Biomedical subjects

M R Brandon

Publications and source records attributed to M R Brandon.

At least 37 records · Page 2Linked to original sources

PubMed1995

Characterization of local antibody responses to the gastrointestinal parasite Haemonchus contortus.

The ability to identify antigens associated with an infection has generally relied on the use of serum antibodies produced by infected or previously exposed individuals. A major drawback with the use of serum is that it does not necessarily reflect the local antibody response at mucosal tissue sites. This study describes an approach that allows the use of antibodies generated close to the infection site to detect the transient expression of stage-specific antigens during infection with the gastrointestinal parasite Haemonchus contortus. This was achieved by infecting immune sheep with H. contortus larvae and removing the abomasal lymph nodes draining the infection site shortly after the challenge infection. Antibody-secreting cell (ASC) probes were generated from these lymph nodes after short-term in vitro culture of cell suspensions, which allowed the accumulation of antibodies secreted by in vivo-induced ASC into the culture supernatant. Lymph node culture supernatants (= ASC probes) from immune sheep challenged 5 days previously were used to probe Western blots of third and fourth stage larval preparations, and revealed distinct reactivity to larval antigens. No antibody reactivity to larval antigen preparations was detected in sheep that were not challenged. The number of antigens identified using ASC probes was significantly restricted compared to either pre- or post-challenge sera. In contrast to the variability of the serum response, the specificity of ASC probes was highly repeatable between different sheep. ASC probes were also used to purify a H. contortus larval antigen by affinity chromatography, which allowed limited biochemical studies to be undertaken. The antigen(s) recognized by the ASC probes were shown to be expressed on the surface of the larvae. These studies illustrate the use of a novel means of studying the local antibody response close to a mucosal infection site in order to identify and isolate stage-specific antigens expressed during infection.

Animals↗

PubMed1994

Antibody secreting cells as specific probes for antigen identification.

Parasite specific antibody probes were prepared by harvesting lymphocytes from infected tissue and incubating them in vitro to allow the spontaneous secretion of antibodies in the culture medium by antibody secreting B cells present in the lymphocyte cultures. The culture supernatant was then used to screen Western blots of parasite antigens and resulted in the detection of antigens specific for the parasite stages present in the tissue at the time of sampling. Similar antigen recognition patterns were also observed when the cells were taken from the draining lymph nodes but the same pattern was not observed with serum taken from the same animal. The use of antibody probes obtained from in vivo induced antibody secreting B cells (ASC probes) offers a unique and universal approach to study local antibody recognition during infection.

Animals↗

PubMed1994

A spectroscopic and equilibrium binding analysis of cationic detergent-protein interactions using soluble and insoluble recombinant porcine growth hormone.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Anilino Naphthalenesulfonates↗

PubMed1994

Membrane dynamics during migration of placental cells through trophectodermal tight junctions in sheep and goats.

Binucleate cells in ruminant trophectodermal epithelium are unique in that they form part of the tight junction as they migrate across it, maintaining the ionic barrier seal to the internal milieu of the fetus. Such participation imposes considerable constraints on the cell migration because membrane cannot flow through a tight junction. We report quantitative ultrastructural immunocytochemical evidence for vesicle membrane insertion into the binucleate cell plasmalemma which allows the cells to form a pseudopodium past the tight junction. This pseudopodium increases continuously in area by vesicle insertion and develops a close apposition to the plasmalemma of the fetomaternal syncytium which constitutes the fetomaternal boundary in the placenta of the sheep and goat. Eventually the apposed membranes of the binucleate cell pseudopodium and the syncytium fuse by vesiculation and the cytoplasm and nuclei of the binucleate cell merge into the fetomaternal syncytium. The binucleate cell plasmalemma remaining on the trophectodermal side of the tight junction is blebbed off into, and phagocytosed by, the uninucleate trophectodermal cells between which the binucleate cell passed. This process permits the delivery of the binucleate cell granules to the maternal side of the placenta but none of the fetal molecules expressed on the plasma membrane of the binucleate cells are exposed to potential maternal immunological rejection.

Animals↗

PubMed1994

Studies on the distribution of immune cells in the uteri of prepubertal and cycling gilts.

To establish the cellular basis for the local immune response in the porcine uterus, immunohistochemical studies using a panel of monoclonal antibodies to pig leukocytes were conducted on uterine tissues from prepubertal and cycling gilts. In prepubertal uteri, neutrophils were the most predominant cell type, while MHC class II+ cells and CD2+ T lymphocytes were also common. At the early-stage of the oestrous cycle, CD2+ T cells were numerous in the endometrium, particularly in the uterine epithelium and subepithelial regions. However, by the mid-stage of the cycle there was a significant and dramatic fall in CD2+ T cells and other lymphocytes expressing the CD4, CD8 and CD1 phenotypes, MHC class II+ cells were predominant throughout the endometrium. During late oestrus there was a dramatic infiltration of neutrophils into the subepithelial stroma. A distinct increase in the CD2+ intraepithelial T lymphocyte population was also observed at this stage of the cycle. It was concluded that in the healthy, non-pregnant pig uterus T lymphocytes, macrophages and neutrophils were the prominent leukocyte cell types and their migration and distribution in the uterus was strongly influenced by the oestrous cycle. These immune cells may play an important interactive role in the cyclic cellular changes in both the structure and function of the endometrium. Furthermore, the leukocyte phenotypes found in the porcine endometrium indicate that a local cellular immune response could be elicited.

Age Factors↗

PubMed1994

Inflammatory response in the pig uterus induced by seminal plasma.

The immunological and physiological influence of seminal plasma on the local uterine environment was investigated by immunohistochemical and flow cytometrical studies on uterine tissues and lymph nodes taken from gilts after mating with a vasectomised boar and from control, unmated gilts. These studies revealed that mating with a vasectomised boar induces an acute transient inflammatory response in the endometrium resulting in marked changes in the presence and distribution of leukocytes and extensive proliferation of the endometrial glands. At the same time there was an increase in CD8L and sIg+ cells and an up-regulation of MHC class II and IL-2 receptor expression in the uterine lymph nodes of mated pigs. This would suggest that seminal plasma deposited in the uterus can activate cells in the local draining lymph nodes. Together, these results demonstrate in utero that pronounced immunological and physiological changes are induced in vivo by seminal plasma.

Animals↗

PubMed1994

The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph.

Changes in lymphocyte traffic in efferent lymph from the prescapular lymph node of sheep were monitored during local primary and secondary infection with blowfly, Lucilia cuprina. During primary infections the response was characterised by an increase in the output of CD4+ T cells over CD8+ T cells for the first 48 h after wound initiation. By 72 h the output of CD8+ T cells exceeded that of CD4+ T cells. During secondary infections the increased output of CD8+ T cells was more pronounced and occurred earlier at approximately 48 h. The percentage of B lymphocytes as measured by sIg, CD45R and MHC class II expression increased at approximately 96-120 h after both primary and secondary infections, with the secondary response being greater than the primary. This increase in B cells corresponded with peak antibody titres recorded in the efferent lymph to a first instar antigen preparation as measured by ELISA. An increase in IFN-gamma and soluble IL-2 receptor was recorded after both primary and secondary infections, with the response after secondary infection being greater than that recorded after primary larval infections.

Animals↗

PubMed1994

Production and application of monoclonal antibodies to ovine interleukin-1 alpha and interleukin-1 beta.

Monoclonal antibodies (mAbs) were raised against recombinant ovine interleukin-1 alpha and beta (ovIL-1 alpha and ovIL-1 beta). Five ovIL-1 alpha specific mAbs and three ovIL-1 beta specific mAbs, all of the IgG1 isotype, were characterized. Four of the five ovIL-1 alpha specific mAbs, designated 10.36, 10.49, 10.82 and 5.16, fell into two distinct groups based on several criteria. MAbs 10.36, 10.49 and 10.82 reacted with recombinant ovIL-1 alpha in Western blot analysis, were potent in neutralizing ovIL-1 alpha biological activity in vitro and bound to the same or a closely related epitope. MAb 5.16 also bound ovIL-1 alpha in Western blot analysis, but was less potent in neutralizing ovIL-1 alpha biological activity and bound to a different epitope. A fifth ovIL-1 alpha specific mAb, 5.01, had some characteristics of antibodies from both groups. While the combination of mAb 5.16 with any of 10.36, 10.49 and 10.82 was suitable for detection of ovIL-1 alpha in a sandwich immunoassay, the most sensitive detection of ovIL-1 alpha utilized mAb 10.82 for capture and a rabbit polyclonal anti-ovIL-1 alpha antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent. This combination of reagents had a detection limit for ovIL-1 alpha of 5 pg ml-1 and could detect both recombinant and native ovIL-1 alpha. Of the three ovIL-1 beta specific mAbs, (designated 2.93, 3.41 and 5.60) 3.41 and 5.60 recognized the same or a closely related epitope while 2.93 recognized an epitope more accessible on denatured ovIL-1 beta and proved most useful in Western blot analysis. Only mAb 3.41 was potent in neutralizing ovIL-1 beta biological activity in vitro. A sandwich immunoassay using mAb 3.41 to capture ovIL-1 beta and a rabbit polyclonal anti-ovIL-1 beta antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent had a sensitivity of 5 ng ml-1. The immunoassays were used to assess the relative proportions of IL-1 alpha and IL-1 beta in the supernatant of lipopolysaccharide stimulated ovine alveolar macrophages with IL-1 beta found to be the predominant secreted species of ovIL-1.

Animals↗

PubMed1994

Production and in vivo use of recombinant ovine IL-1 beta as an immunological adjuvant.

To determine the potential of ovine interleukin 1 (IL-1) as a vaccine adjuvant in sheep, we have expressed and purified recombinant ovine IL-1 beta (rovIL-1 beta) from bacterial cultures using a modified form of the ovine IL-1 beta cDNA. Adjuvant trials using the model protein avidin demonstrated that rovIL-1 beta when administered in association with a compound providing a slow-release mechanism, resulted in significant enhancement of specific serum antibody levels in both mice and sheep. In a dose-response experiment in sheep, intradermal immunization with avidin plus either 10 or 100 micrograms of rovIL-1 beta in aluminium hydroxide resulted in antibody levels four- to eightfold higher than immunizations without rovIL-1 beta. The addition of rovIL-1 beta also resulted in a more severe DTH response to avidin indicating that rovIL-1 beta is able to enhance both humoral and cell-mediated responses to avidin. The highest antibody titres were observed when sheep received rovIL-1 beta in both the primary and secondary immunizations although the addition of rovIL-1 beta in only one of the immunizations still resulted in a significant increase in antibody levels. Additional experiments showed that rovIL-1 beta and avidin must be administered in a site drained by the same lymph node for the adjuvant effect of rovIL-1 beta to be observed.

Adjuvants, Immunologic↗

PubMed1994

Identification of a liver-specific promoter for the ovine growth hormone receptor.

Growth hormone (GH) receptor cDNA clones from several species are characterized by heterogeneity in the 5' untranslated region (5'UT). This has been attributed to different promoters directing the expression of the gene from exons encoding 5'UT's which are alternatively spliced onto a common splice acceptor 11 basepairs (bp) upstream of the initiating AUG on exon 2. The following study identifies exon 1A of the ovine (o) GH receptor gene, corresponding to the 5'UT of a developmentally regulated, liver-specific transcript. Exon 1A spans 206 bp at a position 17 kilobases (kb) upstream of exon 2. Sequencing of the 669 bp region 5' to the transcription initiation site (+1) reveals a TATA box at -31, a CCAAT box at -88, and putative binding sites for several transcription factors involved in liver-specific gene expression. Two repetitive sequence elements are located in the 5' and 3' flanking regions of exon 1A. Functional analysis of the 4.5 kb region upstream of exon 1A was performed by transfecting the human hepatoma cell line HuH7 with luciferase reporter gene constructs. Positive and negative regulatory regions are identified, with basal promoter activity within 473 bp of the transcription initiation site. A 47 bp region containing putative binding sites for the activated glucocorticoid receptor and C/EBP-like proteins, between -180 and -133, is essential for transcriptional activation.

Animals↗

PubMed1994

Strategies for the isolation and characterization of bovine embryonic stem cells.

The practical application of advanced breeding technologies and genetic manipulation of domestic animals is dependent on the efficient and routine isolation of embryonic stem (ES) cell lines from these species. ES cell lines of proven totipotency have thus far been isolated only from the mouse. Murine ES cells can be identified by a number of criteria including morphology and characteristics in culture, the presence of specific markers, differentiative capacity and contribution to chimaeras. Reported cell lines derived from ruminant preimplantation embryos do not stably exhibit these characteristics. As demonstrated for the mouse, primordial germ cells may provide an alternative source for pluripotential cell lines. The isolation, culture and preliminary characterization of bovine primordial germ cell-derived (PGCd) cells are described in this paper. The PGCd cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase. With farm animals, long generation intervals and small numbers of offspring make it important to develop techniques for evaluating chimaeric embryos in vitro before embarking on expensive in vivo programmes. A method for labelling putative pluripotential cells with a fluorochrome marker to follow the fate of such cells was developed. Labelled PGCd cells were injected into blastocysts and the chimaeric embryos were monitored in vitro. Preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1994

Identification of a novel antigen from Corynebacterium pseudotuberculosis that protects sheep against caseous lymphadenitis.

A 40-kDa protein antigen from Corynebacterium pseudotuberculosis has been identified by application of a strategy that employs locally derived antibody-secreting cells (ASC). ASC probes generated by culture of ASC obtained from the lymph node draining the site of infection showed a specificity restricted to a 40-kDa antigen. Analysis of immunoblots with sequential serum samples taken from sheep during the course of experimental primary infection with C. pseudotuberculosis also revealed the 40-kDa antigen as an early immunodominant antigen. Sheep vaccinated with two 100-micrograms doses of a 40-kDa antigen preparation in aluminium hydroxide adjuvant were protected against infection with C. pseudotuberculosis, with an 82% reduction in the proportion of infected sheep and a 98% reduction in lung lesions. Sera from vaccinated sheep exhibited a strong response only to the 40-kDa antigen on immunoblots. These results strongly suggest that the 40-kDa antigen plays a major role in immunity to caseous lymphadenitis.

Animals↗

PubMed1994

Expression and characterization of biologically active ovine FSH from mammalian cell lines.

Stably transfected cell lines expressing the alpha subunit, beta subunit and alpha/beta heterodimer of ovine (o)FSH have been established following the transfection of Chinese hamster ovary cells with alpha and beta subunit cDNA expression vectors. In the absence of the alpha subunit, FSH beta subunit polypeptides were inefficiently secreted and displayed a short intracellular half-life, while free alpha subunits were readily secreted in the absence of the beta subunit. Cotransfection of oFSH alpha and beta subunit cDNAs led to heterodimer assembly and secretion. While alteration of the nucleotide sequence flanking the beta subunit AUG initiation codon did not appreciably enhance heterodimer biosynthesis and secretion, the replacement of the 5' untranslated and signal peptide-coding regions of the beta subunit cDNA with the corresponding sequences from an oGH cDNA clone was associated with a twofold increase in oFSH heterodimer secretion. The recombinant oFSH had a higher molecular weight than pituitary-derived oFSH, and was more acidic than the native hormone when analysed using isoelectric focusing, suggesting a greater degree of sialylation of the recombinant hormone. A comparison of the activities of the recombinant and native hormones in the porcine testis radioreceptor assay and in the in vitro Sertoli cell bioassay revealed that the recombinant oFSH displayed enhanced biological activity in the Sertoli cell assay when compared with the native hormone.

Amino Acid Sequence↗

PubMed1993

Characterization of placentation-specific binucleate cell glycoproteins possessing a novel carbohydrate. Evidence for a new family of pregnancy-associated molecules.

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and characterization of a ruminant interleukin-6 cDNA.

By hybridization with a human interleukin-6 (IL-6) cDNA fragment a corresponding ruminant (ovine) cDNA was isolated from a lipopolysaccharide (LPS)-stimulated alveolar macrophage library. The nucleotide sequence of the cDNA and the predicted amino acid sequence of the protein showed significant homology to the human and murine molecules. Ovine IL-6 cDNA encodes a polypeptide of 208 amino acids that, based on analysis of human IL-6, is processed to a protein of 180 amino acids. Northern blot analysis and the 7TD1 bioassay were used to analyse regulatory aspects of IL-6 production by primary ovine fibroblasts. Both LPS and recombinant ovine IL-1 alpha were shown to induce IL-6 mRNA with peak levels occurring at 1 h post-stimulation and declining thereafter. When fibroblasts were pretreated with cyclohexamide prior to stimulation the level of induction by LPS and IL-1 alpha increased dramatically and peak levels were observed at 5 h post-stimulation. The level of secreted IL-6 increased rapidly over the first 24 h and continued to increase over the next 48 h.

Amino Acid Sequence↗

PubMed1993

Recombinant cytokines as immunological adjuvants.

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin-2 (rovIL-2), interleukin-1 alpha (rovIL-1 alpha) and tumour necrosis factor alpha. These purified proteins had specific activities in appropriate bioassays of 1 x 10(7) 1 x 10(7) and 1 x 10(5) U/mg, respectively. Recombinant ovIL-1 alpha was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL-1 alpha significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 micrograms per sheep enhanced the humoral response to a similar extent. Recombinant ovIL-1 beta had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL-1 beta into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Adjuvants, Immunologic↗

PubMed1993

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes.

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine lambda gt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class II A sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.

Alleles↗

PubMed1993

Monoclonal antibody to CD4+ T cells abrogates genetic resistance to Haemonchus contortus in sheep.

The roles of CD4+ and CD8+ T cells in genetically determined resistance of sheep to Haemonchus contortus (a natural host-parasite relationship) was investigated by selectively depleting genetically resistant merino lambs of their CD4+ or CD8+ T cells by treatment with mouse monoclonal antibody (mAb) specific for the appropriate determinant before and during challenge infection. Administration of anti-CD4 mAb to genetically resistant lambs completely abrogated their expression of genetic resistance as indicated by significantly higher faecal egg output and worm burdens found in the CD4+ T-cell-depleted lambs compared with those of controls. Host responses associated with resistance to H. contortus including mucosal mast cell hyperplasia and tissue eosinophilia were also significantly suppressed in CD4-depleted lambs. The development of anamnestic anti-parasite antibody responses were also significantly inhibited by anti-CD4 mAb. Furthermore, anti-CD4 mAb abolished differences in host responses between genetically resistant and random-bred (susceptible) lambs. In contrast, depletion of CD8+ T cells had no effect on genetic resistance; faecal egg output, worm counts, mast cells and eosinophil responses in CD8-depleted lambs were not significantly different from those in controls. Together, these results suggest that CD4+ T cells play a pivotal role in mediating genetic resistance to H. contortus, and in the generation of mucosal mast cell hyperplasia, tissue eosinophilia and anti-Haemonchus antibody. CD8+ T cells appear to play no protective role. The possible mechanisms by which CD4+ T cells might mediate anti-parasite resistance are discussed.

Animals↗