• Application of immunological methods for the detection of species adulteration in dairy products

      Hurley, Ian P.; Ireland, H. Elyse; Coleman, Robert C.; Williams, John H. H.; University College Chester (Wiley, 2004-10-20)
      A number of enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of milk adulteration in dairy products. Target antigens have been caseins, lactoglobulins, immunoglobulins and other whey proteins. Polyclonal and monoclonal antibodies have been used in a variety of formats including direct, indirect, competitive and sandwich ELISAs. ELISAs have been successfully applied to the detection of cows' milk adulteration of sheep, goat and buffalo milk. Goat milk adulteration of sheep milk has also been detected. A number of ELISAs have also been applied to cheese. It is recommended that ELISA should be used in combination with PCR to ensure compliance with current legislation.
    • A bio-assay for effectors of osteoclast differentiation in serum from patients with bone disease

      Dugard, Marit-Naomi; Sharp, Christopher A.; Evans, Sally F.; Williams, John H. H.; Davie, Michael W. J.; Marshall, Michael J.; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry ; University of Chester ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry (Elsevier, 2005-06)
      Osteoclast differentiation and activity, and hence bone loss, depend on two opposing cytokines. Receptor activator of NF-κB ligand (RANKL) produced by osteoblasts and T-cells stimulates, while osteoprotegerin inhibits. Both of these cytokines are found in serum. Our aim was to develop a functional assay for any factors present in human serum that can affect osteoclast differentiation and to assess whether any such factors vary in diseases in which bone loss occurs.
    • Birth defects and anti–heat shock protein 70 antibodies in early pregnancy

      Child, David F.; Hudson, Peter R.; Hunter-Lavin, Claire; Mukhergee, Sagarika; China, Susnata; Williams, Clive P.; Williams, John H. H.; University of Chester (Hunter-Lavin & Williams) (Springer-Verlag, 2006-03)
    • Bone extracts can stimulate the secrtion of osteoprotegerin in the osteosarcoma cell lines MG-63 and SAOS-2

      Powell, Diane E.; Johnson, William Eustace Basil; Marshall, Michael J.; Williams, John H. H.; Davie, Michael W. J. (American Society for Bone and Mineral Research, 2005)
    • Detection of irradiated food by immunoassay - development and optimization of an ELISA for dihydrothymidine in irradiated prawns

      Tyreman, Anne L.; Bonwick, Graham A.; Smith, Christopher J.; Coleman, Robert C.; Beaumont, Paul C.; Williams, John H. H.; University of Chester ; University of Chester ; University of Chester ; University of Chester ; Homerton College, Cambridge ; University of Chester (Blackwell, 2013-11-12)
      This article discribes the development and use of a competitive enzyme-linked immuno-sorbent assay (ELISA) to detect prawns which have been irradiated.
    • The development of immunoassays to identify and quantify species source of gum Arabic

      Ireland, H. Elyse; Clutterbuck, Abigail L.; Cloquet, Jean-Phillipe; Thurston, Miranda; Williams, Peter A.; Cronk, Quentin C.; Dewey, France M.; Williams, John H. H.; University College Chester (Ireland, Thurston, Williams, J H H) (American Chemical Society, 2004)
    • The effects of a high carbohydrate diet on cortisol and salivary immunoglobulin A (s-IgA) during a period of increase exercise workload amongst Olympic and ironman triathletes

      Costa, Ricardo J. S.; Jones, G. E.; Coleman, Robert C.; Lamb, Kevin L.; Williams, John H. H. (Georg Thieme Verlag, 2005-04-11)
      This article discusses a study of the effects of a 6-day high carbohydrate (H-CHO) diet on salivary cortisol and IgA during a period of increased exercise workload with thirty-two competitively trained male triathletes.
    • Effects of dissociated glucocorticoids on OPG and RANKL in osteoblastic cells

      Humphrey, E. L.; Williams, John H. H.; Davie, Michael W. J.; Marshall, Michael J.; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; University College Chester ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry (Elsevier, 2006-05)
      This article demonstrates that dexamethasone, prednisolone, deflazacort and the dissociated glucocorticoids, RU24858, RU40066, RU24782, AL438-F1 and ZK216348 significantly inhibit osteoprotegerin (OPG) production in two human osteoblastic cell lines (MG63 and hFOB).
    • Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

      Ireland, H. Elyse; Harding, Steve J.; Bonwick, Graham A.; Jones, Michael; Smith, Christopher J.; Williams, John H. H.; University College Chester (Taylor & Francis, 2004-03)
      Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025-10 μg ml-1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.
    • Folate supplementation reduces serum Hsp70 levels in patients with type 2 diabetes

      Hunter-Lavin, Claire; Hudson, Peter R.; Mukhergee, Sagarika; Davies, Gareth K.; Williams, Clive P.; Harvey, John N.; Child, David F.; Williams, John H. H.; University College Chester ; Wrexham Maelor Hospital, North East Wales NHS Trust ; Wrexham Maelor Hospital, North East Wales NHS Trust ; Wrexham Maelor Hospital, North East Wales NHS Trust ; Wrexham Maelor Hospital, North East Wales NHS Trust ; Wrexham Maelor Hospital, North East Wales NHS Trust ; Wrexham Maelor Hospital, North East Wales NHS Trust ; University College Chester (Cell Stress Society International, 2004-10)
      Type 2 diabetes patients are subject to oxidative stress as a result of hyperglycemia. The aim of this study was to determine whether administration of the antioxidant folic acid, previously shown to reduce homocysteine levels, would reduce circulating levels of Hsp70 while improving the condition of type 2 diabetes patients with microalbuminuria. Plasma homocysteine fell from pretreatment values of 12.9 to 10.3 μM (P < 0.0001). The urine albumin-creatinine ratio fell from 12.4 to 10.4 mg/mM (P = 0.38). Pretreatment Hsp70 levels were higher in patients not taking insulin (5.32 ng/mL) compared with those on insulin (2.44 ng/mL) (P = 0.012). Folic acid supplementation resulted in a significant fall in Hsp70 (5.32 to 2.05 ng/mL) (P = 0.004). There was no change in Hsp70 in those receiving insulin. Folic acid supplementation in non–insulin-treated type 2 diabetes patients, therefore, resulted in a fall in Hsp70, reflecting an improvement in oxidative stress. The data shows that improvement in homocysteine status can lead to a reduction in Hsp70, indicating the possibility of its use as a marker for severity of disease.
    • Heat shock proteins form part of a danger signal cascade in response to lipopolysaccharide and GroEL

      Davies, Emma L.; Bacelar, Maria M. F. V. G.; Marshall, Michael J.; Johnson, E.; Wardle, T. D.; Andrew, Sarah M.; Williams, John H. H.; University of Chester ; University of Chester ; Charles Salt Centre, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; Spinal Studies, The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; Countess of Chester Hospital ; University of Chester ; University of Chester (Wiley, 2006-05-26)
      An increasing number of cell types, including peripheral blood mononuclear cells (PBMCs), have been demonstrated to release heat shock proteins (Hsps). This paper investigates further the hypothesis that Hsps are danger signals. PBMCs and Jurkat cells released Hsp70 (0·22 and 0·7 ng/106 cells, respectively) into medium over 24 h at 37°C. Release of Hsp70 was stimulated 10-fold by GroEL (P < 0·001) and more than threefold by lipopolysaccharide (LPS) (P < 0·001). Although Hsp60 could be detected in the medium of cells cultured at 37°C for 24 h, the low rates of release were due probably to cell damage. Significant release of Hsp60 was observed when Jurkat cells were exposed to GroEL (2·88 ng/106 cells) or LPS (1·40 ng/106 cells). The data are consistent with the hypothesis that Hsp70 and Hsp60 are part of a danger signalling cascade in response to bacterial infection.
    • Homocysteine and cognitive decline in healthy elderly

      McCaddon, Andrew; Hudson, Peter R.; Davies, Gareth K.; Hughes, Alan; Williams, John H. H.; Wilkinson, Clare; University of Wales College of Medicine ; Wrexham Maelor Hospital ; Wrexham Maelor Hospital ; Royal Alexandra Hospital, Paisley ; Chester College ; University of Wales College of Medicine (Karger, 2001-09)
      Serum homocysteine is increased, and correlates inversely with cognitive scores, in Alzheimer's disease (AD), vascular dementia and "age-associated memory impairment". Elevated levels might signal accelerated cognitive decline, although this remains to be established. We therefore repeated Mini-Mental State Examinations, together with additional ADAS-Cog assessments, in 32 healthy elderly individuals to determine whether prior homocysteine levels predicted cognitive changes over a 5-year period.
    • Hsp70 release from peripheral blood mononuclear cells

      Hunter-Lavin, Claire; Davies, Emma L.; Bacelar, Maria M. F. V. G.; Marshall, Michael J.; Andrew, Sarah M.; Williams, John H. H.; University of Chester ; University of Chester ; University of Chester ; The Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; University College Chester ; University College Chester (Elsevier, 2004-11-12)
      There are an increasing number of studies reporting the presence of Hsps in human serum. We have investigated the release of Hsp70 into blood and culture medium from peripheral blood mononuclear cells (PBMCs), and whether this release is due to cell damage or active secretion from the cells. Intact Hsp70 was released from cells within whole blood and from purified PBMCs under normal culture conditions. Hsp70 release was rapid (0.1 ng/106 cells/h) over the first 2 h of culture and continued at a reduced rate up to 24 h (<0.025 ng/106 cells/h). Using viable cell counts and lactate dehydrogenase release we were able to confirm that the release of Hsp70 was not due to cellular damage. Hsp70 release was inhibited by monensin, methyl-β-cyclodextrin, and methylamine, but not by brefeldin A. These data suggest that Hsp70 is released from cells via a non-classical pathway, possibly involving lysosomal lipid rafts.
    • The inhibition of osteoprotegerin production in human osteoblast-like cells by dissociated glucocorticoid analoges

      Humphrey, E. L.; Smith, Heather L.; Williams, John H. H.; Marshall, Michael J.; University College Chester (Williams) (American Society for Bone and Mineral Research, 2004-06)
    • Interleukin-1 beta blocks glucocorticoid inhibition of osteoprotegerin production in osteoblastic cells

      Humphrey, E. L.; Smith, Heather L.; Williams, John H. H.; Marshall, Michael J.; University College Chester (Williams) (American Society for Bone and Mineral Research, 2004-06)
    • Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration

      Hurley, Ian P.; Coleman, Robert C.; Ireland, H. Elyse; Williams, John H. H.; University College Chester (American Dairy Science Association, 2004-03)
      The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows’ milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows’ milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 µg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows’ milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.
    • Measuring the secretion of heat shock proteins from cells

      Ireland, H. Elyse; Leoni, Francesca; Altaie, Ala; Birch, Catherine S.; Coleman, Robert C.; Hunter-Lavin, Claire; Williams, John H. H.; University of Chester (Elsevier, 2007-10-03)
      This article outlines procedures, using Hsp70 as the example, to: ensure the status of cells (viable, apoptotic or necrotic); identify the heat shock protein secreted; and quantify the secreted protein. Hsp70 has previously been quantified by ELISA, but newer methods are now being adopted, such as BIAcore and bead-based assays for use by FACS. These methods have the advantages of being more sensitive and requiring less sample than ELISA. The BIAcore has the potential to analyse Hsp70 ligands and provide affinity constants.
    • Monocytes/macrophages express CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation

      Schmutz, Caroline; Cartwright, Alison; Williams, Helen; Haworth, Oliver; Williams, John H. H.; Filer, Andrew; Salmon, Mike; Buckley, Christopher D.; Middleton, Jim F.; Keele University/University of Birmingham ; Keele University ; University of Chester ; University of Birmingham ; University of Chester ; University of Birmingham ; University of Birmingham ; University of Birmingham ; Keele University/University of Bristol (BioMed Central, 2010-08-05)
      Abstract Introduction Monocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers. Methods CCR9 expression on PB monocytes/macrophages was analysed by flow cytometry and in synovium by immunofluorescence. Chemokine receptor CCR9 mRNA expression was examined in RA and non-RA synovium, monocytes/macrophages from PB and synovial fluid (SF) of RA patients and PB of healthy donors using the reverse transcription polymerase chain reaction (RT-PCR). Monocyte differentiation and chemotaxis to chemokine ligand 25 (CCL25)/TECK were used to study CCR9 function. Results CCR9 was expressed by PB monocytes/macrophages in RA and healthy donors, and increased in RA. In RA and non-RA synovia, CCR9 co-localised with cluster of differentiation 14+ (CD14+) and cluster of differentiation 68+ (CD68+) macrophages, and was more abundant in RA synovium. CCR9 mRNA was detected in the synovia of all RA patients and in some non-RA controls, and monocytes/macrophages from PB and SF of RA and healthy controls. CCL25 was detected in RA and non-RA synovia where it co-localised with CD14+ and CD68+ cells. Tumour necrosis factor alpha (TNFα) increased CCR9 expression on human acute monocytic leukemia cell line THP-1 monocytic cells. CCL25 induced a stronger monocyte differentiation in RA compared to healthy donors. CCL25 induced significant chemotaxis of PB monocytes but not consistently among individuals. Conclusions CCR9 expression by monocytes is increased in RA. CCL25 may be involved in the differentiation of monocytes to macrophages particularly in RA.
    • Osteoprotegerin is produced when prostaglandin synthesis is inhibited causing osteoclasts to detach from the surface of mouse parietal bone and attach to the endocranial membrane

      O’Brien, E. A.; Williams, John H. H.; Marshall, Michael J.; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry ; Chester College ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry (Elsevier, 2001-02-13)
      This article tests the hypothesis that osteoprotegerin (OPG) mediates the inhibition of osteoclast activity that occurs with indomethacin in the mouse calvaria.
    • Platelet-derived growth factor stimulates osteoprotegerin production in osteoblastic cells

      McCarthy, Helen S.; Williams, John H. H.; Davie, Michael W. J.; Marshall, Michael J.; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry / University of Chester ; University of Chester ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry ; Charles Salt Centre, Robert Jones and Agnes Hunt Orthopaedic Hospital NHS Trust in Oswestry (Wiley, 2008-11-20)
      This article discusses how osteoprotegerin (OPG) production by osteoblastic cells was stimulated by platelet-derived growth factor (PDGF) in two human osteosarcoma cell lines (MG63, Saos-2), a mouse pre-osteoblastic cell line (MC3T3-E1) and human bone marrow stromal cells (hMSC) by 152%, 197%, 113% and 45% respectively over 24 h. OPG was measured in the cell culture medium by immunoassay. PDGF isoforms AA, BB and AB show similar stimulation of OPG production. Message for OPG was also increased similarly to the increased secretion into the culture medium. Using specific inhibitors of cell signalling the authors demonstrate that PDGF acts through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NF-kB or JNK. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process.