Biomedical subjects

R L Ward

Publications and source records attributed to R L Ward.

At least 73 records · Page 4Linked to original sources

PubMed1997

Osteomyelitis due to Mycobacterium haemophilum in a cardiac transplant patient: case report and analysis of interactions among clarithromycin, rifampin, and cyclosporine.

We describe a case of osteomyelitis due to Mycobacterium haemophilum in a cardiac transplant recipient and review the two other reported cases of M. haemophilum infection in cardiac transplant patients. Our patient had an excellent response to a prolonged course of therapy with clarithromycin and rifampin. We examine in detail the interactions between these two antibiotics and cyclosporine, including the apparently offsetting effects of clarithromycin/rifampin combination therapy on blood levels of cyclosporine.

Adult↗

PubMed1997

Evidence that resolution of rotavirus infection in mice is due to both CD4 and CD8 cell-dependent activities.

The effector functions responsible for resolution of shedding in mice orally inoculated with the murine rotavirus strain EDIM were identified in B-cell-deficient and normal BALB/c mice after monoclonal antibody (MAb) depletion of CD4 and CD8 cells. When depleted of CD8 cells, B-cell-deficient muMt mice resolved their infections more slowly than nondepleted animals, but CD4 cell depletion caused chronic, high-level shedding. This finding indicated that CD4 cell-dependent immunological effectors other than, or in addition to, CD8 cells played roles in rotavirus resolution in muMt mice in the absence of antibody. The roles of CD4 and CD8 cells in resolution of rotavirus shedding were further characterized in immunologically normal BALB/c mice. Depletion of CD4 cells before EDIM inoculation resulted in rapid resolution of most shedding, but chronic, low-level shedding continued for weeks. When the CD4 cell-depleted BALB/c mice were subsequently depleted of CD8 cells, shedding levels increased significantly (P < 0.001), indicating that CD8 cells were responsible for the rapid but incomplete suppression of rotavirus shedding. Further experimentation revealed that little rotavirus antibody was made in CD4 cell-depleted BALB/c mice, and only after CD4 cells were repopulated did antibody production increase and virus shedding fully resolve. Thus, resolution of rotavirus shedding in both muMt and BALB/c mice was associated with CD4 and CD8 cell effector activities.

Animals↗

PubMed1997

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.

Adult↗

PubMed1997

Detection of apoptosis in colorectal carcinoma by light microscopy and in situ end labelling.

OBJECTIVE: An in situ end labelling reaction (ISEL) was recently described for the detection of apoptotic cells in histologic sections. We compared the efficiency and reproducibility of this assay with those of routine light microscopy using hematoxylin and eosin (H&E) in the quantitation of apoptotic cells in colorectal cancer. STUDY DESIGN: Paraffin-embedded archival tissues from 15 colorectal carcinomas were used in the study. ISEL was performed using terminal deoxynucleotidyl transferase, biotinylated deoxyuridine triphosphate and streptavidin-alkaline phosphatase. RESULTS: Apoptotic indices determined by the two assays showed a strong positive correlation (Pearson coefficient 0.82), with the ISEL assay detecting twice the number of apoptotic cells as that seen with H&E staining. Both assays showed significant interobserver and intraobserver variation, while the ISEL assay also showed considerable interassay variability and was less cost-effective than H&E in the detection of apoptotic cells. CONCLUSION: In the light microscopic quantitation of apoptosis in colorectal tumors, the ISEL assay offered no greater reproducibility, and was less cost-effective, than routine H&E staining.

Apoptosis↗

PubMed1996

Apoptosis in v-myc transformation of myelomonocytic cells and its modulation by CSF-1.

c-myc is a proto-oncogene essential for cell growth. When activated, its expression can lead to uncontrolled cell proliferation, transformation and tumorigenesis. The cell line tEMmyc4 is a murine myelomonocytic cell line that was established following transformation by v-myc. It has a high level of v-myc expression and constitutively expresses endogenous CSF-1, the monocytic growth and viability factor. Under growth restricting conditions (high cell density serum deprivation, heat shock, or dexamethasone addition) cells of this line were found to undergo cell death through apoptosis. The induction of apoptosis by dexamethasone was associated with a decrease in constitutive CSF-1 expression without significant change in v-myc expression. Exogenous CSF-1 rescued these cells from dexamethasone induced-apoptosis. In vivo studies showed that tEMmyc4 cells were tumorigenic in syngeneic animals despite exhibiting some spontaneous apoptosis within the tumour mass. Co-administration of dexamethasone with the tumour cells significantly inhibited tumor development and the administration of dexamethasone to mice with established tumors resulted in tumor regression in all mice. This regression was associated with a high level of apoptosis and necrosis in the tumors. This study shows a correlation between the in vitro and in vivo induction of apoptosis and indicates that cancer cells bearing activated oncogenes may be more sensitive to apoptosis induction by chemotherapeutic agents.

Animals↗

PubMed1996

Retrieval of human antibodies from phage-display libraries using enzymatic cleavage.

A combinatorial human IgG1, kappa gene library of 2 x 10(7) clones was constructed from a pericolic lymph node using the phagemid vector pComb3H. Fabs with binding activity against tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH) were isolated from this library, and one such TT binding Fab was used to further evaluate a new phagemid vector for the display of recombinant antibody fragments (MCO1). This vector was designed to incorporate a cleavage site for the enzyme Genenase I, a myc peptide tag, and an amber codon between the heavy chain cloning site and the truncated M13 phage gene III. When MCO1 phage displaying an anti-TT Fab were bound to TT on a solid substrate, elution with Genenase I at concentrations of 5-10 micrograms/ml proved as effective as acid elution in releasing bound phage. Furthermore, enzymatic elution with Genenase I was comparable to acid elution in the enrichment of a TT binding Fab from the pericolic library subcloned into the vector MCO1. Importantly, the use of enzymatic or acid elutions resulted in the retrieval of different anti-TT Fabs from this same library. We conclude that panning of phage-displayed combinatorial antibody libraries can be successfully performed using enzymatic elution, and that this offers a useful alternative to currently available phage elution techniques.

Aged↗

PubMed1996

Characterisation and clinicopathological correlates of serum anti-p53 antibodies in breast and colon cancer.

The humoral immune response to p53 was determined in 54 individuals with colon or breast cancer and 50 healthy subjects, in an attempt to better understand the origin and significance of anti-p53 serum antibodies. The presence of anti-p53 antibodies in serum was determined by enzyme-linked immunosorbent assay using purified recombinant human p53, and results were validated by immunoprecipitation of radiolabelled p53. Immunohistochemical analysis of 28 tumours was performed to detect the accumulation of p53 protein. Antibodies against p53 were significantly more common in patients with colorectal (10 of 42) and breast (2 of 12) cancer than in healthy individuals (2 of 50). They were of both the IgM (7 of 11) and IgG (4 of 11) isotypes. There was no significant difference in prevalence of serum antibodies against p53 with respect to the p53 immunohistochemical status of the tumour or to other pathological features, including the presence of lymph node and distant metastases. These findings provide indirect evidence that, rather than arising as a result of a specific immune response, anti-p53 antibodies in individuals with cancer may represent elevated levels of naturally occurring polyreactive antibodies.

Adult↗

PubMed1996

Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses.

A large placebo-controlled efficacy trial of the rhesus tetravalent (RRV-TV) and serotype G1 monovalent (RRV-S1) rotavirus vaccines was conducted in 1991-1992 at 24 sites across the United States. Protection was 49% and 54% against all diarrhea but 80% and 69% against very severe gastroenteritis for the two vaccines, respectively. Post-vaccination neutralizing antibody titers to the G1 Wa strain, whose VP7 protein is nearly identical to that of the D strain of rotavirus contained in both vaccines, did not correlate with protection against subsequent illness with G1 strains. This result raised the possibility that in infants who developed post-vaccination neutralizing antibody to Wa, breakthrough (i.e., vaccine failure-the occurrence of rotavirus diarrhea after immunization) may have been due to infection by G1 strains that were sufficiently antigenically distinct from the vaccine strain to evade the neutralizing antibodies elicited by vaccination. To test this hypothesis, we initially compared post-vaccination neutralizing antibody titers of vaccinees against Wa and G1 breakthrough strains using sera from subjects who experienced breakthrough. Post-immunization neutralizing antibody titers to Wa elicited by vaccination were significantly (P < 0.001) greater than to the breakthrough strains subsequently obtained from these subjects. This difference did not, however, correlate with lack of protection since similar differences in titer to Wa and breakthrough strains were found using post-vaccination sera from vaccinees who either experienced asymptomatic rotavirus infections or no infections. To determine the genetic basis for these differences, we compared the VP7 gene sequences of Wa with vaccine strain D, 12 G1 breakthrough strains, and 3 G1 control strains isolated during the same trial from placebo recipients. All breakthrough strains were distinct from Wa and D in antigenically important regions throughout the VP7 protein, but these differences were conserved between breakthrough and placebo strains. Furthermore, a comparative analysis of the deduced amino sequences form VP7 genes of G1 rotaviruses from 12 countries indicated that four distinct lineages have evolved. All breakthrough and control strains from the U.S. vaccine trial were in a lineage different from strain D, the serotype G1 vaccine strain. Although the overall results do not support our original hypothesis that immune selection of antigenically distinct escape mutants led to vaccine breakthrough in subjects with a neutralization response to Wa, it cannot be excluded that breakthrough could be partially due to antigenic differences in the VP7 proteins of currently circulating G1 strains.

Amino Acid Sequence↗

PubMed1996

Isolation of a human rotavirus containing a bovine rotavirus VP4 gene that suppresses replication of other rotaviruses in coinfected cells.

Bovine-human reassortant strains containing ten human rotavirus gene segments and segment 4, encoding VP4, of a bovine rotavirus were isolated from the stool of an infected Bangladeshi infant during cell culture adaptation. Two plaque purified variants of this reassortant, one making very large (429-L4) and the other tiny (429-S4) plaques, were further analyzed. The electropherotypes of these variants were identical except for slight mobility differences in segment 4. The predicted sequence of amino acids (aa) 16-280 in VP4 proteins revealed four differences between variants even in this limited region, so no single difference could be linked to plaque size. The small plaque variant S4 was phenotypically unstable and mutated to a large plaque-former within a single cell culture passage. The predicted sequence of aa 16-280 of a large plaque variant derived from S4 revealed six changes, only one of which was common to that of the L4 strain, thus suggesting that multiple amino acid changes in VP4 may affect plaque size. Although the large plaque variant L4 grew faster and was released from cells more rapidly than S4, its replication and that of other rotaviruses tested (i.e. RRV, NCDV and Wa) was suppressed by S4 in coinfected cells. Using an RRV x S4 reassortant containing only RRV segment 4, it was established that suppression was linked to the S4 VP4 protein. This suppression could not be associated with inhibition of viral adsorption and, therefore, appeared to occur following internalization. Thus, a new property of the rotavirus VP4 protein has been identified in a bovine-human rotavirus reas-sortant.

Amino Acid Sequence↗

PubMed1996

Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis.

Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.

Antibodies, Monoclonal↗

PubMed1996

Concurrent oral poliovirus and rhesus-human reassortant rotavirus vaccination: effects on immune responses to both vaccines and on efficacy of rotavirus vaccines. The US Rotavirus Vaccine Efficacy Group.

Interference between oral poliovirus vaccine (OPV) and monovalent (RRV-S1) and tetravalent (RRV-TV) rhesus-human rotavirus vaccines was evaluated. Serum antibody responses to OPV and rotavirus vaccines and efficacy of rotavirus vaccines were compared among control and vaccine groups stratified by number of concurrent OPV and rotavirus vaccinations received. Neutralizing antibody titers to poliovirus type 1 tended to rise more steeply in placebo than RRV-TV recipients, but there were no significant differences in seroprevalence or in geometric mean titers (GMTs) of antibodies to types 1, 2, or 3 among groups. Concurrent OPV resulted in lower IgA GMTs to rotavirus in RRV-S1 but not RRV-TV recipients. Rotavirus gastroenteritis rates among rotavirus vaccines did not differ by number of concurrent OPV doses received, but the sample sizes were too small to rule out any effect. These results suggest OPV and rhesus-human rotavirus vaccines may be given at the same visit in the United States.

Administration, Oral↗

PubMed1996

Mechanisms of protection against rotavirus in humans and mice.

Immune responses following either natural or experimental rotavirus infection provide protection against subsequent rotavirus illnesses, and the mechanisms involved have been examined in humans and animals. In adult volunteers challenged with human rotaviruses, protection has been shown to correlate with serum and intestinal antibodies; however, titers of no specific antibody could be used reliably as a marker of protection, including neutralizing antibody to the challenge virus. Studies in children confirmed these general associations between antibody titers and protection, but the serotype specificity of antibody and its role in protection remained unclear. Studies in mice suggested antibody, CD8 cells, and a third, undetermined, factor as mediators of protection. Antibody appeared to be most important, both in resolution of infection and protection against subsequent infection, but its activity was not serotype specific. CD8 cells helped resolve rotavirus infection but were less important in protection against reinfection. The third factor remains to be identified.

Adult↗

PubMed1996

Comparative evaluation of reactogenicity and immunogenicity of two dosages of oral tetravalent rhesus rotavirus vaccine. US Rhesus Rotavirus Vaccine Study Group.

OBJECTIVE: To compare the safety and immunogenicity of two dosages of tetravalent rhesus rotavirus vaccine (RRV-TV) and the effect of age at dosing. METHODS: A total of 195 infants were stratified by age into 2 groups, 6 to 12 weeks and 16 to 24 weeks, and randomly assigned to receive a single dose of placebo or RRV-TV containing either 4 x 10(5) or 4 x 10(6) plaque-forming units (pfu). Symptoms were recorded for 5 days after vaccination. Anti-rotavirus IgA and neutralizing antibody to human rotavirus serotypes G1 to G4 and RRV were measured in serum obtained pre- and postvaccination. RESULTS: Rates of fever > 38 degrees C (9%), diarrhea (6%) and vomiting (8%) were similar in all groups. IgA (69% vs. 49%, P = 0.02) and RRV (85% vs. 66%, P = 0.004) seroconversion rates were significantly higher in the 4 x 10(6) pfu vaccine group as were antibody titers to RRV (440.2 vs. 263.7, P = 0.04). Older infants demonstrated significantly higher seroconversion rates and antibody titers for IgA (71% vs. 52%, P = 0.03; and 110.6 vs. 54.8, P = 0.004) and RRV (92% vs. 66%, P = 0.05 and 498.3 vs. 205.6, P = 0.01) at either dose level than did the younger infants. There were no significant differences in seroconversion rates or antibody titers to human rotavirus types G1 to G4 between the two vaccination groups. CONCLUSIONS: RRV-TV at a dose of 4 x 10(6) pfu can be safely administered to infants 6 to 24 weeks of age. A single dose of 4 x 10(6) pfu of RRV-TV was significantly more immunogenic than a single dose of 4 x 10(5) pfu but did not improve responses to the human serotypes. Older vaccine recipients demonstrated significantly higher IgA and neutralizing antibody seroconversion rates and antibody titers than younger infants independent of dosage.

Administration, Oral↗

PubMed1996

Serum rotavirus neutralizing-antibody titers compared by plaque reduction and enzyme-linked immunosorbent assay-based neutralization assays.

Comparisons in rotavirus neutralizing-antibody responses were made with sera collected from vaccinated infants. The methods were a plaque reduction assay and a new enzyme-linked immunosorbent assay-based neutralization assay. Agreement of 94% was found in detecting at least fourfold seroresponses, and correlation coefficients between titers obtained by the two methods showed excellent agreement, indicating that either could be used reliably.

Antibodies, Viral↗

PubMed1996

Biological properties of renal oncocytoma cells in culture.

This paper sought to determine biological properties of oncocytoma cells, both in vivo and in vitro, which may serve as useful diagnostic indicators. Cell lines were grown in short-term culture from two renal oncocytomas following enzymatic dissociation, characterised by immunohistochemistry and electron microscopy, and further studied for abnormal p53 or retinoblastoma genes. Cells in culture were shown to maintain the morphological attributes of the primary lesion, including the overproduction of mitochondria. Despite the absence of vimentin staining in the primary tumour, cells in culture were shown to express this intermediate filament. Immunohistochemistry for P53 protein demonstrated an overexpression in one of the tumours, suggesting that mutation had occurred. Restriction fragment length polymorphisms of the retinoblastoma and the p53 genes were not demonstrated. Mutation of the p53 gene does occur in oncocytomas. The maintenance of the phenotype of oncocytoma cells in culture suggests that in vitro studies may be useful in the identification of unique properties of the tumour.

Adenoma, Oxyphilic↗

PubMed1995

Effector functions of antibody and CD8+ cells in resolution of rotavirus infection and protection against reinfection in mice.

The importance of antibody and CD8+ cells in resolution of murine rotavirus (EDIM) infection and protection against reinfection was examined with two strains of B-cell-deficient mice. Following inoculation of one strain (JHD), rotavirus infection was resolved within days, but when later reinoculated with EDIM, these mice again shed rotavirus. Thus, effector mechanisms other than antibody resolved viral shedding in JHD mice but were insufficient to prevent reinfection. EDIM shedding in another B-cell-deficient mouse strain (microMT) diminished but was not fully resolved 93 days after the initial infection, thus demonstrating that antibody could also be important in resolution of rotavirus infection. When depleted of CD8+ cells by monoclonal antibody treatment before EDIM inoculation, JHD mice were unable to resolve shedding. Even though microMT mice did not fully resolve their initial infection, depletion of CD8+ cells 49 days after initial inoculation resulted in a burst of shedding. Thus, CD8+ cells were involved in resolution of the initial EDIM infection in both strains of B-cell-deficient mice. Finally, when microMT mice were depleted of CD8+ cells before the initial EDIM infection, gradual resolution of rotavirus shedding was still observed, suggesting a third effector mechanism was also involved in resolution of rotavirus infection in mice.

Animals↗

PubMed1995

Long-term production of rotavirus antibody and protection against reinfection following a single infection of neonatal mice with murine rotavirus.

It has been found that mice infected with murine rotavirus can be protected against subsequent murine rotavirus infection for up to 2 months. It was also reported that protection against rotavirus infection in adult mice correlated with serum and stool rotavirus IgA titers. The present study was conducted to determine the duration of rotavirus antibody production and protection against rotavirus infection in this mouse model and its possible correlation with rotavirus antibody titers. It was found that protection of mice against subsequent infection following a single oral immunization with the murine rotavirus strain EDIM was 100% effective for at least 14 months, most of the lifetime of a mouse. During this period, serum and stool rotavirus antibody titers which included serum IgA, IgG, and neutralizing antibody to EDIM, as well as stool IgA, remained elevated. Of particular note, stool rotavirus IgA titers gradually decreased to levels that were approximately 10% of their peak at 1 month after infection but did not decrease further, while serum rotavirus IgG titers continuously increased during the 14 months of the study. Serum rotavirus IgA titers varied from month to month but overall remained relatively constant throughout the 14-month period. Thus, both serum and stool rotavirus antibody was retained at substantial levels long after a single rotavirus immunization in the absence of reexposure, and mice remained protected against reinfection.

Animals↗

PubMed1995

Lack of correlation between serum rotavirus antibody titers and protection following vaccination with reassortant RRV vaccines. US Rotavirus Vaccine Efficacy Group.

In a large placebo-controlled efficacy trial of the rhesus tetravalent (RRV-TV) and serotype 1 monovalent (RRV-S1) rotavirus vaccines in multiple sites throughout the United States, protection against rotavirus disease over a 2-year period was found to be 57 and 40%, respectively (Bernstein et al., J. Am. Med. Assoc., 1995, 273, 1191-1196). Sera collected from a subset of subjects during this trial were used to determine possible correlations between rotavirus antibody responses after vaccination and protection. Between 82% (RRV-S1) and 92% (RRV-TV) of the vaccinees seroconverted by at least one of the six antibody assays performed (i.e. rotavirus IgA and neutralizing antibody to RRV and serotype 1-4 human rotaviruses). Rises in neutralizing antibody were due primarily to RRV. The seroconversion rate was only 18-22% to each of the four human rotavirus serotypes following RRV-TV vaccination and was only 43% to serotype 1 human rotavirus after RRV-S1 administration. Furthermore, no correlate of immunity against rotavirus infection or disease was identifiable based on seroconversion to any of the antibodies measured. Likewise, no consistent relationship was found between the titers of any of these six antibodies following vaccination and protection against rotavirus, thus suggesting that serum antibody titers will not be useful markers of protection with these reassortant RRV vaccines. In addition, vaccinated subjects did not develop higher titers of neutralizing antibody to human rotaviruses following a subsequent natural rotavirus illness, a further indication that only weak immune responses to human rotaviruses were stimulated by vaccination with the RRV reassortants.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Viral↗