An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Significance of urinary isolates of coagulase-negative Micrococcaceae.

Of 16,347 urine cultures submitted to the hospital laboratory, 68 (0.4%) specimens from 50 patients yielded greater than 10(4) coagulase-negative staphylococci/ml in pure culture. A total of 62 of 63 organisms available for study were staphylococci: 45 Staphylococcus epidermidis (predominantly subgroup 1), 15 Staphylococcus saprophyticus (subgroup 3), and 2 Staphylococcus aureus. Twenty-one patients had "probable" urine infections. Eight patients had two or more positive urine cultures, and all isolates from the same patients were identical (by morphology, antibiotic susceptibility, and hemolytic pattern). Nine (75%) of the 12 isolates of S. saprophyticus, which were novobiocin resistant and nonhemolytic on the synergistic hemolysis test, were from patients with probable urinary infection. Eight were young women with acute symptoms and pyuria. Differences in the glucose and mannitol fermentation tests with different media may lead to difficulties in identification. Novobiocin resistance cannot be relied upon to differentiate isolates of S. saprophyticus from S. epidermidis.     

Bakterien als Ursache allergischer Asthmaanfälle (Versuche mit Keimen aus der Klebsiella-Gruppe)

Zusammenfassung Keime der Klebsiella-Gruppe, Typ 2, in vollsynthetischem eiweißfreiem Nährmedium gezüchtet, eignen sich zur direkten Sensibilisierung von Meerschweinchen sowie zur nachfolgenden Provokation von Asthmaanfällen und anaphylaktischem Schock.Zur Sensibilisierung wurden Keime verwandt, die bei 60° C mit Phenol, Formol oder Ultraschall abgetötet worden waren. Asthmaanfälle konnten durch Inhalation von abgetöteten, aber auch von lebenden Keimen ausgelöst werden. Die Asthmabereitschaft blieb mehrere Monate lang bestehen. Die Anfälle können durch N-Isopropylnoradrenalin zum Verschwinden gebracht werden. Im Grundsätzlichen entsprachen sie Asthmaanfällen, die durch Hühnereiweiß bedingt waren. Unterschiede gegenüber dem Hühnereiweißasthma zeigten sich in geringerer Schockbereitschaft, vermehrter Dyskrinie und dem gelegentlichen Auftreten von Spätreaktionen.Das anaphylaktogene Klebsiella-Antigen ist ein unlöslicher Bestandteil des Bakterienleibes, vermutlich kommt es in der Grenzphase vor.Die Untersuchungen erbringen den tierexperimentellen Beweis, daß Bakterien alsprimäres Allergen Asthma hervorrufen können.FräuleinTrude Schulte danken wir für die wertvolle Mitarbeit. Die Untersuchungen wurden durch ERP-Mittel ermöglicht, für deren Vermittlung wir Herrn Prof. Dr.Hagen, Bundes-Innenministerium, danken.     

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.     

Motility-indole-lysine-sulfide medium.

A medium designed for the detection of motility, indole, lysine decarboxylase and deaminase reactions, and H2S production was devised and evaluated. Results, using 157 strains of enteric pathogens, were in agreement with reference methods. When 300 isolates from fecal cultures were screened using this medium, Shigella was easily differentiated from Escherichia and more of the Proteus species, especially P. morganii, could be eliminated from further study.     

Long-term preservation of cortically dependent visual function in RCS rats by transplantation.

Cell transplantation is one way of limiting the progress of retinal degeneration in animal models of blinding diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Here we transplanted a human retinal pigment epithelial (RPE) cell line into the subretinal space of one such model, the Royal College of Surgeons (RCS) rat, and showed, using head tracking to moving stripes and pattern discrimination in conjunction with single-unit cortical physiology, that cortically mediated vision can be preserved with this treatment.     

Effect of sunlight exposure on circulating 1,25-dihydroxyvitamin D in hemodialyzed patients and of exogenous parathyroid hormone in anephric patients

Sunshine exposure increased the serum concentration of 25-hydroxyvitamin D (25-OHD) in 9 hemodialyzed patients. Mean 1,25-dihydroxyvitamin D (1,25-(OH)2D) was unchanged, but in two patients with low initial 25-OHD values this increase was accompanied by a rise in circulating 1,25-(OH)2D, although not to normal levels. One hemodialyzed patient developed liver insufficiency with a resultant reduction of serum 25-OHD concentration accompanied by a decrease in serum 1,25-(OH)2D concentration. The results indicate that the circulating levels of 1,25-(OH)2D in patients with end-stage renal failure are to some extent regulated by the serum 25-OHD concentrations. Injection of parathyroid hormone (PTH) induced minor increases in serum concentrations of 1,25-(OH)2D in patients with end-stage renal failure and even in anephric patients, suggesting the existence of an extrarenal PTH-sensitive 1-alpha-hydroxylase. However, the enzyme was stimulated by supraphysiological concentrations of PTH, and therefore not necessarily of importance in the normal regulation of calcium metabolism.     

Efficacy of Amodiaquine in uncomplicated falciparum malaria in Nigeria in an area with high-level resistance to Chloroquine and Sulphadoxine/Pyrimethamine

Falciparum Malaria is hyperendemic in southern Nigeria and chloroquine resistance is an increasing problem. Therefore, the parasitological and haematological response to treatment with amodiaquine was studied in children under 5 years during a 14-day follow-up. Of 105 children who accomplished the study (out of 114 who were enrolled), 95.3% were parasite-negative on thick blood film on day 7, which decreased to 89.5% on day 14. The haemoglobin levels increased on average by 1.3% on day 14 (±1.9) and more pronounced in children with anaemia < 10 g/dl on enrolment. The number of patients with adverse events (mainly pruritus and nausea) was few. This study shows that amodiaquine is effective, safe and affordable in an area with high resistance to chloroquine.     

Radiation dose measurements with alanine/agarose gel and thin alanine films around a 192Ir brachytherapy source,using ESR spectroscopy

Alanine/agarose gel and alanine films in stacks have been used for measurements of absorbed dose around an HDR 192Ir source in a vaginal cylinder-applicator, with and without a 180 degrees tungsten shield. The gel and the films were analysed by means of ESR spectroscopy and calibrated against an ion chamber in a 4 MV photon beam to obtain absolute dose values. The gel serves as both dosimeter and phantom material, and the thin (130 microm) films are used to achieve an improved spatial resolution in the dose estimations. Experimental values were compared with Monte Carlo simulations using two different codes. Results from the measurements generally agree with the simulations to within 5%, for both the alanine/agarose gel and the alanine films.