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R L Ward

Publications and source records attributed to R L Ward.

At least 91 records · Page 5Linked to original sources
PubMed1995

A rapid PCR ELISA for the detection of activated K-ras in colorectal cancer.

Aims-To develop a rapid PCR ELISA procedure for the detection of mutations in K-ras in a microtitre plate format, and to evaluate the assay for the detection of these mutations in human colorectal cancer.Methods-An enriched PCR method was used with labelled primers, and PCR product was captured on GCN4 coated immunoassay plates. Detection of biotinylated mutant product was performed by colorimetric assay with streptavidin-horseradish peroxidase. The assay was used to determine K-ras status in a series of 60 human colorectal neoplasms, together with paired normal colonic mucosa. Results from gel electrophoretic analysis were compared with ELISA results.Results-The assay proved reliable in detecting K-ras mutations in DNA extracted from both fresh and paraffin embedded colorectal tumours. ELISA results were comparable with results from gel electrophoresis. Mutations of K-ras were detected in 16 of 48 adenocarcinomas and five of 12 adenomas but no mutations were detected in normal mucosa. There was a highly significant difference (p<0.0005) between optical density values for carcinomas with mutant K-ras and their paired normal data. Adenomas did not show the clear distinction between positive and negative results seen with carcinomas.Conclusions-This assay provides a rapid and reliable means of detecting mutations in codon 12 of the K-ras oncogene. The single tube format colorimetric analysis in microtitre plates and clear discrimination between mutant and wild type genes makes the assay suitable for automation. The occurrence of intermediate results in the case of adenomas provides support for the hypothesis that mutations of K-ras occur early in the course of colorectal carcinogenesis.

Journal Article↗
PubMed1994

Non-covalent DNA groove-binding by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The cooked meat mutagen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is metabolized in vivo to electrophilic intermediates that covalently bind to DNA guanines. Here we address the mechanism of PhIP's non-covalent interaction with DNA by using spectroscopic and computational methodologies. NMR methodologies indicated that upon addition of DNA, PhIP aromatic protons underwent a small, 0.11-0.12 p.p.m. upfield shift. DNA phosphorus resonances of non-covalent PhIP-DNA complexes broadened and slightly shifted upfield, while DNA base imino proton resonances shifted slightly downfield relative to DNA alone. UV and fluorescence spectra of PhIP titrated with DNA showed no detectable shifting and hypochromism of absorbance or fluorescence bands. In the presence of DNA, PhIP fluorescence was efficiently quenched by acrylamide, but not by silver ion. Further, the NMR spectra suggest that PhIP is in fast exchange with the DNA, and is slightly specific for adenine-thymine (A-T) sequences. Finally, structural arguments based on quantum chemistry calculations suggested that PhIP and its metabolites are unlikely to intercalate into DNA. These data collectively indicate that PhIP non-covalently binds in a groove of DNA.

Computer Simulation↗
PubMed1994

Active immunity against rotavirus infection in mice is correlated with viral replication and titers of serum rotavirus IgA following vaccination.

Using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge with the murine rotavirus strain EDIM. To determine if a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective EDIM strain and a partially protective heterologous rotavirus strain (RRV-G). Reassortants that contained genes for EDIM proteins responsible for protection were anticipated to provide complete protection; however, no EDIM proteins were found to be both necessary and sufficient for full protection. Instead, protection was found to be highly correlated with viral shedding (P = 0.005) and with serum rotavirus IgA titers stimulated by the different reassortants (P < 0.001). This indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of EDIM proteins. This conclusion was supported by the finding that the titers of serum rotavirus IgA, but not IgG, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against EDIM. If these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization.

Animals↗
PubMed1994

Cytokine-mediated induction of HLA antigen expression on human glomerular mesangial cells.

Well-characterized human glomerular mesangial cells were stimulated in vitro with interferon-gamma (IFN-gamma), interleukin-1, and tumor necrosis factor alpha (TNF-alpha) to determine the influence of these cytokines on the expression of class I and II HLA antigens. IFN-gamma induced a dose-dependent increase in expression of both class I and class II HLA antigens. TNF-alpha induced only class I antigens when used alone, but acted synergistically with IFN-gamma in the expression of class I and II antigens. Interleukin-1 had little effect on HLA antigen expression. These results raise the possibility that stimulated mesangial cells may act as antigen-presenting cells and may mediate local immune reactions in the glomerulus.

Antigen-Presenting Cells↗
PubMed1994

Comparison of VP4 and VP7 of five murine rotavirus strains.

The nucleotide and deduced amino acid sequences of outer capsid proteins VP4 and VP7 of five murine rotavirus strains (EW, EB, EC, EL, EHP) were determined. Comparisons of the VP7 amino acid sequences of the five murine rotavirus strains with rotavirus strains representative of G serotypes 1-14 showed that the murine strains were highly homologous to one another and more closely related to strains representing G3 than to any other G type. Analysis of the VP4 amino acid sequences of the murine strains revealed the presence of at least two murine P types. Therefore, sequence analysis would predict that the murine rotaviruses are G3 or G3-like and represent at least two unique P types (tentatively P17 for EW, EB, EC, and EL and P18 for EHP). When we attempted to categorize the murine strains by serum neutralization tests, our results were less clear. Serum to two murine strains, EHP and EW, displayed one-way reactivity by focus-reduction neutralization assays using several prototype G3 strains. The G3 serotyping monoclonal antibody 159 failed to neutralize EHP and EW, while the G3-specific monoclonal YO-1E2 neutralized EHP, but not EW. A reassortant (A15) containing VP7 from EW and VP4 from RRV was neutralized by these two G3-specific monoclonal antibodies to a level 8- to 20-fold more than EW, but was still 64- to 250-fold less than either SA11 and RRV. These results suggest that VP4 can influence the antigenicity of VP7.

Amino Acid Sequence↗
PubMed1994

Naturally occurring dual infection with human and bovine rotaviruses as suggested by the recovery of G1P8 and G1P5 rotaviruses from a single patient.

Culture adaptation of rotaviruses from an infant with severe diarrhea in Cincinnati, Ohio, yielded not only a virus with the original RNA electropherotype (CJN) but also rotaviruses with other electropherotypes, the most dominant of which was called CJN-M [Ward RL, Knowlton DR, Schiff GM, Hoshino Y, Greenberg HB (1988) in J Virol 62: 1543-1549]. RNA-RNA hybridization and sequencing studies indicated that CJN was a typical G1P8 human rotavirus while CJN-M was a G1P5 strain and contained four gene segments (including segment 4) of a bovine rotavirus. Thus, the infant was apparently dually infected with human and bovine rotaviruses.

Animals↗
PubMed1994

Characterization of a novel leucocyte surface membrane antigen recognized by the monoclonal antibody WM65.

WM65 is a murine mAb which recognizes a novel surface membrane antigen present on leukaemic and normal leucocytes. The present study further investigates the nature of this antigen, especially those features which relate to the possible therapeutic applications of the WM65 antibody. There are 1-3 x 10(4) molecules of this antigen present on normal leucocytes, and the same or greater numbers of antigen molecules are present on a variety of leukaemic cells. In vitro data showed that the WM65 antibody is internalized following interaction with its antigen on normal leucocytes. The affinity of this antibody was calculated using an ELISA method which required neither labelling of the antibody nor purification of the antigen and the affinity constant was found to be 3 x 10(7) +/- 2 x 10(7) (mol/L)-1. Further data are presented which suggest that this antigen is a differentiation antigen and an integral membrane protein. Despite the relatively low affinity of the WM65 antibody, a number of characteristics of the antigen suggest the antibody may possibly have therapeutic applications. These characteristics include its cellular distribution, the number of antigen molecules expressed on the cell surface and its ability to internalize in vitro.

Antibodies, Monoclonal↗
PubMed1994

Protection against rotavirus disease after natural rotavirus infection. US Rotavirus Vaccine Efficacy Group.

Determination of protective efficacy after natural rotavirus infection is important as a basis for evaluating rotavirus vaccines. Therefore, placebo recipients in a large 2-year rotavirus vaccine trial conducted across the United States were followed to determine the protection afforded by natural rotavirus infection. Serotype 1 rotaviruses predominated (93% year 1, 66% year 2), but isolates of all four major human rotavirus serotypes circulated during both years. Of the 45 placebo recipients with documented rotavirus illnesses in year 1, 1 developed rotavirus disease in year 2 compared with 29 of the other 235 placebo recipients (P = .03). Serologic data were available for 171 placebo recipients, and 37 of 140 without rotavirus illnesses in year 1 had evidence of asymptomatic rotavirus infection. None of these 37 experienced rotavirus disease in year 2 compared with 22 of the remaining 103 (P < .001). Overall efficacy after natural rotavirus infection was 93% (95% confidence interval, 50%-99%).

Antibodies, Viral↗
PubMed1993

Identification of a new neutralization epitope on VP7 of human serotype 2 rotavirus and evidence for electropherotype differences caused by single nucleotide substitutions.

Neutralizing monoclonal antibodies 2F1 and 1C10, which are specific for VP7 of serotype 2 rotaviruses (G2), were used to select neutralization escape variants of the human serotype 2 rotavirus, DS-1. Neutralization survival patterns generated by monoclonal antibodies 2F1, 1C10, and RV5:3 indicated that 2F1 and 1C10 did not recognize identical epitopes. Direct sequencing of PCR products of gene 8, encoding VP7, revealed that each escape variant possessed only a single nucleotide mutation which resulted in a single amino acid substitution. In one variant, a second nucleotide change occurred, but did not result in an amino acid change. Four independently selected 2F1 mutants showed mutations in four separate sites in antigenic regions A, C, and D. Two independently selected 1C10 variants had mutations in either the A region or an unreported site at amino acid 190. Two of the mutations resulted in the creation of new glycosylation sites which were utilized, but did not appear to greatly affect antigenicity. Of note, three of the mutants also demonstrated alterations in the migration patterns of gene 8 on PAGE. Such electrophoretic mobility shifts caused by single base neutralization escape mutations have not previously been reported for rotavirus. Since multiple mutations were selected with the same monoclonal antibody it appears that the antigenic regions of VP7, although widely separated in the linear sequence, are parts of a single, large and complex neutralization domain which includes amino acid 190 as well as the other previously reported epitopes.

Amino Acid Sequence↗
PubMed1993

Tamoxifen reduces bone turnover and prevents lumbar spine and proximal femoral bone loss in early postmenopausal women.

Although widely used for its anti-estrogen properties tamoxifen has estrogen like effects on a number of tissues including bone and liver. Previous studies suggest a preservation of lumbar spine density in postmenopausal women but the effect on the hip had not been addressed. To determine whether tamoxifen prevents bone loss in the early postmenopausal period bone mineral density at the lumbar spine and femoral neck was measured using dual energy X-ray absorptiometry at presentation and 6 monthly thereafter for 1 year in a prospective controlled study. Also indices of bone turnover, serum osteocalcin and urinary hydroxyproline excretion, were assessed. Fifteen early postmenopausal women with Stage I or II breast cancer treated with tamoxifen and 21 normal postmenopausal women were studied. Sex hormone binding globulin and antithrombin III levels in serum were also measured as indices of the hepatic estrogenic activity. Tamoxifen (20 mg daily) prevented bone loss at the femoral neck and lumbar spine. Median rates of change in bone mineral density (%/year) for the tamoxifen group were +0.09%/year in the lumbar spine and 1.4%/year in the femoral neck compared with -2.3%/year and -1.8%/year for the control group (P = 0.04 and 0.03, respectively). Tamoxifen resulted in a significant decrease in both serum osteocalcin and urinary hydroxyproline by 6 months of treatment and this effect persisted for the 12 months of observation. An increase in sex hormone binding globulin and a decline in antithrombin III levels was also observed. These data indicate that, in recently, postmenopausal women tamoxifen prevented bone loss at both the lumbar spine and femur and reduced bone turnover.

Absorptiometry, Photon↗
PubMed1993

Serotypic and genotypic characterization of human serotype 10 rotaviruses from asymptomatic neonates.

Human rotaviruses were isolated from asymptomatic neonates at various hospitals and clinics in the city of Bangalore, India, and were found to be subgroup I specific and possess long RNA patterns (M. Sukumaran, K. Gowda, P. P. Maiya, T. P. Srinivas, M. S. Kumar, S. Aijaz, R. R. Reddy, L. Padilla, H. B. Greenberg, and C. D. Rao, Arch. Virol. 126:239-251, 1992). Three of these strains were adapted to tissue culture and found by serotype analysis and neutralization assays to be of serotype 10, a serotype commonly found in cattle but infrequently found in humans and not previously identified in neonates. By RNA-RNA hybridization, a high level of relatedness to a serotype 10 bovine rotavirus strain and a low-to-medium level of relatedness to a human rotavirus strain were observed. Since this human isolate shares a genogroup with bovine rotavirus, it is likely that it originated by interspecies transmission. A human rotavirus strain isolated from asymptomatic neonates and similar to bovine rotavirus might represent a good vaccine candidate.

Animals↗
PubMed1993

Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children.

Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.

Antibodies, Viral↗
PubMed1993

Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence.

Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.

Animals↗
PubMed1993

Production of a functional single-chain Fv fragment from the pan-leukocyte antibody WM65 using splicing by asymmetric PCR.

A method of assembly of a single-chain Fv fragment is described, whereby asymmetric polymerase chain reactions (APCR) and primer extension were used to join immunoglobulin heavy and light chain variable region genes via a linker sequence. In this procedure heavy and light chain genes, together with a linker gene containing complementary sequences, were amplified by APCR to generate single-stranded products. The single stranded heavy or light chain genes were hybridized to the relevant single-stranded link product, and extended to produce double-stranded heavy-link and link-light genes. These genes then underwent another round of APCR, resulting in two single-stranded genes (heavy-link and link-light) containing extensive overlapping sequences. Hybridization and extension of these two single-stranded products allowed the formation of the complete heavy chain-link-light chain double-stranded product. Using this method, a functional single-chain Fv fragment based on the pan-leukocyte antibody WM65 was expressed and purified from E. coli. This method of immunoglobulin gene assembly by polymerase chain reaction (PCR) offers an alternative to the current methods of the genetic engineering of antibody fragments.

Antibodies↗
PubMed1992

Salivary antibody titers in adults challenged with a human rotavirus.

To determine whether salivary antibody is a reliable indicator of rotavirus infection and mucosal rotavirus antibody concentrations, salivary rotavirus antibody titers were determined as a function of time following inoculation of 24 adult volunteers with a virulent strain of human rotavirus (CJN). Twenty of the subjects became infected and all produced detectable amounts of salivary rotavirus IgA. These antibody concentrations remained undetectable in the four uninfected subjects. Rises of greater than or equal to fourfold in either salivary rotavirus IgA or neutralizing antibody to the challenge virus were detected in all but two subjects. The titers of both antibodies were maximal at 13 days after inoculation and decreased significantly (p less than .02) by 27 days, a result very similar to that previously found with stool rotavirus IgA in CJN-infected subjects. These results suggest that salivary rotavirus antibody titers are an accurate reflection of mucosal rotavirus antibody and a possible surrogate for intestinal rotavirus antibody.

Adolescent↗
PubMed1992

Active protection against rotavirus infection of mice following intraperitoneal immunization.

Active immunity to rotavirus has been demonstrated following oral inoculation with live virus but little is known about the effects of parenteral immunization. In this study, adult mice were immunized by intraperitoneal (ip) inoculation with live rotaviruses and later orally challenged with murine rotavirus (EDIM) to measure active immunity against infection. Three doses of EDIM (8 micrograms/dose) given intraperitoneally (ip) provided full protection against EDIM infection, whether administered with or without Freund's adjuvant. Only partial protection was found when the quantity of immunogen was reduced to < 2 micrograms/dose. Reduction of the number of doses from three to one (8 micrograms/dose), however, still resulted in protection of all mice. Significant protection was also observed after inoculation with one or three doses (2 micrograms/dose) of heterologous rotaviruses. Protection provided by the heterologous strains did not correlate with neutralizing antibody to EDIM, which indicated that neutralizing antibody to the challenge virus was not required for protection. uv-Inactivated EDIM also provided significant protection against EDIM, thus demonstrating that viral replication was not required for protection. These results suggest that parenteral immunization may be an effective method to vaccinate against rotavirus disease.

Animals↗
PubMed1992

Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody.

Studies were performed to determine whether active immunity against murine rotavirus (EDIM) infection of mice correlated with titers of neutralizing antibody to the challenge virus. Neonatal mice administered either murine or heterologous rotaviruses all developed diarrhea and high titers of serum rotavirus IgG. However, only mice given EDIM, the murine EB, or simian SA11-FEM strains were protected against EDIM infection when challenged 60 days later. Other serotype 3 strains (RRV, SA11-SEM), as well as strains belonging to serotypes 5 and 6 (OSU, NCDV, WC3), were not protective. Serum neutralizing antibody titers to EDIM were almost undetectable after rotavirus infection with any strain and could not, therefore, be correlated with protection. Likewise, intestinal neutralizing antibody titers were extremely low 21 days after EDIM infection, and by 60 days after inoculation, EDIM-infected mice had no greater intestinal neutralizing antibody titers than uninoculated controls. Mice inoculated with SA11-FEM as neonates had much higher serum rotavirus IgG responses than mice inoculated as adults, and only those infected with this virus as neonates were protected. Thus, although immunity to EDIM did not correlate with the presence of neutralizing antibody to EDIM, it did correlate with the overall magnitude of the immune response after inoculation with SA11-FEM.

Animals↗