Biomedical subjects

T J Hill

Publications and source records attributed to T J Hill.

At least 19 recordsLinked to original sources

PubMed2002

Recovery from acidification in the Tillingbourne catchment, southern England: catchment description and preliminary results.

Measurements of acid deposition and streamwater chemistry made in 1979-1982 and 1999-2000 are compared for a small, acid-sensitive catchment in Southeast England. The location, geology, soils, vegetation and hydrology of the catchment are described. The catchment is located on an acidic cretaceous sandstone with a low permeability clay sub-stratum. Soils are predominantly podzol and gley, with some mesotrophic peat. The catchment is forested. Mean volume-weighted concentrations in precipitation have changed approximately in proportion to emission changes. SO4(2-) has declined by 61%, H+ by 75%, both NO- and NH4+ by 37% and Cl- by 26%. Changes in wet deposition are greater, sulfate deposition declined by 69%, non-marine SO4(2-) by 73%, H+ deposition by 75%, NO3- and NH4+ by 50% and Cl- by 41%. Sulfate deposition in throughfall, a surrogate for total deposition measurement, has declined by 82% and non-marine SO4(2-) by 86%. Some of these changes are due to alterations in the tree cover and location of the collectors. In 1979-1982, the flux of NO3- and NH4+ in throughfall was less than in rainfall, 7.5 compared with 11.3 kg N ha(-1) year(-1), showing that N uptake by the canopy was greater than dry deposition of these species. However, in 1999-2000, the throughfall flux of N was greater than rainfall, 19.6 compared to 5.7 kg N ha year(-1), indicating that canopy uptake is not occurring to the same extent. Surface water was sampled at the same locations in the catchment during the two periods. At the catchment exit, mean pH increased, from 3.93 to 4.21 mg l(-1), and SO4(2-) declined from 20.2 to 16.7 mg l(-1) (18%). The decrease in SO4(2-) is much less than the reduction in deposition, suggesting that the predicted recovery is being delayed by release of sulfur from the soil. In contrast, NO3- concentrations in the catchment waters increased from 0.22 to 0.52 mg N l(-1) (133%) despite the reduction in N deposition. NH4+ concentrations were low during both study periods. It is concluded that recovery from acidification is probably occurring, but is possibly being delayed by desorption of soil S. The catchment is also showing signs of increasing N saturation, despite a reduction in N inputs.

Absorption↗

PubMed2001

The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies.

The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (K(d) = 4 +/- 2 microm). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.

Amino Acid Sequence↗

PubMed2001

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

Adjuvants, Immunologic↗

PubMed2001

Cytokine production in a murine model of recurrent herpetic stromal keratitis.

PURPOSE: To determine the pattern of cytokine production in the cornea and its relationship with viral antigens, in our murine model of recurrent ocular herpes simplex virus (HSV)-1 infection. METHODS: Six weeks after corneal inoculation with HSV-1, the eyes of latently infected and control mice were UV irradiated and examined for signs of disease and viral reactivation. The eyes of five mice with recurrent stromal disease and two controls were processed for immunohistochemistry on days 4, 7, 10, and 14 after irradiation. Sections were double stained for viral antigens and one of the following cytokines: interleukin (IL)-1ss, IL-2, IL-4, IL-6, IL-10, IL-12, and interferon (IFN)-gamma. RESULTS: Fifty percent of mice showed signs of recurrent stromal disease, the severity of which peaked on day 10 after UV irradiation. There was a large cellular infiltrate in the stroma of all the corneas with recurrent disease and the predominant cytokines were IL-1ss, IL-6, IL-10, IL-12, and IFN-gamma, all present in large numbers of cells on the days studied. There were very few cells producing IL-2 and IL-4. Control eyes had no significant cytokine-producing cells in the stroma. CONCLUSIONS: These observations suggest that recurrent herpetic stromal keratitis (HSK) may not be characterized by a classic T-helper (Th)1 or Th2 response. However, the large number of IFN-gamma(+) and IL-12(+) cells and the relative absence of IL-4 favors a Th1 response, and despite the numerous IL-10(+) cells, the overall balance of cytokine production appears to be proinflammatory.

Animals↗

PubMed2000

Conditions in grey seal pups (Halichoerus grypus) presented for rehabilitation.

A survey was carried out on the condition of 188 live grey seal (Halichoerus grypus) pups presented for rehabilitation from the coasts of south-west England between 1992 and 1998. The survey was carried out to assess the incidence of malnutrition, hyperthermia, respiratory and gastrointestinal conditions, and also the incidence of traumatic, skin, oral and ocular lesions. Malnutrition was a common finding in pups approaching weaning (mid-moult pups) and those at the point of weaning or postweaning (moulted pups) (62 per cent and 82 per cent, respectively). Separation from the dam, believed to be the main cause of malnutrition in grey seals, was encountered frequently in unweaned pups (91 per cent). Thirty-nine per cent of pups presented with a respiratory condition, 38 per cent with hyperthermia and 9 per cent with diarrhoea. Seven per cent and 14 per cent of pups had confirmed respiratory and gastrointestinal parasitic infestations respectively, although these were probably underestimates. Clinically significant traumatic lesions were seen in 41 per cent, oral ulceration in 26 per cent, ocular conditions in 13 per cent, nail bed infections in 13 per cent and oiling in 10 per cent of pups. Umbilical infections and other skin and oral conditions were also encountered. The incidence of these conditions is compared with data from dead grey seals, and clinical conditions in other pinniped species presented for rehabilitation.

Animals↗

PubMed2000

Heterocomplex formation between metastasis-related protein S100A4 (Mts1) and S100A1 as revealed by the yeast two-hybrid system.

S100A4 (Mts1) is a Ca(2+)-binding protein of the S100 family. This protein plays an important role in promoting tumor metastasis. In order to identify S100A4 interacting proteins, we have applied the yeast two-hybrid system as an in vivo approach. By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family. The non-covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mutational analysis revealed that replacement of Cys(76) and/or Cys(81) of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo.

Animals↗

PubMed2000

Calmodulin-peptide interactions: apocalmodulin binding to the myosin light chain kinase target-site.

Noncovalent binding of the synthetic peptide RS20 to calmodulin in the presence of calcium was confirmed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry to form a complex with a 1:1:4 calmodulin/RS20/calcium stoichiometry. There was no evidence for formation of a calmodulin-RS20-Ca(2) species. The absence of calmodulin-RS20-Ca(2) would be consistent with models in which the two globular domains are coupled functionally. There was evidence that calmodulin, RS20-calmodulin without associated calcium, and calmodulin-RS20-Ca(4) existed together in solution, whereas calmodulin-calcium complexes were absent. It is proposed that calcium binding to form the calmodulin-RS20-Ca(4) complex occurs after an initial RS20-calmodulin binding event, and serves to secure the target within the calmodulin structure. The binding of more than one RS20 molecule to calmodulin was observed to induce unfolding of calmodulin.

Apoproteins↗

PubMed2000

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin.

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25 degrees C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25 degrees C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.

Actin Cytoskeleton↗

PubMed1999

Evidence of noncovalent dimerization of calmodulin.

Calcium-binding proteins, such as S-100, dimerize readily, and this phenomenon plays an important role in their regulation of target enzymes [Krebs, J., Quadroni, M. & Van Eldik, L.J. (1995) Nat. Struct. Biol. 2, 711-714; Kilby, P.M., Van Eldik, L.J. & Roberts, G. C. (1996) Structure 4, 1041-1052]. We have investigated by Fourier-transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium-binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra-high-resolution electrospray ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodulin was electrosprayed from buffer, monomers ions with only 5-10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its alpha-helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding. Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer-monomer equilibrium. The ultra-high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer.

Buffers↗

PubMed1999

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines.

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-alpha from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-alpha+ and most were also IL-6(+). Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-alpha and/or IL-6. IL-4(+) cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2(+) and/or IFN-gamma+ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-alpha and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.

Animals↗

PubMed1998

Induction of mucosal immunity against herpes simplex virus type 1 in the mouse protects against ocular infection and establishment of latency.

Immune responses were assessed after intranasal immunization of mice with a mixture of herpes simplex virus type 1 (HSV-1) glycoproteins with cholera toxin and its B subunit as adjuvant. Antigen-specific serum antibodies, which were largely IgG with IgG1 the major subclass, neutralized virus in vitro with a titer equivalent to that elicited by active infection. Significant levels of antigen-specific IgA were found in mucosal fluids of the eye as well as the vagina. Lymphocytes from draining lymph nodes showed secondary proliferative responses when cultured with HSV-1 in vitro, in immunized mice only, with the production of interleukin-2, interferon-gamma, interleukin-4, and interleukin-5. After ocular challenge, immunized mice were protected against the development of severe eye disease, zosteriform spread, or encephalitis, whereas the incidence of clinical symptoms in mock-immunized mice was 83%, 74%, and 52%, respectively. Finally, the incidence of latency was reduced from 88% to 13% after intranasal immunization.

Adjuvants, Immunologic↗

PubMed1997

Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues.

We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10O, IFNgamma and TNFalpha using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate-lysine-paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54 degrees C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNFalpha, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFNgamma. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.

Animals↗

PubMed1997

Enhancement of the immune response to non-replicating herpes simplex virus type-1 preparations by mucosal administration in the presence of cholera toxin.

Different immunization regimes were compared in order to enhance the immune response following mucosal administration of non-replicating HSV-1 preparations to mice. The serum anti-HSV Ig response following intragastric administration of heat or UV inactivated HSV-1 strain SC16 was compared with that elicited by an attenuated derivative of SC16 (TKDM21). The highest response followed immunization with TKDM21 and this was markedly enhanced by repeated intragastric administration, reaching ca 35% of that elicited following a cutaneous infection with live virus. Repeated doses of killed virus produced only a minimal increase in the response even when given intranasally (i.n.). However, cholera toxin and its B-subunit with UV-inactivated virus or a mixture of purified viral glycoproteins enhanced the anti-HSV response after i.n. immunization and produced antibody levels equivalent to those following intragastric delivery of live TKDM21.

Adjuvants, Immunologic↗

PubMed1997

Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1.

Immunocytochemistry on serial paraffin sections was used to monitor the production dynamics of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) and viral antigens in the trigeminal ganglion (TG) and the central side of the dorsal root entry zone (DRE) of mice, following infection of the cornea with herpes simplex virus type 1. In normal TG, scattered satellite cells were TNF-alpha+ and in the DRE, TNF-alpha+ and/or low numbers of IL-6+ cells were detected. On day 3 after infection, foci of TG neurons with viral antigens were surrounded by large numbers of TNF-alpha+ and/or IL-6+ cells and low numbers of IFN-gamma+ cells. IL-2+ and/or IL-4+ cells appeared later, when viral antigens had almost cleared. In the TG, the most striking changes occurred with TNF-alpha, with respect to its source (satellite cells, Schwann cells and infiltrating cells) and the extent and long duration of its production. TNF-alpha was the predominant cytokine throughout acute and latent infection and even by day 30, numbers of satellite cells expressing this cytokine were three times higher than those in normal ganglia. Moreover, in the DRE, TNF-alpha was the only cytokine detected during virus clearance and again, its production continued, along with that of IL-6, on days 20 to 30, in both infiltrating cells and astrocytes. Thus, cytokines, particularly TNF-alpha and perhaps IL-6, from infiltrating cells and resident glial cells may have a role both in virus clearance and in normal homeostatic mechanisms in the nervous system such as repair and protection of neurons from damage.

Animals↗

PubMed1996

Anxiety-like and depression-like behavior in Maudsley reactive (MR) and non-reactive (NMRA) rats.

1. Female MR ("anxious") and MNRA ("non-anxious") Maudsley rats were tested in the CSD behavioral conflict paradigm (anxiety-like measure) and also in the FST paradigm (depression-like measure). 2. As expected, MNRA rats accepted significantly more shocks in the CSD paradigm than did MR rats (i.e., MNRA rats were less "anxious"), MNRA rats also exhibited significantly less immobility in the FST procedure (i.e., MNRA rats were less easily made "depressed"). 3. When the data were pooled across the two strains, there was a significant correlation between CSD and FST behavioral scores; however, there was no significant correlation between these measures when the data from the two strains were evaluated separately. Multiple regression (independent variables of rat strain and CSD score, dependent variable of FST score) revealed a significant effect of rat strain, but not CSD score, on FST behavior. 4. The relationship of these findings to the apparent relationship between anxiety and depression in humans is discussed.

Animals↗

PubMed1996

Modulation of Langerhans cell phenotype, migration and maturation by agents known to cause herpes simplex virus reactivation in a mouse model.

The factors which control whether an asymptomatic or symptomatic infection in the skin follows reactivation of herpes simplex virus (HSV) in the sensory ganglia remain unclear. Xylene, retinoic acid and dimethylsuphoxide (DMSO) all stimulate similar levels of virus reactivation in the ganglia of latently infected mice, yet give rise to high, moderate and very low incidences of clinical skin disease, respectively. This observation suggests that the chemicals may be capable of affecting the local microenvironment of the skin. In the present study we have investigated the effects of xylene, retinoic acid and DMSO on Langerhans cell (LC) phenotype and function. The results show that none of the chemicals inhibit the phenotypic maturation of Langerhans cells in vitro. However, DMSO induced a dramatic elevation in class II MHC expression on Langerhans cells. In addition, fewer antigen-bearing dendritic cells (DC) were evident in the lymph nodes if xylene was administered to the skin prior to challenge with the contact sensitizer, FITC. None of the chemicals inhibited the accumulation of DC in the lymph nodes after such an antigen-challenge, suggesting that xylene was affecting not migration, but antigen uptake. The inhibition of antigen uptake by xylene together with the activation of LC in terms of class II expression by DMSO may explain, in part, the relative abilities of these chemicals to allow the establishment of recurrent HSV disease in the skin.

Animals↗

PubMed1996

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and immune cell infiltration.

The corneas of latently infected mice were UV irradiated to induce reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG). On days 1 to 4 after irradiation, TG were removed, serially sectioned and double stained to identify immune cells and virus antigens. Virus antigen was detected in small numbers (most commonly one) of neurons per ganglion as early as day 1, confirming the rapidity of reactivation and the neuron as the likely site of this event. The immune response was also rapid and effective since virus antigen was identified in immune cells at day 1 and by day 4 all samples were negative. The predominant infiltrating cells on days 1 and 2, when virus antigen was present and being cleared, were T cells, both CD4+ and CD8+. Later, large numbers of B cells appeared, suggesting that local antibody production may also be involved in controlling the reactivated infection. The observations suggest that a significant proportion of reactivation events do not result in disease of the eye or shedding of virus in the tear film. However, they also suggest that as little as one reactivating neuron in the ganglion may be sufficient to lead to such disease and/or shedding.

Animals↗

PubMed1996

Immune cell infiltration in corneas of mice with recurrent herpes simplex virus disease.

Reactivation of latent herpes simplex virus type 1 (HSV-1) infection was induced by UV irradiation of the corneas of latently infected mice. On days 1-4 after stimulation, infectious virus was sought in nervous and ocular tissue. On days 4, 7 and 10, eyes with either recurrent epithelial or stromal disease and appropriate controls were stained to identify immune cells and HSV-1 antigens. The maximum incidence of infectious virus was on day 2 when 5/10 ophthalmic parts of the trigeminal ganglion yielded HSV. Thus in this mouse model, as in humans, reactivation of virus in the trigeminal ganglion is the likely source of virus producing recurrent disease and shedding in the tear film. On day 4, when virus antigens were still present, granulocytes were the predominant infiltrating cell in corneas with either type of disease. Small numbers of T cells, dendritic cells and cells expressing MHC class II were also present. In stromal disease, the granulocyte infiltrate persisted and T cells remained sparse. In contrast, in epithelial disease, granulocyte numbers rapidly declined and both CD4+ and CD8+ T cells (present at a ratio of 1:1) increased significantly. The secondary immune response to virus antigen is more rapid and vigorous than that during primary corneal infection. Granulocytes may play a role in the initial clearance of virus, however, the other types of cells present early on provide the potential for a local secondary immune response. The high proportion of CD8+ cells in epithelial disease compared with stromal disease suggests that they may be acting as suppressors.

Animals↗