摘要:

The present work describes the purification and characterization of antigen B (AgB), the thermostable lipoprotein fromE. granulosus. Native AgB was purified to homogeneity by a new strategy involving adsorption on DEAE‐Sepharose, followed by immunopurification. The purified antigen was analysed using mapped monoclonal antibodies (MoAbs) and peptide isolation byin situdigestion in gels after SDS‐PAGE. Epitope mapping of 7 MoAbs using PEPSCAN, synthetic peptides and competition studies, revealed that six of them defined epitopes which clustered the N‐terminal extension of a 8 kDa subunit of AgB, whilst the remaining one reacted against the stretch RGLIAEGE, corresponding to the C‐terminus. The epitopes defined by the seven MoAbs were found to be present in all the subunits. Furthermore, the similarities of the peptide finger prints obtained by HPLC analysis and amino acid sequencing of tryptic peptides isolated from the 8, 16 and 24 kDa subunits, indicated that they have most if not all the amino acid sequence in common. We also found evidence that the band representing a component of an apparent molecular weight of 8 kDa in SDS‐PAGE, believed to be the smallest subunit of AgB, contained at least two components, which may constitute the building blocks of the higher molecular

ISSN:0141-9838    

DOI:10.1046/j.1365-3024.1996.d01-38.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Antigen 5, a major parasite‐derived lipoprotein of unilocular hydatid (Echinococcus granulosus) cyst fluid, comprises subunits of 56–70 kDa which dissociate on disulphide bound reduction to two subunits of approximately 25 and 38 kDa on SDS‐PAGE. The 38 kDa component is recognized as a potentially important diagnostic marker for cystic hydatid disease. Single dimensional SDS‐PAGE, two dimensional (2‐D) gel electrophoresis, modified ‘2‐D’ gel electrophoresis, immunoaffinity‐purification and HLPC were employed to purify antigen 5. Subsequent N‐terminal sequencing suggested that antigen 5 isoforms exist due to the identification of a single amino acid sequence with alternative residues at some positions. An 18 amino acid consensus N‐terminal sequence was derived for antigen 5 as a result of comparing the sequences obtained by the different fractionation methods. No homology was revealed to any previously identified protein including putative antigen 5 amino acid sequences. A synthetic peptide, mimicking the N‐terminal consensus sequence, did not bind with patient sera (confirmed positive to antigen 5) or an anti‐antigen 5 MoAb. Protein sequences (16 residues compared) for the 38 kDa subunit from sheep and horse (representing different strains of E.  granulosus) cyst fluids were very similar except for differences at three positions. 2‐D and modified ‘2‐D’ gel electrophoresis, in combination with immunoblot analysis, indicated the presence of more than one protein in the 38 kDa region of

ISSN:0141-9838    

DOI:10.1046/j.1365-3024.1996.d01-42.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

The flagellar fraction (FF) ofTrypanosoma cruzican be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF‐β (Hansenet al.,submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom‐matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice.In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL‐1 and IL‐6. In immunized mice, the FT elicited higher levels of antigen‐specific IgG2athan the FF as well as broader recognition ofT. cruziantigens. Splenocytes from mice immunized with FT proliferated spontaneouslyin vitroand secreted TH1and TH2cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN‐γ. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN‐γ through a TGF‐β

ISSN:0141-9838    

DOI:10.1046/j.1365-3024.1996.d01-40.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Particular immunopathological features and their effects on the vascular permeability of different ocular structures were analysed in two dogs naturally infected byLeishmania infantum.The existence of specific anti‐Leishmaniaimmunoglobulin G(IgG)in the aqueous humour was confirmed by the ELISA technique. There was no correlation between antibody levels in the aqueous humour and the related serum. The histopathological study of the eyes showed the existence of lesions in various ocular structures. The ciliary processes, ciliary body, sclerocorneal limbus, iris and lacrimal duct showed intense inflammatory zones with lymphocyte infiltrates, plasmatic cells and macrophages with amastigote forms ofLeishmania.In addition vasculitis with dilation and thrombi were also detected in both cases, with consequent oedema and hyalinization. The immunohistochemistry analysis revealed the presence of granular and diffuse IgG deposits in the ciliary body, ciliary processes, sclerocorneal limbus and iris. Furthermore, numerous thrombosed vessels were observed in the sclerocorneal zone and iris. Complement 3(C3)fraction deposits were not present in the ocular structures. The present data suggest that the ocular lesions may have an immunopathological origi

ISSN:0141-9838    

DOI:10.1046/j.1365-3024.1996.d01-39.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Peripheral blood mononuclear cells (PBMC) from five chronic schistosomiasis patients, three former patients, a SEA sensitized individual, and normal controls were tested in lymphoblastogenesis assays for their ability to proliferate in response to soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) fromSchistosoma mansoni. Cells from all patients and the SEA sensitized individual gave significantly higher responses than the normal controls when stimulated with SEA and SWAP. However, the chronic patients’ SEA responses were much lower than those of the former patients and the SEA sensitized individual. When cells from the same donors were tested in thein vitrogranuloma assay, all produced significant granulomatous responses except the normal controls. Once it was established that all individuals in the study gave significant lymphoproliferative responses and granulomatous reactions, SEA was subjected to HPLC fractionation to identify immunogenic protein components of SEA. HPLC separation yielded 25 major fractions. SEA responses from the sensitized individual and former patients exhibited broad, unregulated responsiveness including fractions with neutral, less charged proteins while the chronic patients demonstrated a more restricted range of responsiveness. SEA‐HPLC fractions 14, 21, and 22 contain the most immunodominant proteins based on cellular proliferation data from reactive individuals tes

ISSN:0141-9838    

DOI:10.1046/j.1365-3024.1996.d01-41.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY