Specific immunoglobulin E antibodies to saprophytic yeasts in sera of atopic patients allergic to house dust mites.

BACKGROUND: Atopic dermatitis (AD) is a complex disease caused by genetic and environmental interactions. AD impairs skin barrier function, enabling microorganisms to penetrate and interact with the immune system. OBJECTIVES: This study aimed to investigate the presence of specific immunoglobulin (Ig) E antibodies to the yeast species Candida pelliculosa, Candida guilliermondii, Candida famata, and Rhodotorula rubra in the sera of AD patients and to evaluate possible cross-reactivity between yeasts and allergens of Dermatophagoides pteronyssinus (D1) and Dermatophagoides farinae (D2). METHODS: We analyzed serum samples from 14 healthy individuals and 34 AD patients: 19 were positive to D1 and D2 (Immulite) and 15 were negative. Determinations were made using enzyme-linked immunosorbent assay (ELISA), competitive ELISA, and the Immulite inhibition assay. RESULTS: The results of ELISA showed that all house dust mite-positive sera had specific IgE antibodies to the yeast species tested: 42% of these sera reacted with all 4 yeast species. The inhibition study demonstrated partial cross-reactivity between IgE class antibodies with the yeast species. This finding indicates that different Candida species and R rubra have species-specific and cross-reactive antigens with partially overlapping epitopes, thus suggesting cross-reactivity with mite allergens. C pelliculosa protein extract inhibited IgE binding to D1 (63.4%) and D2 (71%) allergens. The inhibition value for D1 showed a significant correlation with the inhibition value for D2 (r = 0.669, P = .03; Spearman rank correlation). CONCLUSION: The results of the present study suggest that C pelliculosa and house dust mites share common antigens.

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies.

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Immunochemical study of human immunoglobulin G Fc region.

Fifteen murine monoclonal antibodies (mAb) were produced that reacted with conformational epitopes present on the Fc portion of all human IgG subclasses (PAN-Fc). Inhibition and sandwich ELISA were used to elucidate overlapping and distinct epitopes. The epitopes were aligned to form a continuous "chain" of overlapping determinants from the CH2-CH3 interface to the direction of hinge. The five more hinge-proximal epitopes demonstrated high lability both in competitive and sandwich assays, being completely or partially destroyed when any of these 15 other mAb bound the IgG first. Heat-inactivation of IgG caused full disruption of these epitopes. In contrast, epitopes situated at the opposite distal of the "chain" were more stable and mAb binding could only be affected by occupying an overlapping epitope. Under heat-inactivation these epitopes were affected, but not completely destroyed. Human IgG class anti-DNA autoantibodies were bound to insolubilized dsDNA and their reaction with PAN-Fc mAb was studied. mAb titration plots on IgG and dsDNA-IgG were compared. Five epitopes proved to be altered by antigen (dsDNA) binding. Two of these were the labile hinge-proximal epitopes and the other three were situated near the CH2-CH3 interface. Cross-reactivity of mAb with xenogeneic IgG was also studied. An "epitope map" of the crystallographic model of human IgG Fc portion drawn was based on these experimental data and printed matter, concerning the location of subclass-specific amino acids and homology regions of human and animal IgG.

Type X collagen expression and hypertrophic differentiation in chondrogenic neoplasias.

Little is known about matrix biochemistry and cell differentiation patterns in chondrogenic neoplasms. This is the first description of the focal expression of collagen type X by neoplastic chondrocytes in situ and its incorporation into the extracellular matrix of cartilaginous tumors. This shows that neoplastic chondrocytes have the potential to undergo the full program of cell differentiation, including hypertrophy, comparable to their physiological counterparts in the growth plate. However, only in benign osteochondromas was a zonal expression of type X collagen found similar to that observed in the growth plate, where the cells immediately above the ossification frontier are selectively positive for type X collagen. In enchondromas and chondrosarcomas, the expression was randomly distributed within the tumors. Surprisingly, in less differentiated chondrosarcomas with spindle-shaped cells and non-cartilaginous extracellular matrix, exceptional expression of collagen type X was observed, which indicates potential uncoupling of collagen type X expression from the differentiated chondrocytic phenotype in neoplastic chondrocytes in vivo.

A rapid ELISA test for detection of human paraproteins.

A rapid ELISA test for detection, characterization and quantification of human paraproteins was developed. The proposed method is a sandwich ELISA, where the capture antibody is specific for a given heavy chain (gamma, alpha or mu) and the labelled antibody is specific either for kappa or for lambda light chain. Both standard and patient sera are tested with all six possible antibody combinations. Each paraprotein produces a significant increase in titre (as compared with standard) only when tested with the relevant pair of antibodies. This enables the determination of the isotype and light chain type of the paraprotein and the evaluation of its relative quantity in patient serum. The accuracy of the assay (relative deviation) varies from 0.04 for gamma lambda to 0.19 for alpha kappa. The cut-off values for each type of polyclonal immunoglobulin were determined with 200 healthy donor sera. 103 patient sera were analysed. ELISA data are in good agreement with M-component and other clinical data.

Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses.

Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.

Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells.

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.

Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling.

Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor-induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1(-/-)Vav-2(-/-) B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-independent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1(-/-)Vav-2(-/-) B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.

Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production.

OBJECTIVE: To investigate whether histone-specific T helper (Th) cells that are able to induce anti-double-stranded DNA (anti-dsDNA) antibodies can be isolated from patients with systemic lupus erythematosus (SLE) and to characterize the cytokine secretion pattern of such Th clones. METHODS: Peripheral blood mononuclear cells from SLE patients and healthy donors were stimulated with autologous apoptotic cell material or purified histones, expanded with interleukin-2 (IL-2), and cloned by limiting dilution. Histone reactivity of clones was examined by histone-specific proliferation and cytokine release. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Induction of anti-dsDNA antibodies was measured in cocultures of autologous B cells and Th clones by ELISA: RESULTS: Numerous histone-specific T cell receptor (TCR) alpha/beta+ Th clones were established from 2 of 3 patients with active SLE and from 1 of 2 healthy individuals. Most Th clones secreted IL-2, interferon-gamma (IFNgamma), and IL-4, whereas some produced predominantly IL-2 and IFNgamma. Th clones that could stimulate the production of anti-dsDNA antibodies were derived from SLE patients and from a healthy individual. CONCLUSION: Th cells specific for histones may play an important role in the pathogenesis of SLE by inducing autoantibodies to dsDNA. Both Th1 and Th2 cytokines may be involved in the pathogenesis of SLE. The presence of histone-specific Th cells in a healthy individual indicates the importance of peripheral tolerance for preventing autoimmunity to nuclear antigens.

The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site.

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.