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Biomedical subjects

R A Williamson

Publications and source records attributed to R A Williamson.

At least 19 recordsLinked to original sources
PubMed2006

Copper induces conformational changes in the N-terminal part of cell-surface PrPC.

Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC.

Amino Acid Sequence↗
PubMed2002

Characteristics of immunity induced by viral antigen or conferred by antibody via different administration routes.

The characteristics of the immunity induced by viral antigens or conferred by antiviral antibody via different routes of administration were evaluated comparatively. C57BL/6 mice were immunized via intranasal, intradermal or enteric routes with a live recombinant vaccinia virus expressing the respiratory syncytial virus (RSV) F glycoprotein (F.rVV) or RSV, and then challenged intranasally with RSV. Inhibition of RSV replication was observed in the lungs of all the mice; however, only intranasal immunization hindered virus replication in the nose. Lung inflammation, characterized by infiltration of neutrophils and of mononuclear cells was strongest in the intradermally immunized mice, but was observed in all F.rVV immunized mice to various degrees. Intranasal administration of a potently neutralizing human anti-RSV antibody Fab fragment to infected mice inhibited RSV replication in the nose and, when combined with intraperitoneal administration, protected both the lung and the nose in the absence of deleterious lung pathology. These data suggest that intranasal immunization with F.rVV reduces RSV replication in the respiratory tract, but still induces pathological lung inflammation, even though this is milder than that observed following intradermal immunization. Local neutralizing antibody is indispensable for protection in the nose.

Administration, Cutaneous↗
PubMed2001

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Animals↗
PubMed2001

Tyrosine 36 plays a critical role in the interaction of the AB loop of tissue inhibitor of metalloproteinases-2 with matrix metalloproteinase-14.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) is potentially an important inhibitor of all known matrix metalloproteinases (MMPs). However, it has been shown to undergo specific interactions with both MMP-2 (gelatinase A) and MMP-14 (MT1-MMP), and it has been proposed that these three proteins function as a cell surface-based activation cascade for matrix metalloproteinases and as a focus of proteolytic activity. In this study, we have carried out mutagenesis and kinetic analyses to examine the unique interactions between the AB loop of TIMP-2 and MMP-14. The results demonstrate that the major binding contribution of the AB loop is due solely to residue Tyr-36 at the tip of the hairpin. From this work, we propose that TIMP-2 may be engineered to abrogate MMP-14 binding, whereas its binding properties for other MMPs, including MMP-2, are maintained. Mutants of TIMP-2 with more directed specificity may be of use in gene therapeutic approaches to human disease.

Amino Acid Sequence↗
PubMed2001

Immobilized prion protein undergoes spontaneous rearrangement to a conformation having features in common with the infectious form.

It is hypothesized that infectious prions are generated as the cellular form of the prion protein (PrP(C)) undergoes pronounced conformational change under the direction of an infectious PrP(Sc) template. Conversion to the infectious conformer is particularly associated with major structural rearrangement in the central portion of the protein (residues 90-120), which has an extended flexible structure in the PrP(C) isoform. Using a panel of recombinant antibodies reactive with different parts of PrP, we show that equivalent major structural rearrangements occur spontaneously in this region of PrP immobilized on a surface. In contrast, regions more towards the termini of the protein remain relatively unaltered. The rearrangements occur even under conditions where individual PrP molecules should not contact one another. The propensity of specific unstructured regions of PrP to spontaneously undergo large and potentially deleterious conformational changes may have important implications for prion biology.

Animals↗
PubMed2001

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Adult↗
PubMed2001

Inhibition of stimulated Jurkat cell adenosine 3',5'-cyclic monophosphate synthesis by the immunomodulatory compound HR325.

HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'(trifluoromethyl)-phenyl]-propenamide) is an immunomodulatory compound through pyrimidine biosynthesis inhibition with antiproliferative properties which was derived from the isoxazol compound A77 1726 [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide]. During studies of the effects on early signal transduction events of this type of compound, it was found that HR325 dose-dependently inhibited adenosine 3',5'-cyclic monophosphate (cAMP) synthesis by Jurkat cells stimulated with prostaglandin E(2), (PGE(2)), cholera toxin (CTX), or forskolin (FKN). The potency of inhibition by HR325 of FKN-stimulated cells (IC(50) 30.4 microM) was approximately 3-fold higher than that of the other agonists (11.6 and 11.7 microM) and was independent of time of preincubation for both PGE(2) and FKN. Interestingly, A77 1726, an analogue of HR325, displayed a markedly different profile of stimulus-dependent potencies. The inhibition of cAMP synthesis by HR325 when stimulated by both PGE(2) and FKN was unaffected by glucose supplementation, in contrast to HR325-inhibited ATP levels, which were restored under such conditions. Further studies revealed that HR325 reduced intracellular ATP levels by uncoupling oxidative phosphorylation, albeit with a 1000-fold lower potency than the antihelmintic drug niclosamide. In addition, glucose supplementation experiments showed that, in contrast to HR325, the niclosamide-mediated reduction of ATP levels was wholly responsible for its inhibition of PGE(2)- and FKN-stimulated cAMP synthesis.

Adenosine Triphosphate↗
PubMed2001

Molecular immunologic strategies to identify antigens and b-cell responses unique to multiple sclerosis.

Identification of the causative agent of multiple sclerosis (MS) has long eluded investigators and has become the "Holy Grail" of researchers in the field. The immune response in cerebrospinal fluid of patients with MS, indicated by an increased IgG level and the presence of specific oligoclonal bands after electrophoresis, strongly parallels that found in various infectious diseases of the central nervous system. To understand the nature of B-lymphocyte activation in MS, 4 laboratories studied the antigen-binding regions of antibodies found in MS brain demyelinative plaques and cerebrospinal fluid. Each analysis revealed (1) limited germline expression, results not expected for a random bystander response; (2) features consistent with a specific antigen-targeted process; and (3) the clonal expansion of populations of B lymphocytes in MS. The screening of libraries expressing protein products derived from chronic MS plaque messenger RNA with antibodies purified from plaques, cerebrospinal fluid, or serum of patients with MS has thus far not revealed the antigenic target(s) of the MS antibody response. Because putative MS antigens could be in low abundance, the screening of large libraries of random peptides expressed on phage surfaces might offer an alternative approach to identify peptide sequences recognized by MS antibodies. New sophisticated molecular immunologic techniques described herein should enhance our ability to identify putative antigen(s) targets in MS.

Antibodies, Monoclonal↗
PubMed2001

Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen.

Experimental evidence indicates Epstein Barr virus (EBV) envelope glycoprotein gp350/220 elicits a potent virus neutralizing response in the infected human host that may play an important role in restricting viral pathogenesis. In this study, we report the molecular cloning in combinatorial phage display vectors, of the IgG1 repertoire of an individual naturally infected with EBV, and describe the recovery and characterization of a monoclonal antibody recognizing gp350/220. A detailed understanding of the human antibody response in EBV infection will identify antibodies of potential use in anti-viral prophylaxis and will advance the production of more effective vaccine candidates.

Amino Acid Sequence↗
PubMed2001

Expanded Möbius syndrome.

A woman presented at 33 weeks gestation with reduced fetal movements and a nonreactive nonstress test. Fetal ultrasound examination revealed a peculiar unilateral arm tremor. At emergency cesarean section, performed for fetal indications, a 1,672-gm male infant was delivered requiring intubation for feeble respiratory effort. After delivery the neonate was transiently hypertonic and later hypotonic. Continuing ventilatory support at minimal settings was necessary. The work-up for aneuploidy, metabolic disorders, and infection was negative. The infant died after being removed from ventilatory support on day 22. Postmortem examination revealed extensive bilateral brain gliosis and mineralization without evidence of inflammation, partial absence of cranial nerve nuclei III-XI, and a total absence of cranial nerve roots VI-XI. Together these finding are compatible with a diagnosis of expanded Möbius syndrome.

Adult↗
PubMed2001

Adenotonsillar histopathology after organ transplantation.

OBJECTIVES: The increasing number of surviving pediatric organ transplant recipients has resulted in a new clinical controversy surrounding the significance of adenotonsillar hypertrophy. The objective of this study is to evaluate adenotonsillar specimens, understand characteristic histopathology, and to examine the frequency and significance of this finding in this population. METHODS: Twenty-one cases of pediatric transplant recipients with adenoidal and/or tonsillar hypertrophy were reviewed retrospectively in a tertiary-care setting. Particular attention was given to the histopathology of their surgical specimens, including any evidence of posttransplantation lymphoproliferative disorders (PTLD). RESULTS: Using morphologic, immunohistochemical, and molecular genetic analyses, 15 (71%) of 21 patients were noted to have Epstein-Barr virus (EBV)-related lymphoid hyperplasia, including 1 case (4.7%) of PTLD. Six (29%) of 21 had evidence of reactive follicular hyperplasia not related to EBV. B-cell and T-cell markers were nearly uniformly positive when tested for, except in the single patient with PTLD, who exhibited polymorphic, polyclonal B-cell morphology. Kappa and lambda light-chain clonality markers were positive in 11 (92%) of 12 patients. CONCLUSIONS: EBV-related lymphoid hyperplasia is frequently associated with adenotonsillar hypertrophy in pediatric organ transplant patients (71% of our cases); 92% of those cases tested exhibit polyclonal B-cell populations. PTLD, an important cause of morbidity and mortality in this population, represented approximately 5% of our cases. The remainder of cases represent follicular hyperplasia unrelated to EBV or lymphoproliferative abnormalities. Characteristic histopathologic findings are presented.

Adenoids↗
PubMed2001

Genetic susceptibility to preeclampsia: roles of cytosineto-thymine substitution at nucleotide 677 of the gene for methylenetetrahydrofolate reductase, 68-base pair insertion at nucleotide 844 of the gene for cystathionine beta-synthase, and factor V Leiden mutation.

OBJECTIVE: The purpose of this study was to evaluate the association between preeclampsia and 3 relatively common mutations that are important in the development of vascular disease and thrombosis; these are similar to conditions observed in pregnancies complicated by preeclampsia. STUDY DESIGN: Deoxyribonucleic acid was extracted from whole blood or cheek swabs of 281 patients with preeclampsia and 360 control subjects (all white). Control subjects consisted of women who had undergone at least 2 term pregnancies unaffected by preeclampsia. Mutation frequencies among patients with preeclampsia and control subjects were compared by standard chi2 analysis, with P <.05 considered significant. RESULTS: Thirty-three of 281 women with preeclampsia (11.7%) and 22 of 193 women with severe preeclampsia (11.4%) were homozygous for cytosine-to-thymine substitution at nucleotide 677 in the gene for methyltetrahydrofolate reductase (MTHFR), versus 41 of 360 control subjects (11.4%; difference not significant). Forty of 258 women with preeclampsia (15.5%) and 22 of 175 women with severe preeclampsia (12.6%) were heterozygous for the insertion of 68 bases at position 844 in the gene for cystathionine beta-synthase (CBS), versus 58 of 332 control subjects (17.5%). Fifteen of 250 women with preeclampsia (6.0%) and 11 of 169 with severe preeclampsia (6.5%) were heterozygous for the Leiden mutation (glycine-to-alanine substitution at nucleotide 1691) in the gene for factor V (F5), versus 12 of 253 control subjects (4.7%; difference not significant). CONCLUSION: In this white population a missense mutation of MTHFR, an insertion mutation of CBS, and a missense mutation of F5 were not found to be associated with an increased risk for preeclampsia, either independently or in combination.

Amino Acid Substitution↗
PubMed2001

Strain-specified relative conformational stability of the scrapie prion protein.

Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrP(Sc)). To explore this postulate, we examined the properties of PrP(Sc) from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrP(Sc) molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrP(Sc) as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have approximately 75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a approximately 170-d incubation period and approximately 1.25 M for the SHa(RML) and 139H isolates with approximately 180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a approximately 320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrP(Sc) conformers encipher the biological properties of prion strains.

Animals↗
PubMed2001

Lipoprotein lipase gene mutations and the genetic susceptibility of preeclampsia.

In the pathogenesis of preeclampsia, endothelial cell activation or dysfunction is a central theme, and marked dyslipidemia may contribute to endothelial cell dysfunction. The objective of this study was to evaluate the association between preeclampsia and mutations within the lipoprotein lipase (LPL) gene. DNA was extracted from whole blood or cheek swabs of 250 preeclamptic patients, 265 control subjects, and 106 offspring of preeclamptic patients (all white). Control subjects were women who had undergone >/=2 term pregnancies unaffected by preeclampsia. All samples were genotyped for 3 LPL polymorphisms with the use of polymerase chain reaction of known allelic variants. The 3 mutations studied were the following: (1) Asp9Asn substitution in exon 2, (2) T-to-G substitution at position -93 of the proximal promotor region (-93T/G), and (3) Asn291Ser substitution in exon 6. Results were analyzed with an chi(2) contingency table. The prevalences of the Asp9Asn mutation, -93T/G promotor mutation, and Asn291Ser mutation were not significantly different among the preeclamptic patients and control subjects (Asp9Asn: patients, 2.8%; control subjects, 4.0%; -93T/G: patients, 4.5%; control subjects, 5.5%; Asn291Ser: patients, 4.0%; control subject, 3.0%). In addition, there was no difference in the frequency of any of the mutations in the offspring of preeclamptic women compared with that observed in the control population. Between a small group of patients with nulliparous HELLP syndrome (a variant of severe preeclampsia: hemolysis, elevated liver enzyme, low platelets) patients (n=12) and control subjects, there was a significant difference in the prevalence of the Asn291Ser mutation (16.7% versus 3.0%, P=0.01). In this large white population, the Asp9Asn mutation, -93T/G promotor mutation, and Asn291Ser mutation were not associated with an increased risk for preeclampsia. In a small subgroup of patients, the Asn291Ser mutation was associated with an increased risk for nulliparous HELLP syndrome.

Female↗
PubMed2001

Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance.

To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.

Adipocytes↗
PubMed2001

In vivo evaluation of 111In-DTPA-N-TIMP-2 in Kaposi sarcoma associated with HIV infection.

Matrix metalloproteinases are the major agents responsible for the degradation of extracellular matrix and are produced at high levels by transformed and tumour cells, where they participate in the metastatic process by allowing local invasion. They are also more active at sites of new normal growth and angiogenesis. In the early stages of Kaposi sarcoma (KS), in vitro studies have demonstrated that vascular invasion can be inhibited by inhibitors of matrix metalloproteinases. Imaging of visceral and cutaneous KS presents a problem and therefore the potential use of a labelled inhibitor of metalloproteinases, N-TIMP-2, with indium-111 was thought to present a possible imaging tool. The biokinetics, dosimetry and potential for imaging with 111In-DTPA-N-TIMP-2 were assessed in five patients with HIV infection and KS. Between 103.1 and 108.0 MBq of this agent was injected into each patient, and the dynamic uptake over the kidneys was assessed, whole body scans were performed and blood samples were obtained. The clearance from the blood was rapid, with a first component half-time of 16.6+/-3.4 min and a second component half-time of 9.68+/-2.68 h. Two out of five patients experienced minor shivering but one of these patients was generally unwell before the study. The last three patients had no such problems. The tracer distributed predominantly to the kidneys and did not localise in other tissues. No KS lesions were clearly identified. 111In-DTPA-N-TIMP-2 can be successfully prepared and administered to patients safely, with a biodistribution and dosimetry which would allow its use as an imaging tracer. It is unlikely to be of use for imaging KS, but may have a role in other tumours that produce matrix metalloproteinases.

Adult↗