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Immunological Characterization (immunological + characterization)
Selected AbstractsAcute lymphoblastic leukemia with the phenotype of a putative B-cell/T-cell bipotential precursorAMERICAN JOURNAL OF HEMATOLOGY, Issue 2 2004Lee Gong Lau Abstract Biphenotypic acute leukemias (BALs) are uncommon. Most are of myeloid-B-cell or myeloid-T-cell lineage. We report herein a 70-year-old man with an unusual acute leukemia where the blasts expressed both B- and T-lymphoid markers. He presented to us with an enlarging cutaneous tumor. The presenting peripheral blood and bone marrow aspirate showed 40% and 90% blasts, respectively, which were negative for the usual cytochemical stains. The flow cytometric analysis revealed that the blasts were positive for CD19, CD20, CD22, cytoplasmic (Cyt) CD79a, CD10, Cyt CD3, CD5, CD7, CD4, HLA-DR, TdT, and were negative for myeloid markers. According to the scoring system from the European Group for the Immunological Characterization of Acute Leukaemias (EGIL), this case was an unequivocal B-cell/T-cell BAL. Conventional cytogenetic analysis revealed 46XY [t(4;11)(q31;q13), add(8)(q24), der(9)del(9)(p21)del(9)(q32q34), ,13, +mar] in all 25 metaphases analyzed. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) for 11q23 rearrangements as well as t(9;22) were negative. PCR for both TCR- , and IgH gene analyses revealed polyclonal rearrangements. We postulate that this case of BAL might have arisen from the putative common lymphoid progenitor cell. Am. J. Hematol. 77:156,160, 2004. © 2004 Wiley-Liss, Inc. [source] Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopesCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009J. Zakzuk Summary Background Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens. Objective To evaluate the differences in the IgE response against two Blo t 12 isoallergens. Methods The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens. Results The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes. Conclusion In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy. [source] Peptide based vaccine design: Synthesis and immunological characterization of branched polypeptide conjugates comprising the 276,284 immunodominant epitope of HSV-1 glycoprotein DJOURNAL OF PEPTIDE SCIENCE, Issue 3 2002Gábor Mez Abstract The importance of the length and conjugation site of a protective epitope peptide (276SALLEDPVG284) from glycoprotein D of herpes simplex virus in branched polypeptide conjugates has been investigated. A new set of peptides, with a single attachment site and truncated sequences, was prepared. The immunogenicity of conjugates and the specificity of antibody responses elicited were investigated in BALB/c, C57/Bl/6 and CBA mice. It was found that the covalent coupling of the peptide comprising the 276,284 sequence of gD through its Asp residue at position 281 did not influence the immunogenic properties of the epitope, while involvement of the side chain of Glu at position 280 almost completely abolished immunogenicity. These results clearly indicated that the conjugation site of the epitope peptide influenced the intensity and specificity of antibody responses. Comparison of the immunological properties of conjugates containing truncated gD peptides revealed the presence of two epitopes within the 276,284 region. One of the proposed epitopes is situated at the N -terminal (276,281) region, while the other is located at the C -terminal end of the sequence (279,284). Binding data demonstrated that some of the peptides comprising these epitopes induced gD-specific responses in their conjugated form and also elicited an immune response that conferred protection against lethal HSV-1 infection. The correlation of peptide- and gD-specific antibody responses with the protective effect of the immune response is discussed. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Molecular and immunological characterization of Asp f 34, a novel major cell wall allergen of Aspergillus fumigatusALLERGY, Issue 8 2009A. G. Glaser Background:, Although fungal spores have been recognized as triggers of respiratory allergy and asthma, only two allergenic fungal cell wall components have so far been described. Methods:, Eighty-one sequences derived from an Aspergillus fumigatus cDNA library encoding putative allergens were examined for the presence of cell wall components. A new allergen (Asp f 34) was evaluated by Western blots, enzyme-linked immunosorbent assay (ELISA), peripheral blood mononuclear cell (PBMC) proliferation assays, and skin prick test (SPT). Results:, The cDNA encoding Asp f 34 contained an open reading frame predicting a protein of 185 amino acids with a molecular weight of 19.38 kDa, showing sequence homology to phiA, an essential protein for the formation of conidia in the genus Aspergillus. The recombinant Asp f 34 was binding IgE from sensitized individuals in Western blots. An ELISA survey showed that 94% of the ABPA and 46% of the A. fumigatus -sensitized individuals tested had Asp f 34-specific serum IgE. Asp f 34 induced allergen-specific proliferation exclusively of PBMCs from patients sensitized to the allergen. Eight patients with anti-Asp f 34 serum IgE tested reacted positively in SPT, whereas four A. fumigatus -sensitized individuals without Asp f 34-specific IgE and eight healthy controls scored negatively. Conclusions:, A cell wall protein of the phialides of A. fumigatus was identified as a major allergen. Asp f 34 belongs to the Aspergillus -specific proteins of the phiA family and has relevant potential for a specific diagnosis of Aspergillus sensitization. [source] Standardization of allergen products: 1.ALLERGY, Issue 7 2009Detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation Background:, Standardization of allergen extracts requires the availability of well-characterized recombinant allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Methods:, Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Results:, Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4°C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Conclusion:, Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1. [source] Molecular and immunological characterization of the C-terminal region of a new Echinococcus granulosus Heat Shock Protein 70PARASITE IMMUNOLOGY, Issue 3 2003E. Ortona SUMMARY By screening an expression library of Echinococcus granulosus with IgE from sera of patients with cystic echinococcosis (CE) and allergic reactions, we isolated the C-terminal region of a new heat shock protein (HSP)70 of E. granulosus. The protein, named Eg2HSP70, has close homology with the C-terminal region of Dermatophagoides farinae and human HSP70. We investigated the humoral and cell-mediated immune responses to this antigen in patients with CE grouped according to the presence of allergic reactions. Immunoblotting detected total IgG, IgG4 and IgE specific to Eg2HSP70 (83% of sera contained IgG, 31% IgG4 and 57% IgE). No significant difference was found in immunoglobulin percentages according to the presence of allergic reactions. Immunoblotting inhibition showed that no IgG or IgE specific to Eg2HSP70 cross-reacted with D. farinae or human HSP70. Eg2HSP70-stimulated PBMC from 26 patients produced significantly greater amounts of TNF-,, IFN-,, and IL-10 than unstimulated cultures in all patients, irrespective of the presence of allergic reactions (P < 0·05). They also produced significantly greater amounts of IL-4 than unstimulated cultures exclusively in patients with allergic reactions (P < 0·05). These findings show that Eg2HSP70 is a new antigenic molecule inducing both B and T cell responses. [source] Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB,MPR649,684PLANT BIOTECHNOLOGY JOURNAL, Issue 2 2009Nobuyuki Matoba Summary Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB,MPR649,684[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB,MPR649,684 expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB,MPR649,684 proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N- glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine,X,serine/threonine (Asn-X-Ser/Thr) N- glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR649,684 moiety. Furthermore, the protein induced mucosal and serum anti-MPR649,684 antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate. [source] Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopesCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009J. Zakzuk Summary Background Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens. Objective To evaluate the differences in the IgE response against two Blo t 12 isoallergens. Methods The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens. Results The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes. Conclusion In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy. [source] Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5CLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2006Ĺ. Marknell DeWitt Summary Background Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein. Objective To clone and characterize timothy grass pollen allergen Phl p 4. Methods Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3,-and 5,-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies. Results Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner. Conclusions Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4. [source] Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cellsCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2000Jacquet Background The major house dust mite allergen Der p 1 elicits strong IgE antibody responses in patients suffering from mite allergy. Objective This study reports the expression and characterization of a recombinant precursor form of Der p 1 secreted as ProDer p 1 from insect cells. Methods The cDNA coding for ProDer p 1 was cloned downstream to the gp67 signal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplified ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mite culture, in terms of glycosylation, enzymatic activity as well as IgG- and IgE-binding capacity. Results Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns were also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydrate analysis clearly indicated that ProDer p 1 expressed from insect cells was glycosylated and that glycan structures were located only in the prosequence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclonal and polyclonal IgG antibodies compared to natural Der p 1. Specific activity measurements using synthetic substrates clearly indicated that, contrary to natural Der p 1, ProDer p 1 was totally enzymatically inactive. Conclusions The expression of an enzymatically inactive and highly antigenic ProDer p 1 zymogen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy. [source] | ||||||||||