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Biomedical subjects

T W Chang

Publications and source records attributed to T W Chang.

At least 73 records · Page 4Linked to original sources
PubMed1986

Determination of human T cell activity in response to allogeneic cells and mitogens. An immunochemical assay for gamma-interferon is more sensitive and specific than a proliferation assay.

T lymphocytes proliferate and secrete lymphokines in response to allogeneic cells, mitogens and other stimuli. Cell proliferation as measured by [3H]thymidine ([3H]Tdr) incorporation into DNA has been routinely used to determine T cell responses in research and clinical laboratories. We have compared the sensitivity of an immunoradiometric assay (IRMA) for human gamma-interferon (IFN-gamma) (Chang et al., 1984), with that of the conventional [3H]Tdr incorporation assay in the measurement of T cell responses to antigens and mitogens in culture. Peripheral blood mononuclear cells (PBMs) were incubated in the presence and absence of phytohemagglutinin (PHA) or mononuclear cells from another individual for various periods of time. The culture fluids were collected for determining IFN-gamma and the cells were assayed for [3H]Tdr incorporation. Results of measurements were expressed in terms of stimulation indices. Both IFN-gamma secretion and thymidine incorporation were measurable in mixed lymphocyte cultures after incubation for 3 days, and in PHA stimulated culture after 24 h of incubation. The stimulation indices reflecting increased gamma-interferon were found to be more pronounced and more consistent than those of [3H]Tdr incorporation.

Biological Assay↗
PubMed1986

Expression of the protein encoded by the 3' open reading frame of human T-cell lymphotropic virus type III in bacteria: demonstration of its immunoreactivity with human sera.

The genome of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the infectious agent etiologically associated with the acquired immunodeficiency syndrome, contains, in addition to the genes for the polymerase, core, and envelope proteins, several open reading frames. To investigate whether the 3' open reading frame (3' orf) located between the envelope gene and the 3' long terminal repeat is a gene expressed in vivo in infected individuals, we inserted a fragment of 3' orf in a prokaryotic expression vector. The protein product synthesized in bacteria was purified and allowed to react with sera from individuals infected with human T-cell lymphotropic virus type III as indicated by seropositivity for other viral proteins. Two-thirds of the sera, regardless of the clinical status of the individuals, reacted with the purified protein indicating that 3' orf is a viral gene the product of which is immunogenic in vivo. A polyclonal rabbit antiserum reacting against the 3' orf gene product was obtained by serial injection of rabbits with the purified bacterial protein. The antiserum recognized a 27-kDa protein in the human T-cell lymphotropic virus type III-infected lymphocytes.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Human T-cell lymphotropic virus type III (HTLV-III) core antigens: synthesis in Escherichia coli and immunoreactivity with human sera.

Fragments of human T-cell lymphotropic virus type III (HTLV-III) proviral DNA carrying the gene for the core antigen (gag) was cloned in the plasmid REV. Several of the recombinants direct high levels of synthesis of the antigens. One clone, pG1, produced a hybrid protein containing 13 amino acid residues of the carboxyl terminus of the 17 kD virion protein, the entire p24, the major core protein of HTLV-III, and 74 amino acid residues of the amino terminal of the 15 kD core ribonucleoprotein. A second clone, pG2, was similar to pG1 except that it contained no p17 sequences and was missing the amino-terminal 77 amino acid residues of the p24. A third clone, pG3, was similar to pG2, except that all but 56 amino acids of the carboxyl terminus of p24 were removed. All three proteins were found to be strongly immunoreactive with anti-HTLV-III antibodies present in sera from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC). In addition, pG1 and pG2, but not pG3, reacted with a monoclonal antibody (M26) specific for the p24 virion core protein. Whereas all three reacted with an anti-p15 monoclonal antibody, none of the clones reacted with an anti-p17 monoclonal antibody. These results provide direct evidence to support the predicted assignment of the coding region of the gag gene of HTLV-III. The product from pG2 was purified and was found to be potentially useful for the detection of anti-p24 antibodies in sera from patients with AIDS or ARC and from individuals at risk from AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Expression in Escherichia coli of open reading frame gene segments of HTLV-III.

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Clostridium difficile colitis mimicking acute peritonitis.

Five patients receiving penicillin V potassium or a cephalosporin antibiotic for 18 hours to 22 days developed fever, marked leukocytosis, and signs and symptoms that suggested right-lower-quadrant peritoneal irritation. All underwent emergency laparotomy, at which dilatation and inflammation of the ascending colon were found. Only one of the patients had profuse diarrhea, and two patients had no diarrhea prior to laparotomy. Postoperatively, Clostridium difficile colitis was diagnosed by stool toxin assay and was confirmed in one case by proctosigmoidoscopic biopsy results. Antibiotic-associated colitis must be considered in any patient who develops peritoneal signs while or after receiving antibiotics. Over a two-year period, the "acute abdomen" presentation accounted for 5.2% of all patients with C difficile colitis at our institutions. Early proctosigmoidoscopy or stool examination for C difficile or its toxin may avoid unnecessary laparotomy in such patients.

Acute Disease↗
PubMed1985

Monoclonal antibodies as structural probes for oligomeric human interferon-gamma.

Monoclonal antibodies (MAb) B1 and B3, specific for human interferon-gamma (IFN-gamma) failed to immunoprecipitate heat-inactivated human IFN-gamma in solution. However, both MAb retained some reactivity with denatured IFN-gamma immobilized on vinyl plates. The two MAb have been employed in a sensitive immunoradiometric assay (IRMA). In this IRMA one MAb was bound to polystyrene beads and used as immunoadsorbent. The second MAb, labeled with 125I, was used as the tracer to quantitate the amount of IFN-gamma bound to the immobilized MAb. Addition of unlabeled MAb B1 did not inhibit the binding of 125I-labeled MAb B3 (and vice versa), indicating that the two MAb react with two different and nonoverlapping epitopes. Yet, when the same MAb was used in IRMA as both immunoadsorbent and tracer, the amount of labeled MAb bound to a given concentration of natural or E. coli-derived recombinant human IFN-gamma was very similar as with two different MAb, indicating that a single IFN-gamma molecule must have two or more identical binding sites for each of the two MAb. These findings show that biologically active natural and recombinant human IFN-gamma exist in oligomeric form.

Antibodies, Monoclonal↗
PubMed1984

Antibodies to hepatitis B surface antigen potentiate the response of human T lymphocyte clones to the same antigen.

Human t-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.

Antibodies↗
PubMed1984

Antigen-induced production of lymphokines by human T cell clones specific for hepatitis B surface antigen.

The secretion of gamma-interferon (IFN-gamma), Interleukin-2 (IL-2), and B cell growth factor (BCGF) by human T cell clones specific for hepatitis B surface antigen (HBsAg) was examined. Antigenic stimulation by HBsAg but not by influenza A virus resulted in IFN-gamma and BCGF synthesis by the T cell clones. No detectable amounts of IL-2 were obtained in the supernatants of any of the HBsAg-specific T cell clones when cultured in the presence or absence of antigen. IFN-gamma and BCGF were produced, even when cell proliferation was inhibited, suggesting that the secretion of these T cell factors occurred regardless of cell proliferation. The significance of the various factors in the immune response against hepatitis B virus infection is discussed.

Clone Cells↗
PubMed1984

Vesiculobullous lesions caused by cytomegalovirus infection in an immunocompromised adult.

Skin involvement in cytomegalovirus (CMV) infection is very rare. Reported cutaneous manifestations are nonspecific and include petechial, purpuric, and morbilliform rashes. We describe an immunocompromised patient who developed a vesiculobullous eruption. A simple Tzanck preparation from the base of a blister revealed multinucleate giant cells suggestive of a viral infection. Subsequent viral cultures and electron microscopic studies confirmed the diagnosis of CMV infection. Consideration of this infection should be included in the differential diagnosis of multinucleate giant cells seen on Tzanck preparations of cutaneous vesicles.

Cytomegalovirus↗
PubMed1984

Use of monoclonal antibodies as sensitive and specific probes for biologically active human gamma-interferon.

Mouse monoclonal antibodies B1 and B3 are specific for natural and Escherichia coli-derived recombinant human gamma-interferon (IFN-gamma). The two antibodies recognize different epitopes of the IFN-gamma molecule and do not compete with each other's binding. We have used these two antibodies to construct a solid-phase, sandwich immunoradiometric assay for human IFN-gamma. Purified antibody B1 was coated on polystyrene beads (0.64 cm in diameter) and used as the solid-phase immunoadsorbent and antibody B3 was labeled with 125I and used as tracer. This assay can be completed in about 4 hr and is capable of detecting IFN-gamma levels in human serum or tissue culture fluids as low as 0.1 NIH reference unit/ml. Recombinant human IFN-gamma derived from E. coli was detectable at a concentration of 0.02 ng/ml. The assay appears to be specific for the biologically active forms of IFN-gamma, since after exposure to pH 2, 37 degrees C, or 56 degrees C, biological activity and reactivity in the immunoradiometric assay decreased in parallel. The immunoradiometric assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule.

Animals↗
PubMed1984

Regulation of T-cell function by antibodies: enhancement of the response of human T-cell clones to hepatitis B surface antigen by antigen-specific monoclonal antibodies.

Eight mouse monoclonal antibodies specific for hepatitis B surface antigen (HBsAg) were examined for their effects on the antigen-induced proliferative response and lymphokine production of human HBsAg-specific T-cell clones in vitro. While all specifically enhanced the T-cell proliferative response, antibodies of the IgG class were generally more effective than those of the IgM class. Both the divalent F(ab')2 and the monovalent Fab fragments of an IgG monoclonal antibody had no effects, indicating that the Fc portion of the antibody molecules was required. Since antigen-presenting cells bear surface receptors for the Fc of IgGs and fewer or none for that of IgMs, the above results also suggest that antibodies enhance the capture of antigens by antigen-presenting cells as a result of the binding of antigen-antibody complexes to the Fc receptors on these cells. In addition to potentiating the proliferation of the T-cell clones, antibodies also increased the antigen-induced production of interferon-gamma by these cells. The present in vitro studies suggest that antibodies may regulate immune responses and do so by enhancing antigen presentation and thus augmenting antigen-induced activation and clonal expansion of T cells.

Animals↗
PubMed1984

Hepatitis B virus-reactive human T lymphocyte clones: antigen specificity and helper function for antibody synthesis.

To study immunity to hepatitis B surface antigen (HBsAg) at the cellular level, lymphocytes were obtained from the peripheral blood of hepatitis B vaccine recipients and were examined for various immune responses to HBsAg in vitro. The peripheral blood mononuclear cells (PBM) from most of the vaccinees did not proliferate to a great extent to HBsAg in vitro. However, HBsAg-reactive lymphocyte lines and clones were obtained from some of these individuals if the PBM were stimulated in vitro with HBsAg and were maintained in the presence of T cell growth supplement. Most of the HBsAg-reactive T cell clones obtained were found to be antigen-specific and some of them provided help in the production of anti-HBsAg antibodies by a cell population enriched for HBsAg-binding cells. These results indicate that HBsAg-specific T and B cells exist in the circulation of hepatitis B vaccine recipients, although they are at limiting concentrations for the in vitro cell proliferation and antibody production assays.

Antibodies, Monoclonal↗
PubMed1984

A cDNA clone encoding a product of activated human T lymphocytes.

A cDNA cloning approach was used to study the regulation of gene expression in human T lymphocytes upon mitogen stimulation. Poly(A)+ mRNA was prepared from phytohemagglutinin A (PHA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA) activated human T cells and a cDNA clone library was constructed. After screening by colony hybridization with [32P]cDNA probes made from resting and activated T cell mRNA, several clones whose mRNA increased at least 10 to 20-fold upon stimulation were isolated. Northern blot analysis of the mRNA from various cell types using these cDNA clones as probes revealed that one of the cDNA clones, pNC5A, encoded a gene expressed only in PHA and TPA-stimulated human T lymphocytes and in a human neoplastic T cell line HUT102-SH9. Less than 20 copies of this mRNA species per cell was detected in resting human T lymphocytes, B lymphocytes and monocytes and in two other T cell lymphoma lines (CEM and MOLT4), two B lymphoblastoid cell lines (WIL2-729-HF2 and HFB-1), a myeloid cell line (HL60) and a human embryonic lung fibroblast cell line (MRC-5). Hybrid selection translation and sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis of the translated product indicated that a polypeptide of 30,000 to 32,000 Mr is encoded by this particular cDNA clone. Thus, this cDNA clone may define a novel gene that is expressed only in activated human T cells.

Base Sequence↗
PubMed1983

Binding of cells to matrixes of distinct antibodies coated on solid surface.

The present studies investigate the potential of simultaneous multiple determinations of specific cell surface antigens in one reaction incubation by employing orderly arranged antibody spots on a solid surface. Antibodies of distinct specificities were coated on very small areas in close proximity forming matrix-like arrays on glass cover slips. These antibody spots were found to be capable of serving as minute specific immunoadsorbents for cells bearing on their surface the antigens with which the antibodies reacted. Antibody spots of 1.0, 0.5 and 0.25 mm diameter could adsorb maximally about 17,000, 4500, and 1100 mononuclear cells. An area of 1 cm2 could be coated with 25, 100, or 400 of these spots, respectively. In matrixes that contained anti-Lyt 2.1 and anti-Lyt 2.2 antibody spots, AKR (Lyt 2.1+) thymocytes adhered only to anti-Lyt 2.1 spots, BALB/c (Lyt 2.2+) thymocytes only to anti-Lyt 2.2 spots, and thymocytes of (AKR X BALB/c) F1 to both spots. The potential of this method for determining allotypes of HLA antigens and for determining in a mixed cell population the proportions of subsets bearing specific differentiation antigens is discussed.

Animals↗