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J T Stapleton

Publications and source records attributed to J T Stapleton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Recombinant hepatitis A virus antigen: improved production and utility in diagnostic immunoassays.

Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.

Animals↗
PubMed1998

Characterization of hepatitis G virus (GB-C virus) particles: evidence for a nucleocapsid and expression of sequences upstream of the E1 protein.

Hepatitis G virus (HGV or GB-C virus) is a newly described virus that is closely related to hepatitis C virus (HCV). Based on sequence analysis and by evaluation of translational initiation codon preferences utilized during in vitro translation, HGV appears to have a truncated or absent core protein at the amino terminus of the HGV polyprotein. Consequently, the biophysical properties of HGV may be very different from those of HCV. To characterize HGV particle types, we evaluated plasma from chronically infected individuals with and without concomitant HCV infection by using sucrose gradient centrifugation, isopycnic banding in cesium chloride, and saline density flotation centrifugation. Similar to HCV, HGV particles included an extremely-low-density virion particle (1.07 to 1.09 g/ml) and a nucleocapsid of approximately 1.18 g/ml. One major difference between the particle types was that HGV was consistently more stable in cesium chloride than HCV. Plasma samples from chronically HGV-infected individuals and controls were assessed by a synthetic peptide-based immunoassay to determine if they contained HGV antibody specific for a conserved region in the coding region upstream of the E1 protein. Chronically HGV-infected individuals contained antibody to the HGV core protein peptide, whereas no binding to a hepatitis A virus peptide control was observed. Competitive inhibition of binding to the HGV peptide confirmed the specificity of the assay. These data indicate that HGV has a nucleocapsid and that at least part of the putative core region of HGV is expressed in vivo.

Amino Acid Sequence↗
PubMed1998

Poor correlation between published methods to predict creatinine clearance and measured creatinine clearance in asymptomatic HIV infected individuals.

The purpose of this study was to evaluate the predictive ability of six published creatinine clearance (CCr) equations in healthy human immunodeficiency virus (HIV) infected individuals. A 24-h urine collection to determine CCr was done on an out-patient basis in 18 subjects. Predicted CCr was compared with the measured values, and the predictive performance was assessed with percentage mean error (bias) and percentage root mean error (precision). Mean +/- standard deviation measured CCr was 107 +/- 35 mL/min/1.73 M2. CCr determined using each of the published equations correlated poorly with measured values. CCr determined using Hull methods was significantly different from the measured values. Though Cockcroft and Gault and Jelliffe methods had the lowest mean bias and greater precision, a significant range of difference from measured CCr was observed (-12 to +28%). All methods over estimated the measured CCr in HIV-infected individuals. Until other approaches are developed, a 24 h urine collection may be the best approach for assessing renal function in HIV-infected individuals, especially in those receiving medications with narrow therapeutic indices that are cleared by the kidney.

Adult↗
PubMed1997

Distribution of hepatitis C virus (HCV) RNA in whole blood and blood cell fractions: plasma HCV RNA analysis underestimates circulating virus load.

Previous experiments using a cationic surfactant to detect hepatitis C virus (HCV) RNA in whole blood (WB) suggested that WB was a more plentiful source of viral RNA than was plasma. The relative HCV RNA titers in WB, plasma, peripheral blood mononuclear cells (PBMC), neutrophils, and red blood cells (RBC)/platelets from 10 patients with chronic HCV infection were compared. WB contained significantly more HCV RNA than plasma, which contained more HCV RNA than PBMC, neutrophils, or RBC/platelets (P < .001). To determine if this increased sensitivity was clinically relevant, results of WB and plasma HCV RNA assays were compared with commercial quantitative and qualitative plasma HCV RNA assay results obtained for patients receiving interferon therapy. WB was significantly more sensitive than commercial plasma reverse transcription-polymerase chain reaction for detecting HCV RNA (P < .005). These data indicate that a significant proportion of HCV RNA in peripheral blood is not identified by standard plasma RNA detection methods.

Blood Cells↗
PubMed1997

Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests.

Reverse transcription-polymerase chain reaction was used to identify hepatitis C virus (HCV) RNA in peripheral whole blood (WB) and plasma samples from 15 patients with chronic, unexplained hepatitis. These patients were serologically negative for hepatitis A, B, and C and were classified as having chronic non-A, non-B, non-C hepatitis (NANBNC). HCV RNA was repeatedly detected in WB samples from 10 (67%). In contrast, plasma samples from only 5 were intermittently positive. Statistically, HCV RNA detection in WB was significantly more sensitive than in plasma. Nucleic acid hybridization and HCV genotypic analysis confirmed the specificity of the HCV RNA assay. Liver biopsies from these patients suggested histopathologic differences between HCV RNA-positive and -negative groups. These data demonstrate that HCV infection is present in patients with unexplained chronic hepatitis more frequently than previously believed. Additionally, WB HCV RNA detection is more sensitive than plasma assays in identifying antibody-negative HCV infection.

Adult↗
PubMed1997

Effect of a single ethanol exposure on HIV replication in human lymphocytes.

BACKGROUND: Alcoholism is known to cause perturbations in cellular and humoral immunity, and some data suggest that acute alcohol ingestion enhances HIV replication in the lymphocytes of drinkers. METHODS: To study the acute effects of alcohol ingestion on HIV replication, oral ethanol (1 g/kg) was administered to 12 healthy volunteers in a controlled clinical setting. In vitro replication of HIV in the subjects' cultured lymphocytes and changes in lymphocyte phenotypes were evaluated. RESULTS: Statistically significant increases in peripheral lymphocytes and natural killer cell numbers were identified after the initial ethanol trial. HIV replication also increased in the isolated lymphocytes of some subjects after ethanol ingestion, but most subjects in the second trial showed essentially no changes in any of these parameters. CONCLUSIONS: Our results are consistent with either a subtle, study-induced stress-related enhancement in HIV replication or significant individual variation in response to ethanol. The results do not provide evidence for a general increase in HIV replication in the lymphocytes of subjects following a single in vivo ethanol dose of 1 g/kg.

Adult↗
PubMed1997

Ethanol and natural killer cells. II. Stimulation of human natural killer activity by ethanol in vitro.

A single ethanol ingestion of 1 g/kg by healthy individuals under controlled conditions does not inhibit and may stimulate fresh natural killer (NK) activity measured 16 hr later. However, ethanol inhibits fresh human NK activity when added to the lytic assay medium, as reported previously by other investigators. In contrast, using the same target (K562 erythroleukemia cells), peripheral blood mononuclear cells cultured 3 days with 50 units/ml of interleukin-2 are no longer inhibited significantly by the same concentration of ethanol that inhibited the fresh cells by 80%. When freshly isolated peripheral blood mononuclear cells, monocyte-depleted lymphocytes, or partially purified NK cells are pre-exposed to ethanol in vitro for 1 to 7 days, washed, and assayed for lytic activity against K562, the lytic activity is increased compared with nonethanol-exposed cells incubated concurrently. This increase is not dependent on accessory cells, added cytokines, or cell growth, and seems to be an intrinsic response of the NK subset to ethanol exposure. The finding of NK stimulation by ethanol, considered together with the observation of NK cell loss in some chronic alcoholics, suggests that loss of NK activity in the chronic alcoholic may result from cell loss rather than direct ethanol inhibition of NK activity.

Adult↗
PubMed1995

T cells and HIV-induced T cell syncytia exhibit the same motility cycle.

Ameboid cells ranging in complexity from Dictyostelium amebas to human polymorphonuclear leukocytes (PMNs) translocate in a cyclical fashion. Using computer-assisted motion analysis, we have analyzed the motility of human lymphocytes of the immortal SupT1 cell line and of a peripheral blood mononuclear cell population highly enriched for CD4-positive cells (CD4-enriched PBMCs) on four substrates--plastic, dehydrated rat tail collagen, hydrated rat tail collagen, and bovine aortic endothelium. In addition, we have analyzed the motility on these substrates of syncytia induced by human immunodeficiency virus (HIV) in cultures of both cell types. It is demonstrated that both SupT1 cells and CD4-enriched PBMCs exhibit a motility cycle with a period of 1.6 min that is independent of substrate, independent of average cell velocity, and similar to the periods of translocating Dictyostelium amebas and PMNs. More surprisingly, it is demonstrated that HIV-induced SupT1 and PBMC syncytia with volumes 10 to 100 times those of single cells exhibit the same motility cycle as their single-cell progenitors. These observations support the generality of the motility cycle in animal cells and, for the first time, demonstrate that the cycle is independent of cell size.

CD4 Antigens↗
PubMed1995

Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells.

Hepatitis C virus (HCV) requires reverse transcriptase-polymerase chain reaction (RT-PCR) or branched DNA signal amplification assays to be detected in patient samples. Although conventional methods of RNA isolation are employed for samples of serum, plasma, and peripheral blood mononuclear cells (PBMCs), whole blood is generally considered an unsuitable source of RNA because of abundant RNases and polymerase inhibitors. Using a cationic surfactant, Catrimox-14, we adapted a procedure for RNA isolation from whole blood, plasma, and PBMCs that yields RNA template suitable for HCV RT-PCR. RNA isolation required less than 2 hr, and HCV sequences were easily detected in sample volumes of 50 microliters whole blood or plasma, and in less than 1 x 10(4) PBMC. Following the addition of blood to Catrimox, HCV RNA was stable in the mixture when incubated for at least 7 days at room temperature prior to RNA extraction. Comparison of whole blood HCV RNA and plasma HCV RNA from individuals with chronic hepatitis suggests that HDV RNA can be more reliably detected in whole blood. Three of 15 HCV antibody positive patients (20%) had HCV RNA present in whole blood but simultaneously obtained plasma samples were negative. Two of the HCV antibody negative individuals with chronic hepatitis contained HCV RNA in whole blood, yet one of these patient's plasma was negative for viral RNA. The Catrimox-14 method of RNA purification is useful for detecting HCV RNA in whole blood and blood subfractions, and provides a practical method of measuring plasma and PBMC HCV RNA from clinical specimens.

Base Sequence↗
PubMed1995

HIV-induced syncytia in peripheral blood cell cultures crawl by extending giant pseudopods.

It was previously demonstrated that HIV-induced syncytia of the immortalized T cell line SupT1 reorganize their cytoskeleton and form a spherical supernuclear complex, thus mimicking the organization, polarity, and morphology of a single SupT1 cell. Then, through extension of a single, giant pseudopod, these syncytia, which grow to more than 100 times the volume of a single SupT1 cell, translocate along a substratum. To verify that syncytium motility is not peculiar to the SupT1 cell line, we have analyzed the cytoskeletal organization and motile capabilities of HIV-induced syncytia formed in peripheral blood cell cultures containing more than 90% CD4-positive cells. The results demonstrate that although peripheral blood T cells differ from SupT1 cells in size and morphology, they are continuously motile and translocate along a substratum in a manner quite similar to that of SupT1 cells, and peripheral blood T cell syncytia induced by HIV-1LAI as well as two additional clinical isolates translocate by the extension of a giant anterior pseudopod in a fashion indistinguishable from that of HIV-induced SupT1 syncytia. Together, these results support the generalization that HIV-induced T cell syncytia are motile.

Adult↗
PubMed1995

Host immune response to hepatitis A virus.

Hepatitis A virus (HAV) is transmitted by the fecal-oral route. The virus crosses through the gastrointestinal tract by an uncharacterized mechanism and travels to the liver, where it replicates in hepatocytes. It is released into the bloodstream and is simultaneously present in the bile and shed in the feces. Fecal shedding and viremia are maximal at the onset of liver function abnormalities and terminate about the time humoral immunity is detected, approximately 28 days after exposure. IgM, IgA, and IgG anti-HAV antibodies are usually present at onset of symptoms. Although the IgM response becomes undetectable usually within 6 months, IgG responses frequently persist for life, providing protection against reinfection. Pre- and postexposure immunization with pooled human serum immunoglobulin (ISG) is approximately 90% effective in preventing hepatitis A. Recipients of ISG have very low levels of detectable anti-HAV antibodies, and vaccines that elicit anti-HAV levels comparable with those produced by ISG should confer similar protection.

Hepatitis A↗
PubMed1993

Synthesis of immunogenic hepatitis A virus particles by recombinant baculoviruses.

Recombinant baculoviruses were constructed which contained the hepatitis A virus (HAV) open reading frame (ORF) under the control of the polyhedrin promoter. Northern blot analysis with an HAV-specific oligonucleotide probe demonstrated a single transcript large enough to include the HAV ORF in Spodoptera frugiperda cells infected with these recombinants. Immunoblots revealed a 220 kDa protein representing the HAV polyprotein. In addition, proteins which co-migrated with HAV capsid proteins, and several proteins of intermediate size were present, consistent with processing intermediates. HAV antigen was present in cells infected with the recombinant baculoviruses when assessed by solid-phase radioimmunoassay. This HAV antigen had a buoyant density in caesium chloride gradients similar to HAV empty capsids, and elicited HAV neutralizing antibodies in mice.

Animals↗
PubMed1993

Parvovirus B19 infection in human immunodeficiency virus type 1-infected persons failing or intolerant to zidovudine therapy.

To determine the incidence of B19 infection in patients with AIDS who were being treated with dideoxyinosine, serial sera (n = 28) taken over a 2-year period from 14 individuals were analyzed with respect to anti-B19 serology and the presence of B19 DNA. All 14 individuals were anti-B19 IgM negative. Nine of 14 had B19 viremia by Southern analysis of polymerase chain reaction product. Five of 9 with B19 viremia had > or = 1 anti-B19 IgG-positive sample; none of 5 without viremia had anti-B19 IgG. Four of 9 viremic individuals had serially positive samples. All 4 had severe anemia (hemoglobin < 8.5 g/dL) while taking zidovudine. A fifth individual whose severe anemia resolved after zidovudine was discontinued did not have B19 viremia. Therefore, a significant proportion of this group of patients with AIDS who developed severe anemia while receiving zidovudine had persistent B19 infection. These results suggest that B19 infection should be considered in anemic patients with AIDS.

AIDS-Related Opportunistic Infections↗
PubMed1993

Antigenic and immunogenic properties of recombinant hepatitis A virus 14S and 70S subviral particles.

Hepatitis A virus (HAV) has an immunodominant neutralization antigenic site. By using a panel of monoclonal antibodies targeted against the HAV neutralization antigenic site, it was shown that three epitopes within this site are present on 14S subunits (pentamers of the structural unit). In contrast, two other epitopes within this site are formed upon assembly of 14S subunits into capsids. Thus, the epitopes recognized by these two monoclonal antibodies are formed either by a conformational change in the antigenic site or by the juxtaposition of epitope fragments present on different 14S subunits during assembly of 14S into 70S particles. Both 14S and 70S particles elicited HAV-neutralizing antibodies in mice; thus, these particles may be useful for HAV vaccine development.

Antibodies, Monoclonal↗
PubMed1992

Passive immunization against hepatitis A.

Administration of human serum immune globulin (Ig) is an effective means of protecting individuals against hepatitis A virus (HAV) infection and disease. Several large field studies have demonstrated that if given before exposure, Ig will prevent infection with HAV. Furthermore, if Ig is given during the incubation period of hepatitis A, the severity of infection may be reduced and potentially clinical infections may be converted into subclinical ones. Although uncommon, infection which occurs in the presence of circulating antibody may occasionally lead to passive-active immunity. Unfortunately, the duration of Ig protection is dose dependent, and high dose administration provides less than six months protection. Ig preparations contain HAV antibodies at levels detectable by commercial immunoassays; however, recipients of Ig do not have detectable levels of HAV antibodies when tested by the same method. Using more sensitive immunoassays and neutralization assays, low titres of HAV antibody can be detected in Ig recipients. Since Ig provides approximately 90% efficacy in preventing hepatitis A, it would appear that very low levels of HAV antibody are needed to prevent infection. Consequently, measurement of HAV antibodies elicited by HAV vaccines should provide a reasonable method to evaluate their immunogenicity and predict their efficacy.

Hepatitis A↗
PubMed1992

Immunoglobulin prophylaxis for hepatitis A.

Studies conducted over the past 45 years have shown that immunoglobulin (IG) prevents 80%-90% of cases of hepatitis A when administered before exposure or shortly thereafter. Protection is short lived and requires early diagnosis and timely administration of IG to contacts. Inactivated and attenuated hepatitis A virus (HAV) vaccines have recently been developed and should be available for clinical use within the next few years. Evaluation of antibodies to HAV in IG and in IG recipients provides one method of determining the immunogenicity of HAV vaccines. The role of IG in the prevention of hepatitis A is reviewed with emphasis on the relationship of antibody response following IG administration to the efficacy of HAV vaccines.

Hepatitis A↗
PubMed1991

The role of secretory immunity in hepatitis A virus infection.

Because the role of intestinal immunity remains uncertain in hepatitis A, samples of feces and saliva from infected primates and humans were tested for virus neutralizing activity. Only two of eight owl monkeys infected by the intragastric route developed neutralizing antibody detectable in extracts of feces collected up to 88 days after viral challenge, although serum neutralizing antibody was present in all monkeys by day 33. Similarly, neutralizing antibody was detected in fecal extracts from none of three experimentally infected human volunteers and only 1 of 15 naturally infected humans. The single positive human specimen contained occult blood. Only 2 of 19 saliva samples from naturally infected humans had significant viral neutralizing activity. In contrast, neutralizing antibody to type 2 poliovirus was present in most human fecal or saliva specimens tested. These data suggest that intestinal immunity does not play a significant role in protection against hepatitis A.

Animals↗