Synthese von selbstaggregierenden Glycopeptidkonjugaten mit (per)fluorierten Membranankern

Für eine effektive Erkennung tumorassoziierter Kohlenhydratantigene durch das Immun-system in der Krebs¬immuntherapie ist eine multivalente Präsentation der Haptene notwendig. In der vorliegenden Arbeit wurde ein neuer Zugang zu einer solch räumlichen Konzentration der Haptene untersucht, indem MUC1-Antigene mit perfluorierten Alkylketten funktionalisiert und in einer geeigneten Lipidmatrix entmischt wurden. Perfluoralkyl-Amphiphile zeichnen sich durch eine hohe Entmischungstendenz in Alkyllipiden aus und bewirken dadurch eine Anreicherung der Erkennungsstrukturen (Haptene) in Analogie zu den natürlichen raft-Domänen auf der Zelloberfläche.rnDazu wurden zunächst verschiedene Membranankersysteme mit unterschiedlichem Fluorierungsgrad entwickelt. Beispielsweise konnte ausgehend von einem zentralen Glycerin-fragment ein Membrananker mit zwei Perfluoralkylketten hergestellt werden. Letztere wurden mittels radikalischer Perfluoralkylierung eingeführt, wobei der Fluorgehalt der Verbindung über die Kettenlänge gesteuert wurde. Daneben konnte ein weiteres Ankersystem, basierend auf der Aminosäure Lysin, synthetisiert werden, dass einen bequemen Einbau der Perfluoralkylketten durch Peptidkupplungen von entsprechenden perfluorierten Aminen bzw. perfluorierten Carbonsäuren erlaubte. In diesem Fall wurde der Fluorgehalt durch die Einführung von Alkyl- bzw. Perfluoralkylketten verändert.rnBeide Systeme konnten für erste Untersuchungen ihres Phasenverhaltens mit polaren Kopf-gruppen ausgestattet werden, wobei neben einem hydrophilen, nicht-immunogenen Triethylenglycolspacer vor allem ein TN-Antigen tragendes Dipeptid zum Einsatz kam. In Gegenwart des Matrixlipids DODAMA konnten in Langmuir-Blodgett-Untersuchungen mit diesen Verbindungen eine Entmischung und die Ausbildung mikroseparierter Bereiche nachgewiesen werden. Auch war es möglich, durch Anbindung eines Fluoreszenzfarbstoffes zu zeigen, dass solche amphiphilen Membrananker auf perfluorierten Oberflächen effektiv und dauerhaft immobilisiert werden können. Damit eröffnet diese Verbindungsklasse interessante Anwendungsmöglichkeiten in der Entwicklung von diagnostischen Microarray-Formaten.rnUm eine Anbindung der fluorierten Membrananker an den N-Terminus eines an fester Phase aufgebauten mucinanalogen Glycopeptids als antigene Einheit zu ermöglichen, wurde ein entsprechendes Ankersystem auf Basis von Glutaminsäure entwickelt. Dabei wurden an diese Verbindung neben dem TN-Antigen noch weitere komplexe tumorassoziierte Kohlenhydrat-antigene des Mucintyps angebunden, wobei der Aufbau der resultierenden amphiphilen Glycolipopeptide vollständig an der festen Phase gelang. Insgesamt konnten so mithilfe des teilfluorierten Lysinankers und des zweifach perfluorierten Glutaminsäureankers erste amphiphile Glycopeptid-Konjugate hergestellt werden, deren antigene Kopfgruppe aus einer 20 Aminosäuren umfassenden Wiederholungseinheit des Mucins MUC1 mit TN-, T- bzw. STN-Antigen-Seitenkette besteht. Derartige Verbindungen stellen reizvolle Bausteine für die Tumordiagnostik und für die Entwicklung von stabilen liposomalen Tumorvakzinen dar, da die verwendeten Perfluoralkylanker die Antigenpräsentation nicht wesentlich beeinflussen und die Bindung des Antikörpers nicht behindern. rn

Pemphigus herpetiformis: analysis of the autoantibody profile during the disease course with changes in the clinical phenotype

Pemphigus herpetiformis (PH) is a rare dapsone-responsive variant of pemphigus, characterized by annular and vesiculopustular cutaneous lesions. Most PH serum samples contain autoantibodies against desmoglein (Dsg)1, but not Dsg3, and the presence of the latter is almost invariably associated with mucosal involvement, as predicted based on the 'Dsg compensation theory'. We describe a patient with features characteristic of PH with histologically eosinophilic spongiosis who repeatedly tested positive for anti-Dsg3 but not anti-Dsg1 autoantibodies by ELISA. To investigate whether the peculiar clinical phenotype was due to a distinct immunological profile, the patient's serum was tested by ELISA and immunoblotting using recombinant forms of Dsg3. Serum samples were found to have low and high reactivity against the EC1 and the EC4 domains of Dsg3, respectively, whereas the autoantibodies belonged predominantly to the IgG1 and IgG4 subclasses. The overall immunological profile was typical of pemphigus vulgaris. The patient finally developed isolated oral erosions 22 months after initial presentation, without significant changes in the autoantibody profile and of the targeted antigenic sites. Our patient presented features characteristic of PH. Although circulating anti-Dsg3 antibodies were present, the patient had only cutaneous involvement for a long period. Our findings indicate that the proposed Dsg compensation theory cannot always explain the clinical phenotype, changes in which may occur without apparent modification of the autoantibody profile and antibody specificity. Hence, additional factors, such as Fcgamma-dependent neutrophil activation, may critically affect the clinical presentation of pemphigus.

Molecular epidemiology of the nasal colonization by methicillin-susceptible Staphylococcus aureus in Swiss children

Nasal carriage of Staphylococcus aureus contributes to an increased risk of developing an infection with the same bacterial strain. Genetic regulatory elements and toxin-expressing genes are virulence factors associated with the pathogenic potential of S. aureus. We undertook an extensive molecular characterization of methicillin-susceptible S. aureus (MSSA) carried by children. MSSA were recovered from the nostrils of children. The presence of Panton-Valentine leukocidin (PVL), exfoliatins A and B (exfoA and exfoB), and the toxic-shock staphylococcal toxin (TSST-1) and agr group typing were determined by quantitative PCR. A multiple-locus variable-number of tandem repeat analysis (MLVA) assay was also performed for genotyping. Five hundred and seventy-two strains of MSSA were analysed. Overall, 30% were positive for toxin-expressing genes: 29% contained one toxin and 1.6% two toxins. The most commonly detected toxin gene was tst, which was present in 145 (25%) strains. The TSST-1 gene was significantly associated with the agr group 3 (OR 56.8, 95% CI 32.0-100.8). MLVA analysis revealed a large diversity of genetic content and no clonal relationship was demonstrated among the analysed MSSA strains. Multilocus sequence typing confirmed this observation of diversity and identified ST45 as a frequent colonizer. This broad diversity in MSSA carriage strains suggests a limited selection pressure in our geographical area.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Escherichia coli variants in periprosthetic joint infection: diagnostic challenges with sessile bacteria and sonication

The diagnostic yield of prosthetic joint-associated infection is hampered by the phenotypic change of bacteria into a sessile and resistant form, also called biofilm. With sonication, adherent bacteria can be dislodged from the prosthesis. Species identification may be difficult because of their variations in phenotypic appearance and biochemical reaction. We have studied the phenotypic, genotypic, and biochemical properties of Escherichia coli variants isolated from a periprosthetic joint infection. The strains were collected from synovial fluid, periprosthetic tissue, and fluid from the explanted and sonicated prosthesis. Isolates from synovial fluid revealed a normal phenotype, whereas a few variants from periprosthetic tissue and all isolates from sonication fluid showed different morphological features (including small-colony variants). All isolates from sonication fluid were beta-galactosidase negative and nonmotile; most were indole negative. Because of further variations in biochemical properties, species identification was false or not possible in 50% of the isolates included in this study. In contrast to normal phenotypes, variants were resistant to aminoglycosides. Typing of the isolates using pulsed-field gel electrophoresis yielded nonidentical banding patterns, but all strains were assigned to the same clonal origin when compared with 207 unrelated E. coli isolates. The bacteria were repeatedly passaged on culture media and reanalyzed. Thereafter, most variants reverted to normal phenotype and regained their motility and certain biochemical properties. In addition, some variants displayed aminoglycoside susceptibility after reversion. Sonication of an explanted prosthesis allows insight into the lifestyle of bacteria in biofilms. Since sonication fluid also reveals dislodged sessile forms, species identification of such variants may be misleading.

Humoral immune response to ADAMTS13 in acquired thrombotic thrombocytopenic purpura

The apparently spontaneous development of autoantibodies to ADAMTS13 in previously healthy individuals is a major cause of thrombotic thrombocytopenic purpura (TTP). Epitope mapping studies have shown that in most patients antibodies directed towards the spacer domain of ADAMTS13 are present. A single antigenic surface comprising Arg(660) , Tyr(661) and Tyr(665) that contributes to the productive binding of ADAMTS13 to unfolded von Willebrand factor is targeted by anti-spacer domain antibodies. Antibodies directed to the carboxyl-terminal CUB1-2 and TSP2-8 domains have also been observed in the plasma of patients with acquired TTP. As yet it has not been established whether this class of antibodies modulates ADAMTS13 activity. Inspection of the primary sequence of human monoclonal anti-ADAMTS13 antibodies suggests that the variable heavy chain germline gene segment VH1-69 is frequently incorporated. We suggest a model in which 'shape complementarity' between the spacer domain and residues encoded by the VH1-69 gene segment explain the preferential use of this variable heavy chain gene segment. Finally, a model is presented for the development of anti-ADAMTS13 antibodies in previously healthy individuals that incorporates the recent identification of HLA DRB1*11 as a risk factor for acquired TTP.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

A Near-Infrared Spectroscopic Study of Young Field Ultracool Dwarfs

We present a near-infrared (0.9-2.4 mu m) spectroscopic study of 73 field ultracool dwarfs having spectroscopic and/or kinematic evidence of youth (approximate to 10-300 Myr). Our sample is composed of 48 low-resolution (R approximate to 100) spectra and 41 moderate-resolution spectra (R greater than or similar to 750-2000). First, we establish a method for spectral typing M5-L7 dwarfs at near-IR wavelengths that is independent of gravity. We find that both visual and index-based classification in the near-IR provides consistent spectral types with optical spectral types, though with a small systematic offset in the case of visual classification at J and K band. Second, we examine features in the spectra of similar to 10 Myr ultracool dwarfs to define a set of gravity-sensitive indices based on FeH, VO, Ki, Nai, and H-band continuum shape. We then create an index-based method for classifying the gravities of M6-L5 dwarfs that provides consistent results with gravity classifications from optical spectroscopy. Our index-based classification can distinguish between young and dusty objects. Guided by the resulting classifications, we propose a set of low-gravity spectral standards for the near-IR. Finally, we estimate the ages corresponding to our gravity classifications.

Effect of repetitiveness on the immunogenicity and antigenicity of Trypanosoma cruzi FRA protein

Repetitive proteins (RP) of Trypanosoma cruzi are highly present in the parasite and are strongly recognized by sera from Chagas' disease patients. Flagelar Repetitive Antigen (FRA), which is expressed in all steps of the parasite life cycle, is the RP that displays the greatest number of aminoacids per repeat and has been indicated as one of the most suitable candidate for diagnostic test because of its high performance in immunoassays. Here we analyzed the influence of the number of repeats on the immunogenic and antigenic properties of the antigen. Recombinant proteins containing one, two, and four tandem repeats of FRA (FRA1, FRA2, and FRA4, respectively) were obtained and the immune response induced by an equal amount of repeats was evaluated in a mouse model. The reactivity of specific antibodies present in sera from patients naturally infected with T. cruzi was also assessed against FRA1, FRA2, and FRA4 proteins, and the relative avidity was analyzed. We determined that the number of repeats did not increase the humoral response against the antigen and this result was reproduced when the repeated motifs were alone or fused to a non-repetitive protein. By contrast, the binding affinity of specific human antibodies increases with the number of repeated motifs in FRA antigen. We then concluded that the high ability of FRA to be recognized by specific antibodies from infected individuals is mainly due to a favorable polyvalent interaction between the antigen and the antibodies. In accordance with experimental results, a 3D model was proposed and B epitope in FRA1, FRA2, and FRA4 were predicted.

Local glucocorticoid production in the mouse lung is induced by immune cell stimulation

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive steroid hormones, mainly produced by the adrenal glands. However, increasing evidence supports the idea of additional extra-adrenal sources of bioactive GC. The lung epithelium is constantly exposed to a plethora of antigenic stimuli, and local GC synthesis could contribute to limit uncontrolled immune reactions and tissue damage.

Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data

OBJECTIVES: We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans. METHODS: Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various beta-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed. RESULTS: Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried bla(OXA-66), bla(ADC-25), bla(TEM-1), aacC2 and IS1133. Strains of ST15 possessed bla(OXA-69), bla(ADC-11), bla(TEM-1) and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of bla(ADC) (one ST10 and one ST12) and/or bla(OXA-66) (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection. CONCLUSIONS: STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.

Emergence of linezolid-resistant Staphylococcus aureus after prolonged treatment of cystic fibrosis patients in Cleveland, Ohio

Linezolid (LZD)-resistant Staphylococcus aureus (LRSA) isolates were monitored from 2000 to 2009 in Cleveland, OH. LRSA first emerged in 2004 only in cystic fibrosis (CF) patients, with 11 LRSA-infected CF patients being identified by 2009. LRSA was isolated from 8 of 77 CF patients with S. aureus respiratory tract infection treated with LZD from 2000 to 2006. Analysis of clinical data showed that the 8 CF patients with LRSA received more LZD courses (18.8 versus 5.9; P = 0.001) for a longer duration (546.5 versus 211.9 days; P < 0.001) and had extended periods of exposure to LZD (83.1 versus 30.1 days/year; P < 0.001) than the 69 with LZD-susceptible isolates. Five LRSA isolates included in the clinical analysis (2000 to 2006) and three collected in 2009 were available for molecular studies. Genotyping by repetitive extrapalindromic PCR and pulsed-field gel electrophoresis revealed that seven of these eight LRSA strains from unique patients were genetically similar. By multilocus sequence typing, all LRSA isolates were included in clonal complex 5 (seven of sequence type 5 [ST5] and one of ST1788, a new single-locus variant of ST5). However, seven different variants were identified by spa typing. According to the Escherichia coli numbering system, seven LRSA isolates contained a G2576T mutation (G2603T, S. aureus numbering) in one to four of the five copies of domain V of the 23S rRNA genes. One strain also contained a mutation (C2461T, E. coli numbering) not previously reported. Two strains, including one without domain V mutations, possessed single amino acid substitutions (Gly152Asp or Gly139Arg) in the ribosomal protein L3 of the peptidyltransferase center, substitutions not previously reported in clinical isolates. Emergence of LRSA is a serious concern for CF patients who undergo prolonged courses of LZD therapy.

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

AIMS: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. METHODS AND RESULTS: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which - in combination with MLST - increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37.5, 33.1 and 8.1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone-sensitive human isolates showed overlapping MLST-flaB types with those of chicken origin, resistant strains showed only 39% of matching types. CONCLUSION: Mainly quinolone-sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.

Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia (EP), an important cause of disease-associated losses in swine production and a role of wild boar in recurrent infections can be supposed. Genotypes of M. hyopneumoniae from wild boar are unknown but could indicate its role as a potential reservoir. Therefore, 34 lung samples being PCR-positive for M. hyopneumoniae from wild boar from the Geneva region in Switzerland were assayed by genotyping using the p146 and multi-locus sequence typing (MLST) approaches and compared to data from outbreak cases from domestic swine in Switzerland. Successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in the samples as assessed by different real-time PCR assays. The p146 genotyping was more successful with 24 samples (70.5%) being typeable whereas only 6 samples (17.6%) could be genotyped using the MLST approach. Variability of genotypes was high but identical types were found in geographically related animals. Genotypes from wild boar showed phylogenetic relatedness to those from domestic pigs but no matching types could be identified. Results show that direct genotyping from wild boar lung samples is possible and provides a promising approach to investigate future EP outbreak related samples from wild boar. (C) 2011 Elsevier B.V. All rights reserved.

A new variant of Actinobacillus pleuropneumoniae serotype 3 lacking the entire apxII operon

Actinobacillus pleuropneumoniae is an important respiratory pathogen causing pleuropneumonia in pig. The species is genetically characterized by the presence of 4 RTX (Repeats in the Structural ToXin) toxin genes: apxI, apxII, and apxIII genes are differentially present in various combinations among the different serotypes, thereby defining pathogenicity; the apxIV gene is present in all serotypes. Polymerase chain reaction (PCR)-based apx gene typing is done in many veterinary diagnostic laboratories, especially reference laboratories. The present report describes the isolation of atypical A. pleuropneumoniae from 4 independent cases from 2 countries. All isolates were beta-nicotinamide adenine dinucleotide (beta-NAD) dependent and nonhemolytic but showed strong co-hemolysis with the sphingomyelinase of Staphylococcus aureus on sheep blood agar. Classical biochemical tests as well as Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequence-based analysis (16S ribosomal RNA [rRNA] and rpoB genes) identified them as A. pleuropneumoniae. Apx-toxin gene typing using 2 different PCR systems showed the presence of apxIV and only the apxIII operon (apxIIICABD). None of the apxI or apxII genes were present as confirmed by Southern blot analysis. The 16S rRNA and rpoB gene analyses as well as serotype-specific PCR indicate that the isolates are variants of serotype 3. Strains harboring only apxIV and the apxIII operon are possibly emerging types of A. pleuropneumoniae and should therefore be carefully monitored for epidemiological reasons.