Biomedical subjects

W F Wade

Publications and source records attributed to W F Wade.

At least 37 records · Page 2Linked to original sources

PubMed1996

Increased inhibition of proliferation of human B cell lymphomas following ligation of CD40, and either CD19, CD20, CD95 or surface immunoglobulin.

Non-Hodgkin's (NHL) B cell lymphomas are growth-inhibited by ligation of their CD40 molecules. This inhibition is not absolute in that approximately 50% of the cells are not inhibited. We conducted studies to see if other signals that have been reported to inhibit B cell lymphoma growth could be used in combination with anti-CD40 signaling to completely inhibit growth. Ligation of surface immunoglobulin (Ig), CD19, CD20, CD37 or CD95 with soluble antibody did not affect growth of the panel of NHL cells examined. Ligation of CD20, CD19 or CD95 was inhibitory for some NHL cell lines if the primary antibody was crosslinked with a secondary antibody. Combining anti-CD40 with anti-CD19, anti-CD20, or anti-Ig resulted in increased inhibition past that produced by anti-CD40 alone. The additive effect of anti-CD40 and other antibodies to selected surface markers was not observed in all NHL cell lines. Crosslinking of CD95 was also growth inhibitory for the majority of the NHL, and when combined with anti-CD40 under conditions that afforded crosslinking of the two receptors, increased inhibition was seen in three of the NHL cell lines. We found that cAMP or sodium butyrate (NaB) were also effective at inhibiting growth of the NHL cells; this was a profound inhibition (approaching 100%) compared to the 50% inhibition seen with anti-CD40 treatment. The potential for anti-CD40 and either cAMP or NaB to be additive was tested and not found to be the case. The ability to inhibit proliferation of the NHL was very dynamic with some antibody combinations being either inhibitory for multiple cells, not having an effect at all, or in some cases being stimulatory. This suggests that the NHL may represent unique stages of B cells that might serve as a model system which could be developed to precisely categorize patient NHL.

Antibodies, Monoclonal↗

PubMed1995

Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP.

K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B cells with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells.

4-1BB Ligand↗

PubMed1995

Truncated MHC class II cytoplasmic and transmembrane domains: effect on plasma membrane expression.

Plasma membrane (PM) expression of major histocompatibility complex (MHC) class II molecule is required for the interaction of antigen (Ag) presenting cells and T lymphocytes. Class II molecules composed of an alpha and a beta chain are highly polymorphic which facilitates their interaction with Ag and Ag-specific T cells. Recently, we have focused on the less polymorphic sequences of class II molecules, the transmembrane (TM) and cytoplasmic (Cy) domains, in an attempt to understand what their function might be. Using site-directed mutagenesis to create truncations in the TM and Cy domains of IAk's alpha or beta chain, or both, we have identified some of the sequence requirements for efficient surface expression of I-Ak molecules. Ak beta TM mutants that are not expressed at the PM are not transported past the medial-Golgi as indicated by in situ staining and Western blot analysis of endoglycosidase-H-treated immunoprecipitates. The lack of transport of TM class II mutants is not due to lack of association with the invariant chain (Ii). Class II molecules with Cy domain truncations in both chains are not efficiently transported to the PM and also have a percentage of molecules that are endoglycosidase-H sensitive. In situ staining of class II in cells expressing Cy domain truncated class II molecules revealed a discrete vesicular pattern compared to the staining of transfectants that expressed wildtype class II molecules. The immunofluorescence data along with the endoglycosidase-H data indicate the Cy domains are required for efficient transport. Immunoprecipitation studies using a panel of I-Ak conformation-specific antibodies revealed that the truncation of the Cy domains of both chains did not effect the conformation of class II. However, further truncation of the Ak beta chain into the TM domain resulted in lack of transport past the ER/medial-Golgi and diminished expression (stability) of mutant class II proteins within the cells. The alpha/beta chains of the TM mutants that did associate bound a panel of conformation sensitive antibodies except for one, 3F12. We conclude that the Cy domain of the alpha and beta chains of MHC class II, as well as sequences in the TM domains of the Ak beta chain are required for efficient class II PM expression. The reason for the lack of PM expression of TM mutants may be the inability to assess a transport competent conformation as defined by the 3F12-specific epitope, while truncation of the Ak alpha Cy domains is proposed to prevent complete masking of the ER retention sequence of the Ii chain.

Animals↗

PubMed1995

Class II cytoplasmic and transmembrane domains are not required for class II-mediated B cell spreading.

B cells cultured on immobilized anti-class II monoclonal antibody (mAb) change from round to flattened cells, with lamellipodia and filopodia. This change in cell morphology, termed 'spiders', occurs within 30 min upon culture and is mediated through either I-A or I-E molecules. Class II molecules that are defective in mediating protein kinase C (PKC) due to the deletions of both alpha and beta chain's cytoplasmic (Cy) domain sequences can induce spider formation. B-cell transfectants that express chimeric MHC class II/class I molecules, where the ectodomains are class II sequences and the transmembrane and Cy domains are class I sequences also form spiders when cultured on anti-class II mAb. The spider morphology is not induced by either anti-immunoglobulin (Ig) or anti-MHC class I mAb. Treatment of B cells to increase intracellular cAMP, a component of the class II signaling pathway also results in spider formation with the same kinetics and percent change in the responding population as that induced by anti-class II mAb. Cytochalasin A treatment which disrupts cytoskeletal actin filaments and the tyrosine kinase inhibitor, genistein, both inhibit spider formation. Actin redistributes from a concentric ring in round cells to the ends of the filopodia in the spiders. The mechanism of spider induction whether resultant from second messengers following class II signaling or from non-signaling-induced physical interactions of class II with intracellular cytoskeletal components only requires the extracellular domains of class II. The biologic relevance of B-cell spiders is currently not known but has been reported to be associated with class II signal transduction and efficient Ag presentation.

Actins↗

PubMed1994

MHC class II molecules that lack cytoplasmic domains are associated with the cytoskeleton.

MHC class II molecules, composed of alpha- and beta-chain heterodimers, are required for Ag presentation. The carboxyl-terminal domains of class II molecules are believed to mediate the location of class II in the plasma membrane and are important for signal transduction and Ag presentation. These domains contain typical transmembrane sequences, and cytoplasmic sequences of 12 or 18 amino acids for the alpha- and beta-chains, respectively. We examined these domains to determine whether they linked class II molecules to the actin-based cytoskeleton. Our analyses of class II-cytoskeleton interactions, such as a colocalization with actin filaments during capping, association with the detergent-insoluble cytoskeleton, and direct binding of filamentous actin, revealed that both the cytoplasmic and transmembrane domains contributed to class II interactions with the cytoskeleton. Detergent-extracted and immunoprecipitated full-length class II molecules had quantitatively stronger interactions with the cytoskeleton than did molecules with deleted cytoplasmic domains. A secondary Ab, which was used to cross-link primary Ab bound to class II, up-regulated the class II-cytoskeletal associations. This association was efficiently inhibited by dihydrocytochalasin B, but only partially disrupted by chlorpromazine. The mechanism of interaction with actin filaments after ligation of class II occurred without a measurable increase in filamentous actin levels. This suggested that enhanced class II-cytoskeleton associations involved a rearrangement of existing actin filaments, possibly through the multiple kinases that are activated after class II transmembrane signaling.

Actins↗

PubMed1994

Distinct structural compartmentalization of the signal transducing functions of major histocompatibility complex class II (Ia) molecules.

Class II major histocompatibility complex encoded proteins (MHC class II or Ia molecules) are principal plasma membrane proteins involved in activation of both B and T cells during antigen-driven immune responses. Recent data indicate that class II molecules are more than simply recognition elements that provide a ligand for the T cell antigen receptor. Changes in B cell physiology that follow class II binding are now recognized as being required not only for the induction of T cell activation, but also for B cell activation and proliferation. It is interesting to note that class II molecules appear to transduce signals via two distinct mechanisms depending upon the differentiative state of the B cell on which they are expressed. While one of these pathways, involving cAMP generation and protein kinase C localization in the cytoskeletal/nuclear compartment, is seen in resting B cells, the second is seen in primed B cells and involves tyrosine kinase activation, inositol lipid hydrolysis, and Ca2+ mobilization. Use of this pathway is correlated with ability of class II to transduce signals leading to B cell proliferation. To begin to address the molecular basis of this unique, activation-dependent, differential coupling of class II to signaling pathways, we conducted mutational analysis of class II structural requirements for signal transduction. Here we report that the cytoplasmic (Cy) domains of I-Ak class II molecules are not required for either receptor-mediated activation of protein tyrosine phosphorylation or Ca2+ mobilization. This is in contrast to the requirement of the Cy domain of beta chain of class II for the alternate signaling pathway and efficient antigen presentation to autoreactive T cell lines. Disparate distribution of functional motifs within the MHC class II molecules may reflect use of distinct receptor associated effector molecules to sustain different modes of signal transduction in various class II-expressing cells.

Amino Acid Sequence↗

PubMed1994

Truncation of the A alpha chain of MHC class II molecules results in inefficient antigen presentation to antigen-specific T cells.

Antigen presenting cells (APC) expressing MHC class II molecules composed of chains with part or all of the cytoplasmic domains deleted are inefficient at presenting hen egg lysozyme peptides to antigen specific T cell hybrids compared with APC that express wild-type MHC class II molecules. This effect is most apparent for mutants in which the alpha chain has been truncated. The inefficiency in antigen presentation can be amplified by pulsing the APC for 4 h with peptide rather than having peptide present throughout the presentation assay. Fixation of antigen-pulsed APC improves the capacity of APC with truncated class II molecules to stimulate T cell hybrids. Fixation of APC prior to exposure to antigen also leads to significant improvement in antigen presentation by the truncated class II molecules. Because the inefficiency of a given hybrid for antigen presentation does not correlate with its ability to transduce a signal as measured by protein kinase C translocation, we suggest that defects in this pathway are not the only cause of impaired antigen presentation. However, because previous studies have demonstrated the need for an intact cytoskeleton for successful antigen presentation, we propose that the carboxy truncated class II molecules are inefficient in antigen presentation because they are unable to generate the signal that ultimately leads to their interaction with the cytoskeleton. These observations underscore the complexity of the events that are required for achieving effective interactions between MHC class II molecules and TCR, and suggest, with regard to efficient antigen presentation, that the physical state of the class II molecules is at least as important as their signal transducing capacity.

Animals↗

PubMed1993

Induction of costimulatory molecule B7 in M12 B lymphomas by cAMP or MHC-restricted T cell interaction.

M12 B lymphomas expressing transfected Ak molecules with truncated cytoplasmic domains have a defect in Ag presentation to some autoreactive T cell hybrids. This defect in Ag presentation is corrected by pretreatment of the B cells with agents that elevate intracellular cAMP. Here we show that dibutyryl-cAMP treatment of M12 B lymphomas leads to cell surface expression of the costimulatory molecule B7. Furthermore, CTLA4Ig, a ligand for B7, inhibits activation of an accessory signal-dependent T hybrid. B7 is also inducible in M12 B lymphomas upon MHC-restricted interaction with T cells that can be activated by the APC, but not by T cells that fail to respond to truncated MHC-bearing M12 cells. Activation of the unresponsive T hybrids with immobilized anti-CD3 confers on them the ability to induce B7 in the APC. Direct engagement by immobilized antibodies of MHC class II on M12 B lymphomas did not induce B7 expression. Taken together, these results imply that during T-B interaction, initial T cell activation events lead to the ability of the T cell to induce costimulatory activity in the B cell, which in turn further activates the T cell. Activated T cell supernatants induced a small amount of B7 but were not nearly as effective as cAMP or as coincubation of T and B cells. These results suggest a role for T-B contact or localized cytokine secretion in the induction of B7 during T-B interaction.

Antigens, Surface↗

PubMed1993

Structural compartmentalization of MHC class II signaling function.

Major histocompatibility complex (MHC) class II molecules are critical restricting elements in the generation of thymus-dependent immune responses. Recent studies indicate that in addition to providing a composite epitope for recognition by T-cell antigen receptors, MHC class II molecules function in signal transduction through interaction with other cellular proteins. Mutational analyses indicate that structural information necessary for these functions is compartmentalized in different aspects of the molecular complex. Here, William Wade and colleagues review the structural basis of this MHC class II function as defined in the I-A alpha and -beta chains.

Amino Acid Sequence↗

PubMed1989

Antigen presentation abrogated in cells expressing truncated Ia molecules.

Oligonucleotide site-directed mutagenesis was used to introduce a premature stop codon in wildtype A beta k and A alpha k cDNA clones to create truncated A beta k and A alpha k molecules lacking the cytoplasmic domain. Transfected B lymphoma cells expressing an I-Ak molecule with a truncated beta-chain or with truncated alpha- and beta-chains showed profound defects in two Ia-related functions: Ia-restricted Ag presentation and intracytoplasmic signaling. The ability of these transfected cell lines to activate autoreactive T hybrids was markedly impaired whereas loss of Ag presentation to nominal Ag-specific T hybrids was more subtle. Ia-mediated transmembrane signaling as measured by PKC translocation from cytosol to nucleus after stimulation with anti-Ak antibody was greatly affected by truncation of the A beta and A alpha cytoplasmic domains. These results indicate an important role for the highly conserved cytoplasmic domain in Ia-mediated responses.

Amino Acid Sequence↗

PubMed1989

Altered I-A protein-mediated transmembrane signaling in B cells that express truncated I-Ak protein.

Recent evidence suggests that the major histocompatibility complex class II molecules of B lymphocytes function as signal-transducing receptors during the generation of T lymphocyte-dependent humoral immune responses. By analogy with other receptors, we postulate that perturbation of the class II molecules is coupled to the generation of intracellular second messengers through interactions involving the transmembrane and/or cytoplasmic domains of the class II molecules. We report a series of experiments that assess which amino acids of the class II molecule I-Ak are required for coupling it to the signal-transduction pathway. We prepared a series of B-lymphocyte transfectants that express I-Ak molecules with COOH-terminal truncations of either the Ak alpha or Ak beta chain or both. The ability of each transfected class II molecule to transduce a signal after being bound by monoclonal antibody was found by monitoring the translocation of protein kinase C from the cytosol to the "nuclear compartment" of the transfected B lymphocyte. Results indicate that the Ak beta chain plays the dominant role in signal transduction and that the 6 cytoplasmic amino acids of Ak beta chain most proximal to the inner plasma membrane are of greatest importance in coupling I-Ak molecules to the molecules of the signaling cascade.

Amino Acid Sequence↗

PubMed1989

Translational diffusion of class II major histocompatibility complex molecules is constrained by their cytoplasmic domains.

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy-terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.

Amino Acid Sequence↗

PubMed1987

Effects of infection with Enterobacteriaceae enteropathogens on subsequent infection with Ascaris suum in the laboratory mouse.

Prior to infection with Ascaris suum, ICR strain mice were inoculated with Salmonella typhisuis intraperitoneally or via gastric gavage. Similarly, Salmonella cholerae-suis var. kunzendorf, Salmonella typhimurium and enterotoxigenic Escherichia coli were administered to mice via gastric gavage 2 weeks prior to A. suum inoculation. Previous inoculation with S. typhisuis, via the intraperitoneal or gastric gavage routes and S. cholerae-suis var. kunzendorf decreased recovery of ascarid larvae from mice lungs. This effect appeared to be due to entrapment of migrating larvae by inflammatory reactions in the liver. This reaction was suspected to be due to non-specific resistance stimulated by the prior exposure to the bacterial pathogen. The number of A. suum larvae in the lungs of mice previously inoculated with S. typhimurium or enterotoxigenic E. coli (ETEC) was variable and in some cases greater in mice which had received the bacterial inoculation.

Animals↗

PubMed1987

Immunochemical characterization of proteins from scrapie-infected hamster brain, using immunoblot analysis.

Preparations of brain plasma membrane from scrapie-infected or noninfected hamsters were extracted with a solvent and were used to inoculate rabbits. Antisera evaluated by immunoblot analysis revealed a protein of 45 kD in scrapie-infected hamster brain that had a greater signal compared with proteins of comparable relative mass in noninfected brain. This 45-kD protein was not increased in scrapie-infected mouse, sheep, or goat brain. Seemingly, the 45-kD protein may be a degradation product of glial fibrillary acidic protein.

Animals↗

PubMed1986

Effect of bacterial flora and mouse genotype (euthymic or athymic) on scrapie pathogenesis.

Euthymic and athymic female BALB/c mice, reared under either germfree or defined flora conditions, were used to investigate the pathogenesis of scrapie after intracerebral or intraperitoneal inoculation. Time in days to onset of clinical signs (Stage I), to endstage (Stage II), and the time interval between Stage I and Stage II were compared among groups. In addition, scrapie agent titers in spleen were determined at 28 and 90 days after infection, as were agent titers in spleen and brain at Stage II. Three-way analysis of variance indicated that the bacterial flora, the presence or absence of a thymus, and the route of agent inoculation interact to produce significant differences in the pathogenesis of disease. The three factors in the experimental design also influenced the spleen titers of scrapie infectivity. The variation in scrapie pathogenesis among the groups of mice is likely to be mediated by differences in their reticuloendothelial systems. These differences may alter the agent's adsorption in spleen and/or route of transport from spleen to brain.

Animals↗

PubMed1986

A tongue biopsy technique for the detection of trichinosis in swine.

A tongue biopsy technique developed for the detection of Trichinella spiralis infection in swine involves taking a deep core biopsy of the tongue musculature, and examination of the sample by digestion. Using this procedure, 31 of 52 (60%) swine from an Indiana herd were found to be infected with T. spiralis. The average biopsy weighed 0.42 g, and the intensity of infection averaged 180 larvae per gram (range 2-1157). The biopsy was quick and easy to perform and the tongues healed well following the procedure. This technique may have applicability for Trichinella detection in epidemiological, control and research studies on swine and other animals.

Animals↗

PubMed1985

Comparison of RNA from healthy and scrapie-infected hamster brain.

Density gradient fractions prepared from healthy or scrapie-infected hamster brain tissue enriched in plasma membrane vesicles were treated with nucleases prior to phenol extraction and ethanol precipitation. The recovered nucleic acids were 3' end-labelled and run on one-dimensional polyacrylamide gels. Autoradiography revealed the presence of low molecular weight RNAs (4S) in both healthy and scrapie samples. Two-dimensional fingerprint analysis indicated that the RNAs isolated from scrapie-infected hamsters contained oligonucleotides that were not present in RNAs isolated from healthy hamsters.

Animals↗

PubMed1985

Inactivation of the scrapie agent by ultraviolet irradiation in the presence of chlorpromazine.

The sensitivity of the scrapie agent to u.v. inactivation was found to be related to the purity of the tissue preparation. Scrapie infectivity associated with membrane vesicles was unaffected when irradiated with 10(4) J/m2. Irradiation of more highly purified preparations from detergent-extracted CsCl gradient fractions reduced scrapie infectivity from 10(7.8) log10 LD50 per ml to as low as 10(4.5). Sensitivity of membrane-associated scrapie infectivity to inactivation by u.v. irradiation could be increased by addition of chlorpromazine, a phenthiazine antipsychotic which penetrates lipid bilayers and induces single-strand breaks in nucleic acids under irradiation. Chlorpromazine without irradiation, and a semiquinone protein-binding radical of chlorpromazine, failed to decrease scrapie infectivity by themselves. A closely related phenthiazine antipsychotic, trifluoperazine, which does not bind to nucleic acids, did not reduce scrapie infectivity. These findings suggest that the target of u.v. radiation for inactivation of scrapie infectivity in the presence of chlorpromazine is an essential nucleic acid.

Chlorpromazine↗