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Biomedical subjects

E C Wang

Publications and source records attributed to E C Wang.

At least 37 records · Page 2Linked to original sources
PubMed1997

Consequences of live poliovirus vaccine administration in chronic fatigue syndrome.

The effect of live oral polio virus vaccination on chronic fatigue syndrome (CFS) patients was examined in a double-blind study. CFS patients were allocated randomly to placebo (N = 7) or vaccine (N = 7) conditions. All controls subjects received the vaccine (9). Vaccine administration was not associated with clinical exacerbation of CFS. However, objective responses to the vaccine revealed differences between patients and controls: increased poliovirus isolation, earlier peak proliferative responses, lower T-cell subsets on certain days post vaccination and a trend for reduced gamma-interferon in the CFS-vaccine group. Polio vaccination was not found to be clinically contraindicated in CFS patients, however, there was evidence of altered immune reactivity and virus clearance.

Adult↗
PubMed1997

The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity.

The S1 binding site of trypsin is cross-linked by the conserved Cys191-Cys220 disulfide bond. The substitution of Cys191 and Cys220 with Ala decreases the activity of trypsin by 20-200-fold as measured by kcat/K(m) for the hydrolysis of amide substrates; in contrast, ester hydrolysis is decreased by < 10-fold. Similar decreases are observed in the hydrolysis of oligopeptide and single amino acid substrates. This decrease in activity results from a decrease in the acylation rate. The substrate binding and deacylation rate are not affected by the loss of the disulfide bond. C191A/C220A binds BPTI with the same affinity as trypsin, although the affinity of benzamidine is decreased 10-fold and the affinity of leupeptin is decreased 1000-fold. The CD spectrum of C191A/C220A displays significant differences from that of trypsin; these differences most likely result from the loss of the disulfide chromophore, although perturbation of enzyme structure cannot be discounted. The loss of the Cys191-Cys220 disulfide has no effect on the stability of trypsin as measured by urea denaturation. Single and double substitutions of Ser at positions 191 and 220 have a similar activity to C191A/C220A. These results indicate that the Cys191-Cys220 disulfide bond is not essential for the function, structure or stability of trypsin.

Acylation↗
PubMed1995

CD8highCD57+ T lymphocytes in normal, healthy individuals are oligoclonal and respond to human cytomegalovirus.

CD8+CD57+ lymphocytes from normal peripheral blood are divided into T cells expressing high levels of CD8 and NK cells expressing low levels of surface CD8. Increased numbers of CD8highCD57+ T cells correlate with previous exposure to human cytomegalovirus (HCMV) infection, but no virus-specific function or function for CD8highCD57+ cells has been recorded. We have studied the TCR repertoire and responses of the CD8highCD57+ population induced by virus-infected fibroblasts in healthy individuals. Using three-color flow cytometry of PBL, we detected restricted TCRBV usage in the CD8highCD57+ subset of 11/15 subjects, compared with 1/15 in the CD8+, CD57- subset. The results of anchored PCR and sequencing also showed oligoclonality of TCR; 40-70% of CD8highCD57+ lymphocytes (10-20% of total CD8+ T cells) were derived from single clones. Such expansions were stable with time and detected in one subject over a 2-yr period. Functionally, CD8highCD57+ lymphocytes proliferated strongly to HCMV-, but not HSV-, VZV-, or influenza-infected fibroblasts in an MHC-unrestricted manner in vitro, including preferential augmentation of particular in vivo oligoclonally expanded subpopulations. HCMV-specific MHC-restricted CTL were detected, but limiting dilution analysis showed that these were a minority (< 10%) and not the oligoclonal subsets. In contrast, depletion of oligoclonally expanded CD8highCD57+ subpopulations, resulted in the increase of HCMV-specific CTL, suggesting functional heterogeneity in these cells.

Adult↗
PubMed1995

Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment.

The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.

Alleles↗
PubMed1995

The role of CD8+, CD57+ cells in human cytomegalovirus and other viral infections.

Peripheral blood lymphocytes expressing CD8 and CD57 determinants are a small (1-15%) subset in healthy humans. CD8+, CD57+ peripheral blood lymphocytes may be divided by the level of CD8 expression, into CD8+high (CD57+) T-cells and CD8+low (CD57+) natural killer (NK) cells. CD8+high (CD57+) T-cell numbers are increased in human cytomegalovirus (HCMV)-seropositive subjects, and there is substantial evidence that HCMV is integral in the development of this subset in health and disease. Furthermore, the CD8+high (CD57+) subset is clonally derived, expressing a limited range of T-cell receptors, and are therefore likely to have restricted antigen specificity. Functionally, CD8+low(CD57+) cells exhibit NK activity, while CD8+high(CD57+) T-cells from healthy subjects mediate contact-dependent suppression in several in vitro systems including: (i) pokeweed mitogen-induced proliferation and immunoglobulin synthesis, and (ii) generation of antiviral MHC-restricted cytotoxic T-lymphocytes. This is distinct from the nonspecific, soluble factor-mediated suppression exhibited from a phenotypically similar subset in human immunodeficiency virus (HIV) and bone marrow transplant recipients. This suggests an important immunoregulatory, suppressive role for CD8+high(CD57+) T-cells that may be potentiated by HCMV and altered in diseases associated with higher numbers of this subset including HIV, allograft recipients and rheumatoid arthritis.

Biomarkers↗
PubMed1994

CD8high (CD57+) T cells in normal, healthy individuals specifically suppress the generation of cytotoxic T lymphocytes to Epstein-Barr virus-transformed B cell lines.

We have previously identified two subsets of CD8+, CD57+ lymphocytes in normal peripheral blood: i) T cells expressing high levels [CD8high(CD57+)] and ii) natural killer cells expressing low levels of surface CD8[CD8low(CD57+)]. We investigated the cytotoxic and suppressive function of CD8high(CD57+) T lymphocytes from normal, healthy individuals using standard chromium-release assays and limiting dilution analysis. In normal, healthy subjects, this cell subset suppressed the generation of cytotoxic T lymphocytes (CTL) to autologous, Epstein-Barr virus (EBV)-transformed B cell lines (BCL). Depletion of CD8high(CD57+) T lymphocytes from peripheral blood mononuclear cells (PBMC) resulted in a three- to sevenfold rise in CTL precursor frequency to autologous EBV-transformed BCL, but not allogeneic PBMC or BCL by LDA. Replacement of CD8high(CD57+) T lymphocytes in limiting dilution cultures led to the dose-dependent suppression of EBV-specific, but not allogeneic, CTL generation. Supernatant from CD8high(CD57+) T lymphocytes cultured with autologous BCL did not exhibit suppression, suggesting that soluble factors were not responsible. As CD8high(CD57+) T lymphocytes did not, themselves, exhibit cytotoxicity against autologous BCL, removal of BCL stimulator cells in co-culture was not the mechanism of suppression. Furthermore, while the CD8high(CD57+) T lymphocytes from healthy subjects suppressed the generation of CTL to autologous BCL, they did not suppress the cytotoxic activity of established mixed lymphocyte reactions or peptide-specific CTL clones, as has been reported in bone marrow transplant recipients and human immunodeficiency virus patients. This suggests that CD8high(CD57+) T lymphocytes from healthy subjects suppress the generation of, rather than killing by, CTL in a contact-dependent manner. To our knowledge, this is the first identification of a phenotypically distinct subset of human CD8+ T cells that can suppress generation of antigen-specific major histocompatibility complex class I-restricted CTL.

Adult↗
PubMed1994

Incidence of postoperative epilepsy in children following subfrontal craniotomy for tumor.

Thirty-one children who underwent 36 subfrontal craniotomies were retrospectively studied to determine the incidence of postoperative epilepsy and the effectiveness of antiepileptic drugs for seizure prophylaxis. The incidence of postoperative epilepsy following a subfrontal craniotomy did not exceed 12% when examined at various time periods during a 3-year postoperative course. Antiepileptic drugs were not warranted to reduce the incidence of postoperative seizures after the 1-month postoperative period and should not be used for long-term prophylactic therapy in children following a subfrontal craniotomy.

Anticonvulsants↗
PubMed1993

5-Benzylbarbituric acid derivatives, potent and specific inhibitors of uridine phosphorylase.

5-Benzylbarbituric acid derivatives were synthesized as a series of new, specific, and potent inhibitors of uridine phosphorylase. Among these, 5-(m-benzyloxy)benzyl-1-[(2-hydroxyethoxy)methyl] barbituric acid (5-benzyloxybenzylbarbituric acid acyclonucleoside, BBBA) was found to be the most potent with Ki values of 1.1 +/- 0.2 and 2.6 +/- 0.3 nM with uridine phosphorylase from human and mouse livers, respectively. BBBA exhibited competitive inhibition with uridine phosphorylase from both human and mouse livers. The 5-benzylbarbituric acid derivatives are specific inhibitors of uridine phosphorylase, as they had no effect on thymidine phosphorylase (EC 2.4.2.4), thymidine kinase (EC 2.7.1.21), uridine-cytidine kinase (EC 2.7.1.48), orotate phosphoribosyltransferase (EC 2.4.2.10), orotidine 5'-monophosphate decarboxylase (EC 4.1.2.23), and dihydrouracil dehydrogenase (EC 1.3.1.2). These compounds are more potent, easier to synthesize, and have better water solubility than their uracil counterparts as inhibitors of uridine phosphorylase. Furthermore, the 5-benzylbarbituric acids were found to be better inhibitors of human uridine phosphorylase than the murine enzyme, whereas the reverse holds true for the 5-benzyluracil derivatives. The 5-benzylbarbituric acid derivatives have potential usefulness in the therapy of cancer and AIDS, as well as other pathological and physiological disorders.

Animals↗
PubMed1993

Subsets of CD8+, CD57+ cells in normal, healthy individuals: correlations with human cytomegalovirus (HCMV) carrier status, phenotypic and functional analyses.

Two different subsets of CD8+, CD57+ cells have been defined, one expressing high levels (CD8high+(CD57+)), the other expressing low levels of surface CD8 (CD8low+(CD57+)). Increased numbers of CD8high+(CD57+) cells correlated with previous HCMV infection. By three-colour fluorescence analysis, the CD8high+(CD57+) population expressed T cell markers such as CD3 and CD5, and most were alpha beta T cell receptor (alpha beta TCR)-positive. A significant proportion also expressed CD71 (transferrin receptor) and MHC class I, although little if any CD25 (IL-2R-p55). Some (> or = 40%) co-expressed CD45RA and CD45RO. The CD8low+(CD57+) population expressed classical natural killer (NK) cell markers--CD2, CD16 and CD56. The two subsets were also functionally distinct; CD8high+(CD57+) cells suppressed pokeweed mitogen (PWM)-driven, but not phytohaemagglutinin (PHA)-driven proliferation and immunoglobulin production; CD8low+(CD57+) cells exhibited NK cytotoxic activity which was not increased by interferon-alpha (IFN-alpha). Supernatant from cultured CD8high+(CD57+) cells suppressed PWM-driven immunoglobulin production, but not proliferation, and this effect was abrogated by physical separation with tissue culture inserts. Thus, a T cell subset expressing activation and memory T cell markers with direct non-specific suppressor activity was present in peripheral blood mononuclear cells (PBMC) of healthy subjects with asymptomatic HCMV infection.

Antigens, CD↗
PubMed1993

Analysis of the human T-cell response to picornaviruses: identification of T-cell epitopes close to B-cell epitopes in poliovirus.

Little is known about the nature and specificity of T-cell-mediated responses to picornaviruses in humans. In this study, the nature of the T-cell response to seven picornaviruses, including polioviruses, coxsackieviruses B3 and B4, human rhinovirus 14, and encephalomyocarditis virus, was determined. Twenty-nine individuals responded to poliovirus type 3, coxsackievirus B3, and encephalomyocarditis virus by proliferation of T cells, and from such cultures, 130 virus-specific T-cell lines were established. T-cell lines generated in response to encephalomyocarditis virus were exclusively strain specific. However, the majority of T-cell lines established in response to viruses, other than encephalomyocarditis virus, were cross-reactive to each other. Their cross-reactivity was confirmed in 2 of the 30 picornavirus-specific clonally derived T-cell lines from two subjects, but the majority of these lines were serotype specific. T-cell epitopes adjacent to each of the B-cell antigenic sites in VP1 of poliovirus type 3 were identified. The response to the region adjacent to B-cell antigenic site 1 (residues 97 to 114) was dominant between individuals. The localization of this major CD4 T-cell epitope may permit the construction of chimeric viruses utilizing the natural picornavirus T-cell response to augment production of antibody specific for inserted sequences.

Amino Acid Sequence↗
PubMed1981

Deoxyribonucleic acid single-strand-specific endonucleases in human cells: partial purification of a salt-resistant endonuclease with an acidic isoelectric point.

A second endonuclease with DNA single-strand specificity has been purified from KB cells, a continuous line of hunan epithelial cells. In contrast to other mammalian enzymes that cleave single-stranded DNA, this enzyme has an acidic isoelectric point (6.5 +/- 0.2). Its pH optimum is 9.5, it requires Mg2+ of Mn2+ for activity, and it has a sedimentation coefficient of 3.2 S, based on sucrose gradient centrifugation. The enzyme specifically catalyzes the endonucleolytic cleavage of synthetic DNA homopolymers and denatured viral DNA but does not attack linear duplex viral DNA. The rate of hydrolysis of poly(dT) is approximately 8-fold greater than that observed with denatured DNA. The relative rates of hydrolysis of homopolymers by the endonuclease are poly(dA) greater than poly(dT) greater than poly(dC) greater than poly(dG). Unlike other DNA single-strand-specific endonucleases isolated from human cells, this endonuclease is relatively insensitive to inhibition by KCl.

Cell Line↗
PubMed1978

Purification and characterization of a DNA single strand specific endonculease from human cells.

An endonuclease with DNA single-strand specificity has been purified from KB cells. The enzyme has a pH optimum at 9.2, requires Mg2+ for activity, and is inhibited by mono- or divalent cations. Its sedimentation coefficient of 4.6 S is based on sucrose gradient sedimentation, and it has a molecular weight of 54 000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme specifically catalyzes the endonucleolytic cleavage of denatured DNA, yielding acid-soluble oligonucleotides which contain 5'-phosphoryl termini. The rate of hydrolysis of poly(dT) is approximately eightfold greater than that observed with denatured DNA, although the Km for both substrates is 1.74 X 10(-5) M. The relative rates of hydrolysis of homopolymers by the endonuclease are: poly(dG) greater than poly(dT) greater than poly(dA) greater than poly (dC). Purified enzyme preparations also hydrolyze poly(U), releasing acid-soluble products. This activity cosediments in sucrose gradients with the DNA endonuclease activity, suggesting that both activities are contained in the same enzyme molecule.

Cations, Divalent↗
PubMed1977

Mammalian endonuclease, DNase V. Purification and properties of enzyme of calf thymus.

An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.

Amino Acids↗