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G F Newlands

Publications and source records attributed to G F Newlands.

At least 19 recordsLinked to original sources
PubMed2001

The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants.

Rapid developments in molecular biology have had an enormous impact on the prospects for the development of vaccines to control the major nematode and trematode infestations of livestock. Vaccine candidates are purified using conventional protein chemistry techniques but the limitations imposed by the scarcity of parasite material provide an insurmountable barrier for commercial vaccine production by this means. The ability to purify mRNA from different parasite life-cycle stages and to prepare cDNA expression libraries from it has proven central to the identification of immunogenic parasite proteins. Potentially, protective parasite antigens can now be produced in recombinant form in a variety of vectors and this represents a key breakthrough on the road to commercial vaccine production. The contribution of molecular biology to this process is discussed using several examples, particularly in vaccine development against the pathogenic abomasal nematode of sheep and goats, Haemonchus contortus, and the liver fluke of sheep and cattle, Fasciola hepatica. The difficulties of producing recombinant proteins in the correct form, with appropriate post-translational modification and conformation, are discussed as well as emerging means of antigen delivery including DNA vaccination. The opportunities offered by genome and expressed sequence tag analyses programmes for antigen targeting are discussed in association with developing microarray and proteomics technologies which offer the prospect of large scale, rapid antigen screening and identification.

Animals↗
PubMed2001

Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus.

A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatin. Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.

Amino Acid Sequence↗
PubMed2000

Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortus.

Jacalin lectin was used as a ligand to isolate a fraction containing two distinct protective antigens from detergent extracts of membranes from Haemonchus contortus. The first antigen was identified as a complex which appeared very similar to Haemonchus galactose-containing glycoprotein (H-gal-GP), which is a previously described protective protease complex, except that it was substantially depleted of one of the main H-gal-GP components, a 230 kDa metallopeptidase-containing band. The new complex was termed Haemonchus sialylated galactose-containing glycoprotein (H-sialgal-GP), because it bound to jacalin but not to peanut lectin and only jacalin will bind the sialylated form of galactosyl (beta-1, 3) N-acetylgalactosamine. Two protection trials with sheep showed that H-sialgal-GP and H-gal-GP were equally efficacious, reducing numbers of Haemonchus eggs by between 86% and 93% and worms by between 52% and 75%, respectively. The second jacalin-binding protective antigen fraction was separated from H-sialgal-GP by ion exchange and gel filtration chromatography. It was greatly enriched for two proteins termed p46 and p52 according to their apparent molecular weights. Immunization of sheep with these proteins gave protection values of 78% for eggs and 33% for worms, which are significantly lower than those obtained with either H-gal-GP or H-sialgal-GP. N-terminal amino acid sequence data from p46 and p52 showed that both proteins were closely related to a previously described 45 kDa Haemonchus membrane protein, which had conferred protection against Haemonchus in guinea-pigs.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus.

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a 'proteinase-enriched' Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT-PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

Animals↗
PubMed1999

Cloning and characterization of a beta-galactoside-binding protein (galectin) from the gut of the gastrointestinal nematode parasite Haemonchus contortus.

A cDNA encoding a beta-galactoside-binding lectin (galectin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a membrane protein complex (H-gal-GP) derived from the parasites' gut. The cDNA sequence, exhibiting a tandem repeat structure and designated Hco-gal-2, showed significant levels of similarity with galectins from several species of nematode as well as mammalian galectin type 4. Native galectin was preferentially extracted from the H-gal-GP complex and also from an insoluble membrane fraction prepared from adult worms using lactose-agarose affinity chromatography. The affinity-purified material had apparent molecular mass of around 35 kDa with 3 distinct bands visible on SDS-PAGE. All 3 bands were identified as galectins by reaction with antiserum raised to recombinant Hco-GAL-2 on Western blot. To determine whether H. contortus galectins have any protective capacity against infection lambs were vaccinated with affinity-purified galectin and subsequently given a single challenge infection. Vaccination did not confer any protection against infection with H. contortus as judged by faecal egg output or worm counts.

Amino Acid Sequence↗
PubMed1999

Mucosal mast cell responses and release of mast cell protease-I in infections of mice with Hymenolepis diminuta and H. microstoma: modulation by cyclosporin A.

The dynamics of intestinal mucosal mast cells and the major mucosal mast cell protease were followed during the course of laboratory infections of mice with Hymenolepis diminuta and H. microstoma. The effects of the drug cyclosporin A (CsA), which is both immunosuppressive and selectively anthelmintic depending upon dose regime, were determined. In H. diminuta infections worm expulsion occurred around day 9 and coincided with peak mastocytosis and peak mMCP-I concentrations in tissues and serum. Immunosuppressive treatment with CsA prevented worm expulsion, permitting some individuals to reach maturity, and abrogated mast cell proliferation and mMCP-I production and release. By contrast, H. microstoma infections persisted for 64 days in spite of a considerable mastocyosis in both intestine and bile duct tissues accompanied by a high level of mMCP-I in tissues and serum. A subimmunosuppressive regime of CsA had only limited effects on worms and mast cell numbers and activity. Together these data shed light on the variable mast cell response to gastrointestinal infections and on the potential significance of parasite location in evasion of mast cell action. Use of CsA reveals the contributions of both T cell-dependent mechanisms, including mast cell proliferation and activation, and T cell-independent events in regulating intestinal helminth infections.

Animals↗
PubMed1999

Further immunization and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus.

Immunization of sheep with a protective antigen complex from the intestinal cells of Haemonchus contortus in Freund's adjuvant stimulated individually variable antibody responses but still conferred significant protection against parasite infection. Correlation between antibody concentration and degree of protection was suggestive of antibody being the effector mechanism. The antigen is known as Haemonchus galactose-containing glycoprotein complex (H-gal-GP) because it binds to lectins with a specificity for N-acetyl-galactosamine. Polypeptide composition analysis by polyacrylamide gel electrophoresis indicated an apparent molecular weight of about 1000 kDa and SDS gels revealed four major polypeptides, containing between 2 and 5 disulphide linked subunits, nearly all being glycosylated. N-terminal amino acid sequence was obtained from 12 subunits, ten showing homologies with cDNAs from Haemonchus encoding either pepsin, metalloprotease or cysteine protease-like enzymes. pH optima, inhibitor and various substrate studies confirmed that the native complex possessed proteolytic activities in agreement with the sequence data. Although the cDNAs predicted water soluble enzymes, little of the complex was solubilized from worm membranes without the use of a detergent, such as Triton X-100. It is hypothesized that H-gal-GP is a gut membrane associated multiprotease complex which is involved in the digestion of the blood meal and which can be neutralized by specific antibodies with drastic consequences for the parasite.

Amino Acid Sequence↗
PubMed1997

Mast cell specific proteases in rat brain: changes in rats with experimental allergic encephalomyelitis.

Mast cell populations were identified within brain parenchyma by their specific proteases, using antibodies for immunohistochemistry and ELISAs, and riboprobes were developed for in situ hybridisation. Connective tissue mast cells expressing rat mast cell protease I (RMCPI) mRNA and immunoreactivity were observed in thalamus and showed no degranulation at 3, 8 and 13 days after induction of experimental allergic encephalomyelitis (EAE). Mucosal-like mast cells were clearly demonstrated in control rats by measuring RMCPII and by visualising cells expressing RMCPII mRNA and immunoreactivity. At day 13, but not 3 and 8 post immunisation, the number of RMCPII-expressing cells markedly increased in the EAE-induced group, mainly within brainstem and spinal cord close to inflammed blood vessels. The markers of histaminergic neurons were marginally affected 13 days after immunisation and the increase of [3H] histamine synthesis elicited by the H3-receptor antagonist, thioperamide, was not modified in any region of the brain. It is concluded that the cerebral RMCPII-expressing mast cells could play a role during EAE.

Animals↗
PubMed1997

Constitutive expression of mouse mast cell protease-1 in normal BALB/c mice and its up-regulation during intestinal nematode infection.

Rodent intestinal mucosal mast cells (IMMC) store and secrete soluble granule serine proteases, the beta-chymases, which may promote epithelial permeability during intestinal hypersensitivity reactions. The beta-chymase mouse mast cell protease-1 (mMCP-1) is generally considered to be expressed late in the in vitro differentiation of mast cells. The purpose of this study was to determine the kinetics of mMCP-1 transcription and expression in vivo during nematode-induced IMMC hyperplasia. Concentrations of mMCP-1 in blood and jejunum of BALB/c mice were quantified by enzyme-linked immunosorbent assay before and at various stages after infection with the intestinal nematode Nippostronglyus brasilliensis. Mature mMCP-1 enzyme was detected in jejunal homogenate (194 ng/mg soluble protein) and in blood (8.3 ng/ml serum) from normal uninfected BALB/c mice. Maximal IMMC hyperplasia occurred 7-14 days post infection and was significantly correlated with increased levels of mMCP-1 in jejunum (r = 0.58, P < 0.001) and with raised concentrations of mMCP-1 in serum (r = 0.66, P < 0.001). Transcription of the mMCP-1 gene was detected by RNA blotting in normal, uninfected jejunum, but transcription was up-regulated after infection with maximal transcription occurring on days 7 and 14. In conclusion, mMCP-1 transcription, storage and secretion occur constitutively in normal BALB/c jejunum but this basal secretion is up-regulated during nematode infection, suggesting both a physiological and pathological function for this protease.

Animals↗
PubMed1997

Mast cell and mast cell granule phenotypes in normal and Nippostrongylus-infected rats. A qualitative laser confocal microscopic study.

In the rat, the individual mast cell secretory granules may be divided into three subpopulations based on the presence of the specific proteases RMCP-1, RMCP-2, or a variable combination of these two proteases. Mast cells in the tongue only express RMCP-1, both in normal and infected animals, whereas in the other tissue locations studied (lung, intestinal mucosa and submucosa, tracheal epithelium and submucosa) the mast cells contain all three granule subtypes in a wide variation of combinations. These studies demonstrate that there is wide heterogeneity in protease expression in rat mast cells, which may be influenced by local stimulation with environmental tissue factors.

Animals↗
PubMed1997

Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode-infected and normal BALB/c mice.

Soluble granule chymases in rodent intestinal mucosal mast cells (IMMCs) may play an important role in altering epithelial permeability during immediate hypersensitivity reactions. Using a monoclonal antibody against the chymase mouse mast cell protease-1 (mMCP-1), we have shown that it is constitutively expressed in < or = 20% of esterase-positive (esterase+) IMMCs but not in esterase+ gastric mucosal mast cells (GMMCs) in normal BALB/c mice. Intestinal infection with mouse- or rat-adapted strains of Nippostrongylus brasiliensis resulted in IMMC hyperplasia with 100% of esterase+ IMMCs expressing mMCP-1. In contrast, there was a variable response in terms of numbers of GMMCs and of the proportion expressing mMCP-1. Esterase+ mast cells in the gastric submucosa, muscularis, ear pinna, lung parenchyma, major airway submucosa, and peritoneal cavity did not express mMCP-1. The few airway esterase+ mast cells expressing mMCP-1 were, like the great majority of IMMCs and GMMCs, located intraepithelially. In conclusion, mMCP-1 is predominantly expressed by intraepithelial mucosal mast cells but not in all sites; the immunological stimulus associated with intestinal nematodiasis substantially up-regulates mMCP-1 expression by mast cells in the jejunum but not in the stomach; IMMCs and GMMCs in BALB/c mice are phenotypically and possibly functionally distinct.

Animals↗
PubMed1996

Rat cerebral mast cells undergo phenotypic changes during development.

The evolution of rat cerebral mast cell phenotype during development was studied using antibodies against the granule chymases, rat mast cell protease I (RMCP-I) and rat mast cell protease II (RMCP-II) and their gene transcripts, as markers for serosal and mucosal mast cells, respectively. In situ hybridization using specific oligoprobes for RMCP-II permitted visualization of RMCP-II mRNA-containing cells as early as day 15 of embryonic development (E15). From E19 to day 4 postpartum (D4) their number increased whilst they migrated from the pia mater to the choroid fissure; at D8 cells expressing RMCP-II gene transcripts were no longer observed. The 3'-end untranslated nucleotide sequence of the RMCP-I cDNA was established in order to design selective cDNA probes for Northern blot analysis of both enzymes. Northern blot analysis revealed a strong expression of RMCP-I and RMCP-II mRNAs at D2. At D4, RMCP-I mRNA expression was still high, whereas that of RMCP-II was decreased. In adult brain, mRNA expression for both proteases was low, but detectable. Quantification of both proteases by ELISA showed that, from E19 to D4, levels of RMCP-II were maximal at E19 and remained constant until D4, whereas RMCP-I increased as a function of age. Thereafter, levels of both proteases decreased progressively, but were still present in the adult brain, with RMCP-II being uniformly distributed and RMCP-I concentrated in the thalamus. Immunohistochemical staining showed RMCP-II-immunoreactive cells within the pia mater at E19; on D2 and D4, cells with both RMCP-I and RMCP-II immunoreactivities were found within the choroid fissure and from D8, only RMCP-I-immunoreactive mast cells were observed. In the thalamus of adult rats, the latter had a perivascular localization. This study shows that in the adult, both types of mast cells are present, although in small numbers, except for RMCP-I-immunoreactive mast cells which are abundant in the thalamus. The changes in the number and phenotype of cerebral mast cells may result from the influence of a number of growth factors during development.

Amino Acid Sequence↗
PubMed1996

Phenotypic and quantitative changes in mast cells after syngeneic unilateral lung transplantation in the rat.

1. Lung transplantation causes a total interruption of the inneration and vascularization within the transplanted organ, followed by repair processes. This is frequently associated with bronchial hyper-responsiveness. A common feature of tissue repair is an increase in the number of mast cells. Three phenotypically distinct mast cell subsets, with respect to their protease content, have been identified in rat lung, and it is probable that mast cells of differing protease phenotype fulfil different functions. 2. We have compared the number, protease phenotype and distribution of mast cells in left lung from transplanted and control Lewis rats 1 month after syngeneic unilateral left lung transplantation, without interference of inflammation, graft rejection or of any treatment. Connective and mucosal-type mast cell phenotypes were characterized using antibodies directed against their specific rat mast cell proteases, RMCPI and RMCPII, respectively. 3. After transplantation, RMCPI and RMCPII tissue concentrations increased by 172% and 239%, respectively, compared with controls (13.1 +/- 1.2 and 5.6 +/- 1.0 micrograms/g). 4. Localization of mast cell phenotypes was studied by immunohistochemistry after double immunostaining. The number of mast cells increased after transplantation: the increase in the number of RMCPI-immunoreactive mast cells (RMCPI+) was significant around bronchioles and arterioles, around large vessels and in the pleura. The number of RMCPII+ mast cells also significantly increased around bronchioles and arterioles, as well as in the smooth muscle layer of large airways. Some mast cells stained for the presence of both RMCPI and RMCPII, supporting the existence of co-expressing phenotype in rat lung. The number of mast cells of the RMCPI+/II+ phenotype significantly increased around bronchioles and arterioles and in the pleura. Moreover, the distribution of the mast cell phenotypes was modified in the different areas after transplantation. 5. This indicates a local differentiation/maturation of mast cells after transplantation.

Animals↗
PubMed1996

Stem cell factor enhances immunoglobulin E-dependent mediator release from cultured rat bone marrow-derived mast cells: activation of previously unresponsive cells demonstrated by a novel ELISPOT assay.

Mucosal mast cells (MMC) are important effector cells in the immune response against gastrointestinal nematodes. We used cultured rat bone marrow-derived mast cells (BMMC) as an in vitro model of MMC to study the effects of the multifunctional cytokine stem cell factor (SCF) on immunoglobulin E (IgE)-dependent secretion of granule mediators. SCF (< or = 1000 ng/ml) was not a direct secretagogue for these cells, but it significantly enhanced IgE-mediated secretion of the granule constituents rat mast cell protease-II (RMCP-II) and beta-hexosaminidase from mature BMMC in a dose-dependent manner (> 10 ng/ml). Maximum up-regulation of secretion occurred after cells were pretreated with SCF (50 ng/ml) for 5 minutes before challenge with anti-IgE, but the effect then declined and was absent in cells incubated with the cytokine for 3 to 24 h. In a novel ELISPOT assay developed to identify individual BMMC secreting RMCP-II, the proportion of mature BMMC responding to anti-IgE was significantly increased by treatment with SCF. To investigate this effect further, the percentage release of RMCP-II and beta-hexosaminidase from populations of mature BMMC was directly compared to the proportion of individual cells releasing RMCP-II as detected by ELISPOT. The release of both mediators was enhanced by SCF, and the increased percentage release reflected both an increased proportion of secreting cells, and enhanced mediator release from individual cells. These results suggest that SCF can enhance IgE-dependent mediator release from BMMC not only by augmenting the secretory response from individual cells, but also by activating previously unresponsive cells.

Animals↗
PubMed1996

Rat bone marrow-derived mast cells co-cultured with 3T3 fibroblasts in the absence of T-cell derived cytokines require stem cell factor for their survival and maintain their mucosal mast cell-like phenotype.

When cultured without fibroblasts, rat bone marrow-derived mast cells (BMMC) contain abundant rat mast cell proteinase type II (RMCP-II), and exhibit survival and proliferation when maintained in mesenteric lymph node conditioned medium (CM). When BMMC were co-cultured with 3T3 fibroblasts in the absence of CM, BMMC numbers increased for 7 days and the BMMC survived for up to 23 days. There was a gradual loss of stored RMCP-II in BMMC that were co-cultured with 3T3 cells, but the fibroblast microenvironment did not induce a detectable increase in the low levels of the connective tissue mast cell (CTMC)-associated proteinase, RMCP-I, in the BMMC. Nor did 3T3 cell co-culture induce significant heparin synthesis in BMMC as judged by the cells' reactivity with the fluorescent heparin-binding dye, berberine sulphate. These results suggest that rat BMMC, unlike murine BMMC, do not have the potential to develop multiple CTMC-like characteristics upon co-culture with 3T3 cells. However, when BMMC and fibroblast co-cultures were treated with an antibody to recombinant rat stem cell factor (rrSCF), mast cell survival was completely abrogated. This result suggests that endogenous, fibroblast-derived SCF is essential for the maintenance of rat BMMC viability in the absence of CM. On the other hand, prior treatment of the fibroblasts with the anti-rrSCF antibody did not affect the adherence of BMMC to the monolayer, implying that (an) other molecule(s) is(are) involved in the attachment process. The demonstration that rat BMMC survival on fibroblasts in vitro is dependent upon SCF may indicate an important mechanism by which tissue mucosal cells can be maintained in vivo in the absence of T-cell derived factors.

Animals↗
PubMed1996

Evidence against a significant role for mast cells in blastocyst implantation in the rat and mouse.

Rats were treated with the highly potent stabilizer of mast cells, FPL 55618, before and during the first seven days of pregnancy to establish whether stabilization of mast cells resulted in impaired blastocyst implantation. There was no significant reduction in either the number of ovulations or the number of implantation sites in treated rats compared with controls; 11 of 15 treated rats were pregnant compared with 5 of 6 control rats. The distribution of mast cells was examined in uterine tissues, implantation sites and interimplantation sites in both rats and mice using highly sensitive immunohistochemical techniques. Virtually all of the mast cells in rat uterine tissue stained for rat mast cell protease-I (RMCP-I; connective tissue type), whereas few stained for RMCP-II (mucosal type). Most of the mast cells were present in the myometrium with very sparse distribution in the endometrium and there were no differences in numbers of mast cells between implantation and inter-implantation sites on Day 7 of pregnancy. In tissue sections of mouse uteri sampled from Day 1 to Day 8 of pregnancy there were virtually no mast cells in the endometrium or deciduum adjacent to implantation sites. Mouse uterine mast cells also stained predominantly for the connective tissue-type mast cell protease MMCP-4, the murine equivalent of RMCP-I. Thus, mast cells and their products appear to play little, if any, role in blastocyst implantation in murid rodents. Since mast cells are a prominent feature of human endometrium, this study emphasizes the important consideration of species differences when choosing animal models for implantation studies.

Animals↗
PubMed1996

A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth.

In common with many intestinal nematode infections, Trichinella spiralis infections in mice are associated with a pronounced intestinal mast cell hyperplasia. The expulsion of the parasite from the gut is temporally associated with intestinal mastocytosis and mast cell function reflected by the secretion of mast cell protease into tissue and serum. In vivo, mucosal mast cell production is highly dependent upon T cell-derived cytokines including IL-3 and IL-4. We present data here to show that intestinal mast cell hyperplasia induced by helminth infection is also dependent upon the production of stem cell factor (SCF). Neutralization of SCF by anti-SCF or anti-SCF receptor mAb completely abrogated the mast cell hyperplasia generated by T. spiralis infection. Moreover, worm expulsion was dramatically delayed in treated mice and a reduced intestinal eosinophilia was observed. These effects did not appear to be mediated through alteration of Th cell responses and the parasite-specific serum antibody response was not affected. The reduction in the mast cell response and worm expulsion observed after SCF neutralization were reversible following cessation of monoclonal treatment. The data presented here clearly demonstrate a major role for SCF in the generation of intestinal mastocytosis and the host protective immune response following parasitic infection.

Animals↗