 by enzyme immunoassay and scanning densitometry. Validation by skin-prick testing

Ole e 1 is an important allergen in Olea europaea pollen extracts. This study describes the development of two new methods that can be used to estimate the Ole e 1 content in olive tree pollen extracts. They are based on (1) an enzyme immunoassay that uses rabbit polyclonal, monospecific antibodies and purified Ole e 1, and (2) scanning densitometry of SDS-PAGE gels. Twelve extracts were evaluated by in vivo and in vitro methods. The in vivo biological potency was estimated by prick skin testing 17 allergic individuals; the in vitro allergenic potency by direct IgE and IgE inhibition assays. The enzyme immunoassay showed an operative range of 0.03-100 microg/ml and demonstrated to be specific for Ole e 1. The Ole e 1 content ranged from 1% to 5% of the total protein in the 12 extracts. The amount of Ole e 1, assessed by gel scanning densitometry significantly correlated with the Ole e 1 content obtained by the immunoassay (r = 0.92; p < 0.001). The Ole e 1 content showed a significant correlation with the total allergenic potency of the extracts, evaluated by direct IgE, specific IgE inhibition and skin-prick testing. These two methods can be used to determine the Ole e 1 content in olive pollen extracts. The content of Ole e 1 can vary from 1% to 5% of the total protein in the extracts.     

Expression of proinflammatory cytokines by hepatic macrophages in acute classical swine fever

Fourteen pigs were inoculated with the 'Alfort 187' strain of classical swine fever (CSF) virus and killed in pairs at 2, 4, 7, 9, 11, 14 or 17 days post-inoculation for histopathological, ultrastructural and immunohistochemical examination. For the latter method, the antibodies used were those against viral antigen Gp55, porcine myeloid marker SWC3, IL-1alpha, IL-6, TNF-alpha and Factor VIII-related antigen. Activation and increase in the number of hepatic macrophages was observed following viral detection in liver, as well as an increase in IL-1alpha and IL-6 production, mainly by Kupffer cells. Maximum detection of viral antigen was observed in the middle stage of the experiment coinciding with overexpression of the three cytokines studied, with IL-6 production by interstitial macrophages prominent at the end. Additionally, the labelling of platelets for Factor VIII-related antigen and the ultrastructural study of the sinusoids revealed activation and aggregation of thrombocytes close to Kupffer cells at the beginning of the infection. The liver seems to play a prominent role in the origin of the thrombocytopenia that occurs in CSF and contributes to the overexpression of proinflammatory cytokines considered responsible for the disorders observed during the course of the disease.     

5-Azacytidine produces differential undercondensation of alpha,beta and classical human satellite DNAs

Fluorescencein situ hybridization employing human alphoid, beta and classical satellite DNA probes was performed on 5-azacytidine treated and untreated chromosomes obtained from human lymphocytes. The individual used in this study presented a polymorphism of constitutive heterochromatin of chromosomes 1 and 9 as revealed byin situ digestion with the restriction endonucleaseAlul. Neither the alphoid nor the beta satellite DNA domains were susceptible to condensation-inhibition by 5-azacytidine. Only the classical satellite localized on chromosome 9 was affected. The constitutive heterochromatin size polymorphism was shown to depend mainly on variations of the classical satellite DNA domain. Therefore, condensation-inhibition, as a phenomenon which may modify the natural folding of the chromatin fibre, regionally affects human constitutive heterochromatin and seems to be dependent on the heterochromatic family.     

Prediction of postoperative morbidity after lung resection using an artificial neural network ensemble

OBJECTIVE: To propose an ensemble model of artificial neural networks (ANNs) to predict cardio-respiratory morbidity after pulmonary resection for non-small cell lung cancer (NSCLC). METHODS: Prospective clinical study was based on 489 NSCLC operated cases. An artificial neural network ensemble was developed using a training set of 348 patients undergoing lung resection between 1994 and 1999. Predictive variables used were: sex of the patient, age, body mass index, ischemic heart disease, cardiac arrhythmia, diabetes mellitus, induction chemotherapy, extent of resection, chest wall resection, perioperative blood transfusion, tumour staging, forced expiratory volume in 1s percent (FEV(1)%), and predicted postoperative FEV(1)% (ppoFEV(1)%). The analysed outcome was the occurrence of postoperative cardio-respiratory complications prospectively recorded and codified. The artificial neural network ensemble consists of 100 backpropagation networks combined via a simple averaging method. The probabilities of complication calculated by ensemble model were obtained to the actual occurrence of complications in 141 cases operated on between January 2000 and December 2001 and a receiver operating characteristic (ROC) curve for this method was constructed. RESULTS: The prevalence of cardio-respiratory morbidity was 0.25 in the training and 0.30 in the validation series. The accuracy for morbidity prediction (area under the ROC curve) was 0.98 by the ensemble model. CONCLUSION: In this series an artificial neural network ensemble offered a high performance to predict postoperative cardio-respiratory morbidity.     

Time adaptive denoising of single trial event-related potentials in the wavelet domain

We present a new wavelet-based method for single trial analysis of transient and time variant event-related potentials (ERPs). Expecting more accurate filter settings than achieved by other techniques (low-pass filter, a posteriori Wiener filter, time invariant wavelet filter), ERPs were initially balanced in time. By simulation, better filter performance could be established for test signals contaminated with either white noise or isospectral noise. To provide an example of real application, the method was applied to limbic P300 potentials (MTL-P300). As a result, variance of single trial MTL-P300s decreased, without restricting the corresponding mean. The proposed method can be regarded as an alternative for single-trial ERP analysis.     

Investigation for a more virulent variant among the c:2b:P1.2,5 Spanish meningococcal epidemic strains by molecular epidemiology

A rise in the incidence of meningococcal disease has occurred in Spain in recent years, especially in some regions in the north-west of the country. Most cases have been caused by meningococci characterised as Neisseria meningitidis C:2b:P1.2,5. A total of 107 C:2b:P1.2,5 meningococcal isolates (60 from patients and 47 from carriers) and 12 isolates showing related antigenic combinations (C:2b:NST, C:2b:P1.2, C:2b:P1.5, C:NT:P1.2,5) was analysed by pulsed-field gel electrophoresis to determine the genetic variability of the epidemic and related strains. Endonucleases BglII and NheI were used to cut chromosomal DNA. When BglII was used, most of the C:2b:P1.2,5 isolates showed the same pulsotype regardless of whether they were from clinical cases or carriers. Isolates showing the principal profile after digestion with endonuclease BglII were analysed with NheI. Four pulsotypes were identified, of which two were found in only one isolate each. The major profiles (1 and 2) showed differential distribution among clinical and carrier isolates; pulsotype 1 was the most frequent among clinical isolates. However, the proportions of isolates showing profiles 1 and 2 were similar among carrier isolates. This could indicate that there are two variants of the C:2b:P1.2,5 strain with differing pathogenicity.     

Diagnosis ofMycoplasma pneumoniae infection by microparticle aggluination and antibody-capture enzyme-immunoassay

The performance of two new commercial assays for the serological diagnosis ofMycoplasma pneumoniae infection (microparticle agglutination and antibody-capture enzyme-immunoassay) was studied using a panel of 169 serum samples from patients withMycoplasma pneumoniae pneumonia and a control group. Both assays were shown to be sensitive and specific for diagnosis. The performance of the capture immunoassay, however, decreased in older patients, probably due to its inability to detect cases of reinfection without IgM antibody response.     

Improved approach to identify cancer-associated autoantigens

The failure to identify biomarkers of clinical significance for cancer diagnosis and prognosis generated a great deal of skepticism in regard to the usefulness of autoantibody-based methods. SEREX was a major advancement in immunoscreening that resulted in the identification of a large group of autoantigens recognized by cancer sera. However, few SEREX-defined autoantigens have proven to have definitive diagnostic value in clinical practice. Often, the identified antigens are patient-specific rather than tumor-specific and many tumor-associated antigens are rare in expression libraries made from non-autologous cells. Since autoantibodies are part of the normal immune response, it can be difficult to single out tumor-associated antibodies from the scores of irrelevant patient-specific responses. In our view, any practical approach for identifying cancer-related autoantigens must include an integral strategy for demonstrating tumor relevance early in the screening process. Care must also be taken not to exclude potentially important autoantibodies by pre-screening manipulations to patient sera. We have introduced substantial modifications in SEREX, designed to minimize confounding effects of unrelated autoantibodies and to eliminate steps that preclude the identification of cancer-related autoantigens commonly recognized by cancer sera. In addition, we incorporate methodology to identify candidate antigens that have potential diagnostic or prognostic value prior to their molecular cloning and characterization.