Shinichi Oka

Hydrogen peroxide activates IκB kinases through phosphorylation of serine residues in the activation loops

The cellular redox state regulates nuclear factor‐κB (NF‐κB) signaling systems. We investigated the effects of H2O2 on inhibitor of NF‐κB (IκB) kinases (IKKα and IKKβ), which phosphorylate IκB leading to its degradation and NF‐κB activation. Tumor necrosis factor (TNF) stimulation increased IKK activity within 10 min, and then IKK activity decreased gradually within 30 min in HeLa cells. Stimulation of the cells with H2O2 induced a slight activation of IKK within 30 min. Furthermore, co‐stimulation with TNF suppressed the downregulation of IKK and sustained the activation for more than 30 min. H2O2 also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H2O2 enhanced TNF‐induced NF‐κB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKα and Ser181 of IKKβ, in the activation loops was essential for the H2O2‐mediated activation of IKK. H2O2‐induced activation of IKKα and IKKβ was reduced by IKKβ and IKKα kinase‐negative mutants, respectively, indicating that IKKα and IKKβ were stimulated by H2O2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF‐κB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops.

N‐Acetylcysteine suppresses TNF‐induced NF‐κB activation through inhibition of IκB kinases

Here, we used a reductant, N‐acetyl‐L‐cysteine (NAC), to investigate the redox‐sensitive step(s) in the signalling pathway from the tumor necrosis factor (TNF) receptor to nuclear factor κB (NF‐κB). We found that NAC suppressed NF‐κB activation triggered by TNF or by overexpression of either the TNF receptor‐associated death domain protein, TNF receptor‐associated factor 2, NF‐κB‐inducing kinase (NIK), or IκB kinases (IKKα and IKKβ). NAC also suppressed the TNF‐induced activation of IKKα and IKKβ, phosphorylation and degradation of IκB, and nuclear translocation of NF‐κB. Furthermore, NAC suppressed the activation of IKKα and IKKβ triggered by the overexpression of NIK. These results indicate that IKKα and IKKβ are subject to redox regulation in the cells, and that NAC inhibits NF‐κB activation through the suppression of these kinases.

Quantitative analysis of human immunodeficiency virus type-1 DNA in asymptomatic carriers using the polymerase chain reaction

A method for detecting human immunodeficiency virus type 1 (HIV-1) provirus DNA in lymphocytes with improved sensitivity and reproducibility was developed using the polymerase chain reaction (PCR). Amplified HIV-1 DNA was hybridized with a 32P-labeled probe and quantitated with a beta-scanner after electrophoresis. A linear relationship was obtained between the common logarithms of the counts detected and the number of HIV-1 DNA copies applied to the PCR. Detectability was from 3 copies/10(5) lymphocytes, and linearity was maintained from 10 to 10(3) copies. HIV-1 DNA was detected in all 9 asymptomatic carriers tested (18 to 2,857 copies/10(5) CD4+ T lymphocytes). The viral burden was inversely related to the CD4+ lymphocyte count, suggesting that quantitation of provirus levels may serve as a predictor of progress in early HIV infection.

Risk and prognostic significance of tuberculosis in patients from The TREAT Asia HIV Observational Database

BackgroundTo assess the risk and the prognostic significance of tuberculosis (TB) diagnosis in patients from The TREAT Asia HIV Observational Database, a multi-centre prospective cohort of HIV-infected patients receiving HIV care in the Asia-Pacific region.MethodsThe risk of TB diagnosis after recruitment was assessed in patients with prospective follow-up. TB diagnosis was fitted as a time-dependent variable in assessing overall survival.ResultsAt baseline, 22% of patients were diagnosed with TB. TB incidence was 1.98 per 100 person-years during follow up, with predictors including younger age, lower recent CD4 count, duration of antiretroviral treatment, and living in high TB burden countries. Among 3279 patients during 6968 person-years, 142 died (2.04 per 100 person-years). Compared to patients with CDC category A or B illness only, mortality was marginally higher in patients with single Non-TB AIDS defining illness (ADI), or TB only (adjusted HR 1.35, p = 0.173) and highest in patients with multiple non-TB AIDS or both TB and other ADI (adjusted HR 2.21, p < 0.001).ConclusionThe risk of TB diagnosis was associated with increasing immunodeficiency and partly reduced by antiretroviral treatment. The prognosis of developing TB appeared to be similar to that following a diagnosis of other non-TB ADI.

TREAT Asia Quality Assessment Scheme (TAQAS) to standardize the outcome of HIV genotypic resistance testing in a group of Asian laboratories.

The TREAT Asia (Therapeutics, Research, Education, and AIDS Training in Asia) Network is building capacity for Human Immunodeficiency Virus Type-1 (HIV-1) drug resistance testing in the region. The objective of the TREAT Asia Quality Assessment Scheme - designated TAQAS - is to standardize HIV-1 genotypic resistance testing (HIV genotyping) among laboratories to permit rigorous comparison of results from different clinics and testing centres. TAQAS has evaluated three panels of HIV-1-positive plasma from clinical material or low-passage, culture supernatant for up to 10 Asian laboratories. Laboratory participants used their standard protocols to perform HIV genotyping. Assessment was in comparison to a target genotype derived from all participants and the reference laboratorys result. Agreement between most participants at the edited nucleotide sequence level was high (>98%). Most participants performed to the reference laboratory standard in detection of drug resistance mutations (DRMs). However, there was variation in the detection of nucleotide mixtures (0-83%) and a significant correlation with the detection of DRMs (p<0.01). Interpretation of antiretroviral resistance showed approximately 70% agreement among participants when different interpretation systems were used but >90% agreement with a common interpretation system, within the Stanford University Drug Resistance Database. Using the principles of external quality assessment and a reference laboratory, TAQAS has demonstrated high quality HIV genotyping results from Asian laboratories.

Cycloprodigiosin hydrocloride suppresses tumor necrosis factor (TNF) α-induced transcriptional activation by NF-κB

Cycloprodigiosin hydrochloride (cPrG·HCl) obtained from a marine bacterium Pseudoalteromonas denitrificans induces apoptotic cell death in various cancerous cell lines. cPrG·HCl alone caused a little cytotoxicity in HeLa cells, but it enhanced the apoptotic process progressively when co‐administered with tumor necrosis factor (TNF)α. Here we studied the effect of cPrG·HCl on TNFα‐induced activation of the transcription factor nuclear factor κB (NF‐κB). Luciferase gene reporter assays revealed that cPrG·HCl potently suppressed the TNFα‐ and the phorbol myristate acetate‐induced activation of NF‐κB. The suppression occurred in the presence of imidazole, indicating that it was not related to the intracellular acidification resulting from the intrinsic H+/Cl− symporter activity of cPrG·HCl. cPrG·HCl inhibited neither the TNFα‐induced phosphorylation and degradation of inhibitor of nuclear factor‐κB, nor the subsequent nuclear translocation and DNA binding of NF‐κB. cPrG·HCl also suppressed NF‐κB‐enhanced gene expression induced by Rac1, Cdc42, MEKK1, inhibitor of nuclear factor‐κα (IKKα), IKKβ, and a subunit of NF‐κB, p65. These results indicate that cPrG·HCl suppresses NF‐κB‐dependent gene expression through the inhibition of transcriptional activation.

Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells

Long‐term, persistent infection by HIV‐1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV‐1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV‐1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi‐quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV‐1‐seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus‐infected PBMC. These data are consistent with a mechanism of HIV‐1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.

Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay

Following HIV infection, there is a window period of 6-8 weeks, during which HIV antibodies are not detectable and the infection cannot be diagnosed by methods for detecting HIV antibodies. However, HIV antigens are detectable in the latter part of the window period, although the level of HIV antigens declines as the level of HIV antibodies increases. We developed an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for the simultaneous detection of both p24 antigen of HIV-1 and antibody IgGs to p17 and reverse transcriptase of HIV-1 in a single assay tube and tested 11 HIV-1 seroconversion serum panels and serum samples randomly collected from 79 HIV-1 seropositive subjects and 100 HIV-1 seronegative subjects. The simultaneous detection was shown not only to shorten the window period significantly as compared with conventional methods for HIV-1 antibody detection but also to make possible a reliable diagnosis of HIV-1 infection from the time of seroconversion until late stages of the infection.

WHOLE SALIVA DRIED ON FILTER PAPER FOR DIAGNOSIS OF HIV-1 INFECTION BY DETECTION OF ANTIBODY IGG TO HIV-1 WITH ULTRASENSITIVE ENZYME IMMUNOASSAY USING RECOMBINANT REVERSE TRANSCRIPTASE AS ANTIGEN

Whole saliva samples collected from HIV‐1 seropositive subjects by simple spitting without using any devices were dried on filter paper strips, from which filter paper discs of 3‐mm diameter were punched out. The eluates of the discs were subjected to the immune complex transfer enzyme immunoassay for antibody IgG to HIV‐1 using recombinant reverse transcriptase of HIV‐1 as antigen and a two‐site enzyme immunoassay for whole IgG. The signals for antibody IgG to HIV‐1 and the amounts of whole IgG obtained with one disc per assay tube were 126–290% of those obtained with 1 μl of whole saliva samples, provided that filter paper strips were treated with nonspecific rabbit serum prior to drying whole saliva samples and that filter paper discs were tested within a few days after drying whole saliva samples. From these results, diagnosis of HIV‐1 infection was indicated to be possible with whole saliva samples dried on filter papers, since the diagnosis was previously shown to be possible with 1 μl of whole saliva samples. The test for HIV‐1 infection with whole saliva samples dried on filter papers was suggested to be useful for various purposes.

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

ABSTRACT For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.