Biomedical subjects

E S Ward

Publications and source records attributed to E S Ward.

At least 37 records · Page 2Linked to original sources

PubMed2000

Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-) rats.

Previous studies have demonstrated that phenobarbital treatment impairs the biliary excretion of acetaminophen glucuronide (AG), although the transport system(s) responsible for AG excretion into bile has not been identified. Initial studies in rat canalicular liver plasma membrane vesicles indicated that AG uptake was stimulated modestly by ATP, but not by membrane potential, HCO(3)(-), or pH gradients. To examine the role of the ATP-dependent canalicular transporter multidrug resistance-associated protein 2 (Mrp2)/canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of AG, the hepatobiliary disposition of acetaminophen, AG, and acetaminophen sulfate (AS) was examined in isolated perfused livers from control and TR(-) (Mrp2-deficient) Wistar rats. Mean bile flow in TR(-) livers was approximately 0.3 microl/min/g of liver ( approximately 4-fold lower than control). AG biliary excretion was decreased (>300-fold) to negligible levels in TR(-) rat livers, indicating that AG is an Mrp2 substrate. Similarly, AS biliary excretion in TR(-) livers was decreased ( approximately 5-fold); however, concentrations were still measurable, suggesting that multiple mechanisms, including Mrp2-mediated active transport, may be involved in AS biliary excretion. AG and AS perfusate concentrations were significantly higher in livers from TR(-) compared with control rats. Pharmacokinetic modeling of the data revealed that the rate constant for basolateral egress of AG increased significantly from 0.028 to 0.206 min(-1), consistent with up-regulation of a basolateral organic anion transporter in Mrp2-deficient rat livers. In conclusion, these data indicate that AG biliary excretion is mediated by Mrp2, and clearly demonstrate that substrate disposition may be influenced by alterations in complementary transport systems in transport-deficient animals.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2000

Probenecid-associated alterations in valproic acid pharmacokinetics in rats: can in vivo disposition of valproate glucuronide be predicted from in vitro formation data?

Previous investigations have suggested that probenecid (PRB) alters the in vivo disposition of valproic acid (VPA), perhaps by inhibiting hepatic formation of valproate glucuronide (VG). Because VPA and PRB bind moderately to plasma proteins, protein binding also is a potential locus of interaction. The purpose of this investigation was to determine whether in vitro systems could accurately predict PRB-associated perturbations in the hepatobiliary disposition of VPA and VG in vivo. VPA and PRB were coadministered to rats for 60 min at various infusion rates to examine steady-state VPA disposition. PRB did not alter the binding of VPA in serum or hepatic cytosol. However, PRB decreased the apparent intrinsic clearance of VPA (1.81 +/- 0.58 versus 1.23 +/- 0.23 ml/min; P =.025) by competitively inhibiting VPA elimination. In a separate study, rat hepatic S9 fractions were incubated with VPA (7.2-721 microg/ml) and PRB (0-2850 microg/ml). VG formation (V(max) = 0.80 +/- 0.06 microg/min/mg of protein; K(m) = 173 +/- 28.8 microg/ml) was impaired by PRB in a competitive manner (K(i) = 876 +/- 559 microg/ml), consistent with the in vivo data. Despite inhibition of phase II metabolism of VPA to VG by PRB, the VG biliary excretion rate at similar unbound VPA concentrations in hepatic cytosol was not lower in PRB-treated rats. These results indicate that VG disposition in the presence of PRB cannot be predicted accurately based solely on in vitro inhibition of glucuronidation and emphasize the complexity of processes associated with the hepatobiliary system.

Algorithms↗

PubMed1999

The influence of signal noise on the accuracy of kinetic constants measured by surface plasmon resonance experiments.

An analysis is carried out to investigate the accuracy of kinetic parameters obtained using surface plasmon resonance methodology with a BIAcore instrument. The Cramer Rao lower bound for the least possible variance of an estimator of the kinetic parameters is determined. Using simulations it is shown that the standard least-squares estimation technique provides estimates that achieve this bound. The theoretical and simulation results are compared with experimental data obtained from an analysis of the interaction of the myc peptide with the anti-myc antibody, 9E10. This investigation indicates that the accuracy of the results depends on the signal level which has particular relevance to the design of experiments with low signal levels. It is shown how the accuracy of the estimates of the kinetic constants depends on the kinetic constants themselves and how the accuracy of the association constants depends on the concentration of the analyte that is used in the experiment. In addition, the effects of increasing the number of data points in the analysis of dissociation data on the accuracy of the estimates are quantitated. It is also demonstrated that signal averaging of data derived from repeat sensorgrams can result in a significant decrease in the standard deviation of the estimates.

Animals↗

PubMed1999

The choice of reference cell in the analysis of kinetic data using BIAcore.

An important step in the analysis of sensorgram data for BIAcore experiments is the subtraction of reference cell data to remove the effects of the bulk shift on the sensorgram of interest. It is shown that this step can introduce errors in the measured kinetic constants. This phenomenon is investigated both theoretically and with experimental data.

Animals↗

PubMed1999

Molecular requirements for T cell recognition by a major histocompatibility complex class II-restricted T cell receptor: the involvement of the fourth hypervariable loop of the Valpha domain.

The role of two central residues (K68, E69) of the fourth hypervariable loop of the Valpha domain (HV4alpha) in antigen recognition by an MHC class II-restricted T cell receptor (TCR) has been analyzed. The TCR recognizes the NH2-terminal peptide of myelin basic protein (Ac1-11, acetylated at NH2 terminus) associated with the class II MHC molecule I-Au. Lysine 68 (K68) and glutamic acid 69 (E69) of HV4alpha have been mutated both individually and simultaneously to alanine (K68A, E69A). The responsiveness of transfectants bearing wild-type and mutated TCRs to Ac1-11-I-Au complexes has been analyzed in the presence and absence of expression of the coreceptor CD4. The data demonstrate that in the absence of CD4 expression, K68 plays a central role in antigen responsiveness. In contrast, the effect of mutating E69 to alanine is less marked. CD4 coexpression can partially compensate for the loss of activity of the K68A mutant transfectants, resulting in responses that, relative to those of the wild-type transfectants, are highly sensitive to anti-CD4 antibody blockade. The observations support models of T cell activation in which both the affinity of the TCR for cognate ligand and the involvement of coreceptors determine the outcome of the T cell-antigen-presenting cell interaction.

Antigen Presentation↗

PubMed1999

Anti-Vbeta8 antibodies induce and maintain staphylococcal enterotoxin B-triggered Vbeta8+ T cell anergy.

The mechanism involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy is poorly understood. We demonstrated earlier that B cells play an important role in the maintenance of SEB-induced T cell anergy in vivo and in vitro. Here, we demonstrate that B cells are not essential in SEB-induced T cell activation, but are important for the maintenance of T cell memory phenotype and anergy in vivo. Studying the activated B cell repertoire, we observe that SEB treatment increases serum anti-Vbeta8 antibody titer as detected by enzyme-linked immunosorbent assay using soluble Vbeta8 chains as antigens, and by staining of a Vbeta8-expressing thymoma. These antibodies disappear gradually after immunization with SEB, whereas the capacity of the T cells to respond to SEB in vitro is restored. Anti-Vbeta8 monoclonal antibody treatment causes Vbeta8+ T cell unresponsiveness to SEB in vitro (anergy), without affecting CD4Vbeta8+ T cell frequency. Together, these results suggest a new mechanism to explain the maintenance of SEB-induced T cell anergy, which is dependent on B cells and on anti-Vbeta8 antibody that specifically interacts with Vbeta8+ T cells.

Animals↗

PubMed1999

Identification and function of neonatal Fc receptor in mammary gland of lactating mice.

In addition to its proposed function in regulating serum IgG levels, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a role in IgG transfer across rodent yolk sac and neonatal intestine. In contrast to humans, for which transplacental transfer of IgG appears to be the only mechanism of maternal IgG delivery, the transmission of IgG in mice occurs both antenatally (yolk sac) and neonatally (transport from mother's milk across intestinal epithelial cells). In the current study, a possible role for FcRn in regulating IgG transfer into milk has been investigated. FcRn has been shown to be present in functional form in the mammary gland of lactating mice, and is localized to the epithelial cells of the acini. Analysis of the transfer of Fc fragments and IgG which have different affinities for FcRn indicate that, unexpectedly, these proteins are transferred in inverse correlation with their binding affinity for FcRn. Thus, in the lactating mammary gland FcRn appears to play a role in recycling IgG in a mode that may have relevance to FcRn trafficking during the maintenance of constant serum IgG levels.

Animals↗

PubMed1999

Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn.

The analysis of the pharmacokinetics of wild-type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half-life in mice. Reduced serum half-life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I-related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half-life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half-life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2-CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.

Animals↗

PubMed1999

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.

Amino Acid Substitution↗

PubMed1998

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases.

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.

Animals↗

PubMed1998

Comparative studies of rat IgG to further delineate the Fc:FcRn interaction site.

Recent data have indicated that the MHC class I-related receptor, FcRn, regulates the half-lives of serum IgG in addition to its known role in transferring IgG from mother to young. In the current study, the activity of rat IgG (rIgG) isotypes in FcRn-mediated functions has been analyzed. The serum half-life and maternofetal transfer in mice decreased in the order rIgG2a > rIgG1 > rIgG2c > rIgG2b. This decrease in activity correlates well with reduced binding affinity for soluble mouse FcRn, and site-directed mutagenesis of a recombinant Fc-hinge fragment has been used to investigate the molecular basis for the differences in activities of the rIgG. Analysis of the serum half-lives of the mutated Fc-hinge fragments demonstrated that, in addition to Ile253, His310, His435 and His436 that were identified in earlier studies, amino acids at positions 257, 307 and 309 play a role in building the FcRn interaction site of IgG. The study also excludes the involvement of amino acids in a fourth loop located at the CH2-CH3 domain interface that encompasses residues 386-387 in FcRn binding. Sequence differences at positions 257, 307 and 309 between rIgG most likely account for the reduced affinity of rIgG2b and IgG2c relative to rIgG1 and rIgG2a for binding to FcRn.

Animals↗

PubMed1998

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice.

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.

Amino Acid Sequence↗

PubMed1998

Dental and gingival pain as side effects of niacin therapy.

Two 65-year-old white men with coronary heart disease, given niacin therapy for dyslipidemia for 5 months, developed intense dental and gingival pain that was associated with increases in dose and that was relieved with discontinuance of niacin treatment. One individual who took crystalline niacin had beneficial effects on lipid levels, while the other person who took a delayed release preparation had little lipid effect. The cause of these previously unreported side effects of niacin therapy is uncertain but may be related to prostaglandin-mediated vasodilatation, hyperalgesia of sensory nerve receptors, and potentiation of inflammation in the gingiva with referral of pain to the teeth.

Aged↗

PubMed1997

Recombinant T cell receptor molecules can prevent and reverse experimental autoimmune encephalomyelitis: dose effects and involvement of both CD4 and CD8 T cells.

Autoimmune diseases are often characterized by spontaneous remission followed by relapses. Although the mechanism(s) controlling pathogenic self-reactive T cells are not fully understood, recent data in experimental autoimmune encephalomyelitis (EAE), a prototype for CD4 T cell-mediated autoimmune diseases, indicate that spontaneous recovery is mediated by regulatory T cells (Treg) specific for peptides derived from the beta-chain of the TCR. Here we have tested whether recombinant single-chain TCRs (scTCRs) containing Vbeta domains can be used as vaccines for efficient priming of Treg. A single injection of mice with these recombinant proteins leads to efficient in vivo priming of Treg and almost complete protection from Ag-induced EAE. Significantly, administration of scTCRs during ongoing disease at a 10-fold lower dose than that required for prophylactic treatment also reverses established EAE. However, if a higher dose of scTCR is administered during ongoing disease, paralytic symptoms become exacerbated and the majority of treated animals die from severe and chronic EAE. Furthermore, we demonstrate that regulatory determinants are processed and presented from scTCRs resulting in the recruitment of both CD4 and CD8 regulatory T cells which are required for efficient regulation induced by scTCR. Reversal of established disease following an optimum dose of recombinant TCRs suggests that proteins expressing appropriate Vbeta domains could be used for the treatment of a variety of T cell-mediated pathologic conditions.

Animals↗

PubMed1997

Dual conformations of a T cell receptor V alpha homodimer: implications for variability in V alpha V beta domain association.

The crystal structure of a mutant T cell receptor (TCR) V alpha domain containing a grafted third complementarity-determining region (CDR3) from a different V alpha was determined at 2.3 A resolution by molecular replacement using the wild-type V alpha structure as a search model. Like the wild-type V alpha domain, the mutant crystallized as a homodimer very similar to TCR V alpha V beta and antibody V(L)V(H) heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant V alpha homodimer differs from that in the wild-type by a rotation of 14 degrees such that the buried surface area in the dimer interface of the mutant is 140 A2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the V alpha and V beta domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.

Amino Acid Sequence↗

PubMed1997

Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgG1.

The MHC class I-related receptor, FcRn, is involved in both the transcytosis of serum gamma-globulins (IgGs) and in regulating their serum persistence. The interaction site of FcRn on the Fc region of rodent IgG has been mapped to residues at the CH2-CH3 domain interface using site-directed mutagenesis and x-ray crystallographic analyses. In the current study, the role of individual residues (H310, H433, and N434) at this interface in mediating the Fc-FcRn interaction has been investigated using recombinant, mutated Fc hinge fragments derived from mouse IgG1. In addition, two highly conserved Fc histidines (H435 and H436) have been mutated to alanine, and the resulting mutated Fc hinge fragments were analyzed in both transcytosis and pharmacokinetic studies in mice and in competition binding assays using recombinant, soluble FcRn. The analyses indicate that mutation of H310, H435, and, to a lesser extent, H436 to alanine results in reduced activity of the Fc hinge fragments in both in vivo and in vitro assays. Thus, in addition to the previously defined role of 1253 in the FcRn-IgG interaction, these histidines play a key role in mediating the functions conducted by this Fc receptor. The effects of these mutations on binding of Fc hinge fragments to staphylococcal protein A have also been analyzed and demonstrate a partial, but not complete, overlap of the FcRn and staphylococcal protein A interaction sites on mouse IgG1.

Amino Acids↗

PubMed1997

FcRn: the MHC class I-related receptor that is more than an IgG transporter.

Recent data have indicated that the major histocompatibility complex (MHC) class I-related receptor FcRn plays a role in regulating serum IgG levels, in addition to transferring maternal IgGs across the rodent neonatal gut. The isolation of a human homolog of FcRn from placenta suggests that the studies in rodents have relevance to understanding similar processes in humans. This has implications for the engineering of improved antibodies for therapy.

Animals↗

PubMed1997

Biophysical and structural studies of TCRs and ligands: implications for T cell signaling.

The availability of soluble alphabeta TCRs and the individual chains has now made it possible to carry out structural studies of these molecules and analyze their molecular interactions with peptide-MHC ligands. Recent X-ray crystallographic structures of TCR alpha and beta chains have finally established their structural similarity with the lg molecules. Kinetic measurements of the interaction between TCRs and their ligands have provided strong evidence in favour of an affinity/avidity model for T cell activation in the periphery as well as during development in the thymus.

Animals↗