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Biomedical subjects

R M Parkhouse

Publications and source records attributed to R M Parkhouse.

At least 19 recordsLinked to original sources
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↗
PubMed1992

A longitudinal study of porcine serological responses to experimental infections with T-1 and T-3 Spanish Trichinella isolates.

Comparison of antibody response and antigen recognition was made by ELISA and western-blot analysis in pig experimental infections by T-1 and T-3 Spanish Trichinella isolates. Two groups of Iberian pigs were experimentally infected with 150 larvae/kg body weight of GM-1 and C-76 Spanish Trichinella isolates as representatives of T-1 and T-3 gene pools respectively. Antibody levels and antigen recognition were measured on days -14, 0, 6, 16, 20, 27, 34, 49, 63 and 82 after infection by ELISA and western-blotting assays. Antibody response against C-76 infection was significantly delayed and lower than against GM-1. The two Trichinella isolates were indistinguishable, however, by western blotting analysis, although recognition of larval antigens was quantitatively higher than adult ones. Interestingly, the principle larval antigenic components recognized by pigs were those recognized by the monoclonal anti-sera NIM-M1. Finally, there were no serological patterns indicative of the stage of infection ("antibody windows") discriminating, for example between early versus late infections.

Animals↗
PubMed1992

Immunological cross-reaction between an Onchocerca paramyosin-like molecule and a microfilaria surface antigen.

A monoclonal antibody (2A5B9), previously shown to be reactive with a 14 kD surface associated antigen of Onchocerca microfilariae, was found to recognise a 92 kD molecule present in an adult worm extract. The antibody was used to select cDNA clones with a coding capacity larger than 14 kD, from a lambda gt11 library of O. volvulus. Nucleotide sequencing of the cDNA of one such clone revealed extensive homology to the myosin (unc-54) and paramyosin (unc-15) genes of Caenorhabditis elegans, similarly to myosin and paramyosin genes of Onchocerca volvulus, Brugia malayi, Dirofilaria immitis and Schistosoma mansoni. The immunological implications of antigenic cross-reactivity between a surface molecule and paramyosin, a known protective antigen, are discussed.

Amino Acid Sequence↗
PubMed1992

Relative expression of surface IgM, IgD and the Ig-associating alpha(mb-1) and beta(B-29) polypeptide chains.

Membrane immunoglobulins are associated with a transmembrane disulphide-linked heterodimer composed of an alpha-chain (mb-1) and a beta-chain (B-29). The relative surface expression of all of the polypeptide chains comprising the Ig-alpha beta complex has been investigated using surface labelling coprecipitation analysis and two-colour flow cytometric analysis. The main conclusions are that mb-1 and B-29 are B-cell surface markers on immature and mature B cells, and that all components of the surface Ig-alpha beta complex are expressed in stoichiometrically equivalent amounts. Thus the complex was quantitatively precipitated from digitonin lysates of 125I-surface-labelled cells with anti-B-29, anti-mb-1 or anti-Ig. Secondly, by two-colour FACS analysis there was a proportionality between the relative amounts of cell surface mb-1 or B-29 and surface IgM or IgD, but not other B-cell markers (class II, B220, FcR gamma, FcR epsilon). Finally there was an insignificant number of B cells expressing membrane Ig without alpha- and beta-chains, and vice versa. Thus there appears to be a closely controlled relative synthesis and surface expression of all components of the B-cell receptor complex.

Animals↗
PubMed1991

DNA polymorphisms within Spanish Trichinella isolates.

A total of 13 Spanish Trichinella isolates were characterised by DNA analysis. Genomic DNA cross-hybridisation tests revealed two distinct groups that exhibited weak cross-reactions. Further diagnostic subdivision was attempted according to restriction-fragment polymorphism using a cloned T. spiralis DNA repetitive fragment as a probe, but no major difference between the two Trichinella groups was observed.

Animals↗
PubMed1991

Surface antigens of male worms and microfilariae of Onchocerca gibsoni.

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelled surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity of microfilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

Animals↗
PubMed1991

Studies on a 14-kilodalton surface protein of Onchocerca microfilariae.

A 14-kDa antigen present on the surface of uterine microfilariae of Onchocerca spp. has been identified using monoclonal antibodies. The antigen was also found in skin microfilariae, but in a masked or cryptic form. A complementary DNA clone encoding the epitope recognised by one of the monoclonal antibodies was identified in a lambda gt11 library. Nucleotide sequencing revealed that the 233-bp cDNA fragment codes for the carboxy-terminus of the antigen. The deduced amino acid sequence consists of three hydrophobic domains with high potential for beta-sheet formation. The amino-terminal hydrophobic domain is followed by 4 positively charged residues (positions 22-25) which contribute to the rather basic character of the protein. Another interesting feature of the polypeptide is its richness in phenylalanine (12.7%). From the sequence information, a synthetic peptide was synthesised which was recognised by one of the monoclonal antibodies directed against the 14-kDa antigen and a small number of sera from patients with onchocerciasis. The relevance of this to vaccination is discussed.

Amino Acid Sequence↗
PubMed1991

A species-specific oligonucleotide DNA probe for the identification of Meloidogyne incognita.

A genomic library of Meloidogyne incognita Race 1 has been prepared in the bacteriophage lambda gt10 and screened for specific DNA sequences by hybridization with radio-isotope labelled total genomic DNA from a number of Meloidogyne species. One clone isolated (MR1 #15), although not totally species specific, clearly showed preferential hybridization to M. incognita. Following subcloning and sequencing of the 255 bp insert, four stretches of the sequence corresponding to oligonucleotides of approximately equal length (approximately 70 bp) were synthesized and examined for specificity. One of them, MR1 #15.2, showed the necessary specificity to be used as a diagnostic tool.

Animals↗
PubMed1990

Cloning of a species-specific DNA probe from Onchocerca gibsoni.

A genomic library of Onchocerca gibsoni has been prepared in the vector lambda-gt10 and has been screened for specific DNA sequences by hybridization with radiolabelled total genomic DNA from a number of Onchocerca species. A clone--fOGI--has been isolated which does not interact with DNA prepared from O. gutturosa, O. lienalis, O. ochengi, O. cervicalis or O. volvulus (both Liberian and Mexican isolates). In addition, no hybridization is observed with host (cattle) DNA. fOGI can detect as little as 100-200 pg of O. gibsoni DNA. It is thus concluded that fOGI has the sensitivity to detect microfilariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.

Animals↗
PubMed1990

Association between circulating antigen and parasite load in a model filarial system, Acanthocheilonema viteae in jirds.

Jirds (Meriones libycus) were infected with various numbers of Acanthocheilonema viteae L3 stage parasites. During the course of the ensuing 16 weeks, blood samples were collected at 2 weekly intervals and the amount of the major parasite excretory-secretory product (E-S 62) and antibodies directed against it measured. After 16 weeks, animals were sacrificed and the size of the mature worm burden established. In spite of interaction between E-S 62 and host antibody, a statistically significant relationship was found to exist between the amount of E-S 62 present in the bloodstream and the size of the parasite load. It is suggested that the detectable antigen level is more influenced by the size of the worm burden than the presence of antibody and that antibody is only likely to affect adversely antigen measurement in situations where the amount released is relatively low. Examples of this are early in infection and in low-level infections. These ideas are discussed in relation to the development and assessment of serological assays which attempt to predict parasite burden in human filarial infections.

Animals↗
PubMed1990

Differential diagnosis of Taenia saginata and Taenia solium with DNA probes.

A size selected genomic DNA library was constructed using DNA extracted from Taenia saginata. The DNA was digested using the restriction enzyme EcoR1 under star conditions and the 2-4 kbase fraction, selected following sucrose density-gradient separation, was cloned in the bacteriophage lambda gt 10. A panel of cestode DNAs including Taenia saginata, Taenia solium, Taenia taeniaeformis, Taenia crassiceps, Echinococcus granulosus and DNAs of bovine, porcine and human origin were used in conjunction with hybridization analysis to identify two recombinant bacteriophages. The first probe, designated HDP1, reacted specifically with T. saginata DNA. The second, designated HDP2, reacted with DNA from both T. saginata and T. solium but not the other DNA samples and thus provided a general reagent for positive identification of fragments of Taenia spp. proglottides of human faecal origin. If used in conjunction the two DNA probes allow positive identification of T. saginata. In the clinical situation it is important to be able to distinguish T. saginata and T. solium infections and DNA probes such as these may be useful in such differentiation.

Animals↗
PubMed1990

New approaches in the diagnosis of Taenia solium cysticercosis and taeniasis.

Taenia solium cysticercosis is now recognized as a priority in Mexico and a number of other developing countries, both in public health and in economic terms. Recognition of the problem has been greatly aided in recent years by new developments in molecular diagnostics. In this paper data are presented on ELISA for the detection of anti-cysticercus antibodies and of parasite antigens in patients with neurocysticercosis and in cysticercotic pigs. Also, several biological fluids were evaluated: cerebrospinal fluid (CSF), serum and saliva, all of which have proved useful. CSF is, however, the most appropriate for detection of antibodies and antigens in patients and serum in pigs. In addition, saliva may be especially used in epidemiological surveys. The electroimmuno-transfer blot technique (EITB) for antibody detection in patients and pigs has also proved highly sensitive and due to the use of an enriched fraction of glucoproteins, EITB is also highly specific. Cloned cDNA sequences from T. solium are now being assessed as an alternative source of antigens for immunodiagnosis. Two methods for the diagnosis of the adult stage of T. solium are also undergoing standardization. These are an ELISA for the detection of parasite antigens in fecal samples and DNA hybridization techniques for the detection of eggs in stools. Both assays have promising results and should now be assessed in larger numbers of clinical and epidemiological samples.

Animals↗
PubMed1990

Specific and cross-reacting antibodies in human responses to Onchocerca volvulus and Dracunculus medinensis infections.

Immunoelectroblotting and enzyme-linked immunosorbent assay were used to identify non-cross-reacting antigenic components of Dracunculus medinensis and the filarial worms Onchocerca volvulus, Loa loa, Wuchereria bancrofti, Brugia malayi, and Mansonella ozzardi. Parasite specific serodiagnostic ELISA systems for onchocerciasis and dracunculiasis were devised based on these findings. Phosphate buffered saline extracts of adult worms were passed through a column of monoclonal antibodies to phosphorylcholine (PC). Crude and PC-depleted extracts were reacted on ELISA plates with individual sera from subjects infected with a range of nematodes. Binding of total antibody (Ig) or IgG class antibody and IgG4 subclass antibody was revealed using goat antihuman-Ig-phosphatase conjugate, or appropriate mouse monoclonal antihuman-Ig-type-specific reagents, followed by goat antimouse-Ig-phosphatase conjugate. Specificity of ELISA was improved by restricting reaction to the host's IgG4 antibody subclass, and/or by removing PC determinants from crude antigens. In parallel immunoelectroblots, crude and PC-depleted extracts probed with pooled sera showed potentially useful diagnostic antigens, including a 12 kDa protein from D. medinensis and 14, 18, and 27 kDa proteins from O. volvulus. Two Onchocerca specific ELISA systems non-reactive with antibodies to D. medinensis were devised.

Animals↗
PubMed1990

Cloning of specific diagnostic antigens of Onchocerca volvulus.

Specific, serological diagnosis is one of the main goals in onchocerciasis research. To date this objective has been hampered by (a) scarcity of parasite material, and (b) antigenic cross-reaction between Onchocerca volvulus and other nematode species. In order to obtain specific antigens, and in amounts suitable for study, molecular biological techniques have been adopted. A lambda gt11 cDNA expression library prepared from O. volvulus adult female worms was screened using infected human sera from onchocerciasis patients and rabbit hyperimmune sera raised against Onchocerca and genus-specific Onchocerca antigen extracts. Five clones were selected and their inserts expressed as beta-galactosidase fusion proteins. The fusion proteins were examined using individual sera from patients with O. volvulus or Wuchereria bancrofti infections. Three of the fusion proteins were recognised by more than 80% of O. volvulus sera and exhibited weak reactivity with a few W. bancrofti sera. One of these three clones was recognised to a significantly greater degree by sera from sowda than from generalised onchocerciasis patients.

Animals↗
PubMed1990

Three B-cell surface molecules associating with membrane immunoglobulin.

Three cell-surface molecules associated with surface Ig on murine B cells have been identified. Surface-iodinated resting B cells were lysed in digitonin or Nonidet P-40. The lysates were then examined by immunocoprecipitation with anti-Ig reagents and SDS-PAGE. By this procedure, a 73,000 MW heterodimer, composed of 35,000 and 38,000 MW disulphide-linked subunits, was found to be associated with both mIgM and mIgD. This association was detectable in the detergent digitonin, but not Nonidet P-40. As an alternative approach, resting B cell mIg was capped and internalized by anti-Ig induced mIg cross-linking. The resulting 'modulated' cells were then screened for surface reactivity with a panel of monoclonal antibodies. Most antibodies tested (anti-class I, -class II, -LFA, -FcR gamma, -FcR epsilon) reacted equally well with control and modulated B cells. With two, however, there was a significant reduction in surface representation on modulated B cells. One determinant corresponds to a previously undescribed 90,000 MW murine surface marker, and the other was a polydisperse glycoprotein recognized by the mAb J11D.

Animals↗