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A comprehensive study of p53 transcriptional activity in thymus and spleen of gamma irradiated mouse: high sensitivity of genes involved in the two main apoptotic pathways.

PURPOSE: Gamma-irradiation leads to activation of p53 tumour suppressor gene and to p53-dependant stimulation of a large panel of cellular genes including proapoptotic genes involved in intrinsic and extrinsic pathways. Most in vivo published data referred to high (lethal) irradiation doses. The present study was performed to analyse the p53-dependent response to more relevant low irradiation doses. MATERIALS AND METHODS: Mice were whole body exposed to irradiation doses decreasing from 5 - 0.05 Gy. Gene expression was estimated by real time reverse transcriptase polymerase chain reaction measurements on RNA extracted from thymus and spleen. Apoptosis was evaluated by the percentage of either annexin V positive or sub-G1 cells. RESULTS: A 0.1 Gy irradiation dose already gives a significant stimulation of Puma (p53 up-regulated modulator of apoptosis), and 0.2 Gy of Bax (Bcl-2-associated X protein) and Killer/DR5 (Death Receptor 5). The expression of genes involved in the two apoptotic pathways was induced as soon as 1 h post-irradiation and reached a maximum at 3 h, the induction level depending on both the gene and the organ. A significant increase in the number of apoptotic cells is already detectable at 0.5 Gy with a maximum of induction at 6 h. CONCLUSIONS: Our results reveal the high in vivo sensitivity of p53-dependent transcriptional activation of genes involved in the two main apoptotic pathways, their stimulation preceding the induction of apoptosis.

Animals↗

Measurement of human immunodeficiency virus type 1 plasma virus load based on reverse transcriptase (RT) activity: evidence of variabilities in levels of virion-associated RT.

Virus load based on levels of functional reverse transcriptase (RT) was measured in plasma from 50 human immunodeficiency virus (HIV) type 1-infected persons, in 87 samples from 10 HIV-1 seroconversion panels, and in 100 uninfected persons by use of Amp-RT, an ultrasensitive RT assay. Of the 50 clinical samples, 38 (76%) were Amp-RT positive, while all uninfected controls were negative. Pearson's correlation coefficient of RNA and RT levels was .73 for all samples, .86 for seroconversion samples, and .49 for clinical samples. Calculated ratios of RT activity to virion RNA varied widely during both early and late stages of infection. Mean RT:RNA ratios in 8 seroconversion panels and in 12 (34.3%) of 35 individual clinical samples were significantly lower than the ratio for a reference virus. However, ratios were stable in individual seroconversions over time. These data demonstrate that RT activity can be used to quantitate plasma virus load and provide evidence of different levels of virion-associated RT among HIV-1-infected persons.

Acquired Immunodeficiency Syndrome↗

The European panel on the appropriateness of gastrointestinal endoscopy guidelines colonoscopy in an open-access endoscopy unit: a prospective study.

BACKGROUND: The demand for gastrointestinal endoscopy is increasing in most developed countries, resulting in an important rise in overall costs and waiting lists for endoscopic procedures. Therefore, adherence to appropriate indications for these procedures is essential for the rational use of finite resources in an open-access system. AIM: To assess indications and appropriateness of colonoscopy according to the European Panel on the Appropriateness of Gastrointestinal Endoscopy (EPAGE) criteria. METHODS: From May to June 2004, all consecutive patients referred to our Unit for open-access colonoscopy were considered for inclusion in this prospective study. Appropriateness of each colonoscopy was established according to the EPAGE criteria. In order to evaluate whether appropriateness of use correlated with the diagnostic yield of colonoscopy, relevant endoscopic findings were also recorded. RESULTS: A total of 350 consecutive patients were included in the study. In 38 of them, the colonoscopy indication was not listed in the EPAGE guidelines and, consequently, they were not evaluated. In the remaining 312 patients, the indication for the procedure was considered inappropriate in 73 (23%) patients. Both referring doctor characteristics (specialty and health care setting) and patient data (age) correlated with appropriateness of endoscopy. The diagnostic yield was significantly higher for appropriate colonoscopies (42%) than in those judged inappropriate (21%) (P = 0.001). CONCLUSIONS: A noteworthy proportion of patients referred for colonoscopy to an open-access endoscopy unit are considered inappropriate because of their indication, with significant differences among specialties. These results suggest that implementation of validated guidelines for its appropriate use could improve this situation and, considering the correlation between appropriateness and diagnostic yield, even contribute to improve the prognosis of patients with colorectal diseases.

Adult↗

Seroreactivity to and avidity for recombinant antigens in toxoplasmosis.

To improve serodiagnostic methods for the diagnosis of acute toxoplasmosis during pregnancy, a new test system has been developed and evaluated based on the use of recombinant antigens. Five recombinant Toxoplasma gondii antigens (ROP1, MAG1, SAG1, GRA7, and GRA8) were cloned in Escherichia coli, purified, and applied directly onto nitrocellulose membranes in a line assay (recomLine Toxoplasma). A panel of 102 sera from 25 pregnant women with supposed recent toxoplasmosis and from two symptomatic children was compared to a panel of 71 sera from individuals with past infection. Both panels were analyzed using a recombinant line assay for immunoglobulin G (IgG), IgM, and IgA antibodies and a reference enzyme-linked immunosorbent assay. Within the IgM-positive samples, antibodies against ROP1 were predominant regardless of the infection state. In IgG analysis a characteristic antibody pattern was found for very recent infections. This pattern changed to a different one during the time course of infection: antibodies against GRA7 and GRA8 were characteristic for very early IgG, whereas antibodies against SAG1 and MAG1 appeared significantly later. These results were further confirmed by determination of the IgG antibody avidity for every single recombinant antigen. In the time course of infection, IgG antibodies against the early recognized antigens matured significantly earlier than those directed against the later antigens did. The IgA patterns did not give reliable information about the infection time points. The data revealed that the recombinant line assay provides valuable information on the actual state of infection, especially during the early infection time points.

Animals↗

Appropriate use of upper gastrointestinal endoscopy--a prospective audit. Steering Group of the Upper Gastrointestinal Endoscopy Audit Committee.

Work by this group has shown that there is a wide range of opinion as to patients' suitability for endoscopy. In a recent study, 1297 questionnaires were sent to a random selection of doctors, including 350 general physicians, 400 surgeons, 477 gastroenterologists, and 70 general practitioners. The respondent was asked to indicate whether or not he would refer the patient described by each case vignette for endoscopy. Depending on the indication, the positive referral rate varied from 4.5% to 99% overall, and from 4.5% to 63.8% for all those clinical situations that the working party felt to be inappropriate. A second study examined the appropriateness of 400 consecutive cases referred from four units within one health region; these cases were judged independently, and without conferring, by a panel of seven gastroenterologists. The same cases were rated by software that incorporated American opinion (the Rand criteria). Although only 45 (11%) of the cases were classed as inappropriate by the British panel, 120 cases (31%) assessed by the American software were rated inappropriate. These differences occurred largely because in the USA it is recommended that one month's antiulcer treatment be tried before considering endoscopy for dyspepsia and thus many referrals were seen as inappropriate by the American database. Of the 45 cases found to be inappropriate by the British doctors no important abnormality was found at endoscopy; whereas of 120 cases judged inappropriate by the Rand criteria, three duodenal and two gastric ulcers, and one gastric cancer were diagnosed at gastroscopy. This study attempts a quantitative assessment of inappropriate use and serves to encourage further work to define appropriateness.

Adult↗

Quality control assessment of influenza and RSV testing in Europe: 2000-01 season.

The Quality Control Assessment (QCA) was initiated to evaluate the quality of the influenza and respiratory syncytial virus (RSV) testing in the national reference centres belonging to the European Influenza Surveillance Scheme (EISS) network. Samples were coded and sent in two panels of 12 samples within a two week interval to 16 laboratories during the 2000-01 winter season. The antibodies titration by HI test was reported by 60% of the laboratories (n=16), and the results were correct for 56% of them. One false detection of influenza B antibodies was reported by one laboratory, and for the others the sensitivity varied widely. The sensitivity of the tests for the detection of influenza virus varied for A(H3N2) from 10 to 100,000 TCID50/ml. The influenza A subtyping was performed by 87% of the laboratories, and 31% gave correct results. The characterisation of the variants was undertaken by six laboratories and half of them fully achieved it. Fifty six percent of the laboratories used RT-PCR for the diagnosis; the results were specific and the sensitivity equivalent to the cell culture.

Antigens, Viral↗

Parallel solution synthesis of a "Carbohybrid" library designed to inhibit galactose-binding proteins.

Parallel solution S-alkylations of a 1-thio-beta-D-galactopyranoside derivative with Michael acceptors and alpha-chloroketones, followed by ketone reductions, reductive aminations, and acylations were developed to yield a library of 1-thio-beta-D-galactopyranosides carrying small and diverse polar-neutral, hydrophobic, aromatic, cationic, or anionic non-carbohydrate aglycon structures. Screening of the library against a panel of galactose recognizing plant lectins revealed microM inhibitors of toxin A of A. precatorius superior to the reference ligands lactose and N-acetyl lactosamine. Such small, monosaccharide based inhibitors are attractive lead-molecules for therapeutic development, since they are low-molecular, hydrolytically stable and more hydrophobic than natural oligosaccharides.

Calcium-Binding Proteins↗

Pluripotent and lineage-committed CD34+ subsets in leukapheresis products mobilized by G-CSF, GM-CSF vs. a combination of both.

To define an optimal regimen for mobilizing blood-derived progenitor cells from healthy donors for allogeneic transplantation, we have studied the early and lineage-committed CD34+ subsets in the leukapheresis products after mobilization with G-CSF (10 micrograms/kg/d), GM-CSF (10 micrograms/kg/d), and the combination of G-CSF and GM-CSF (G/GM, 5 micrograms/kg/d of each). We used three color and five dimensional flow cytometry with a panel of monoclonal antibodies against CD3, CD7, CD10, CD11b, CD15, CD33, CD34, CD38, CD45, CD61, and CD71. As reference, we also analyzed CD34+ subsets in samples from umbilical cord blood (UCB) and from adult bone marrow (BM). The level of total CD34+ cells was 0.04 +/- 0.03% (mean +/- SD) in peripheral blood at baseline, and reached a maximum on day 5 or day 6 of administration of growth factors. The percentages of CD34+ cells in the leukapheresis products were 1.06 +/- 0.37% (mean +/- SD) with G-CSF mobilization, 0.35 +/- 0.24% with GM-CSF, and 0.92 +/- 0.61% with the combination of both. Among the CD34+ subsets, the percentage of cells that were CD34+/CD38- was highest in UCB (7.18 +/- 5.58%) and lowest in G-CSF mobilized peripheral blood (0.80 +/- 0.22%), whereas GM-CSF or G/GM mobilized products gave rise to intermediate levels (4.43 +/- 3.40%, 3.61 +/- 2.42%, respectively). The differences between G/GM and G-CSF, between UCB and G-CSF, or between UCB and BM are significant. The absolute numbers of CD34+/CD38- and CD34+/CD38-/HLA-DR+ subsets are also significantly higher in the G/GM mobilized products than in G-CSF products. The cloning efficiency of G/GM mobilized CD34+ cells was 2 times higher than that of G-CSF mobilized CD34+ cells, albeit the difference was statistically marginal. The profile of CD34+ subsets mobilized by the combination of G/GM approaches that found in UCB. Our data illustrate that different growth factors and regimens can preferentially mobilize different CD34+ subsets from normal donors, and that the combination of G-CSF and GM-CSF might be an optimal regimen.

Antigens, CD34↗

Identification of the in vivo topically applied "human-identical" ceramides included in negatively charged liposomes.

Among the different classes of human epidermal lipids, Ceramides are definitely one of the most important and studied class judging from the significant amount of scientific information accumulated over the years, specially after being established its crucial role in skin physiology and pathophysiology. However, a clear demonstration of its usefulness resulting from the regular use of these substances included in topical formulations, either in a therapeutical or in a cosmetological perspective have not been entirely achieved. Many difficulties are normally present, from its origin to formulation and protocol design, and to the experimental demonstration of the relative efficacy of these compounds. In the present paper, the authors developed a systematic approach to the problem, using "human-identical" ceramides included into negatively charged liposomes, which were topically applied in a normalised panel of human healthy volunteers. Biological analysis were conducted with non-invasive methodologies by means of bioengineered reference methods (Epidermal capacitance, Evaporimetry; Light Transmission Profilometry; and High Resolution Ultrasonography). The experimental methodologies included the study of acute effects as well as the study of the effects resulting from continuous repeated application of formulations, basically expressed in terms of epidermal water content and skin surface profile. Additionally, an eventual protective "barrier" reinforcement resulting from ceramide application was also assessed by means of a SLS (5% solution) challenge-test. This systematic although preliminary approach indicated that these compounds do have a considerable impact on human skin physiology, justifying further research to clarify their potential use in human cutaneous health.

Adult↗

DNA typing of human platelet antigen systems 1, 2, 3 and 5 in B-lymphoblastoid cell lines of the International Histocompatibility Workshop.

Alloimmunization against human platelet antigens (HPA) can cause thrombocytopenia in different clinical settings, including neonatal alloimmune thrombocytopenia, post transfusion purpura, and refractoriness to platelet transfusion. Recently, DNA based methods have been described for analysis of the polymorphism of the human platelet antigens. However, until now, no reference material for standardization purposes is available. We thus determined the DNA polymorphism of the human platelet antigen (HPA) systems 1, 2, 3 and 5 in B-lymphoblastoid cell lines collected for the International Histocompatibility Workshops. DNA typing of the HPA systems was done by PCR amplification with sequence-specific primers (PCR-SSP). A new enzyme (AmpliTaq Gold) was introduced to enhance the specificity of the PCR. We present a panel of five B-lymphoblastoid cell lines, representing all heterozygous and homozygous genotypes of the tested HPA systems. This work thus provides the reference material required for DNA based diagnosis of human platelet antigens.

Antigens, Human Platelet↗

Analytical bias for cholesterol and the percent of the population deemed at risk for coronary heart disease.

The effect of methodological bias on the population at risk is dependent on the location of the reference value in the distribution of the population. We fitted the cumulative distribution for cholesterol to a rational function and calculated the apparent reference values for four biased methods: Technicon SMAC (2.6%), DuPont aca (4.0 to 4.8%), Kodak DT-60 (-2.0 to -5.5%), and BMD Reflotron (-7.4 to -7.8%). With the true and apparent reference values for cholesterol and the rational function, we determined the percentage increase or decrease in the population deemed at risk for coronary heart disease. The population at risk increased by as much as 48% for methods with positive bias, and decreased by as much as 54% for methods with negative bias. If we restrict the percentage of the population incorrectly diagnosed to 3% and use reference values (cut points) recommended by the National Cholesterol Education Program, the maximum allowable methodological bias would be 1.6% for positive bias and -1.55% for negative bias. Therefore, an absolute methodological bias of 3% (as recommended by the Laboratory Standardization Panel) may be too liberal.

Adult↗

Reference values of total serum IgE and their significance in the diagnosis of allergy in young European adults.

Allergic sensitization mediated by immunoglobulin E (IgE) is the basis of allergic diseases, and elevated total IgE, in spite of some well-known limitations, is frequently included as a diagnostic criterion for allergic diseases. The reference value of total IgE (IgE-t) in the literature (1.5-144 kU/l) was established almost 2 decades ago. The aim of this study was to establish IgE-t reference values, establishing an updated cutoff value able to identify atopic subjects, defined as a positive CAP-radioallergosorbent test to at least one of a panel of common allergens, among young European adults. The study included 6,670 subjects from 10 Western European countries within the framework of the European Community Respiratory Health Survey II. IgE-t and specific IgE (IgE-s) were measured for the main inhalant allergens; IgE-s in class 0 for all allergens (66.2%) characterized non-atopy. The reference values were estimated by means of linear regression using a 50% random subsample of non-atopic subjects. Two non-atopic subsamples were examined so that one subsample could be used to establish reference IgE-t values, and these values were compared to those in the second non-atopic subsample to validate the findings. Sensitivity and specificity for atopy were assessed on the other 50% of non-atopic and on all atopic subjects. The 95th percentile of IgE-t reference values in non-smokers was 148 kU/l in women and 169 kU/l in men, while it was 194 and 220 kU/l in female and male smokers, respectively: serum IgE-t above the 95th percentile identifies <32% and above the 99th percentile <20% of atopic adults (low sensitivity), but a serum IgE-t below the 95th percentile identifies >90% and below the 99th percentile identifies >95% of non-atopic adults (good specificity). Due to the adequate specificity, IgE-t values exceeding the normal limits confirm a suspected atopic status; however, because of the low sensitivity, values below the cutoff seem not to exclude an atopic status with sufficient accuracy.

Adult↗

Methodological approach: American College of Chest Physicians guidelines for the prevention and management of postoperative atrial fibrillation after cardiac surgery.

Atrial fibrillation remains a common and challenging problem following cardiac surgery. The American College of Chest Physicians, through the Health and Science Policy Committee, established a panel to develop a set of clinical practice guidelines for the management or prophylaxis of atrial fibrillation or flutter in patients undergoing coronary artery bypass surgery. The panel based its guidelines on a systematic review of the literature that included a computerized search of PubMed and CENTRAL, the Cochrane Collaboration database, as well as a search of selected journals and references in key articles. Studies were eligible for review if they were controlled trials. Paired reviewers assessed the quality of each eligible study and extracted relevant data. The resulting data were assembled into evidence tables organized by key management questions. The panel derived recommendations that were based on this review of evidence and were formulated according to the ACCP protocol for grading evidence and strength of recommendations.

Atrial Fibrillation↗

Overdiagnosis of multiple sclerosis and magnetic resonance imaging criteria.

Retrospectively, we assessed the specificity of two proposed magnetic resonance imaging (MRI) criteria for multiple sclerosis (MS) in patients suspected to have MS but who ultimately receive another diagnosis. Brain MRIs of 28 patients mixed with 28 MRIs of MS patients from the same cohort of 377 consecutively referred patients were scored by a neuroradiologist masked to the final diagnosis. The criteria for dissemination in space incorporated in the McDonald International Panel showed good specificity (89%). However, the more sensitive criteria proposed by a Subcommittee of the American Academy of Neurology resulted in a lower specificity (29%), indicating an increased risk of a false-positive diagnosis.

Adult↗

Enzyme immunoassays for total and allergen specific IgE in population studies.

OBJECTIVE: Extensive IgE serology in occupational or environmental health studies is often hampered by a lack of technical facilities and finance. The use in population studies of relatively simple and inexpensive enzyme immunoassays (EIAs) was therefore evaluated for the assessment of total serum immunoglobulin E (IgE), and of specific IgE reactions with various common (house dust mites, grass and birch pollen, and cat) or occupational (fungal alpha-amylase and rat urinary protein) allergens. METHODS: Total IgE was measured with a sandwich EIA, calibrated with commercially available IgE standards. Reproducibility was studied by testing pooled normal human serum samples in each of a large series of test plates. A panel of 156 children's serum samples with known IgE values was used to compare the assay with other total IgE assays. A previously developed EIA for anti-yeast IgE was adapted for the measurement of IgE reacting with various common and occupational allergens. Binding of IgE to microwells coated with commercially available allergen extracts, or allergen preparations from our own laboratory, was measured with a monoclonal anti-human IgE antibody and subsequent incubations with biotinylated rabbit anti-mouse Ig and avidin-peroxidase. Panels of serum samples from school children (n = 116), bakery workers (n = 126), and laboratory animal workers (n = 52) were used to study sensitivity and specificity, with reference to skin prick tests as the standard, and to compare the EIAs with commercially available test kits. RESULTS: The detection limit of the EIA for total IgE was 0.5-1 kU/l for undiluted serum samples, and the coefficient of variation between assays was less than 15% at serum concentrations between 1 and 150 kU/l. Results obtained with the panel of 156 children's serum samples were strongly correlated (r2 = 0.86) with IgE concentrations measured previously by radioimmunoassay. The results of the EIA for various occupational allergens correlated very well, both qualitatively and quantitatively, with the results of commercial test kits. Sensitivity and specificity of the EIA results as a predictor of skin prick test reactivity towards common allergens (house dust mite, grass pollen, birch pollen, and cat) were remarkably high (> 80%-90%) in the series of 116 children's serum samples. In a population of bakery workers the specificity of the EIAs was also very high (> 90%). The sensitivity was notably lower (30%-70%) in this adult population, which is, however, in agreement with results reported for conventional IgE tests. CONCLUSION: As the costs were estimated to be at least five to 10-fold lower than those of commercial test kits, the EIAs for total and specific IgE may be very useful tools in epidemiological studies of atopic respiratory or other disorders.

Adult↗

Comparison of MicroScan MICroSTREP, PASCO, and Sensititre MIC panels for determining antimicrobial susceptibilities of Streptococcus pneumoniae.

The MicroScan MICroSTREP MIC panel was compared with PASCO and Sensititre systems against 157 isolates of Streptococcus pneumoniae chosen to include penicillin-susceptible, intermediate, and resistant strains. Arbitration testing was performed by microbroth dilution using National Committee for Clinical Laboratory Standards guidelines. Overall essential agreement of 94-97% and categorical agreement of 91-94% with the reference method was achieved for the three systems. There were 8 very major errors (false susceptibility) for PASCO, 10 for Sensititre, and 9 for MICroSTREP; 4 major errors (false resistance) each for PASCO and MICroSTREP, and 6 for Sensititre. Most of these errors occurred with trimethoprim/sulfamethoxazole. Minor errors (susceptible or resistant versus intermediate) totaled 47 for PASCO, 69 for Sensititre, and 53 for MICroSTREP. Minor interpretive errors were most common with penicillin and ceftriaxone. This study showed that all three MIC panels provided interpretive results comparable to one another and to the reference method.

Adult↗

Characterization of adenovirus hexons by their epitope composition.

For the investigation of epitope composition of different adenovirus hexon types sixty-one mouse ascitic fluids containing monoclonal antibodies (MAbs) developed in three different panels were used. The distinction and marking of the different epitopes recognized by the MAbs were carried out by the determination of the composite cross-reactivity pattern, the titer and the correlation coefficient of all the 61 MAbs with 21 different hexon types representing all the six human subgenera, as well as different bovine and simian adenoviruses. The distinct epitopes were marked by two numbers referring the homologous hexon type to which the MAbs were directed and the serial number of the epitope specified by the different members of the given panel of the MAbs. The three panels of MAbs recognized 22 epitopes on the 21 hexon types among them a genus and three type specific ones and 18 different bi- and multilateral intertype (IT) specific epitopes that grouped adenoviruses within the genus, independently from the subgenus they belong to. Considering that the type specific epitope could be present only on the homologous hexon type, the largest number of the different epitopes distinguishable by the MAbs used could be 20 on the homologous hexon and 19 on the heterologous ones. It was found that the total number of IT specific epitopes on the hexons varied between 2 and 18. The distribution of the distinct specific epitopes on the different hexon types was different, as expected. The antigenic structure of the individual hexon types were characterized by the determination of their IT specific epitope spectrum. By pairwise analysis ten human hexon types formed three epitope clusters (types 4 and 19; types 8, 9, 9/13 and 10; as well as all types of subgenus C) showing identical epitope spectra. No clustering was found with human type 7, 12, 13, 18, 26, 27, 35 and 41, as well as with bovine and simian adenovirus hexons studied. However, they displayed a closer or looser antigenic relationship among each other and to members of the epitope clusters. The degree of antigenic relationship could be expressed by the similarity/dissimilarity percentage calculated from the number of the identical and different epitopes present on any two given hexon types.

Adenoviruses, Human↗

Enzyme-linked immunoassay for anti-HLA antibodies--an alternative to panel studies by lymphocytotoxicity.

In order to provide a simple and specific assay for the detection and quantitation of IgG and IgM anti-HLA antibodies in sera, HLA antigens purified from a pool of 240 random donor platelets were used to develop a solid-phase enzyme-linked immunoassay (EIA). The reference values for identifying the presence of IgG or IgM anti-HLA antibodies were determined by assaying sera from 39 healthy individuals without prior HLA alloimmunization. The assay was evaluated by studying sera from 122 patients who had been characterized previously for panel reactive antibodies by the lymphocytotoxicity assay (LCA). A significant linear correlation between two assays was noted (r = 0.8, P = 0.0001). Further analyses of the data demonstrated that the newly developed EIA has 100% specificity and 95.3% sensitivity as compared with the LCA. Additional studies revealed that patients whose PRA increased or decreased over time were in parallel with antibody levels measured by EIA. When the EIA was used to measure anti-HLA antibody titers, it was more sensitive than the LCA. Since the EIA is sensitive, specific, and technically less demanding, it should provide an useful alternative to reduce the number of the more laborious panel studies for monitoring anti-HLA antibody status in candidates for organ transplantation and recipients of blood transfusions.

Adult↗