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Biomedical subjects

D C Wong

Publications and source records attributed to D C Wong.

At least 37 records · Page 2Linked to original sources
PubMed1995

Efficacy of an inactivated hepatitis A vaccine in pre- and postexposure conditions in marmosets.

A two-part challenge study was done in marmoset monkeys to confirm the efficacy of an inactivated hepatitis A vaccine. In part 1 (preexposure), 7 marmosets received a single low dose of vaccine (360 ELISA units [EL.U.]) and were challenged orally with wild type hepatitis A virus (HAV) either 1 or 6 months later. In part 2 (postexposure), 8 marmosets were challenged orally with HAV and then half each were inoculated with a single dose of 360 or 1440 EL.U. of vaccine 2 days later. The suboptimal immune response elicited by the low vaccine dose in the preexposure group was sufficient to induce complete protection against oral challenge with heterologous HAV in all marmosets that had responded serologically. In the postexposure group, the 360-EL.U. dose of vaccine resulted in partial protection against hepatitis A disease, whereas the 1440-EL.U. dose of vaccine elicited complete protection against disease and virus excretion.

Animals↗
PubMed1995

Infantile hepatic haemangioendothelioma.

Three cases of infantile hepatic haemangioendotheliomas are described. Two demonstrate the natural history of regression of this benign tumour with no associated complication. The last case demonstrates life-threatening complications, which included cardiac failure, thrombocytopenia, disseminated intravascular coagulation and bleeding. Imaging modalities with plain films, ultrasound, computed tomography and angiograms are presented. Pathological findings in two of the cases are also described.

Biopsy↗
PubMed1994

[Ca2+]i inhibition of Ca2+ release-activated Ca2+ influx underlies agonist- and thapsigargin-induced [Ca2+]i oscillations in salivary acinar cells.

Inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases by thapsigargin elicits [Ca2+]i oscillations in rat salivary gland (parotid) acinar cells which are similar to those activated by agonists but are nevertheless independent of inositol 1,4,5-trisphosphate (IP3) or IP3-sensitive Ca2+ stores (Foskett, J. K., Roifman, C. M., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). Neither bafilomycin alone or together with monensin or chloroquine inhibited thapsigargin-induced [Ca2+]i oscillations, ruling out the involvement of vacuolar-type proton pumps or organellar acidity in the mechanisms underlying them. Acute inhibition of plasma membrane Ca(2+)-ATPase by 1 mM La3+ inhibited the decline of [Ca2+]i during the falling phase of the oscillation. Acute inhibition of plasma membrane Ca2+ influx by removal of extracellular Ca2+, membrane depolarization, or inorganic channel blockers immediately abolished oscillations, even when applied during the [Ca2+]i rising phase of the cycle. Ca2+ influx rate oscillated during [Ca2+]i oscillations, varying 1.5-13-fold during a cycle. Modification of the rate of Ca2+ influx, by titrating the extent of depletion of IP3-sensitive stores or manipulating extracellular [Ca2+], indicated that oscillations depended on a high rate of Ca2+ influx. In thapsigargin- or carbachol-treated cells which did not exhibit a sustained [Ca2+]i rise or [Ca2+]i oscillations, inhibition of Ca2+ influx activated plasma membrane Ca2+ permeability. Thus, agonist- and thapsigargin-induced [Ca2+]i oscillations in parotid acinar cells appear to be generated by plasma membrane-based mechanisms which involve periodic inactivation by [Ca2+]i of the Ca2+ release-activated Ca2+ influx pathway.

Animals↗
PubMed1994

Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization.

Hepatitis C virus (HCV) is the most important etiologic agent of non-A, non-B hepatitis and is a major cause of chronic liver disease and hepatocellular carcinoma. Development of an effective vaccine would be the most practical method for prevention of the infection, but whether infection with HCV elicits protective immunity in the host is unclear. Neutralization of HCV in vitro was attempted with plasma of a chronically infected patient, and the residual infectivity was evaluated by inoculation of eight seronegative chimpanzees. The source of HCV was plasma obtained from a patient during the acute phase of posttransfusion non-A, non-B hepatitis, which had previously been titered for infectivity in chimpanzees. Neutralization was achieved with plasma obtained from the same patient 2 yr after the onset of primary infection but not with plasma obtained 11 yr later, although both plasmas contained antibodies against nonstructural and structural (including envelope) HCV proteins. Analysis of sequential viral isolates from the same patient revealed significant genetic divergence as early as 2 yr after infection. However, the HCV recovered from the patient 2 yr after the infection had a striking sequence similarity with the HCV recovered from one of the chimpanzees inoculated with the acute-phase virus, suggesting that the progenitor of the new strain was already present 2 yr earlier. This evidence, together with the different sequences of HCV recovered from the chimpanzees that received the same inoculum, confirms that HCV is present in vivo as a quasispecies. These results provide experimental evidence in vivo that HCV infection elicits a neutralizing antibody response in humans but suggest that such antibodies are isolate-specific. This result raises concerns for the development of a broadly reactive vaccine against HCV.

Animals↗
PubMed1994

After-hours radiology.

After-hours radiological procedures are essential but expensive in any major teaching hospital. Using the criteria that radiological procedures performed after-hours should have a reasonable probability of influencing or changing patient management acutely, a study of the justification of after-hours radiological procedures was performed at the Princess Alexandra Hospital. Computer print-outs were used to select the patients based on the dates and times of the procedures. Their charts were then reviewed. The study found that most after-hours radiological procedures were justified. However, it also identified certain procedures that need not be performed or at least could be reduced after-hours. The procedures were: (i) computed tomography (CT) head--epilepsy, meningitis; (ii) CT abdomen--trauma (intubated)--unable to examine abdomen; (iii) CT spine--obvious unstable fractures; (iv) Ultrasound abdomen--biliary colic (afebrile); and (v) Ultrasound kidneys--renal failure (afebrile).

Humans↗
PubMed1993

Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.

Actins↗
PubMed1993

Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen.

The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 10(3.5) chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B approximately 1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant of HBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.

Amino Acid Sequence↗
PubMed1992

Lack of protective immunity against reinfection with hepatitis C virus.

Some individuals infected with hepatitis C virus (HCV) experience multiple episodes of acute hepatitis. It is unclear whether these episodes are due to reinfection with HCV or to reactivation of the original virus infection. Markers of viral replication and host immunity were studied in five chimpanzees sequentially inoculated over a period of 3 years with different HCV strains of proven infectivity. Each rechallenge of a convalescent chimpanzee with the same or a different HCV strain resulted in the reappearance of viremia, which was due to infection with the subsequent challenge virus. The evidence indicates that HCV infection does not elicit protective immunity against reinfection with homologous or heterologous strains, which raises concerns for the development of effective vaccines against HCV.

Acute Disease↗
PubMed1992

Effect of pili-specific antibodies on the adherence of Haemophilus influenzae type b to human buccal cells.

Different strains of Haemophilus influenzae type b (Hib) produce antigenically distinct pili that mediate adherence to human buccal epithelial cells. This study determined the ability of antibodies specific for the LKP3 pili of Haemophilus influenzae type b to inhibit the adherence of Hib strain Eagan (p+). Antiserum was prepared by immunization of rabbits with pili purified from Hib strain Eagan (p+). The presence of pili-specific antibodies in the immune serum was shown by selective immunoprecipitation of pilin and by electron microscopy of cells labeled with immunogold. Immune serum, affinity-purified antibody, and Fab fragments from immune serum significantly inhibited (P less than .001) adherence of strain Eagan (p+) to human buccal epithelial cells whereas preincubation with preimmune rabbit serum or Fab fragments from preimmune serum had no significant effect on adherence. Dilution of the immune serum, affinity-purified antibodies, or Fab fragments from immune serum resulted in decreased inhibition of Hib adherence. These results indicate that antibodies specific for LKP3 pili can effectively inhibit adherence of Hib strain Eagan (p+) and probably other strains that express this pilus serotype.

Agglutination Tests↗
PubMed1992

The natural history of infection with hepatitis C virus (HCV) in chimpanzees: comparison of serologic responses measured with first- and second-generation assays and relationship to HCV viremia.

The sensitivity of first- and second-generation tests for antibody to hepatitis C virus (HCV) and the relationship among the patterns of antibody response and HCV viremia were examined in serial serum samples from 6 chimpanzees experimentally infected with HCV and followed less than or equal to 3 years. HCV infection was transient in 4 chimpanzees and became chronic in 2. All chimpanzees developed antibodies to HCV detectable by second-generation assays, while only 5 of the 6 became positive by first-generation assay. Second-generation were consistently more sensitive than first-generation assays for the early diagnosis of primary HCV infection. The pattern observed with second-generation assays was not influenced by the outcome of HCV infection, since antibodies remained persistently detectable throughout follow-up regardless of whether viremia was transient or persistent. In contrast, the first-generation antibody response was variable: It usually disappeared after loss of viremia, whereas its presence paralleled HCV viremia in chimpanzees with chronic infection.

Animals↗
PubMed1990

Early events in hepatitis C virus infection of chimpanzees.

The cytoplasmic antigen and ultrastructural changes we described previously for chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) or with hepatitis D virus have recently been shown to be indirect measures of viral replication and appear to represent a host response to the expression or action of interferon. The time of appearance of these changes in hepatocytes during HCV infection, when compared with similar changes in hepatitis D virus infection, suggests a very early replicative phase for HCV. To investigate the early events in HCV infection, we infected two chimpanzees with HCV and obtained blood and liver biopsy samples from them daily during the first 10 days of infection. The early stage of infection with regard to HCV replication, antigen expression, and ultrastructural changes was similar in both chimpanzees. When tested by cDNA/polymerase chain reaction, HCV sequences became detectable in the serum as early as 3 days after inoculation and remained positive through the peak of aminotransferase elevations. In one chimpanzee the peak of virus production appeared to be 7 weeks after inoculation, which was coincident with rising enzyme values. The cytoplasmic antigen, detected by immunofluorescence, and ultrastructural changes, detected by electron microscopy, became positive in hepatocytes 3 and 6 days, respectively, after HCV sequences were first detected in serum. Circulating anti-HCV appeared 13 weeks and 32 weeks after inoculation, respectively, in the chimpanzees. These data indicate a very early replicative phase for HCV and a potentially long period of infectivity before the appearance of anti-HCV.

Animals↗
PubMed1989

Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli.

To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.

Amidohydrolases↗
PubMed1989

Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli.

Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗
PubMed1982

Natural and experimental infection of woodchucks with woodchuck hepatitis virus, as measured by new, specific assays for woodchuck surface antigen and antibody.

Solid-phase radioimmunoassays for woodchuck hepatitis virus (WHV) surface antigen (WHsAg) and antibody to it (anti-WHs) were developed. The test for WHsAg could detect as little as 10 ng/ml. In both tests it was necessary to employ radiolabeled WHsAg instead of anti-WHs as the probe because the latter appeared to be labile to the conditions of labeling. The tests were used to characterize naturally acquired and experimental WHV infections of woodchucks. Forty-three of 72 wild-caught woodchucks had serological evidence of WHV infections; 16 of these resulted in chronic infection, and the remainder were self-limiting. All chronically infected animals were positive for WHsAg and DNA polymerase activity. During 3 years of observation, 11 of the 16 WHsAg-positive animals and 3 of the 27 anti-WHs-positive animals, but none of the 21 uninfected animals developed hepatocellular carcinoma. Seroconversion, possibly resulting from infection with WHV, was documented in a chimpanzee inoculated with WHV. An immune adherence hemagglutination test for WHsAg was also developed by using anti-WHs of chimpanzee origin as a reagent, but the test was not useful for detecting anti-WHs of woodchuck origin because of the lability of the latter.

Animals↗
PubMed1981

Serodiagnosis of viral hepatitis A by a solid-phase radioimmunoassay specific for IgM antibodies.

A solid-phase radioimmunoassay for detecting specific IgM antibodies to hepatitis A virus (HAV) was developed and characterized. The test utilized microtiter plates coated with anti-IgM to specifically absorb the IgM antibodies from the test serum. The anti-hepatitis A IgM antibodies are measured by the specific consecutive binding of hepatitis A antigen and radiolabelled anti-hepatitis A antibodies (anti-HA). In 6 chimpanzees infected with HAV, IgM anti-HA was detected from about the first date of elevated transaminases and was positive for about 3 months. The usefulness of the test was confirmed by testing acute phase sera of 30 patients from a common source outbreak of epidemic hepatitis, and negative sera from 2 control groups. A collection of serum specimens from 190 patients with sporadic HBsAg-negative hepatitis in Brazil was also tested and an etiologic association with HAV was confirmed in the majority of these cases.

Alanine Transaminase↗