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Biomedical subjects

D K Wong

Publications and source records attributed to D K Wong.

At least 19 recordsLinked to original sources

An in vivo probe based on mechanically strong but structurally small carbon electrodes with an appreciable surface area.

Physically small carbon electrodes were fabricated by pyrolyzing acetylene in a nitrogen atmosphere using pulled quartz capillaries as the supporting substrate. A carbon disk geometry was obtained when a parallel flow of acetylene (50 kPa) and nitrogen (10 mL min(-1)) was introduced into the system. Further, carbon was found to deposit at the tip and on the shank of the quartz capillaries when the nitrogen flow rate was increased (80 mL min(-1)), yielding an approximately cylindrical geometry. A series of electrochemical and spectroscopic analyses was carried out to examine the type of carbon surface obtained by pyrolysis of acetylene. The results suggested that a surface consisting of an almost defect-free highly oriented pyrolytic graphite type structure was formed by the pyrolyzed acetylene. However, this contradicts the kinetically reversible electron transfer observed for dopamine oxidation at these electrodes. Meanwhile, the nonpolar and relatively oxygen-free characteristics indicate that these electrodes also behave similarly to a hydrogenated carbon surface. The formation of a hydrogenated carbon-type surface may be plausible as a result of the attack on the carbon surface by a surplus of hydrogen produced by the pyrolysis of acetylene to form graphitic carbon. These characteristics are expected to aid in reducing electrode fouling, which is often encountered in electrochemical detection of neurotransmitters in vivo. In conjunction with a miniature physical dimension, their appreciable surface area and enhanced mechanical strength make these carbon electrodes well suited to the detection of neurotransmitters in vivo.

Acetylene↗

The plasmid-stabilizing ytl2 protein coats DNA in a sequence-independent manner.

Plasmids carrying the ytl2 gene from the large resident plasmid pSLT of Salmonella typhimurium were stabilized >10(5)-fold (compared to control ytl2-free plasmids) in S. typhimurium cells. Purified Ytl2 protein was localized in the cell cytosol and bound to DNA in a sequence-independent manner to form a high-molecular-weight complex, suggesting cooperative binding to the DNA. A mutant ytl2 gene, with a modified C-terminus, did not mediate plasmid stabilization and the mutant Ytl2 protein did not bind cooperatively to DNA. In vivo, while a plasmid carrying the ytl2 gene was stabilized, another plasmid (lacking ytl2) coexisting in the same cell was not. This result suggests that the Ytl2 protein, newly synthesized in a transcription-translation complex, binds preferentially to DNA of the replicon which encodes it and that this binding initiates subsequent cooperative DNA coating by more Ytl2 molecules. Plasmid-encoded newly synthesized Ytl2 protein is thus unavailable to stabilize coresident plasmid DNA, which does not contain the ytl2 gene.

Animals↗

Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV.

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.

Biopsy↗

Analysis of successful immune responses in persons infected with hepatitis C virus.

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-gamma, a "stunned" phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

Acute Disease↗

Spontaneous release of large packets of noradrenaline from sympathetic nerve terminals in rat mesenteric arteries in vitro.

Continuous amperometry was used to monitor noradrenaline (NA) release from sympathetic nerves supplying rat mesenteric arteries in vitro. During electrical stimulation the amplitude of oxidation currents evoked by successive stimuli varied over a small range, with occasional events of larger amplitude. In the absence of stimulation, spontaneous oxidation currents (s.o.cs) were recorded. The frequency of s.o.cs was increased by alpha-latrotoxin (1 nM). This agent also increased the frequency of spontaneous excitatory junction potentials (s.e.j.ps), which monitor the packeted release of adenosine 5' triphosphate (ATP). The frequency of s.o.cs recorded 20-25 min after applying alpha-latrotoxin was about four times the control value, but that of s.e.j.ps was about 30 times the control value. The findings suggest that continuous amperometry can detect the spontaneous packeted release of NA, probably from large dense-cored vesicles. In contrast, s.e.j.ps monitor spontaneous release of neurotransmitter (ATP) from a different store, most likely the small dense-cored vesicles.

Animals↗

Salmonella enterica serovar typhi uses type IVB pili to enter human intestinal epithelial cells.

DNA sequencing upstream of the Salmonella enterica serovar Typhi pilV and rci genes previously identified in the ca. 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139-146, 1997) identified a further 10 pil genes apparently forming a pil operon. The product of the pilS gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of serovar Typhi either lacking the whole pil operon or with an insertion mutation in the pilS gene were constructed, as was a strain in which the pilN to pilV genes were driven by the tac promoter. The pil(+) strains synthesized type IVB pili, as judged by (i) visualization in the electron microscope of thin pili in culture supernatants of one such strain and (ii) the presence of PilS protein (smaller than the prePilS protein by removal of the leader peptide) on immunoblotting of material pelleted by high-speed centrifugation of either the culture supernatant or sonicates of pil(+) strains. Control pil mutants did not express the PilS protein. A pilS mutant of serovar Typhi entered human intestinal INT407 cells in culture to levels only 5 to 25% of those of the wild-type strain, and serovar Typhi entry was strongly inhibited by soluble prePilS protein (50% inhibition of entry at 1.4 microM prePilS).

Amino Acid Sequence↗

An indirect perfluorosulfonated ionomer-coated electrochemical immunosensor for the detection of the protein human chorionic gonadotrophin.

The development of an amperometric immunosensor for the detection of human chorionic gonadotrophin (hCG) is described. In this immunosensor, Nafion was used to immobilize an anti-hCG monoclonal antibody onto a glassy carbon electrode. A systematic study on the effects of experimental parameters such as the quantity of ethanol present in the Nafion solution, the percentage composition of Nafion, the pH of the immobilization buffer, and the concentration of antibody used for entrapment experiments on the binding between the immobilized antibody and 125I-labeled hCG has been carried out. Two immobilization methods, coimmobilization and adsorption immobilization, have then been attempted. A binding of approximately 3% was obtained in the former method, while 5.5% binding was achieved in the latter. On the basis of these results, adsorption immobilization was employed to entrap antibody on the electrode surface. A sandwich assay was then developed for hCG in which the enzyme horseradish peroxidase was conjugated to a second anti-hCG monoclonal antibody. The activity of the enzyme was determined electrochemically by the reduction of benzoquinone to hydroquinone. Binding of hCG to immobilized antibody determines the quantity of enzyme-conjugated antibody at the electrode surface, permitting the quantification of hCG. By a standard additions calibration method of hCG performed in blank human serum samples, the immunosensor exhibits a limit of linearity at 200 mIU mL-1 and a detection limit of 11.2 mIU mL-1 (based on twice the standard deviation of the blank solution).

Adsorption↗

Long-term follow-up of a randomized trial of interferon therapy for chronic hepatitis B in a predominantly homosexual male population.

BACKGROUND/AIMS: Extended follow-up of a previously published therapeutic trial with interferon alfa is now available to further clarify the long-term outcome of HIV-negative and HIV-positive subjects with chronic hepatitis B virus infection after interferon alfa therapy. METHODS: Forty-five subjects with compensated liver disease and chronic hepatitis B infection with evidence of active hepatitis B replication were studied. These subjects were enrolled between 1986 and 1991 and had been randomized, stratified by HIV status, to either receive interferon therapy (10 MU/m2 of lymphoblastoid interferon alfa 3 times per week for 12 weeks) or no treatment. Hepatitis B serology, serum hepatitis B viral DNA and alanine aminotransferase were measured on an annual to biannual basis. CD4-positive T lymphocyte counts and HIV RNA concentration were also obtained. RESULTS: From 9 months post-interferon alfa treatment to the end of the extended follow-up (4 to 9 years), the relative risk of seroconverting to anti-HBe positive for subjects who had received interferon alfa therapy compared to those who did not was not significant in either HIV-negative (p = 0.80) or HIV-positive (p = 0.62) subjects. CONCLUSIONS: Unlike the first 9 months following interferon alfa therapy, the rate of elimination of markers of hepatitis B virus replication, regardless of HIV status, was not increased above the natural rate beyond 9 months following interferon alfa therapy.

Adult↗

Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry.

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 microM) and high-iron (70 microM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.

Aconitate Hydratase↗

Characterization of exochelins of the Mycobacterium bovis type strain and BCG substrains.

Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of Mycobacterium bovis, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of M. bovis, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The M. bovis type strain, five substrains of M. bovis BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent Mycobacterium tuberculosis all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in M. tuberculosis, intermediate in M. bovis, and smallest in M. bovis BCG.

Animals↗

Liver-derived CTL in hepatitis C virus infection: breadth and specificity of responses in a cohort of persons with chronic infection.

Hepatitis C virus (HCV)-specific CTL have been found within the inflammatory infiltrate of the liver of chronically infected individuals, but the breadth and specificity of the CTL response in relation to viral load are less well characterized. In this study, we analyzed the intrahepatic CTL response in liver biopsy specimens from 44 chronically infected subjects. Liver-infiltrating lymphocytes were expanded polyclonally in bulk cultures, and multiple clones were derived by limiting dilution. HCV-specific CTL responses directed at genotype 1a structural proteins were assessed in all subjects, and 22 subjects were tested more comprehensively using vectors expressing all structural and nonstructural HCV Ags. CTL responses were further characterized to determine the HLA restriction and optimal epitopes recognized. In those persons screened for recognition of all HCV Ags, HLA class I-restricted CTL were detected in 45%. Nineteen different CTL epitopes were identified, which were distributed throughout the genome; only one epitope was targeted by more than one person. In those persons with CTL responses, the breadth of response ranged from one to five epitopes. There was no correlation between the presence of a detectable CTL response and viral load. These results indicate considerable heterogeneity in detectable HCV-specific CTL responses in chronically infected persons. The mechanisms by which HCV persists during chronic infection remain to be clarified.

Cells, Cultured↗

Hepatitis C virus-specific cytolytic T lymphocyte and T helper cell responses in seronegative persons.

Hepatitis C virus (HCV) is a common infection worldwide, and in most persons, it leads to persistent viremia and liver damage. Efforts to identify the correlates of protective immunity are hampered by this high rate of persistent infection in both infected humans and the only animal model, the chimpanzee. Peripheral blood mononuclear cells from seronegative persons were stimulated with synthetic peptides that represent epitopes recognized by HCV-specific cytotoxic T lymphocytes (CTL) after natural infection. In addition, CD4+ proliferative responses to recombinant HCV proteins were examined in these same persons. CTL responses directed against a peptide epitope of HCV and proliferative responses in 2 HCV-seronegative persons with possible occupational exposure to HCV were found. These otherwise healthy persons were not viremic, suggesting that they may have recovered from acute HCV infection. Characterization of virus-specific immune responses in exposed but seronegative persons may provide important clues as to the nature of protective immunity in HCV.

Candida↗

Biliary tract cytology in specimens obtained by direct cholangiographic procedures: a study of 74 cases.

A retrospective review of bile (BL) and biliary tract brushings (Br) obtained by endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous transhepatic cholangiography (PTC) was undertaken to determine the sensitivity and specificity of cytology in the diagnosis of pancreaticobiliary malignancies. A total of 104 cytologic specimens (PTC-BL 15, PTC-Br 13, ERCP-BL 8, ERCP-Br 68) received between 1990 and mid-1994 from 77 patients who had undergone ERCP and/or PTC primarily for biliary stricture were reviewed. Specimens were unsatisfactory/ inadequate in 11 (10.6%), benign in 41 (39.4%), suspicious in 25 (24%), and positive for malignant cells in 27 (26%). Follow-up was available in 74/77 patients; 46 (59.7%) had tissue confirmation while 28 (32.5%) had adequate clinical follow-up based on chart review. Of those with histologic confirmation, there were 32 malignant and 14 benign cases. The overall sensitivity and specificity of PTC- and ERCP-obtained cytologic specimens were 88.9 and 95.7% respectively. There was only one false positive case (ERCP-Br). Overall positive predictive value was 96% negative predictive value 88%, and accuracy 96%. PTC had a significantly lower sensitivity rate (42.8%) and higher rate for unsatisfactory specimens (21%) compared with ERCP-obtained material (100 and 1.9%). Bile obtained by PTC or ERCP appeared less sensitive in detecting malignancies compared with endoscopic brushing using either technique (BL 50% vs. Br 100%). All three false negative cases were PTC-BL specimens. Of the 17 suspicious cases, eight were confirmed histologically as malignant, four were clinically consistent with malignancy, and five showed marked inflammatory atypia on biopsy. Positive predictive value and accuracy rate of a "suspicious cytology" diagnosis were 69 and 80.5%, respectively. Inadequate specimen, poor cellular preservation, and cells obscured by bile all interfere with proper cytologic evaluation. Experience is necessary to appreciate subtle malignant changes in well differentiated carcinomas. Communication between the cytopathologist and the clinician is critical in the accurate interpretation and proper management of the patients.

Adult↗

Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes.

Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above. The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes.

Amino Acid Sequence↗

Iron acquisition by Mycobacterium tuberculosis: isolation and characterization of a family of iron-binding exochelins.

Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores called exochelins. Exochelins are thought to transfer iron to another type of high-affinity iron-binding molecule in the bacterial cell wall, mycobactins, for subsequent utilization by the bacterium. In this paper, we describe the purification of exochelins of M. tuberculosis and their characterization by mass spectrometry. Exochelins comprise a family of molecules whose most abundant species range in mass from 744 to 800 Da in the neutral Fe(3+)-loaded state. The molecules form two 14-Da-increment series, one saturated and the other unsaturated, with the increments reflecting different numbers of CH2 groups on a side chain. These series further subdivide into serine- or threonine-containing species. The virulent M. tuberculosis Erdman strain and the avirulent M. tuberculosis H37Ra strain produce a similar set of exochelins. Based on a comparison of their tandem mass spectra, exochelins share a common core structure with mycobactins. However, exochelins are smaller than mycobactins due to a shorter alkyl side chain, and the side chain of exochelins terminates in a methyl ester. These differences render exochelins more polar than the lipophilic mycobactins and hence soluble in the aqueous extracellular milieu of the bacterium in which they bind iron in the host.

Amino Acid Sequence↗

Interferon alfa treatment of chronic hepatitis B: randomized trial in a predominantly homosexual male population.

BACKGROUND/AIMS: It has been suggested that human immunodeficiency virus (HIV) coinfection and male homosexuality predict poor response to interferon alfa therapy of chronic hepatitis B. The aim of this study was to examine the effect of HIV coinfection on the response of chronic hepatitis B virus (HBV) infection to interferon alfa therapy in a predominantly homosexual male population. METHODS: Fifty patients (82% male homosexuals, 50% HIV positive) with evidence of chronic HBV infection were randomized, stratified by HIV status, to undergo either treatment with interferon alfa (10 MU/m2 three times weekly for 12 weeks) or no treatment. Response was predefined as loss of serum HBV DNA, loss of hepatitis B e antigen, and the appearance of antibody to hepatitis B e antigen. HIV status and the interferon alfa-associated enzyme, 2',5'-oligoadenylate synthetase, were evaluated as potential predictors of response to therapy. RESULTS: Six treated patients responded with development of antibodies to hepatitis B e antigen (P < 0.05). HIV-positive patients were about one-fifth as likely to respond to interferon alfa therapy (relative risk, 0.22; 95% confidence interval, 0.03-1.78). Pretreatment alanine aminotransferase levels were significantly higher in responders than in nonresponders (P = 0.0005). Pretreatment 2',5'-oligoadenylate synthetase levels did not predict response. CONCLUSIONS: Interferon alfa, 10 MU/m2 three times weekly for 12 weeks, is effective in eradicating HBV replication in a predominantly homosexual male population not coinfected with HIV.

2',5'-Oligoadenylate Synthetase↗

Primary small bowel lymphoma manifested as obstructive jaundice in a patient with AIDS.

We describe a patient with acquired immunodeficiency syndrome (AIDS) and non-Hodgkin's lymphoma of the duodenum manifested as obstructive jaundice. Endoscopic retrograde cholangiography led to the diagnosis and was used to relieve the obstruction. The patient received chemotherapy and is in remission 21 months after the diagnosis, thus justifying an aggressive approach toward diagnosis and treatment in this patient population.

Acquired Immunodeficiency Syndrome↗