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W V Williams

Publications and source records attributed to W V Williams.

At least 55 records · Page 3Linked to original sources
PubMed1994

T cell receptor analysis in rheumatoid arthritis: what have we learnt?

Many clues point to a role for T lymphocytes in the pathogenesis of rheumatoid arthritis (RA), although the importance of these cells and their position within the rheumatoid pathogenic scheme remain unknown. Encouraged by data from animal models of T-lymphocyte-mediated autoimmunity, a major focus of research into the role of T lymphocytes in RA has been the usage of T cell receptor V genes in rheumatoid synovitis. Despite many methodologic problems, involving choice of patients and controls, choice of specimens, and technical factors, several conclusions can be drawn from the published research. In particular, synovial T lymphocyte populations, as a whole, frequently show biased V gene usage and restricted clonality within those T lymphocyte subsets that utilize over-represented V gene families. Continued research into these synovial T lymphocyte subsets should provide important insights into the pathogenesis of RA, particularly if solutions to the identified methodologic problems are implemented.

Arthritis, Rheumatoid↗
PubMed1994

Granulocyte-macrophage colony-stimulating factor mimicry and receptor interactions.

Development of small molecular mimics of larger polypeptide ligands is an important approach to pharmacophore design. One strategy for the development of such mimics is analysis of alternative ligands that bind to the same site as the native ligand. These allow examination of the structural and chemical constraints for binding within the setting of diverse backbone geometries. The use of antireceptor antibodies as alternative ligands has allowed the development of biologically active peptides in several ligand-receptor systems. This technology has been applied to the study of interactions between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR). GM-CSF is one of a family of signal-transducing cytokines and growth factors characterized by a four-helix bundle core structure. The GM-CSFR is comprised of an alpha-chain (GM-CSFR alpha) specific for GM-CSF, and a beta-chain (beta c) shared with the interleukin-3 and interleukin-5 receptors. At least two sites on GM-CSF have been implicated in the GM-CSF-GM-CSFR alpha/beta c ternary complex. In studies summarized here, synthetic peptide analogs of GM-CSF sequences were designed and used to map neutralizing epitopes. One neutralizing epitope corresponded to the A helix of GM-CSF, and a synthetic analog displayed biological activity as a GM-CSF antagonist in vitro, suggesting interaction with the GM-CSFR alpha/beta c complex. A second peptide comprising the B and C helices was recognized by monoclonal neutralizing antibodies and similarly displayed antagonist activity. Recombinant antibody (rAb) technology was also employed. An expression library of rAbs from mice immunized with neutralizing anti-GM-CSF antibodies was developed and screened with a neutralizing anti-GM-CSF monoclonal antibody. One clone which displayed receptor binding activity exhibited structural similarity with epitopes on GM-CSF previously implicated as interaction sites with the neutralizing monoclonal antibody. A synthetic peptide analog of the rAb inhibited GM-CSF bioactivity. Critical contact residues were predicted on the basis of structural similarity of the rAb peptide and GM-CSF. These studies indicate the feasibility of using rAbs in bioactive peptide design, providing lead compounds and information regarding contact residues for drug design.

Amino Acid Sequence↗
PubMed1994

Immunotherapeutic strategies targeting rheumatoid synovial T-cell receptors by DNA inoculation.

Immunotherapy against autoreactive T-cell receptors (TCRs) has been reported to have promise in several animal models of autoimmune diseases. Facilitated DNA inoculation has many potential advantages as a modality for development of specific immune responses. Specifically, this technology is able to deliver exogenous antigens for processing via both the endogenous pathway, with subsequent presentation by class-I major histocompatibility (MHC) antigens, and the exogenous pathway, with subsequent presentation by class-II MHC antigens. This allows for induction of both arms of the cellular immune system. These cellular immune responses may be particularly important in targeting and controlling pathogenic cell populations. The application of this technology to the treatment of human autoimmune diseases depends on the availability of readily manipulated systems for the evaluation of specific interventions. Here we report the full length cloning and expression of TCRs from rheumatoid arthritis synovial tissue. These were developed by recombinant polymerase chain reaction, cloning and retroviral transduction into a TCR-alpha/beta-negative murine T-cell hybridoma. Reconstitution of CD3 expression was confirmed by flow cytometry. Similar constructs have been developed for TCR-based immunotherapy by facilitated inoculation of DNA intramuscularly. Preliminary analysis of immune responses in mice indicates that these constructs elicit anti-TCR responses. These studies indicate the ability to reconstitute expression of potentially autoreactive human TCRs in a model system wherein specific immune responses elicited against these TCRs by various immunogens can be evaluated.

Amino Acid Sequence↗
PubMed1994

Structural properties of a subset of nephritogenic anti-DNA antibodies.

Structural analysis of lupus autoantibodies is beginning to provide clues to the molecular basis for antigenic specificity and pathogenicity. The present analysis indicates that multiple light and heavy chains contain residues which can facilitate DNA binding, reaffirming the notion that there are multiple ways that different amino acids combine to form an antigen-binding pocket with affinity for dsDNA and ssDNA. Furthermore, this analysis suggests that these conformations and contact residues are intrinsic to germline sequences, although amino acid changes at critical locations (somatically introduced) modulate antigen binding, and appear to influence the capacity of individual immunoglobulin to form immune deposits. Analysis of additional individual immunoglobulins with closely related V-region sequences and differing pathogenic properties will be required to resolve the contribution of specific motifs to pathogenecity.

Amino Acid Sequence↗
PubMed1994

Cloning and biological characterization of human single-chain Fv fragments that mediate neutralization of HIV-1.

OBJECTIVE: To develop recombinant single-chain Fv fragments against HIV-1 gp120. METHODS: A panel of human monoclonal antibody Fv fragments were generated against the HIV-1 gp120 by affinity selection from an antibody library expressed on the surface of filamentous phage. The library was prepared from peripheral blood lymphocytes of an asymptomatic HIV-1-infected mother with a high neutralization titer. This mother did not transmit HIV-1 to her offspring (non-transmitter). Heavy and light chains were initially amplified separately and combined by splicing by overlap extension to generate Fv fragments. RESULTS: Several clones expressing single-chain Fv fragments bind strongly to HIV-1 gp120 and several were found to neutralize cell-free HIV-1IIIB. Gross epitope mapping suggest that different clones bound to different functional regions on the envelope. The clones also exhibited sequence diversity. CONCLUSIONS: This strategy of cloning resulted in the development of functional human-derived antibody reagents with different anti-HIV-1 biological properties in vitro. These recombinant Fv fragments have potential utility as immune reagents, as well as in the design of potential immunotherapeutics. In addition, these antibody reagents may provide information on the relationship between humoral immunity and maternal-fetal (vertical) HIV-1 transmission.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.

Adsorption↗
PubMed1994

Prolactin receptor levels on lymphocytes vary with menstrual cycle in women.

Prolactin exerts effects on the immune system, potentiating T-cell growth in response to various stimuli. These effects are felt to be mediated by specific receptors present on lymphocytes for prolactin. While the presence of prolactin receptors (PRLRs) on lymphocytes is well accepted, potential fluctuations in these receptor levels in individuals have not been studied. Here we present evidence that lymphocyte prolactin levels vary throughout the menstrual cycle in females. Lymphocyte PRLR levels varied up to 10-fold as determined by radioreceptor assay on the different days of the menstrual cycle. A semi-quantitative PCR technique was used to measure message levels, and these generally correlated with radioreceptor results. We conclude that lymphocyte PRLR levels vary throughout the menstrual cycle and that these variations are due at least in part to variations in PRLR mRNA levels.

Adult↗
PubMed1994

Isolated VH4 heavy chain variable regions bind DNA characterization of a recombinant antibody heavy chain library derived from patient(s) with active SLE.

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibodies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. While some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V regions to bind DNA in isolation has not been investigated. We have utilized a bacterial vector for cloning and expressing isolated antibody heavy chain variable regions. RNA was extracted from peripheral blood mononuclear cells of patients with active SLE, cDNA synthesized and heavy chain V regions amplified with VH specific oligonucleotide primers. The VH fragments were cloned into a bacterial expression plasmid including the pelB leader peptide to direct appropriate expression. Recombinant antibodies were screened for binding to 32P-labeled double-stranded plasmid DNA and later also characterized for binding to single-stranded DNA. Binding was confirmed by standard ELISA methodology. Sequence analysis of seven DNA binding VH fragments revealed that they utilized the VH gene family previously described to be associated with autoimmune responses, with a JH6 segment. On VH sequence analysis only one residue substitution in the consensus sequence is needed to form a VH4 germline gene. Potential contact residues with DNA were delineated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of VH regions can be examined in the absence of light chain. DNA binding specificity appears to be a property of the germline VH4 gene. Analysis of such V regions can aid in the identification of hypervariable region contact residues important for DNA binding.

Amino Acid Sequence↗
PubMed1994

DNA inoculation induces protective in vivo immune responses against cellular challenge with HIV-1 antigen-expressing cells.

Direct DNA inoculation induces immune responses through the delivery of nonreplicating transcription units that drive the synthesis of specific foreign proteins within the inoculated host. These proteins are processed within host cells and through association with relevant MHC antigens that can become the subject of immune surveillance and elicit immune responses against pathogens. Direct introduction of DNA into mice has been reported to be antigenic as demonstrated by the use of this technique to develop immune responses against human growth hormone, influenza proteins, as well as HIV-1 proteins. Most recently the demonstration of the use of this technology to produce anti-HIV-1 immune responses has been reported in nonhuman primates. Accordingly a more detailed analysis of this technology could generate important insight into the generality of this approach for immune therapy or vaccine design. In this article we further our investigation of direct DNA inoculation as a tool for induction of relevant immune responses against HIV-1 in vivo. We demonstrate expression of HIV-1 antigens in the inoculated muscle of animals. Inoculated animals demonstrate significant cytotoxic T cell responses against HIV-1 antigen-expressing targets. Furthermore, using a novel challenge system, we demonstrate that the majority of immunized animals can reject lethal, HIV-1 antigen-expressing cell challenge in an antigen-specific manner. This technology has relevance for the development of immunization strategies against HIV as it provides for specific antigen production in vivo without the use of infectious agents.

AIDS Vaccines↗
PubMed1993

Gene inoculation generates immune responses against human immunodeficiency virus type 1.

Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.

Amino Acid Sequence↗
PubMed1993

Induction of cell differentiation by human immunodeficiency virus 1 vpr.

Cell lines from rhabdomyosarcomas, which are tumors of muscle origin, have been used as models of CD4-independent HIV infection. These cell lines can be induced to differentiate in vitro. We report here that the vpr gene of HIV1 is sufficient for the differentiation of the human rhabdomyosarcoma cell line TE671. Differentiated cells are characterized by great enlargement, altered morphology, lack of replication, and high level expression of the muscle-specific protein myosin. We have also observed the morphological differentiation and inhibition of proliferation of two other transformed cell lines. vpr-transfected cells remain fully viable in culture for extended periods. These observations elucidate a potential role for vpr in the virus life cycle and raise the possibility that some aspects of HIV-induced pathologies may be caused by a disturbance of cells by vpr.

Cell Differentiation↗
PubMed1993

Subset-specific effects of sex hormones and pituitary gonadotropins on human lymphocyte proliferation in vitro.

Clinical observations on patients with systemic lupus erythematosus and rheumatoid arthritis and studies in murine models of autoimmune diseases suggest an important role for the sex and pituitary hormones in immune function. Estrogen and testosterone have been shown to modulate B cell functions in vitro. Prolactin (PRL) has been shown to have immunomodulatory functions. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) have homology to thioredoxin, a potentiator of T-cell growth. We therefore studied the proliferative response of peripheral blood mononuclear cells (PBMC) from normal adult males to various stimuli in the presence or absence of estradiol (E2), testosterone (Te), PRL, FSH, and LH and the effect of these hormones on T-cell subsets by flow cytometry. Unfractionated PBMC were stimulated with 1% phytohemagglutinin (PHA) (polyclonal activation), anti-CD3 (T-cell receptor stimulation), or recombinant interleukin 2 (IL-2) (late stages of activation). We assessed the effects of E2 (0.3-30 ng/ml), Te (3-300 ng/ml), PRL (2-200 ng/ml), FSH (1-100 mIU/ml), and LH (1-100 mIU/ml). E2 and Te had no consistent effect on PBMC proliferation in response to any of the stimuli. E2 significantly decreased the percentage of CD4+ cells following PHA stimulation (P = 0.022), while significantly enhancing the percentage of CD8+ cells following IL-2 stimulation (P = 0.007). Te significantly increased the percentage of CD4+ cells following IL-2 stimulation (P = 0.03). PRL enhanced proliferation in response to IL-2 and PHA without subset-specific effects. FSH and LH both enhanced IL-2-induced proliferation, particularly in physiological doses (10 mIU/ml). FSH at 100 mIU/ml significantly decreased the percentage of CD4+ cells in unstimulated cultures (P = 0.003), while it enhanced the percentage of CD8+ cells following PHA stimulation (P = 0.004). The enhancement in CD8+ cells appeared the most marked in the CD8+CD28+ subset. LH at 10 mIU/ml significantly enhanced the percentage of CD4+ cells following IL-2 stimulation (P = 0.009) and at higher doses (100 mIU/ml) enhanced the percentage of CD4+ cells following PHA stimulation (P = 0.011). Thus, sex steroids and adenohypophyseal hormones (PRL, FSH, and LH) have subset-specific effects on T-cell activation which may influence sex-related differences in immune response.

Adult↗
PubMed1993

The galactosyl ceramide/sulfatide receptor binding region of HIV-1 gp120 maps to amino acids 206-275.

Our recent studies have indicated that galactosyl ceramide (GalCer) or sulfatide (sul) may serve as an alternate receptor for human immunodeficiency virus (HIV) in neural cells. In this paper, we describe the mapping of GalCer/sul binding region of HIV env glycoprotein gp120. Deglycosylated gp120 binds to GalCer, suggesting that the amino acids of glycoprotein gp120 and not the carbohydrates are responsible for the observed binding. Specific regions of gp120 responsible for the binding were analyzed by using varying-length truncations of gp120 expressed in Escherichia coli and vaccinia virus. Purified recombinant gp120 containing amino acids 200-295 of gp120 bind to GalCer/sul, whereas recombinant env proteins that deleted this region did not bind. These recombinant proteins also bind to SK-N-MC-derived neuroblastoma cells, the binding of which is inhibited by anti-GalCer. In addition, 125I-labeled gp120 binding to GalCer is inhibited by these proteins. Studies using lysates containing truncated gp120 expressed in vaccinia virus also gave similar results. By eliminating the overlapping regions that do not bind, we conclude that the amino acids responsible for GalCer/sul binding reside between amino acids 206 and 275. The significance of this mapping is discussed in relation to the neurotropism of HIV.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, expression, and biological characterization of an HTLV-II envelope glycoprotein: HIV-1 expression is permissive for HTLV-II-induced cell fusion.

The human T cell leukemia virus II (HTLV-II) is a type C retrovirus closely related to the human transforming retrovirus HTLV-I. In contrast to HTLV-I, the role of HTLV-II in human disease is controversial. However, HTLV-II infection has been documented in several cases of a clinically benign hairy cell leukemia and has also been suggested as a cofactor for HIV-1 disease progression. We report that an HTLV-II isolate (designated FLW) derived from a serum-positive white male can induce cell fusion and significant cytopathic effects in tissue culture. This HTLV-II isolate induced syncytium formation with human T and B cell lines, several human fibroblast cell lines, and, interestingly, HIV-1- and HIV-2-infected cell lines. To elucidate the role in the FLW envelope in this phenomenon, we have cloned the envelope glycoproteins gp46 and gp21 of this isolate. The envelope glycoproteins expressed in the absence of the rest of the viral genome were sufficient to drive syncytium formation in vitro, and preserved the cellular tropism for syncytium formation observed with the native retroviral isolate. Amino acid (aa) sequence analysis demonstrated 88% overall similarity with other HTLV-II envelope glycoproteins. Interestingly, only cells infected by HIV-I, but not parental H9 cells, form syncytia with FLW env-transfected cells as well as with HTLV-II/FLW-infected BJAB-WH cells. Furthermore, antibodies directed at the CD4 receptor failed to inhibit the induction of giant cell formation, implying that the FLW envelope protein was responsible for driving syncytium formation in this system. These observations may be important for the understanding of the processes involved in human retroviral-mediated syncytium formation and may suggest a mechanism whereby HTLV-II could influence the disease process in individuals dually infected with HIV-1 and HTLV-II.

Amino Acid Sequence↗
PubMed1993

Conserved motifs in rheumatoid arthritis synovial tissue T-cell receptor beta chains.

Rheumatoid arthritis is genetically linked to major histocompatibility complex (MHC) molecules (HLA-DR4 and related molecules) and characterized pathologically by high levels of HLA-DR expression and infiltration of proliferative of synovial tissue with CD4+ T lymphocytes. T-lymphocyte activation is driven by specific signaling through polymorphic alpha/beta T-cell receptors (TCRs) that are reactive with antigen-MHC complexes present at the sites of inflammation. We are interested in characterizing rheumatoid TCRs molecularly to ascertain potential binding surfaces for antigen+MHC in synovial tissue. Accordingly, we have recently investigated the TCR alpha and beta chain heterogeneity in a series of 10 rheumatoid synovia obtained at the time of joint surgery. The most frequently detected V beta families were V beta 12, 14, and 17, each of which was found in 80% of specimens. We report here the molecular cloning and sequence analysis of 20 cloned V beta segments amplified with a V beta 14 family-specific TCR primer, and six cloned V beta segments amplified with a V beta 17 family-specific TCR primer from four rheumatoid synovia. Comparison with the data base revealed that these sequences belonged to the closely related V beta 3, V beta 14, and V beta 17 families. Dominant clones were apparent in two of the individuals by the presence of identical V-D-J regions, suggesting an antigen-driven process. Amino acid sequence analysis revealed a conserved motif in the putative fourth hypervariable region or CDR4. Molecular modeling of this epitope suggests that charged side chains are available for binding to ligand structures (e.g., antigen, MHC, or superantigen). We suggest this epitope may play a role in the molecular pathogenesis of rheumatoid arthritis.

Amino Acid Sequence↗
PubMed1993

Genetic infection induces protective in vivo immune responses.

Drug-induced abortive retroviral infection has been reported to induce both T-cell and B-cell immunity in vivo. We sought to analyze if replication-incompetent retroviruses could induce the development of similarly protective in vivo immune responses in a more desirable fashion. To evaluate retroviral transduction vaccination (genetic infection), a plasmid encoding human CD4 in a retroviral vector was transfected into the pA317 amphotropic retroviral packaging system. The resulting replication defective retrovirus was used to transduce BALB/c mice prior to tumor challenge with human CD4. Immunization elicited specific humoral and cellular anti-human CD4 responses. We evaluated anti-cell responses using a tumor model system. We observed that BALB/c mice challenged with SP2/0 lymphoma cells develop lethal tumors and die within 7 weeks of challenge. Cloned SP2/0 cells stably transfected with the human cell-surface antigen CD4 also develop tumors in naive mice and succumb to the tumors in a similar manner to SP2/0 inoculated animals. In contrast, CD4 retrovirus-transduced animals, when challenged with the CD4-expressing SP2/0 cells, demonstrated a low incidence of tumors and significantly enhanced survival compared to the mice immunized similarly with human CD8 retrovirus. These results establish an in vivo tumor challenge system with relevance to the development of protective in vivo immune responses, and indicate that genetic infection is a useful technique for inducing protective immunity.

Animals↗