PubMed Health⌕ Search

Biomedical subjects

M R Brandon

Publications and source records attributed to M R Brandon.

At least 55 records · Page 3Linked to original sources
PubMed1993

Characterization of the ovine interleukin-2 receptor-alpha chain: differential induction on precultured alpha beta and gamma delta T cells.

A combination of concanavalin A (Con A)-stimulated ovine lymph node (LN) cells and Chinese hamster ovarian (CHO) cells stably transfected with the ovine interleukin-2 receptor-alpha (IL-2R alpha) chain cDNA (CHO IL-2R cells) were used in a differential immunization strategy to generate several monoclonal antibodies (mAb) against the ovine IL-2R alpha chain. The specificity of one of the mAb, designated mAb 9-14, for the ovine IL-2R alpha chain was demonstrated by its reactivity with Con A-stimulated LN cells and CHO IL-2R cells, immunoprecipitation of a 47,000 MW protein from CHO IL-2R cells and inhibition of IL-2-dependent proliferation of Con A-stimulated ovine LN cells. Examination of IL-2R alpha chain expression on resting lamb peripheral blood lymphocyte populations showed a high frequency of IL-2R alpha chain expression on CD4 T cells but not on CD8 T cells, CD45RA+ cells or gamma delta T cells, which comprise up to 60% of lamb peripheral blood T cells. The kinetics of IL-2R alpha chain induction on Con A-stimulated peripheral blood alpha beta and gamma delta T cells was compared. A rapid induction of IL-2R alpha chain expression on precultured gamma delta T cells but not alpha beta T cells was observed within 6 hr of Con A stimulation. A preculturing period was required to 'prime' gamma delta T cells for rapid responsiveness to Con A. Using appropriate inhibitors, we demonstrated that both transcription and translation events were required for rapid IL-2R expression on precultured gamma delta T cells and therefore the 'priming' of gamma delta T cells by in vitro culture did not involve an accumulation of IL-2R alpha chain mRNA or preformed receptors within these cells.

Animals↗
PubMed1992

Production of methionyl-minus ovine growth hormone in Escherichia coli and one-step purification.

The overproduction of ovine growth hormone (oGH) in Escherichia coli is described, achieved in part by alteration of the codon usage for nine of the first 15 amino acids (aa) of the mature hormone. Recombinant oGH (re-oGH), representing 12% of the total cellular protein, was isolated from inclusion bodies by solubilisation using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The hormone was refolded and subsequently purified to greater than 95% homogeneity in a single step using preparative reverse phase high performance liquid chromatography. The aa sequence analysis revealed that the N-terminus of the E. coli-derived polypeptide was identical to that of pituitary-derived oGH, and re-oGH displayed potent somatotropic activity in vivo.

Amino Acid Sequence↗
PubMed1992

Solubilization of growth hormone and other recombinant proteins from Escherichia coli inclusion bodies by using a cationic surfactant.

Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations. The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone. The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein. In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations. It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies.

Animals↗
PubMed1992

Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production.

Regulation of ovine alveolar macrophage function by recombinant interferon gamma (rIFN gamma) and lipopolysaccharide (LPS) was investigated. Ten units per millilitre of rIFN gamma increased surface expression of MHC class I and class II (DR alpha, DP alpha, and DQ alpha) molecules but not other surface antigens examined. The upregulation of MHC class II expression was specifically blocked by rIFN gamma specific monoclonal antibodies and determination of a dose/response curve established that the minimum concentration of rIFN gamma required for increased class II expression was 0.1 U ml-1 and for increased class I expression, 1 U ml-1. Northern blot analysis indicated that rIFN gamma mediated increases in surface MHC class I and class II expression were due to increased levels of specific mRNA. Using Northern blot analysis and homologous human cDNA probes we failed to detect mRNA encoding the cytokines IL-1 alpha, IL-1 beta, and TNF alpha in RNA extracted from freshly isolated macrophages or macrophages cultured in medium alone. Exposure of macrophages to LPS increased production of all three cytokines although kinetics of upregulation varied. TNF alpha mRNA was induced to maximal levels within 1 h, declining thereafter. IL-1 alpha mRNA was detected at 1 h post stimulation with a maximal level at 5 h, but none at 24 h. In contrast, IL-1 beta mRNA was not detected until 5 h after stimulation with a low level remaining at 24 h. Dose response analysis indicated that LPS concentrations of 100 pg ml-1 induced detectable levels of TNF alpha mRNA while levels as low as 10 pg ml-1 induced secretion of bioactive IL-1. Analysis of the kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages indicated that levels peaked at 24 h post stimulation.

Animals↗
PubMed1992

Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae.

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.

Animals↗
PubMed1992

Local immunity in the mammary gland.

The mammary glands of pregnant and non-pregnant sheep were stimulated by infusion of killed Staphylococcus aureus, and the lymphoid cell response delineated with a panel of monoclonal antibodies. Seven days after antigen infusion, the mammary glands of both pregnant and non-pregnant sheep displayed a striking feature, characterised by the presence of numerous CD45R+ MHC class II+ B cells in the periductal connective tissues. These cells were seen to be clustering around blood capillaries with very prominent endothelial cell lining. Some CD5+ CD4+ lymphocytes were scattered among the B-cell clusters, whereas a few CD8+ lymphocytes were seen mainly at the periphery of the B-cell clusters. Fourteen days after antigen infusion, numerous plasma cells were observed, most of them being of the IgA isotype. Seven days after parturition (approximately 40 days after antigen infusion) the number of lymphocytes and plasma cells in the infused glands had declined dramatically. These data indicate that B cell and helper T-cell interaction can take place at the local sites of antigen stimulation in the mammary gland.

Animals↗
PubMed1992

Effects of tumour necrosis factor-alpha on growth hormone and interleukin 6 mRNA in ovine pituitary cells.

The inflammatory cytokines tumour necrosis factor-alpha (TNF alpha), interleukin 1 (IL-1) and interleukin 6 (IL-6) have been demonstrated to influence pituitary hormone synthesis directly and via the hypothalamus. Furthermore, IL-6 is produced by some anterior pituitary cells suggesting a paracrine/autocrine role for this cytokine. We show that TNF alpha induces dispersed ovine pituitary cells to produce increased levels of growth hormone (GH) and IL-6 mRNA and secreted IL-6 in a dose and time dependent manner. TNF alpha at concentrations between 1-1000 U/ml increased GH and IL-6 mRNA, relative to control levels, by 5 h post-stimulation. For IL-6, TNF alpha increased specific mRNA at 5 h and 12 h but not 24 h post-stimulation. TNF alpha also induced secreted IL-6 to levels above that spontaneously secreted at all time points from 5 h to 48 h. Levels of common glycoprotein alpha-subunit and follicle stimulating hormone-beta (FSH beta) subunit mRNA were unaffected by TNF alpha. We conclude that TNF alpha can regulate both GH and IL-6 synthesis in dispersed ovine pituitary cells. The implications for paracrine/autocrine control of pituitary hormone synthesis in acute and chronic disease are discussed.

Animals↗
PubMed1992

Functional expression of an ovine growth hormone receptor in transfected Chinese hamster ovary cells.

Structural heterogeneity has been demonstrated for growth hormone (GH) receptors from a number of species, and both high and low affinity art receptors have been characterised by ligand binding studies. In the present study, we have transfected Chinese hamster ovary (CHO-K1) cells with a cDNA clone encoding a full-length transmembrane ovine (o) GH receptor, under the regulatory control of the human metallothionein IIA promoter. A stably transfected cell line was established (GHR9.5) which expresses on the cell surface a single class of receptor which binds 220,000 [125I]oGH molecules at high affinity (Kd = 0.30 nM) which is comparable to the affinity established for endogenous oGH receptors in postnatal sheep liver microsomes (Kd = 0.27 nM, Freemark et al. (1987) Endocrinology 120, 1865-1872). The expressed receptor also binds ovine placental lactogen (oPL, 205,000 binding sites per cell) with high affinity (Kd = 0.76 nM). The presence of two species of oGH receptor was detected in GHR9.5 cells using affinity cross-linking analysis (M(r) 148,000 and M(r) 73,000) and given that the oGH receptor cDNA codes for a non-glycosylated receptor of M(r) 69,914, it is likely that these cross-linked species correspond to homodimeric and monomeric forms of the oGH receptor, each binding to a single molecule of GH. Parallel cross-linking studies with sheep liver microsomes also demonstrated two oGH receptor species (M(r) 133,000 and M(r) 58,000), the difference in relative molecular weights between the transfected and endogenous receptors presumably resulting from tissue-specific post-translational modifications. In the presence of oGH, the GHR9.5 cells respond by increasing total cellular protein synthesis by 27% relative to non-GH-exposed GHR9.5 cells, indicating the functionality of the expressed receptor. We also demonstrate unequivocally that oPL, through a specific interaction with the transfected oGH receptor, is able to mediate a similar cellular response (38% protein synthesis induction). Responsiveness to oGH and oPL in the GHR9.5 cells is dependent on serum starvation prior to oGH exposure and occurs only with prolonged exposure (greater than 2 h) to oGH. This cellular stimulation occurs independently of c-fos transcription which has previously been shown to be one of the earliest events associated with GH action in tissues expressing endogenous GH receptors (Doglio et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1148-1152; Slootweg et al. (1990) J. Mol. Endocrinol. 4, 265-274).

Animals↗
PubMed1992

Cellular immune responses in the skin of sheep infected with larvae of Lucilia cuprina, the sheep blowfly.

Cellular immune responses were observed in the skin of sheep after primary and secondary infection with sheep blowfly (Lucilia cuprina) larvae. Both primary and secondary infections resulted in a massive cellular infiltration within 48 h of wound initiation, with the majority of cells having the CD45 phenotype. Neutrophils comprised the major cell type at the skin surface. In the dermis, the number of CD4+ T helper, gamma delta-TCR+ cells and T19+ (CD4-, CD8-) T cells also increased significantly in skin during both primary and secondary infections compared with control sites. However, there was no significant difference in the numbers of these cells between primary and secondary infections. An increase in the expression of the CD1 antigen on Langerhans/dendritic cells was observed, along with an apparent increase in the number of these cells in secondary lesions, with the majority of the cells being concentrated in the upper dermis and epidermis. While there was no increase in mast cells, eosinophils increased significantly during infection compared with control sites. The cellular infiltration observed following primary and secondary infections suggests polyclonal activation of T cells and their selective recruitment to the lesion site.

Animals↗
PubMed1992

Quantitative and qualitative changes in the intraepithelial lymphocyte population in the uterus of nonpregnant and pregnant sheep.

PROBLEM: Previous studies have revealed the presence of a unique population of CD45R+ granulated cells in the sheep uterine epithelium. In the present study, dramatic changes in this cell population and in the nongranulated lymphocytes in the uterine and endometrial glandular epithelium of non-cycling, cycling, pregnant, and postparturient sheep are described. In noncycling and cycling sheep, the granules in the granulated intraepithelial cells were small. From days 55 to 134 of pregnancy, the granules in these cells were large, and there was a significant increase (P < 0.01) in the proportion of this cell population in the uterine epithelium but not in the endometrial glandular epithelium located in the deeper region of the stroma. The number of these cells declined dramatically (P < 0.01) from 2 to 15 days after parturition. Both the tissue distribution and the time of activation of these cells suggests they are different from the granulated lymphocytes described in placentae of mice and man. CONCLUSIONS: It is concluded that this unique population of granulated cells is derived from lymphocytes, and that these cells become metabolically active from mid- to late-pregnancy and may play a physiological role during pregnancy or birth. In contrast, the number of nongranulated intraepithelial lymphocytes were suppressed throughout pregnancy and they probably do not play a role in pregnancy.

Animals↗
PubMed1992

Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum.

Mycoplasma gallisepticum cell membranes were used to immunize mice to produce monoclonal antibodies to cell surface proteins. Three monoclonal antibodies were chosen for further characterization. All three reacted in immunoblots with an M. gallisepticum protein band of M(r) approximately 67,000 (designated pMGA). By using immunoelectron microscopy, pMGA was shown to be located on the cell surface. When M. gallisepticum whole cells were treated with up to 250 micrograms of trypsin per ml for 30 min, the only major protein lost from the cell surface as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot transfer was pMGA. Two of the pMGA-specific monoclonal antibodies inhibited hemagglutination of chicken erythrocytes by M. gallisepticum S6, suggesting a role for pMGA in the attachment of M. gallisepticum to chicken erythrocytes. Sequencing the amino terminus of pMGA yielded 17 amino acids with no significant homology with the Mycoplasma pneumoniae attachment protein P1 or any other protein in the GenBank, Swiss-Prot, and EMBL data bases.

Amino Acid Sequence↗
PubMed1992

Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.

The lentiviruses of sheep, goats, and horses cause chronic multiorgan disease in which macrophages are highly permissive for viral replication. Monocytes, which mature into macrophages, are thought to be latently infected with lentivirus, but the extent to which other leukocytes are infected is unknown. Dendritic cells have not been studied separately from monocytes and T-cell subsets have not been examined in previous attempts to identify infected cells in peripheral blood mononuclear cells (PBMC). We found no evidence of T-cell tropism using an animal-passaged, pathogenic ovine lentivirus. Phytohemagglutinin-stimulated infectious PBMC produced 20-fold less virus than differentiated macrophages, and cocultivation of infectious PBMC with fresh, uninfected phytohemagglutinin blasts did not facilitate virus replication. Furthermore, central lymph cells, the best in vivo source of purified lymphocytes, lacked virus and did not yield virus upon in vitro cultivation. In contrast, cultivated blood-derived macrophages were highly permissive for viral replication. To identify the latently infected PBMC, PBMC from infected sheep were selectively depleted of monocytes and B cells by passage over nylon wool and then of nonadherent cells bearing CD4, CD8, T19, gamma delta T-cell receptor, CD45RA, or major histocompatibility complex class II antigens by panning. Removal of adherent monocytes and B cells or of adherent cells and the three major T-cell subsets (CD4+, CD8+, T19+) did not decrease the infectivity of PBMC. The richest sources of infected cells in fresh PBMC were CD45RA+ and major histocompatibility complex class II+ nonadherent cells, which are three characteristics of dendritic cells. Thus, the dendritic cell, and not the monocyte or the CD4+ cell, is probably the predominant infected cell type in blood.

Animals↗
PubMed1992

In vivo inhibition by a monoclonal antibody to CD4+ T cells of humoral and cellular immunity in sheep.

The ability of intravenously injected anti-CD4 and anti-CD8 monoclonal antibody (mAb) to deplete specific lymphocyte subsets in vivo and their effects on antibody responses to ovalbumin (OVA) and Brucella abortus, and skin reactivity to T-cell mitogens was examined in merino lambs. Repeated administration of anti-CD4 or anti-CD8 mAb caused a specific and sustained depletion of target cells from peripheral blood. Anti-CD4 mAb significantly inhibited the in vivo antibody response to OVA but had no effect on the antibody response to LPS of B. abortus. In contrast, antibody responses to both OVA and B. abortus lipopolysaccharides (LPS) remained unaffected in lambs depleted of their CD8+ T lymphocytes. These results confirm the T-cell dependence and independence of antibody responses to OVA and LPS, respectively. Skin reactions elicited by intradermal injections of phytohaemagglutinin (PHA) and concanavalin A (Con A) were also significantly suppressed in lambs depleted of their CD4+ T cells, but treatment with anti-CD8 mAb had no effect on skin responsiveness. Together, these results suggest that mAb can be extremely effective at selectively depleting lymphocyte subsets in vivo and can be used for studying various aspects of immunoregulation and immunity in sheep.

Animals↗
PubMed1992

Molecular cloning, expression and characterization of the ovine IL-2R alpha chain.

Interleukin-2 (IL-2) stimulates the proliferation of activated antigen-specific T cells through its interaction with high affinity receptors. This event is largely regulated by the inducible expression of the alpha-chain (CD25) which, in combination with the beta-chain and possibly additional chains, forms the high affinity IL-2 receptor (IL-2R) complex. From a concanavalin A (Con A)-activated ovine T-cell complementary DNA (cDNA) library we have isolated two cDNA clones which together constitute a 2650 base pair (bp) messenger RNA (mRNA) species encoding the ovine IL-2R alpha chain. The nucleotide sequence has high homology with analogous cDNA from other species and predicts a mature protein of 254 amino acids. In addition to the predominate 2.6 kilobase (kb) ovine IL-2R alpha chain mRNA species. Northern blot analysis of activated T-cell RNA revealed two larger mRNA species. The ovine IL-2R alpha chain cDNA was transfected into CHO cells and low affinity binding of human recombinant IL-2 demonstrated. Polyclonal antisera generated against the transfected cells cross-reacted with Con A-activated ovine lymphocytes. In addition these antisera were used to immunoprecipitate a unique 50,000 MW protein from the transfected cells. It is likely that this protein represents the expressed ovine IL-2R alpha chain cDNA which is heavily glycosylated as distinct from the 30,869 MW primary translation product. Southern blot analysis of ovine genomic DNA suggests that the ovine IL-2R alpha chain is encoded by a single copy gene.

Animals↗
PubMed1991

Nucleotide sequence, polymorphism, and evolution of ovine MHC class II DQA genes.

The nucleotide sequence of all exons and introns, excluding exon 1, of the ovine major histocompatibility complex (MhcOvar) genes analogous to the HLA-DQA1 and -DQA2 genes has been determined and the gene structure found to be similar to that reported for other species. The predicted amino acid sequences of the Ovar-DQA genes have been compared with the equivalent DQA genes in man, mouse, rat, rabbit, and cattle and used to determine the evolutionary relationships of the sheep class II genes to these other species. Northern blot analysis of sheep mRNA using exon specific probes for each of the two Ovar-DQA genes show that both genes are transcribed, whereas in humans there is no evidence that HLA-DQA2 is transcriptionally active. Restriction fragment length polymorphisms (RFLPs) have been used to define a polymorphic series of alleles in both Ovar-DQA genes and have indicated that the number of DQA genes is not constant in sheep as it is in humans, but varies with the haplotype.

Amino Acid Sequence↗
PubMed1991

The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB.

The nucleotide sequences of one Ovar-DQB gene, excluding exon 1 and parts of the introns, and one Ovar-DRB pseudogene are presented. The structure of the Ovar-DQB gene is typical of a major histocompatibility complex (MHC) class II B gene and demonstrates considerable sequence similarity with that of humans including such characteristics as the less common polyadenylation signal, ATTAAA. The ovine sequence has a typical 5' acceptor splice signal for exon 5, thus potentially encoding a full length cytoplasmic tail. The Ovar-DRB gene identified in this study was found to be a pseudogene, lacking a defined exon 2 and containing premature termination codons in both exons 3 and 4. The 3' donor splice site of exon 3 is also atypical. A purine-pyrimidine microsatellite repeat, (dC.dA)15, in the 3' region of the pseudogene may be a hotspot for recombination within the ovine DR subregion.

Amino Acid Sequence↗