Search PubMed⌕ Search

Biomedical subjects

G J Silverman

Publications and source records attributed to G J Silverman.

At least 19 recordsLinked to original sources
PubMed1998

B cell superantigens: possible roles in immunodeficiency and autoimmunity.

Lymphocyte antigen receptors have evolved for the discrimination of self and non-self structures and consequently the unconventional binding activities of superantigens pose a special set of potential hazards and opportunities for the immune system. This review presents recent evidence that certain naturally occurring proteins have the properties of B cell superantigens by virtue of their unconventional variable-region mediated interactions with soluble and membrane-bound immunoglobulins. The implications for B lymphocyte activation and clonal selection are discussed and we speculate how superantigens may influence the development of lymphocyte repertoires and the immune responses associated with certain autoimmune and immunodeficiency diseases.

Animals↗
PubMed1997

B-cell superantigens: molecular and cellular implications.

B cell superantigens are proteins that are capable of immunoglobulin variable region mediated binding interactions with the naive B cell repertoire at frequencies that are orders of magnitude greater than occur for conventional antigens. Within this review we discuss recent observations regarding the molecular basis of these interactions and the distribution of superantigen binding capacities in different human B cell populations. These findings and current predictions regarding the relevance of these proteins to the physiologic development of immune repertoires are also discussed.

Aging↗
PubMed1996

Repertoire cloning of lupus anti-DNA autoantibodies.

To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.

Amino Acid Sequence↗
PubMed1996

An endogenous sialoprotein and a bacterial B cell superantigen compete in their VH family-specific binding interactions with human Igs.

Staphylococcal protein A (SpA), a bacterial membrane protein, and protein Fv (Fv binding protein (pFv)), a human sialoprotein involved in gut-associated immunity, have both recently been shown to have unconventional V(H) family-restricted binding interactions with Igs. To determine whether these Ig binding proteins interact with related structures, we performed a series of comparative binding studies. The results confirmed that both molecules are bound by most V(H)3 IgM, but pFv is also recognized by V(H)3 and V(H)6 Ig that do not interact with SpA. We discovered that pFv and SpA (or a single domain of SpA) can compete for binding to a V(H)3 Ab, which suggests that they can recognize the same (or adjacent) V(H) sites. For both SpA and pFv, binding is less frequent among IgG than IgM. However, V(H)3 IgG more commonly possess Fab-mediated binding activity for pFv than for SpA. Binding studies of denatured Ig suggested that both pFv and SpA interact with conformationally dependent V(H) sites, although in certain cases pFv binding is more permissive than SpA binding. Taken together, these results indicate that the superantigen properties of SpA, a microbial protein, and those of pFv, an endogenous sialoprotein, involve binding interactions with overlapping and at times functionally equivalent sites in the V(H) domain, indicating that self and foreign proteins can employ highly conserved strategies to create superantigens for the Ag receptors of B lymphocytes.

Antigens, Bacterial↗
PubMed1996

Differential binding avidities of human IgM for staphylococcal protein A derive from specific germ-line VH3 gene usage.

Human IgM that express the variable region genes of the VH3 family bind staphylococcal protein A (SPA). We previously reported that the SPA-binding IgM can be divided into two groups based on the differential binding avidities for solid-phase SPA. To study the molecular basis for these differences, we cloned B cells from human blood by EBV transformation. The nucleotide sequences of the expressed Ig heavy chain genes were determined on 20 B cell clones that produce SPA-binding IgM. The germ-line VH3 gene usage in IgM with high avidities for SPA were distinct from the germ-line VH3 genes used in IgM with low avidities for SPA. There was no correlation in the usage of D or JH genes or in the usage of light chains in IgM according to the SPA binding avidity. These results suggest that the differential binding avidities for SPA are at least partly due to specific germ-line VH3 gene usage. An investigation of direct binding of SPA to the synthetic peptides corresponding to the portions of the variable regions of SPA-binding and non-SPA-binding IgM showed that the peptides corresponding to the VH3 family specific framework region 3 sequences had significant SPA binding capacities, while the peptides corresponding to the other subdomains and those corresponding to framework region 3 of the reported VH3 sequences from non-SPA-binding IgM showed little or no binding. It is of interest that the Ig-framework region 3 subdomain corresponds to the fourth hypervariable region, which in the TCR-beta chain has been implicated as a critical site for T cell superantigen binding.

Adult↗
PubMed1996

Complement activation by a B cell superantigen.

Staphylococcal protein A (SpA), acting as a B cell superantigen, binds to the Fab region of human VH3+ Igs. Using SpA abrogated of its IgG Fc binding activity (Mod SpA) as a model B cell superantigen, we determined whether such an interaction causes complement activation. Addition of Mod SpA to human serum led to complement consumption and the generation of C3a. To determine whether this complement activation 1) was due to an interaction between VH3+ Igs and the Fab binding site of SpA and 2) proceeded via the classical complement pathway, we tested a panel of monoclonal IgM proteins for the ability to hind C1q following interaction with SpA. C1q binding was restricted to SpA-reactive, VH3+ IgM proteins. To formally determine whether the binding of SpA to the reactive VH3+ IgM proteins led to complement activation, we reconstituted the serum from a hypogammaglobulinemic patient with monoclonal IgM proteins and measured complement consumption and C3a generation following the addition of Mod SpA. We observed complement consumption and C3a production only in Mod SpA-treated serum reconstituted with a VH3+, SpA-binding, IgM protein. Taken together, these results provide compelling evidence that the interaction of the Fab binding site of SpA and VH3+ Igs can lead to complement activation via the classical pathway. This novel interaction may have significant implications for the in vivo properties of a B cell superantigen.

Adult↗
PubMed1996

Immunostimulatory DNA sequences necessary for effective intradermal gene immunization.

Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-alpha, interferon-beta, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

Amino Acid Sequence↗
PubMed1995

VH3 family antibodies bind domain D of staphylococcal protein A.

Staphylococcal protein A (SpA) is a 45-kDa bacterial membrane protein that can interact with either Fc gamma, a constant region portion of IgG, or with the Fab portion that also mediates conventional Ag binding. In recent reports, SpA has been shown to specifically interact with Fab derived from the VH3 family and is little affected by VH CDR3, JH, or light chain usage. To identify a site on SpA responsible for VH3 Fab binding, we cloned and expressed in Escherichia coli the 61 amino acid sequence of SpA that represents domain D, and this small protein exhibited both the VH3 Fab and Fc gamma binding specificities. Surface plasmon resonance measurements demonstrated that domain D and native SpA had the strongest binding interactions with an IgM-kappa encoded by the germline configuration of the VH3 gene VH26c. In contrast, the apparent affinities for Fc gamma binding were at least fivefold weaker. A variant of domain D was also created that is devoid of the three-codon insertion that distinguishes domain D from all other domains in SpA. Although this deletion did not significantly affect the VH3 Fab-mediated SpA binding activity, it did improve the affinity of Fc gamma binding by an order of magnitude. These observations characterize a site on SpA responsible for binding interactions with B cell Ag receptors that are highly analogous to that of superantigens for T cell receptors.

Amino Acid Sequence↗
PubMed1995

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.

Amino Acid Sequence↗
PubMed1995

Preferred usage of specific immunoglobulin gene segments in chronic lymphocytic leukaemia cells of three HLA-identical sisters.

We report three family members, including a set of identical twins, who developed CD5 positive B-CLL. The patients are female Ashkenazi Jews sharing an identical HLA phenotype. Two of the HLA loci (B35 and Cw4) were common with those already described as being shared by Ashkenazi Jews with an increased incidence of CLL. The rearranged immunoglobulin genes of the malignant cells of all three patients were found to express genetically related VH regions belonging to the VH3 subgroup, and chromosomal studies suggest a process of clonal evolution in one of the twins.

Aged↗
PubMed1995

Superantigen properties of a human sialoprotein involved in gut-associated immunity.

Protein Fv (pFv) is a recently described 175-kD gut-associated sialoprotein with a potent capacity for augmentation of antibody-dependent immune functions. To investigate the molecular basis for Fab-mediated binding of pFv, we evaluated a panel of 52 monoclonal IgM and found that approximately 40% bound pFv. Whereas the majority (> or = 75%) of V H3 and V H6 IgM strongly bound pFv, only a small minority (< 20%) of IgM from other V H families bound pFv, and these antibodies had weaker binding interactions. Inhibition studies suggested that all binding occurred at the same (or overlapping) site(s) on pFv. Surface plasmon resonance studies demonstrated binding affinity constants up to 6.7 x 10(8) M-1 for pFv. Biopanning of IgM and IgG Fab phage-display libraries with pFv preferentially selected for V H3 and V H6 antibodies, but also obtained certain V H4 IgM. V H sequence analyses of 36 pFv-binding antibodies revealed that binding did not correlate with CDR sequence, JH, or L chain usage. However, there was preferential selection of pFv binders with V H CDR3 of small size. These studies demonstrate that a protein which enhances immune defense in the gut has structural and functional properties similar to known superantigens.

Amino Acid Sequence↗
PubMed1993

Age-associated changes in binding of human B lymphocytes to a VH3-restricted unconventional bacterial antigen.

We have recently demonstrated that there is a site on Staphylococcal protein A (SpA) that interacts with B cell Ig receptors in a manner comparable with known T cell superantigens, because this binding specificity is restricted to Fab with VH3 H chains and most VH3 Ig bind SpA. In the present studies, SpA was used as a phenotypic marker for VH3 expression by human lymphoid cells. As expected, this Fab-mediated binding specificity was completely inhibited by certain VH3 antibodies but not by antibodies from other VH families. In multiparameter flow cytometric analyses, this binding activity was demonstrated to be highly prevalent among B cells (14 to 54%), and was more common among IgM-bearing B cells compared with IgG-bearing B cells. In all studies, Fab-mediated binding of SpA was uniformly expressed by a greater proportion of CD5-positive B cells than CD5-negative B cells. The proportion of B lymphocytes with this VH3-restricted binding capacity was found to undergo age-associated changes, because a large proportion of the peripheral B cells of neonates (mean +/- SD, 46.0 +/- 2.9%) bind this site, but two 10-mo-old subjects and older children had significantly lower binding levels (29.0 +/- 3.5%) that were the same as binding levels by adult peripheral B lymphocytes (30.2 +/- 3.3%). In immunohistochemical studies, tonsilar B cells that bind this site on SpA were shown to be common in mantle zones and germinal centers of secondary follicles. We speculate that Fab-mediated SpA binding represents a fundamental and primitive binding capacity that is part of the human preimmune repertoire, and we discuss the implications for the observed age-dependent shift in Fab-mediated binding of SpA by peripheral blood B cells.

Adult↗