Biomedical subjects

R M Parkhouse

Publications and source records attributed to R M Parkhouse.

At least 19 recordsLinked to original sources

PubMed1995

Signaling through murine CD38 is impaired in antigen receptor-unresponsive B cells.

CD38 is a 42-kDa membrane associated enzyme which converts NAD into cyclic ADP-ribose (cADPR), a Ca(2+)-mobilizing second messenger, and ADP-ribose (ADPR). Agonistic antibodies to murine CD38 deliver a potent growth co-stimulus to mature splenic B lymphocytes. In this report we demonstrate a striking relationship between CD38-mediated mitogenesis and the ability of surface IgM to promote B cell proliferation. Tolerized B lymphocytes obtained from a double-transgenic mouse model of B cell tolerance do not proliferate in response to antigen stimulation through the Ig receptor or to agonistic anti-CD38 antibodies. Similarly, B-1 cells isolated from the peritoneal cavity of normal mice, and splenic B cells isolated from newborn mice were also unresponsive to both anti-IgM and anti-CD38 stimulation. All of these CD38-unresponsive B cells expressed normal levels of cell surface CD38 and responded to numerous other stimuli. CD38 immunoprecipitated from these B cell populations was normal in size and effectively hydrolyzed NAD, suggesting that the defect in CD38 signaling likely occurs downstream of CD38 itself. Signaling through CD38 and IgM does not always have identical effects on B cells since anti-CD38 cannot deliver inhibitory growth or differentiation signals to normal B cells or immature B cell lines. Nevertheless, the correlative data with these multiple B cell models of unresponsiveness suggests that the signaling pathway utilized by CD38 and IgM intersect, possibly sharing at least one of the crucial components of the Ig receptor signaling cascade.

ADP-ribosyl Cyclase↗

PubMed1995

Murine CD38: an immunoregulatory ectoenzyme.

CD38 is an ectoenzyme that utilizes NAD+ and is expressed by many cells of hematopoietic origin. Antibodies to CD38 potentiate many biological activities on lymphocytes, including induction of murine B-cell proliferation. In this article, Frances Lund and colleagues summarize information concerning the expression, enzymatic activity and signal transduction pathway utilized by murine CD38.

ADP-ribosyl Cyclase↗

PubMed1995

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

Amino Acid Sequence↗

PubMed1995

CD38 unresponsiveness of xid B cells implicates Bruton's tyrosine kinase (btk) as a regular of CD38 induced signal transduction.

CD38 is a 42 kDa membrane-associated ectoenzyme expressed by a large proportion of human and mouse lymphocytes. Agonistic antibodies to CD38 induce a strong proliferative response in lymphocytes additionally co-stimulated with other growth co-factors such as IL-4, IL-2 plus accessory cells or sub-mitogenic doses of endotoxin. We show here that B lymphocytes from unstimulated X-linked immunodeficient (xid) mice are unresponsive to CD38 stimulation, both in terms of proliferative response and surface antigen modulation. This CD38 unresponsiveness is evident in the presence of excess quantities of, and normal responses to, the accessory growth co-stimulants required for this response. CD38 molecules expressed on xid B cells are normal in terms of expression levels, size and enzymatic activity, suggesting that CD38 unresponsiveness reflects a down-stream signaling defect. In light of the recent proposal that the xid gene encodes a tyrosine kinase called Bruton's tyrosine kinase (btk), these data suggest that btk is either an integral component or an indirect regulator of the CD38-induced signal transduction pathway.

ADP-ribosyl Cyclase↗

PubMed1995

African swine fever virus gene j13L encodes a 25-27 kDa virion protein with variable numbers of amino acid repeats.

The African swine fever virus (ASFV) j13L gene encodes a 177 amino acid protein (19.0 kDa) with a putative transmembrane domain between residues 32 and 52. There is a potential signal peptide cleavage site at residue 54 and several possible motifs for phosphorylation and myristylation. Rabbit antisera raised against a synthetic peptide from the C terminus of the j13L ORF identified proteins of 25-27 kDa in cells infected with a recombinant vaccinia virus expressing the j13L ORF, in ASFV-infected cells and in purified extracellular ASF virions. In ASFV-infected cells the j13L protein was expressed late during infection and exhibited size variation (25-27 kDa) between the different ASFV strains. Nucleotide sequence analysis of the gene in these strains showed that these size differences were due to variation in the number and sequence of tandemly repeated amino acid repeats. Although ASFV-infected animals made antibodies to the j13L protein, no protection was observed when pigs were vaccinated with a recombinant vaccinia virus expressing the j13L ORF.

African Swine Fever Virus↗

PubMed1994

Mechanism and subcellular localization of secretory IgM polymer assembly.

The predominant functions of secreted IgM, complement fixation and transcytosis across epithelial barriers via the poly(Ig) receptor, are properties of IgM polymers; thus, the mechanisms that regulate oligomerization are critical to immune function. We have developed methods to assess the mechanism and subcellular location of IgM polymer formation. Using denaturing agarose/SDS-polyacrylamide gel electrophoresis and sucrose gradient fractionation, we demonstrate that IgM polymers are assembled in a stepwise fashion in which primary intermediates, including heavy chain-light chain complexes, are sequentially incorporated. Assembly intermediates include both covalent and non-covalent components, suggesting that IgM subunit interactions precede covalent assembly. IgM polymers are assembled from primary intermediates containing immature N-linked oligosaccharides. Polymerization is insensitive to brefeldin A and occurs at temperatures that inhibit protein transport along the secretory pathway. Together, these data demonstrate that IgM polymerization occurs early in the secretory pathway, most likely in the endoplasmic reticulum. These results are consistent with a model in which secretory IgM represents an oligomeric protein that is retained in the endoplasmic reticulum and does not mature along the secretory pathway until complete assembly is achieved.

Animals↗

PubMed1994

Biosynthetic radiolabelling of excretions-secretions of adult male Onchocerca gibsoni.

Maintenance of adult male worms of the bovine filarial parasite Onchocerca gibsoni in vitro, in the presence of radioactive precursors, resulted in the time-dependent excretion-secretion of radiolabelled parasite macromolecules (ES). Molecules labelled with amino acids ([35S] methionine, [3H] leucine) covered a wide range of molecular weights, whereas labelling with [3H] glucosamine produced predominantly molecules of high molecular weight. Many of the products were recognized by antibodies in two serum pools from humans infected with Onchocerca volvulus. O. gibsoni ES may therefore provide a substitute source of material for studies on the ES of the less readily available human parasite.

Animals↗

PubMed1994

Monoclonal antibodies recognising differentiation antigens on porcine B cells.

Nineteen monoclonal antibodies (mAbs) submitted to the porcine CD Workshop were analysed on porcine B cells from different lymphoid tissues by flow cytometry and immunohistochemistry. Six mAbs only bound to pig B cells and three of these were assigned cluster numbers, 76-7-4 (No. 001):CD1, K231-3B2 (No. 027):CD25 and CC51 (No. 100):CD21. Of the three remaining B cell positive mAbs, one was assigned to a swine cluster, CC55 (No. 101):SWC7, a second, LIG4 (No. 123), recognised cell surface IgM and the third, MUC106A (No. 072), remained unassigned.

Animals↗

PubMed1994

IgM hexamers?

There has been a universal tendency to regard IgM antibodies as pentameric molecules comprising five immunoglobulin monomeric subunits joined by a single J-chain protein. Is this the only form of secreted IgM, or are the possibilities more complex? In this article, Joseph Brewer and colleagues propose that the IgM polymers secreted in primary immune responses may be more heterogeneous than previously believed and that, as a consequence, the biological activity of IgM may have considerable flexibility.

Animals↗

PubMed1994

The differentiation of parasitic nematodes using random amplified polymorphic DNA.

DNA from species and races of plant parasitic nematodes (Meloidogyne, Globodera and Heterodera) and a human parasitic nematode (Trichinella) were subjected to polymerase chain reaction amplification using one arbitrary primer (M-10). This technique results in relatively simple DNA profiles that include polymorphic markers known as random amplified polymorphic DNA (RAPDs). The RAPD profiles of the plant nematode species of Meloidogyne made possible the identification of M. incognita and M. hapla, but no differences were found between the patterns of M. javanica, M. arenaria and M. graminicola. Moreover, the four races of M. incognita were indistinguishable by this primer. In contrast, when races of the plant nematode Globodera rostochiensis (Ro1 and Ro2/3) were studied under the same RAPDs conditions, a race specific profile allows these two most devastating races to be differentiated. When DNAs of eight Trichinella isolates were subjected to RAPD studies, four different patterns were identified, corresponding to the four Trichinella clusters previously defined by isozyme polymorphism.

Animals↗

PubMed1994

Murine B-cell activation via CD38 and protein tyrosine phosphorylation.

CD38 has been implicated in the regulation of both proliferation and rescue from apoptosis of B cells. The signalling events associated with CD38-mediated activation of murine B cells are, as yet, not well defined but it is clear that ligation of CD38 by a mitogenic antibody, NIMR-5, induces a calcium influx in resting B cells. Interestingly, however, cross-linking of CD38 does not mobilize intracellular stores of calcium. We now provide a rationale for these findings by demonstrating that CD38 is not coupled to the generation of inositol phosphates in resting B cells. We do, however, show that CD38 ligation stimulates one, or more, protein tyrosine kinase activities which may play a central role in the transduction of CD38-mediated signals leading to B-cell activation.

ADP-ribosyl Cyclase↗

PubMed1993

Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38.

CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.

ADP-ribosyl Cyclase↗

PubMed1993

Expression cloning of a cDNA encoding a novel murine B cell activation marker. Homology to human CD38.

A rat mAb (NIM-R5) has recently been prepared against a novel murine B cell activation marker. We report here isolation of a cDNA (1-19) encoding the B cell-derived protein recognized by NIM-R5 antibody. This cDNA contains an open reading frame that encodes a polypeptide of 304 amino acids with a predicted molecular weight of 34,500. The existence of a 22-amino acid hydrophobic region located 23 amino acids from the amino terminal of the deduced protein, together with four potential N-linked glycosylation sites, characterize the deduced protein encoded by I-19 cDNA as a typical type II transmembrane glycoprotein. Although I-19 cDNA appears to encode a novel murine protein, its nucleotide sequence and deduced amino acid sequence show approximately 70% homology to the previously reported sequence of human CD38, suggesting that I-19 cDNA encodes either the mouse homologue of CD38 or a closely related protein. Northern blot analysis of the expression of this cDNA product in a variety of cell types, together with immunoprecipitation of the recombinant protein expressed in BaF3 cells, indicated that I-19 cDNA encodes not only the epitope recognized by NIM-R5 but also a protein that is indistinguishable biochemically and in terms of distribution from the murine B cell activation marker recognized by NIM-R5 antibody. Chromosomal mapping studies have localized this locus to the proximal region of mouse chromosome 5. We anticipate that the availability of probes for the murine B cell activation marker recognized by NIM-R5, and the recombinant protein itself, will greatly aid efforts to define the role of this molecule in murine B cell development.

ADP-ribosyl Cyclase↗

PubMed1993

A B lymphocyte surface molecule mediating activation and protection from apoptosis via calcium channels.

A rat mAb (NIM-R5) was prepared against a 42-kD B cell activation Ag (p42). The expression of p42 is increased upon activation. NIM-R5 induces an increase of intracellular Ca2+, due to influx from the exterior milieu via calcium channels. This stimulation does not prejudice further stimulation with anti-Ig, and thus p42 constitutes an activation signal independent of membrane Ig. The antibody induces increased expression of class II molecules on resting B lymphocytes and prepares the cells for "spreading" when interacted with immobilized anti-class II antibody. The antibody alone is weakly mitogenic and comitogenic with IL-4 on resting B cells. Of particular interest, NIM-R5 induces proliferation and rescue from apoptosis in B cells activated in vitro. In conclusion, NIM-R5 induces an Ig-independent activation and proliferation of resting and activated B cells. This antibody does not recognize other known B cell activation Ag such as CD23, CD40, or CD72. We therefore propose that the p42 Ag is a glycoprotein with an important role in the regulation of B lymphocyte activation and survival.

ADP-ribosyl Cyclase 1↗

PubMed1993

Organization of the murine Cd22 locus. Mapping to chromosome 7 and characterization of two alleles.

Murine CD22 (mCD22) is a B cell-associated adhesion protein with seven extracellular Ig-like domains that has 62% amino acid identity to its human homologue. Southern analysis on genomic DNA isolated from tissues and cell lines from several mouse strains using mCD22 cDNA demonstrated that the Cd22 locus encoding mCD22 is a single copy gene of < or = 30 kb. Digestion of genomic DNA preparations with four restriction endonucleases revealed the presence of restriction fragment length polymorphisms (RFLP) in BALB/c, C57BL/6, and C3H strains vs DBA/2J, NZB, and NZC strains, suggesting the presence of two or more Cd22 alleles. Using a mCD22 cDNA clone derived from the BALB/c strain, we isolated genomic clones from a DBA/2J genomic library that contained all the exons necessary to encode the full length mCD22 cDNA. Fifteen exons, including exon 3 that encodes the translation start codon, were identified. Each extracellular Ig-like domain of mCD22 is encoded by a single exon. A comparison between the nucleotide sequences of the BALB/c CD22 cDNA and the exons of the DBA/2J CD22 genomic clones revealed an 18-nucleotide deletion in exon 4 (encoding the most distal Ig-like domain 1 of mCD22) of the DBA/2J genomic sequence in addition to a number of substitutions, insertions, and deletions in other exons. These nucleotide differences were also present in a cDNA clone isolated from total RNA of LPS-activated DBA/2J splenocytes by reverse transcription-polymerase chain reaction. The Cd22 locus was mapped to the proximal region of chromosome 7, a region sytenic to human chromosome 19q, close to the previously reported loci, Lyb-8 and Mag (a homologue of Cd22). An antibody (CY34) against the Lyb-8.2 B cell marker reacted with a BHK transfectant expressing the full length mCD22 cDNA, thus demonstrating that Lyb-8 and Cd22 loci are identical. Furthermore, a rat anti-mCD22 mAb, NIM-R6, bound to sIgM+ DBA/2J B cells, confirming the expression of a CD22 protein by the Cd22a/Lyb-8a allele.

Animals↗

PubMed1993

Identification of bovine B cell reactive and B cell specific monoclonal antibodies.

All monoclonal antibodies (mAbs) submitted to the workshop panel were screened for reactivity with bovine surface immunoglobulin (sIg)+ cells (gated small dense lymphocytes from peripheral blood) by fluorescence activated cell sorter (FACS) analysis. Eighteen temporary clusters--TCs 1-12, 15, 16, 18, 19, 25 and 26--contained mAbs reactive with sIg+ cells. mAb BAS21A (unclustered) and CC92 (TC25) were also reactive with sIg+ cells. Further FACS analysis with B cells from peripheral blood lymphocytes and mesenteric lymph nodes, and B and T lymphoma cell lines, indicated that the majority of mAbs within TCs 2, 4, 15, 18 and 26 reacted specifically with bovine B cells. Bovine B cell specific mAbs within these clusters were TH14B, IL-A55, CACT101A, MUC76A from TC4, VPM30, GC65A, CACT65A from TC15, IL-A58, CC56, CC70, IL-A65 from TC18, and CC57 and 26A9 from TC26. Three mAbs--IL-A65, CC70, and BAQ15A--within TC18 defined WC3; mAbs TD9 and CC56 may also be related to WC3.

Animals↗

PubMed1992

Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule.

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.

Amino Acid Sequence↗

PubMed1992

J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5.

Two functional polymeric forms of IgM can be produced by antibody-secreting B cells. Hexameric IgM lacks detectable J (joining) chain and activates complement 17-fold better than pentameric IgM, which usually contains one J chain per pentamer. Using the inducible B-cell lymphoma CH12, we determined if the synthesis of a particular polymeric form of IgM is a fixed property of B cells or can be altered. Lipopolysaccharide (LPS)-stimulated CH12 cells produced mixtures of IgM hexamers and pentamers, resulting in antibody with high complement-fixing activity. In contrast, interleukin-5-stimulated CH12 cells secreted predominantly pentameric IgM, with a correspondingly lower lytic activity. Differences in lytic activity were due only to the amount of hexameric IgM in the secreted antibody. Interleukin 5 stimulated higher production of J chain RNA and protein than LPS, while LPS induced the highest levels of the secretory form of mu protein. The amount of hexameric IgM secreted was therefore inversely proportional to the level of intracellular J chain protein in the responding B cells. We conclude that the biologic function of IgM produced by B cells differs depending on how they are stimulated and that this difference may be regulated by the relative availabilities of J chain and secretory mu proteins during IgM polymerization.

Animals↗