RESUMO
We present the case of an infant born with scrotoschisis and evidence of meconium periorchitis and peritonitis. A scrotal defect was noted with exposure of the left testis and spermatic cord. Meconium peritonitis and periorchitis were confirmed on operative exploration. Given the history, cystic fibrosis was suspected, but initial screening and diagnostic tests were negative.
Assuntos
Fibrose Cística/diagnóstico , Perfuração Intestinal/complicações , Orquite/diagnóstico , Peritonite/etiologia , Escroto/anormalidades , Doenças Testiculares/congênito , Testículo/anormalidades , Diagnóstico Diferencial , Humanos , Recém-Nascido , Perfuração Intestinal/patologia , Perfuração Intestinal/cirurgia , Masculino , Mecônio , Orquiectomia , Orquite/etiologia , Orquite/patologia , Peritonite/cirurgia , Radiografia Abdominal , Escroto/cirurgia , Doenças Testiculares/patologia , Doenças Testiculares/cirurgia , Testículo/cirurgiaRESUMO
Over the past several decades, techniques for surgery of the aortic arch have undergone significant evolution. In addition, there have been refinements in the mechanism of cerebral protection utilized intraoperatively. However, significant practice variations in the strategy of antegrade selective cerebral perfusion continue to persist. Here, we describe a simple and easily reproducible technique for selective antegrade cerebral perfusion, utilizing axillary cannulation and retrograde coronary sinus balloon catheters.
Assuntos
Cateterismo Cardíaco/métodos , Cateteres Cardíacos , Circulação Cerebrovascular , HumanosRESUMO
We have investigated the adsorption of randomly annealed polyampholytes containing [2-(dimethylamino)ethyl methacrylate)] (DMAEMA), methacrylic acid (MAA), and [3-(2-methylpropionamido)propyl] trimethylammonium chloride (MAPTAC) with various molar compositions. The adsorption was performed from dilute aqueous solutions onto silicon substrates. The adsorbed layers were characterized by reflectivity techniques such as reflectometry, ellipsometry, and neutron specular reflection. As expected for annealed polyampholytes, the adsorption was found to depend strongly on the pH, with a maximum within the isoelectric domain of the polyampholyte. The monomer volume fraction profiles of the adsorbed layers were determined from neutron specular reflection measurements. In the isoelectric domain, the polyampholyte chains adopt a compact conformation, with a layer thickness of about 60 A. The polyampholyte layer is as dense as the adsorbed layer of fully charged polyelectrolyte but much thicker. Finally, we found that changing the ratio of neutral units along the polyampholyte chain in the isoelectric domain had no significant effect on the concentration profile of the adsorbed layer.
RESUMO
The most exciting potential of phage displayed peptide libraries is to obtain small peptide molecules that mimic an antigen, at least with respect to a particular epitope. In addition to their interest as research tools, such mimotopes could in principle be useful as diagnostic tools or for eliciting antibodies to a predefined epitope. However, the reduction of the phage insert sequence to a short peptide that can compete with the antigenic and in particular with the immunogenic properties of the natural antigen faces considerable difficulties. This review assesses critically the antigenicity of phage displayed peptides as free peptides and in different molecular environments. The difficulties to use mimotopes to induce antibodies that bind to the natural antigen (crossreactive immunogenicity) and the considerable discrepancy between antigenicity and immunogenicity of phage-derived peptides are discussed. Peptides selected with antibodies from phage displayed random peptide libraries have raised considerable expectations as low molecular weight substitutes of the natural antigen. This review will focus on the results of phage displayed random peptide libraries screened with antibodies specific for proteins, carbohydrates and nucleic acids and critically examine how the above expectations have been met.
Assuntos
Antígenos/genética , Bacteriófagos/genética , Antígenos/química , Técnicas de Química Combinatória , Mimetismo MolecularAssuntos
Infecções por HIV/genética , HIV-1/fisiologia , Receptores de Citocinas/genética , Receptores de HIV/genética , Adulto , Receptor 1 de Quimiocina CX3C , Progressão da Doença , Genótipo , Infecções por HIV/fisiopatologia , Humanos , Masculino , Polimorfismo de Fragmento de Restrição , Receptores de Citocinas/classificação , Receptores de HIV/classificaçãoRESUMO
Phage-displayed peptide libraries represent an efficient tool to isolate peptides that bind a given target molecule. After several selection rounds, generally a large pool of target binding phages is obtained. Conventional analysis of the selected phage population involves extensive sequencing of many clones, most of which can be identical. We have adapted the Heteroduplex Mobility Assay (HMA) for pre-screening of phage inserts that were amplified by direct colony PCR of ELISA-positive clones. This strategy allowed for the rapid and reproducible assignment of insert sequences to different 'heteroduplex migration groups'. Sequence analysis of only one representative of each HMA migration group then completes the characterisation of the binding phage population. In our model experiments, only 16% of HMA pre-screened clones required further sequence analysis.
Assuntos
Análise Heteroduplex/métodos , Biblioteca de Peptídeos , Animais , Anticorpos Monoclonais , Sequência de Bases , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Reação em Cadeia da PolimeraseRESUMO
The sequence H236-256 of the measles virus (MV) hemagglutinin (H) contains the sequential epitope of the neutralizing and protective monoclonal antibody (mAb) BH129 with the minimal epitope E(245)L-QL(249). Using this mAb, we have recently developed 7mer mimotopes binding up to 135x better than the corresponding 7mer epitope H244-250. In this study, we combined T cell epitopes (TCE) with either highly crossreactive 7mer mimotopes, 13mer mimotopes or less crossreactive MV-derived B cell epitopes (BCE). Antigenicity of these TBB, TTB and TTBB peptides was determined with BH129 in a competition ELISA against MV. We found that chimeric peptides including mimotopes were up to 80x better binders to the mAb than peptides containing the original BCEs. All peptides irrespective of their antigenicity were used for immunization to compare their virus- crossreactive immunogenicity. Unexpectedly, none of the highly antigenic mimotope-based peptides induced MV-crossreactive antibodies. In contrast, a number of peptides with the viral BCE sequence that did not bind to the mAb, induced MV-crossreactive and even neutralizing antibodies. This report describes a striking example of disparity between antigenicity and crossreactive immunogenicity and casts considerable doubt on the predictive value of antigenicity in immunogenicity studies, considerably complicating the selection of potential vaccine candidates.
Assuntos
Anticorpos Monoclonais/imunologia , Reações Cruzadas , Epitopos , Vacinas Sintéticas , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologiaRESUMO
A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 micrograms/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 +/- 12.7 micromol/L and 34.8 +/- 8.59 micromol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.
Assuntos
Colágeno/metabolismo , Peptídeos/genética , Peptídeos/farmacologia , Fator de von Willebrand/metabolismo , Animais , Bacteriófagos , Humanos , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
Antigenicity and conformational propensities of synthetic peptides corresponding to the sequential epitope H236-255 of the measles virus hemagglutinin protein were investigated. This epitope corresponds to the neutralising and protective monoclonal antibody BH129 and includes Arg243, implicated in CD46-down-regulation and Arg253 that has been mapped to the putative enzymatic site. Fine mapping with truncation-, elongation-, Gly- and Ala-substitution analogues defined EL-QL as the critical residues of the minimal epitope S244ELSQL249. CD spectra of peptides, comparison with the 3D-structure of homologous sequences, and prediction algorithms suggested a helical structure with the contact residues E245L-QL249 located on the protein surface. Mimotopes obtained with a 6-mer phage display library contained a consensus Pro (important for binding) instead of Ser247 of the wild-type sequence (irrelevant for binding). The kink induced by Pro seemed to be essential to bring the 4 contact-residues in the mimotopes and in the corresponding short peptides together. CD analysis and prediction algorithms suggested that non-helical conformations of the phage insert and of the peptides may favourably mimic the antigenic helical turns of the wild-type sequence, resulting in an up to 135 times higher antigenicity of the mAb towards the mimotope peptides.