Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induce IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine σ-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.     

Characterization of an RTX toxin from enterohemorrhagic Escherichia coli O157:H7.

A hemolytic determinant of enterohemorrhagic Escherichia coli O157:H7 is encoded on a 90-kbp plasmid (pO157). This enterohemorrhagic E. coli toxin (Ehx) is a newly described RTX cytotoxin. The prototype RTX toxin is the E. coli hemolysin (Hly) associated with extraintestinal E. coli infections. We expressed Ehx from E. coli K-12 strains harboring either pSK3, a pO157 derivative marked with Tn801 unlinked to Ehx, or a recombinant plasmid containing an 11.9-kbp subclone (pEO40) of pSK3. The Ehx activities and antibody reactivities were compared with those of Hly. Little Ehx was secreted extracellularly from the strain harboring pSK3; however, when the Hly transport genes hlyBD were supplied in trans, both intracellular and extracellular levels of Ehx were enhanced more than 15-fold. The strain harboring pEO40 secreted at least 140-fold more Ehx than did the strain harboring pSK3, and neither intracellular nor extracellular levels were significantly enhanced by the addition of hlyBD in trans. Polyclonal anti-HlyA antiserum and several anti-HlyA monoclonal antibodies, including the monoclonal antibody A10, which is panreactive for nearly all RTX toxins, reacted with EhxA antigen by immunoblot analysis. In hemolysis and 51Cr release assays, Ehx demonstrated similar efficiencies in lysis of BL-3 cells (cells from a bovine lymphoma cell line) and sheep and human erythrocytes. Surprisingly, it demonstrated very little activity against two human lymphoma cell lines. In contrast, Hly lysed all five cell types tested, each to a greater extent than that demonstrated by comparable amounts of Ehx. As with other RTX toxins, Ehx activity was calcium dependent and heat labile.     

Analysis of cell type-specific responses mediated by the type IV secretion system of Helicobacter pylori

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.     

Filamentous phage morphogenetic signal sequence and orientation of DNA in the virion and gene-V protein complex

The circular single-stranded viral DNA (ssDNA) of filamentous phage is oriented in the virus with a hairpin forming sequence called mos at the leading end of the virion-the end that emerges from the cell first. In the experiments that originally defined mos it was also found to enhance virion production 100-fold, though in other circumstances it has no such effect. Using a new electron-microscopic method, we have ascertained the orientation of ssDNA in phage having mutations involving mos and other viral functions, and also in the intracellular precursor to the virion-a rod-shaped complex between the ssDNA and the phage-encoded gene-V protein, pV. The results show (1) that the ssDNA is oriented in the complex as in the virion, with mos at one end; (2) that orientation is maintained even if assembly is not mediated by the complex; (3) that orientation is manifested in polyphage--abnormal particles in which many unit-length ssDNA molecules are sheathed in a single, extremely long capsid; (4) that orientation is imposed by mos itself (or something very nearby) since it disappears in a mos deletion; and (5) that a 229-base segment including the minus strand of mos is also effective at imposing orientation. On the basis of our findings, we speculate that mos determines orientation in two stages, one imposing an axis on the ssDNA loop during formation of the pV/ssDNA complex, the other imposing a direction on the loop during initiation of particle assembly. The sequence requirements for the two stages may be different.     

The Topography of Non-Linear Cortical Dynamics at Rest, in Mental Calculation and Moving Shape Perception

Differential cortical activation by cognitive processing was studied using dimensional complexity, a measure derived from nonlinear dynamics that indicates the degrees of freedom (complexity) of a dynamic system. We examined the EEG of 32 healthy subjects at rest, during a visually presented calculation task, and during a moving shape perception task. As a nonlinear measure of connectivity, the mutual dimension of selected electrode pairs was used. The first Lyapunov coefficient was also calculated. Data were tested for non-linearity using a surrogate data method and compared to spectral EEG measures (power, coherence). Surrogate data testing confirmed the presence of nonlinear structure in the data. Cognitive activation led to a highly significant rise in dimensional complexity. While both tasks activated central, parietal and temporal areas, mental arithmetic showed frontal activation and an activity maximum at T3, while the moving shape task led to occipital activation and a right parietal activity maximum. Analysis of mutual dimension showed activation of a bilateral temporal-right frontal network in calculation. The Lyapunov coefficent showed clear topographic variation, but was not significantly changed by mental tasks (p<.09). While dimensional complexity was almost unrelated to power values, nonlinear (mutual dimension) and linear (coherence) measures of connectivity shared up to 37% of variance. Data are interpreted in terms of increased cortical complexity as a result of recruitment of asynchronously active, distributed neuronal assemblies in cognition. The topography of nonlinear dynamics are related to neuropsychological and neuroimaging findings on mental calculation and moving shape perception.     

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.      

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Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.     

Differences in host susceptibility to disease progression in the human challenge model of Haemophilus ducreyi infection

With human volunteers inoculated at two sites with Haemophilus ducreyi, outcomes for a subject were not independent. In a reinfection trial, 2 of 11 previous pustule formers and 6 of 10 previous resolvers resolved all sites of infection. There was no correlation between serum bactericidal or phagocytic activity and outcome in the trial. These data indicate that different hosts are differentially susceptible to disease progression versus resolution in the model.