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Biomedical subjects

G Baier

Publications and source records attributed to G Baier.

At least 73 records · Page 4Linked to original sources
PubMed1995

Immunoregulatory activity of a T-cell receptor alpha chain demonstrated by in vitro transcription and translation.

Previous studies from our laboratory and those of others suggested the possibility that the T-cell antigen receptor alpha (TCR alpha) chain from some T cells can be released in a soluble form and can have antigen-specific immunoregulatory activity. We have analyzed this phenomenon by in vitro transcription and translation (IVTT) of a cDNA encoding a TCR alpha chain (A1.1 TCR alpha) suspected of having such activity. We found that TCR alpha, but not TCR beta, protein produced in this way showed antigen-specific regulatory activity in an in vitro immune-response assay. Protein derived from truncated forms of the A1.1 TCR alpha cDNA had activity providing that, in addition to the variable (V) and joining (J) regions of the alpha chain (VJ alpha), at least the first 25 amino acids of the alpha chain of the constant (C) region (C alpha) were present. Addition of an irrelevant protein sequence to the VJ alpha failed to impart activity to the molecule, suggesting that the C alpha requirement is not simply for stabilization of the resulting protein. These results are discussed in the context of other recent studies on the immunoregulatory activity of soluble TCR alpha molecules, and the possible physiological relevance of these observations is considered.

Amino Acid Sequence↗
PubMed1995

The role of p56lck in CD4-mediated suppression of CD3-induced early signaling events in T lymphocytes.

Crosslinking of the CD4 coreceptor can inhibit subsequent T-cell activation via the T-cell antigen receptor (TCR)/CD3 complex. The ability of the human immunodeficiency virus (HIV) envelope protein, gp 120, to cause similar inhibition has implicated this inhibitory signal in the induction of T-cell anergy and apoptosis observed in the acquired immunodeficiency syndrome (AIDS). In order to clarify the biochemical basis of this inhibition, we analyzed the effect of CD4 ligation on early signaling events induced by subsequent CD3xCD4 co-crosslinking. By comparison with CD3 crosslinking alone, CD3xCD4 co-crosslinking of a CD3+CD4+ human T-cell leukemia line (SupT1) resulted in an enhanced increase in free intracellular calcium concentration and tyrosine phosphorylation of several cellular substrates, including the prominent phosphorylation of an unidentified 120-kDa protein (p120). Prior CD4 ligation inhibited these responses. Similar results were obtained with A3.01, another CD3+CD4+ T leukemic line. However, P120 was only minor phosphorylated on tyrosine upon receptor crosslinking in A2.01/CD4(-cyt401), a derivative line expressing a truncated CD4 coreceptor lacking its cytoplasmic domain which binds the p56lck protein tyrosine kinase (PTK). Furthermore, prior CD4 ligation failed to inhibit in this line the increased tyrosine phosphorylation induced by subsequent CD3xCD4 co-crosslinking. Thus, prior CD4 crosslinking, or expression of truncated CD4, are both associated with reduced p120 phosphorylation. These results suggest that p120 is a p56lck substrate playing an important role during T-cell activation.

CD3 Complex↗
PubMed1995

[Cicatricial pemphigoid--a therapeutic problem].

Cicatricial pemphigoid is an autoimmune subepidermal blistering disease of the skin and mucous membranes. We report on eight patients in whom the clinical diagnosis was confirmed by direct immunofluorescence. The patients' age averaged 69 years; seven were female and one male. Initial symptoms were erosions of the oral mucous membranes in four patients, skin blisters in three patients, and in one patient an ocular involvement was the first manifestation of the disease. Indirect immunofluorescence on NaCl-split human skin revealed circulating IgG antibodies binding to the roof of the artificial blister in three patients. Immunoblotting of epidermal and dermal extracts disclosed binding of IgG antibodies of one of these patients to an epidermal 230-kD protein, whereas IgA-antibodies showed no specific binding. In four of five patients with a strong ocular involvement IgA deposits were found by direct immunofluorescence. These studies were done on biopsies of perilesional skin or oral mucous membranes, and they were positive in one patient even before the first ocular lesions appeared. Therefore, finding of IgA deposits by direct immunofluorescence may be taken as a prognostic criterion allowing selection of the proper treatment. We treated six patients with a dexamethasone-cyclophosphamide pulse therapy, while two patients received dapsone orally. The involvement of skin and oral mucous membrane responded well to both regimens, whereas the ocular lesions were progressive, except in one patient. On the basis of our eight cases, we discuss both options and limitations in the treatment of cicatricial pemphigoid and review new aspects of the pathogenesis of this disease.

Aged↗
PubMed1995

A phage display system for detection of T cell receptor-antigen interactions.

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.

Amino Acid Sequence↗
PubMed1995

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway.

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Animals↗
PubMed1995

Immunogenic targeting of recombinant peptide vaccines to human antigen-presenting cells by chimeric anti-HLA-DR and anti-surface immunoglobulin D antibody Fab fragments in vitro.

To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system.

AIDS Vaccines↗
PubMed1994

Expression and biochemical characterization of human protein kinase C-theta.

In this study, the recently identified human protein kinase C-theta (PKC-theta) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-theta identified an 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC-theta was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC-theta with a representative member of the Ca(2+)-dependent PKC enzymes, full-length cDNAs encoding PKC-theta or PKC-alpha were transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC-theta and PKC-alpha. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC-derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC-alpha, however, full activation of PKC-theta did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelin-basic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-alpha, was a poor substrate for PKC-theta. Similar to PKC-alpha, transient PKC-theta overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbol-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-theta suggest that it may play a specialized role in T-cell signaling pathways.

Animals↗
PubMed1994

p56lck-independent activation and tyrosine phosphorylation of p72syk by T-cell antigen receptor/CD3 stimulation.

Activation of resting T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins. Protein-tyrosine kinases (PTKs) of the src family are known to be important, but the mechanism of their recruitment and their interactions with PTKs of other families are incompletely understood. We show that a member of another family of PTKs, the p72syk kinase, is constitutively bound to the TCR/CD3 complex and becomes tyrosine phosphorylated and activated within 1 min after TCR/CD3 stimulation. This activation did not depend on the presence of p56lck in T cells and in transfected COS cells. In both cases, however, the phosphorylation of cellular substrates was augmented by src family PTKs. We propose that p72syk may act as an immediate receptor-activated kinase upstream of the related p70zap PTK and the src family PTKs p56lck and p59fyn in T cells and that these src family PTKs act as signal amplifiers.

Amino Acid Sequence↗
PubMed1994

Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells.

Ag receptor triggering in B cells stimulates the activity of receptor-associated tyrosine protein kinases (TPK), leading to tyrosine phosphorylation of several cellular substrates, one of which is the Vav proto-oncogene product. We have recently determined that Vav is a TPK-regulated guanine nucleotide exchange factor for Ras in T cells. Here, we show that B cell extracts or Vav immunoprecipitates contain a Ras GDP/GTP exchange activity that is stimulated upon surface Ig (slg) triggering. The receptor-mediated stimulation of Vav exchange activity was blocked by the TPK antagonist, herbimycin A. Furthermore, immunodepletion of Vav from the B cell extracts removed approximately 80% of the Ras GDP/GTP exchange activity. These findings indicate, first, that B cell-derived Vav possesses GDP/GTP exchange activity for Ras; second, that the exchange activity of Vav is accelerated by a slg-triggered, herbimycin A-sensitive TPK and, third, that Vav accounts for most of the receptor-stimulated Ras GDP/GTP exchange activity. Thus, Vav may serve as a critical component in slg-mediated signal transduction pathways by coupling receptor-associated TPK to the activation of Ras proteins.

Animals↗
PubMed1994

Activation of phosphatidylinositol-3-kinase in Jurkat T cells depends on the presence of the p56lck tyrosine kinase.

Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56lck, JCaM1, these responses were absent. We also show that p56lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56lck phosphorylates three tyrosine residues in the p85 alpha subunit of PI3K and two in p110 of PI3K. Our results suggest that p56lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.

Enzyme Activation↗
PubMed1994

Construction and characterization of lck- and fyn-specific tRNA: ribozyme chimeras.

Two src-family protein tyrosine kinases (PTKs), p56lck, and p59fyn, are thought to play an important role in the antigen-specific T cell receptor (TCR)/CD3-initiated signaling pathway, but their relative contribution to these events is not clearly defined. Here, we have explored the potential of catalytic RNA molecules, or ribozymes, as tools for selectively inhibiting expression of the corresponding target genes in T cells. Several lck- or fyn-specific hammerhead ribozymes were synthesized, cloned into a bacterial transcription vector, and found to display specific catalytic activity in vitro. In order to achieve stable high-level ribozyme expression in intact cells, selected ribozymes were subsequently cloned into a retroviral vector (DC-T5T) immediately downstream of a tRNA(met) transcription unit. Upon retroviral transduction of a human leukemic T cell line (Jurkat), two out of four chimeric tRNA:ribozymes, fyn-1 and lck-1, were stably expressed at levels of approximately 10,000 or approximately 25,000 copies/cell, respectively. Ribozyme expression was associated with a reduction of up to 80% (lck) or 61% (fyn) in endogenous target mRNA by comparison to the corresponding transcript levels in control clones transfected with vector alone. By contrast, expression of the corresponding target proteins was not reduced, suggesting a post-transcriptional compensatory mechanism that increases translation or stability of the p56lck and/or p59fyn proteins.

Base Sequence↗
PubMed1994

Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

3T3 Cells↗
PubMed1994

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Alkaloids↗
PubMed1994

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

Amino Acid Sequence↗
PubMed1994

An efficient expression, purification and immunodetection system for recombinant gene products.

We describe a modification of the mammalian expression vector pRc/CMV, which drives expression of inserted genes from either the human cytomegalovirus (CMV) immediate-early promoter or the bacteriophage T7 RNA polymerase promoter. The modification is designed to allow expression, simple purification and specific immunodetection of recombinant fusion proteins. The modified plasmid, termed pTag/CMV-neo, encodes a Kozak consensus ribosome-binding site (RBS) and a 30-amino acid fusion tag peptide. This peptide consists of a metal ion-binding site, (His)6, for single-step affinity purification using Ni(2+)-chelating resin and a multi-purpose HIV-1-derived peptide (p18HIV). This viral epitope can be used to identify, detect and characterize target fusion proteins in conjunction with a specific monoclonal antibody H902 that does not display cross-reactivity with cellular proteins. The H902 production hybridoma cell line is reagent #521 from the NIH AIDS Research and Reference Program.

Amino Acid Sequence↗
PubMed1994

Transcellular activation of the HTLV promoter by human hematopoietic cells.

We examined the ability of hematopoietic cells to transactivate the HTLV promoter by a transcellular mechanism. HeLa cells containing a CAT reporter gene driven by the HTLV-2 promoter were cocultivated with hematopoietic cells of the B-(Raji), T-(HuT78, Jurkat) and monocyte/promyelocytic (THP-1, U937 and HL60) lineages. Cocultivation with U937 and HuT78 cells constitutively and significantly transactivated the HTLV-2 promoter, while no effect was observed with the other lines. However, activation of other T-cell lines (CEM, Jurkat, Molt-3 and MT-4) with a combination of phorbolester and phytohemagglutinin also resulted in potent transactivation. Supernatant from HuT78 cells exhibited detectable transactivating activity, suggesting that the activation is mediated by a secreted factor(s). This factor also transactivates the HTLV-1 promoter. We used a panel of HTLV-1 LTR deletion mutants to map the responsive elements to this factor(s). Unlike the response element to the HTLV transactivator protein, Tax, which can be mapped to a small region in the enhancer, maximal transactivation by the cellular factor(s) required the complete U3 sequence. Transcellular activation of the HTLV promoter by activated T-cells may play a role in the development of leukemia in HTLV infected individuals.

Deltaretrovirus↗