Biomedical subjects

S K Smith

Publications and source records attributed to S K Smith.

At least 19 recordsLinked to original sources

PubMed2006

Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus.

Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1-4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5-9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity.

Amino Acid Sequence↗

PubMed2006

Evaluation of broad-spectrum anthelmintic activity in a novel assay against Haemonchus contortus, Trichostrongylus colubriformis and T. sigmodontis in the gerbil Meriones unguiculatus.

The gerbil Meriones unguiculatus, infected with three species of nematodes, each located in a separate part of the gastrointestinal tract, provided a reliable laboratory assay for the evaluation of broad-spectrum anthelmintic activity. Gerbils harbouring 6-day-old infections of Haemonchus contortus, Trichostrongylus colubriformis and T. sigmodontis were given selected broad-spectrum anthelmintics by gavage. Three benzimidazoles, thiabendazole, oxfendazole and albendazole, a tetrahydropyrimidine, morantel, an imidazothiazole, levamisole hydrochloride, a macrocyclic lactone, ivermectin and an experimental natural product, paraherquamide, were active against all three nematodes at various dosages. Trichostrongylus colubriformis was most sensitive to levamisole hydrochloride, morantel, thiabendazole and paraherquamide whereas ivermectin, oxfendazole and albendazole were more effective against H. contortus. All compounds were active against the caecal nematode T. sigmodontis although it was less sensitive than T. colubriformis. Haemonchus contortus was more sensitive than T. sigmodontis to all anthelmintics tested except thiabendazole.

Albendazole↗

PubMed2005

An immunogenic cathepsin F secreted by the parasitic stages of Teladorsagia circumcincta.

Teladorsagia circumcincta is a common, pathogenic abomasal nematode of sheep. In order to improve disease control in parasite isolates resistant to several anthelmintics, alternative methods must be sought. Sheep develop acquired immunity to T. circumcincta so vaccination is a valid option for control. For this reason, we are investigating parasite excretory/secretory products for molecules, which have potential to invoke protective immunity against T. circumcincta. Here, we describe experiments in which we identified a novel, immunogenic cathepsin F secreted by L4 T. circumcincta. This protease, initially identified by mass spectrometry analysis, is the most abundant molecule in excretory/secretory products released in vitro by T. circumcincta harvested at 5, 6 or 9 days p.i. and is a target of specific, local IgA responses in sheep which are immune to challenge infection. The full-length cDNA encoding this secreted protease was isolated. Sequence and phylogenetic analyses indicated that the protease (designated T. circumcincta cathepsin F-1, Tci-CF-1) belongs to the cathepsin F class and exhibits greatest identity (>60%) to expressed sequence tags present in the Ostertagia ostertagi and Haemonchus contortus expressed sequence tag databases. Tci-CF-1 also displays high identity to hypothetical proteins identified in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae, both proteins having been described as cathepsin F enzymes. Specific inhibitor binding assay of larval excretory/secretory products confirmed the classification of this excretory/secretory component as a cathepsin F. Reverse transcription-PCR studies indicated that Tci-cf-1 is developmentally regulated and is particular to the host parasitic stages of T. circumcincta. The abundance, immunogenicity and temporal expression pattern of Tci-CF-1 make this a potential vaccine candidate for teladorsagiosis.

Amino Acid Sequence↗

PubMed2005

Protection induced by vaccinating sheep with a thiol-binding extract of Haemonchus contortus membranes is associated with its protease components.

Previous work has shown that a protein extract enriched for cysteine protease activity (TSBP) prepared from adult Haemonchus contortus using thiol sepharose affinity chromatography confers substantial protection against a single challenge infection. TSBP comprised proteases and other proteins. Here, TSBP were further fractionated using anion-exchange chromatography and fractions pooled on the basis of containing (1) protease activity, (2) a prominent non-protease peptide and (3) material which did not bind to the column. A protection trial showed that only the protease-enriched material conferred protective immunity and this was comparable to that observed in a TSBP-immunized positive control group. Immunization stimulated a marked IgG response with the IgG2 isotype predominating.

Animals↗

PubMed2004

Nimesulide, a COX-2 inhibitor, does not reduce lesion size or number in a nude mouse model of endometriosis.

BACKGROUND: Women with endometriosis have elevated levels of cyclooxygenase-2 (COX-2) in peritoneal macrophages and endometriotic tissue. Inhibition of COX-2 has been shown to reduce inflammation, angiogenesis and cellular proliferation. It may also downregulate aromatase activity in ectopic endometrial lesions. Ectopic endometrial establishment and growth are therefore likely to be suppressed in the presence of COX-2 inhibitors. We hypothesized that COX-2 inhibition would reduce the size and number of ectopic human endometrial lesions in a nude mouse model of endometriosis. METHODS: The selective COX-2 inhibitor, nimesulide, was administered to estrogen-supplemented nude mice implanted with human endometrial tissue. Ten days after implantation, the number and size of ectopic endometrial lesions were evaluated and compared with lesions from a control group. Immunohistochemical assessment of vascular development and macrophage and myofibroblast infiltration in control and treated lesions was performed. RESULTS: There was no difference in the number or size of ectopic endometrial lesions in control and nimesulide-treated nude mice. Nimesulide did not induce a visually identifiable difference in blood vessel development or macrophage or myofibroblast infiltration in nude mouse explants. CONCLUSION: The hypothesized biological properties of COX-2 inhibition did not influence lesion number or size in the nude mouse model of endometriosis.

Adult↗

PubMed2004

Identification of genes regulated by leukemia-inhibitory factor in the mouse uterus at the time of implantation.

The endometrium is prepared for implantation by the actions of estradiol (E2) and progesterone (P4). In mice the luminal epithelium (LE) only becomes fully receptive to the attaching blastocyst in response to the nidatory estrogen surge on d 4 of pregnancy. The cytokine leukemia-inhibitory factor (LIF) is rapidly induced by nidatory estrogen and has been shown to be the primary mediator of its action. Implantation fails in the absence of LIF, and injection of LIF on d 4 of pregnancy can substitute for the nidatory estrogen. In this study, we sought to identify genes regulated by LIF in the uterine epithelium. We used oligonucleotide microarrays to compare the transcript profiles of paired uterine horns from LIF-deficient MF1 mice after intraluminal injection of LIF or PBS on d 4 of pseudopregnancy. IGF-binding protein 3 was identified as a gene up-regulated by LIF; this was confirmed by RT-PCR. In situ hybridization showed that the primary site of IGF-binding protein 3 expression is the luminal epithelium (LE), the known site of LIF action in the uterus. We identified two other genes: amphiregulin and immune response gene-1, the expression of which were also up-regulated by LIF. Immune response gene 1 has recently been shown to be essential for implantation. Expression of all three of these genes in the LE is known to be regulated by P4. The expression of osteoblast-specific factor 2 and leukocyte 12/15 lipoxygenase, which are also expressed in LE under the control of P4, were not increased by LIF. This suggests that one of the actions of LIF on LE may be to enhance the expression of a subset of P4-regulated genes.

Amphiregulin↗

PubMed2004

The role of replicative senescence in cancer and human ageing: utility (or otherwise) of murine models.

Replicative senescence has the potential both to act as an anti-tumour mechanism, and to contribute to age-related changes in tissue function. Studies on human cells have revealed much, both about the nature of cell division counters, some of which utilize the gradual erosion of chromosomal telomeres, and the downstream signalling pathways that initiate and maintain growth arrest in senescence. A powerful test of the hypothesis that senescence is linked to either ageing or tumour prevention now requires a suitable animal model system. Here we overview the current understanding of replicative senescence in human cells, and address to what extent the senescence of murine cells in culture mirrors this phenomenon. We also discuss whether examples of telomere-independent senescence, such as those seen in mouse embryonic fibroblasts (MEFs) and several human cells types, should be viewed not as a consequence of "inadequate growth conditions", but rather as a powerful potential model system to dissect the selective pressures that occur in the early stages of tumour development, ones that we speculate lead to the observed high frequency of abrogation of p16INK4a function in human cancer.

Aging↗

PubMed2004

Determination of poultry feed-through insecticide activity in an in vivo anticoccidial assay.

The ability of an in vivo anticoccidial assay to identify potential feed-through insecticides was demonstrated using first-instar Lucilia sericata on droppings from chicks fed medicated diets. Cyromazine, a commercially available feed-through insect growth regulator, ivermectin, diflubenzuron, fipronil, permethrin, and 2 experimental compounds were effective in varying degrees in killing L. sericata larvae. Eleven coccidiostats were effective against the protozoan parasites (Eimeria spp.) at commercially used levels, whereas they were ineffective against Lucilia larvae.

Animal Feed↗

PubMed2004

The Hymenolepis diminuta-golden hamster (Mesocricetus auratus) model for the evaluation of gastrointestinal anticestode activity.

A novel laboratory anticestode assay was developed using Hymenolepis diminuta in the hamster. The commercial anticestode compounds, praziquantel, bunamidine, and niclosamide were active against patent infections of Hymenolepis diminuta in golden hamsters (Mesocricetus auratus) when given orally at 3.125, 100, and 200 mg/kg, respectively. The gastrointestinal nematode anthelmintics, cambendazole and mebendazole, were active at 50 mg/kg. Rafoxanide (fasciolicide) was active at 25 mg/kg, the lowest level tested. The coccidiostat, nicarbazin, was active at experimental levels (800 mg/kg and up). The anthelmintic-ectoparasiticide (endectocide), ivermectin, was inactive against the tapeworm at 0.5 mg/kg, as expected.

Administration, Oral↗

PubMed2004

[Study of the profile of gene expression in the endometrium].

Implantation in humans is a complex process that is temporally and spatially restricted. Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium. In order to understand this critical aspect of endometrial physiology, Smith group undertook a genome-wide analysis of transcript abundance and changes in transcript level between normal endometrium in the proliferative and secretory phases of the menstrual cycle, between normal and ectopic endometrium (endometriosis) and between normal and RU486-exposed endometrium. A high-density, oligonucleotide gene array was used to define the gene expression profiles of endometrium. Results from the arrays were verified using real-time PCR. Several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were identified. High density oligonucleotide microarray technology is a valid technique to investigate global changes in gene expression in human endometrium.

Embryo Implantation↗

PubMed2003

Expression of the VEGF and angiopoietin genes in endometrial atypical hyperplasia and endometrial cancer.

Angiogenesis is critical for the growth and metastasis of endometrial cancer and is therefore an important therapeutic target. Vascular endothelial growth factor-A (VEGF-A) is a key molecule in angiogenesis, but the identification of related molecules and the angiopoietins suggests a more complex picture. We investigated the presence of transcripts for VEGF-A, VEGF-B, VEGF-C, VEGF-D, Angiopoietin-1 and Angiopoietin-2 in benign endometrium, atypical complex hyperplasia (ACH) and endometrioid endometrial carcinoma using in situ hybridisation. We confirmed the presence of VEGF-A mRNA in the epithelial cells of cancers examined (13 out of 13), but not in benign endometrium or ACH. We also demonstrate, using quantitative polymerase chain reaction, that levels of VEGF-B mRNA are significantly lower in endometrial cancer than benign endometrium. We conclude that loss of VEGF-B may contribute to the development of endometrial carcinoma by modulating availability of receptors for VEGF-A.

Adult↗

PubMed2003

Vascular endothelial growth factor-D is an independent prognostic factor in epithelial ovarian carcinoma.

We assessed the presence of vascular endothelial growth factor (VEGF)-C, VEGF-D and their receptor VEGFR-3 by immunohistochemistry in 59 epithelial ovarian carcinomas, 11 borderline tumours and 20 benign cystadenomas. VEGF-C and VEGF-D were generally expressed in tumour cells and also in endothelia adjacent to tumour nests which showed a strong staining for them. VEGFR-3 was expressed in lymphatic and vascular endothelial cells adjacent to tumour nests. Immunoreactivity was significantly more frequent as lesions progressed from a benign tumour to advanced carcinoma. A strong correlation was found between VEGF-C and VEGF-D detected in carcinoma and VEGFR-3 detected in neighbouring endothelial cells. Increased expression of VEGF-C, VEGF-D and VEGFR-3 was significantly associated with lymph node metastasis and peritoneal metastasis outside the pelvis. There was a significant correlation between the high levels of VEGF-C and VEGF-D proteins, and poor survival. The presence of VEGF-D was an independent prognostic indicator by multivariate analysis. We conclude that VEGF-C, VEGF-D and VEGFR-3 play an important role in lymphatic spread and intraperitoneal tumour development in ovarian carcinoma. Since VEGF-D was found to be an independent predictor of poor outcome, its measurement, together with other prognostic markers may improve prospective identification of patients with a poor prognosis.

Adenocarcinoma, Clear Cell↗

PubMed2003

Metalloendopeptidases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep.

Substantial protection against the economically important nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of this parasite. This fraction has been termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP) since it was originally isolated through its selective binding to lectins with a specificity for N-acetylgalactosamine. A major component of this highly protective antigen complex is a family of four zinc metalloendopeptidases, designated MEPs 1-4. Various combinations of these MEPs were evaluated in immunization-challenge trials in sheep. In two experiments a combination of all four MEPs, separated from the rest of the complex by gel filtration in 8 m urea, significantly reduced H. contortus egg counts by 45 and 50%, an effect not significantly different from that conferred by 8 m urea treatment of H-gal-GP itself. Similarly, MEP3 alone or MEPs 1, 2 and 4 in combination, electroeluted from the complex following SDS gel electrophoresis, each reduced egg counts by some 33%. The MEPs are therefore protective components of H-gal-GP and from previously published findings, it appears that MEP3 is the most effective member of this metalloendopeptidase family. However, there was no significant protection when sheep were immunized with fully reduced and denatured H-gal-GP or with bacterially expressed recombinant forms of MEP 1 or the principal domains of MEP3, suggesting that conformational epitopes on the MEPs are required for immunity.

Animals↗

PubMed2003

The effect of RU486 on the gene expression profile in an endometrial explant model.

Administration of RU486 in vivo during the receptive phase rapidly renders the endometrium non-receptive to the implanting embryo. In order to identify key pathways responsible for endometrial receptivity we have used cDNA arrays to monitor gene expression changes in short-term endometrial explants in response to RU486. Endometrial biopsies from five normal fertile women at mid-secretory phase were cultured in the presence of estradiol and progesterone with or without RU486 for 12 h. cDNA arrays were produced containing approximately 1000 sequence-verified clones which included genes known to be important in angiogenesis, apoptosis, cell signalling, extracellular matrix remodelling and cell cycle regulation. cDNA probes from the paired endometrial samples were hybridized to the arrays and hybridization signals were quantified. A total of 12 genes displayed significant changes in expression; six were up-regulated and six down-regulated following RU486 treatment. For five of these genes this is the first report suggesting that they are regulated by steroids in the endometrium. JAK1 and JNK1 were two of the genes shown by the arrays to be down-regulated in RU486-treated endometrial explants. This was confirmed by real time RT-PCR. JAK1 immunoreactivity was localized to both glandular epithelium and the stroma of normal endometrium and staining was much stronger in the luteal phase of the cycle. These results show that components of two important signalling pathways in endometrium-the JAK/STAT pathway, and the JNK pathway-are altered by RU486. Genes whose expression is controlled by these pathways are likely to be involved in the mechanism by which steroids render the endometrium receptive to the implanting embryo.

Adult↗

PubMed2003

Lack of functional promoter polymorphisms in genes involved in glutamate neurotransmission.

OBJECTIVES: The regulation of genes involved in glutamatergic function is thought to be a critical for many central nervous system processes including memory, learning, synaptic maintenance, and many pathological states. As part of a larger survey into the key regulatory elements in genes of neuro-psychiatric interest, we sought to identify the promoter regions of genes in this broad family, and to identify sequence variants that alter gene expression. METHODS: Mutation analysis was carried out on the promoters of 20 genes encoding 13 glutamate receptor subunits, four transporters and three metabolizing enzymes using denaturing high performance liquid chromatography. Thirty-nine different promoter haplotypes were cloned into a luciferase reporter gene vector and tested for differences in their ability to drive transcription in both HEK293t and TE671 cell lines. RESULTS: We have identified a total of 48 sequence variants in six glutamate receptor subunits, four glutamate transporters and two enzymes. Interestingly, seven promoter sequences gave three or more haplotypes from a single individual, indicating gene duplication. No differences in expression greater than 1.35-fold were found between haplotypes originating from the same or paralogous genes. CONCLUSION: The lack of common functional polymorphisms in any of these promoters indicates that expression of glutamate receptors and transporters is unusually tightly controlled, and suggests the possibility that non-coding polymorphisms in these genes are rare and may be unlikely to contribute in a major way to neuro-psychiatric phenotypes. This study represents the world's largest survey of the any group of promoters yet performed for any gene system.

Amino Acid Transport System X-AG↗

PubMed2002

Placental vessel adaptation during gestation and to high altitude: changes in diameter and perivascular cell coverage.

The objective of this study was to determine how human placental vascular structures change during gestation and whether this would be altered by external factors such as reduced ambient oxygen. To achieve this, several experiments were carried out: Vessel profile diameter was measured and the presence of perivascular cells (pericytes or smooth muscle cells) noted. This was carried out in normal human first trimester and term placentae, and in term placentae obtained from high altitude and an ethnically matched lowland population. In addition, to characterize endothelial cells in human placenta a panel of endothelial markers anti-CD 105, CD31, CD34, Von Willebrand factor (vWF), Ulex europaeus agglutinin 1 (UEA I), Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Bandeieraea simplicifolia agglutinin 1 (BS 1) was used. The proportion of vessels associated with perivascular cells rises during gestation from 37 per cent in the first trimester to 63 per cent at term (P<0.0001) and vessels with perivascular cells have a larger median diameter at term. In placentae obtained at high altitude, the vessels are dilated and are less frequently associated with perivascular cells. The absence of perivascular cells may allow remodelling of capillaries and this is likely to be physiological important in the first trimester but also under physiological or pathological stress.

Actins↗

PubMed2002

Soluble gp130 is up-regulated in the implantation window and shows altered secretion in patients with primary unexplained infertility.

Members of the IL-6 family of cytokines, which includes leukemia inhibitory factor (LIF) and IL-11, play important roles in implantation. The activity of these cytokines is modified by soluble receptors such as the IL-6 receptor (sIL-6R). gp130 is a signal transduction molecule common to the receptor complexes of this family, and its soluble form (sgp130) antagonizes their actions. The purpose of this study was to determine whether secretion of IL-6, LIF, sIL-6R, and sgp130 was different in the endometrium of women with primary unexplained infertility compared with normal fertile women. Endometrial biopsies were taken between d LH+6 and +13 and cultured in serum-free medium for 4 h. Secretion of IL-6, LIF, sIL-6R, and sgp130 was measured in the supernatant by ELISA. We also measured the secretion of IL-6, sIL-6R, and sgp130 by endometrial biopsies taken throughout the menstrual cycle in normal fertile women. Secretion of sgp130 increased 20-fold between d 20 and 26 of the cycle, coinciding with the implantation window (proliferative phase, median, 27.0 pg/ml.mg; range, 23-36; d 20-26, median, 501.5 pg/ml x mg; range, 26.1-1344; P = 0.03). RT-PCR showed that none of the known splice variants of gp130 were present in endometrium, indicating that sgp130 is produced by proteolytic cleavage of the membrane-bound form. IL-6 secretion varied considerably between patients and was greatest during the secretory phase and at menstruation. No significant change was seen in sIL-6R during the cycle. Between LH+6 and +13, secretion of sgp130 was significantly reduced in the infertile group (median, 93.1 pg/ml.mg; range, 28.5-256; compared with the fertile group, median, 223 pg/ml x mg; range, 63-534; U-statistic = 37; P = 0.017). Secretion of IL-6, LIF, and sIL-6R did not differ between the two groups. Immunolocalization of gp130, IL-6R, and the LIF receptor showed that the glandular epithelium and also endothelial cells are targets for IL-6 and LIF. These findings show that during a normal menstrual cycle, sgp130 secretion is greatly increased between d LH+6 and +13, due to proteolytic cleavage of membrane-bound gp130. Infertile patients show reduced secretion of sgp130 compared with fertile controls during this period, which coincides with the implantation window.

Adult↗

PubMed2002

Expression of CD105 (endoglin) in arteriolar endothelial cells of human endometrium throughout the menstrual cycle.

The cellular mechanisms underlying normal and pathological endometrial bleeding are not well understood, although abnormalities in the structure of endometrial blood vessels may lead to menstrual disorders. Endothelial cells in different organs are heterogeneous and differ in structure, function, antigen composition, metabolic properties and responses to growth factors. Immunostaining was performed with anti-CD105, CD31, CD34 and von Willebrand factor (vWF), and lectin binding with Ulex europaeus agglutinin 1 (UEA 1), Bandeieraea simplicifolia agglutinin 1 (BS 1), Dolichos biflorus agglutinin (DBA) and Peanut agglutinin (PNA) to characterize endothelial cells in human endometrium throughout the menstrual cycle. Serial sections fixed with formalin were stained with primary antibodies and lectins after antigen retrieval. Positive staining for CD31, CD105 and vWF was confined to the vascular endothelium. Endothelial expression of CD31 was observed in all types of vessel, including single cells, and strong staining was found during the early proliferative and mid-secretory phases. Anti-vWF stained arterioles and veins, but there was little positive staining of capillaries. In contrast, staining for CD105 was confined to the arterioles. Although anti-CD34 strongly stained endothelial cells of small vessels and capillaries, staining was also observed on some non-endothelial stromal cells. Strong positive staining for UEA 1 was observed in endothelial cells of all types of vessel throughout the menstrual cycle. Binding of PNA, DBA and BS 1 was confined to the apical region of glandular epithelial cells. This study demonstrates that the differential binding of anti-CD31, CD34, CD105, vWF and UEA 1 distinguishes between endometrial populations of endothelial cells.

Adult↗