Naruhito Otani

Interferon-γ release assay: A simple method for detection of varicella-zoster virus-specific cell-mediated immunity

Herpes zoster is closely related to decreased varicella-zoster virus (VZV)-specific cell-mediated immunity. We validated a new assay for measuring VZV-specific immunity. We cultured the whole blood of healthy subjects with live attenuated VZV vaccine. Cultured supernatants were harvested at 24-h intervals and assayed for interferon-gamma (IFN-gamma) by an enzyme-linked immunosorbent assay (ELISA). The 48-h culture was suitable for estimating IFN-gamma release. IFN-gamma production was stable after standing for at least 4h at room temperature. IFN-gamma production was observed in whole blood from subjects with recent VZV infection, but not in blood from subjects naïve to the virus. Thus, the IFN-gamma release assay may be useful as a new surrogate assay for measuring VZV-specific immunity.

Immune reconstitution to parvovirus B19 and resolution of anemia in a patient treated with highly active antiretroviral therapy

Immune reconstitution inflammatory syndrome (IRIS) is an unsolved problem in the treatment of human immunodeficiency virus (HIV)-1 infection. Despite the high seroprevalence of parvovirus B19 (PVB19) among HIV-1-positive patients, reports on PVB19-induced anemia, especially that associated with PVB19-related IRIS, in these patients are limited. We present the case of a man with acquired immunodeficiency syndrome who developed severe transfusion-dependent anemia and was seropositive and borderline positive for immunoglobulin-M and IgG antibodies against PVB19, respectively. PVB19-DNA was also detected in his serum. The patient was diagnosed with pure red cell anemia (PRCA) caused by a primary PVB19 infection and was treated with periodical blood transfusions. However, he subsequently tested negative for IgG antibodies and developed chronic severe anemia with high levels of PVB19 viremia. This indicated a transition from primary to persistent infection. After initiation of highly active antiretroviral therapy, the patient showed an inflammatory reaction with rapid deterioration of anemia and seroconversion of the IgG antibody to PVB19. Subsequently, PRCA was completely resolved, but the patient’s serum still contained low levels of PVB19-DNA. Thus, this was a case of IRIS associated with PVB19 infection. Our report highlights the significance of seroconversion to PVB19 in the diagnosis of IRIS and re-emphasizes the finding that persistently high levels of PVB19 viremia after primary infection are probably because of the lack of protective antibodies.

Varicella-zoster virus-specific cell-mediated immunity in subjects with herpes zoster

Though cell-mediated immunity (CMI) against varicella-zoster virus (VZV) is critical for prevention of the onset of herpes zoster (HZ), clinicians currently lack a simplified procedure to monitor CMI. We have recently developed an assay, called the IFN-γ release assay, and showed that it is a simple and reliable method to determine VZV-specific CMI. In the present study, we applied an IR assay to measure the VZV-specific CMI of patients with HZ. VZV-specific CMI levels were significantly high at the onset of the disease, but were decreased several weeks later. In contrast, CMI VZV-specific antibody titers increased in convalescent phase compared to those in acute phase. Thus, this technology is likely to be very useful in monitoring ongoing VZV-specific immune status.

Evaluation of VZV‐specific cell‐mediated immunity in adults infected with HIV‐1 by using a simple IFN‐γ release assay

The development of herpes zoster is associated with reduced varicella zoster virus (VZV)‐specific cell‐mediated immune (CMI) reactions. In this study, VZV‐specific CMI reactions in 42 anti‐VZV‐IgG antibody‐positive adults infected with HIV‐1 were evaluated by measuring the IFN‐γ production levels in whole blood in response to stimulation with ultraviolet light‐inactivated live attenuated VZV vaccine. The median VZV‐specific IFN‐γ production level in all patients was 63 pg/ml. Antiretroviral therapy (ART)‐naïve patients with an AIDS‐defining illness (HIV classification category C) had significantly lower IFN‐γ production than ART‐naïve patients in categories A and B and patients receiving ART (P = 0.0194 and P = 0.0046, respectively). IFN‐γ production increased significantly in patients within 1 month of the onset of recurrent VZV disease and at more than 1 year from onset, compared with patients who had never had recurrent VZV disease (P = 0.0396 and P = 0.0484, respectively). In multivariate analyses, category C and history of recurrent VZV disease were significant factors affecting IFN‐γ production. Levels of IFN‐γ were measured before and after ART in seven ART‐naïve patients with no history of recurrent VZV disease, and no significant changes were observed. The results indicate that VZV‐specific CMI reactions were reduced in patients with an AIDS‐defining illness and enhanced in patients with a history of recurrent VZV disease, but not enhanced by ART alone. Vaccination may be necessary to inhibit the development of herpes zoster in patients receiving ART; this IFN‐γ releasing assay is one useful method for evaluating VZV‐specific CMI reactions in clinical settings. J. Med. Virol. 85:1313–1320, 2013.

Human Herpesvirus 6 Infection of CD4+ T‐Cell Subsets

The immune system includes CD4+ regulatory T (Treg) cells that play a role in self‐tolerance and demonstrate functional variations that govern immune responses. HHV‐6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T‐cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV‐6 to specific T‐cell populations such as CD4+CD25high, which includes the majority of Treg cells, and CD4+CD25–. These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV‐6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real‐time PCR. The effects of virus infection on cytokine production from these T‐cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV‐6 replication. Virus infection enhanced the production of both Th1‐ and Th2‐type cytokines from CD4+CD25– T cells; however, only Th2‐type cytokine release was augmented from viral‐infected CD4+CD25high T cells. Further, while virus‐infected CD4+CD25high T cells shift their antiviral immunity toward Th2 dominance by producing IL‐10, the role of virus‐infected CD4+CD25– T cells remains obscure.

Evaluation of influenza vaccine-immunogenicity in cell-mediated immunity.

The immunological effect of influenza vaccines cannot be evaluated accurately using an antibody titer. Therefore, we used a new method that measures cell-mediated immunity to investigate changes in the amount of interferon-gamma (IFN-γ) produced after vaccination in response to the vaccine antigen. The study was conducted during the 2014-2015 influenza season in 23 adults, using a vaccine that contained three types of antigen. The IFN-γ level increased by at least 1.5 times in 65% (15/23) of cases in response to the H1N1 antigen, in 57% (13/23) of cases in response to the H3N2 antigen, and in 57% (13/23) of cases in response to the B antigen. During the study period, 4 subjects developed type A influenza. Our data showed that the IFN-γ level did not increase by 1.5 times in these subjects. We propose that the efficacy of influenza vaccines may be evaluated by measuring changes in the level of IFN-γ produced in response to influenza vaccine.

Storage conditions for stability of offline measurement of fractional exhaled nitric oxide after collection for epidemiologic research

BackgroundThe measurement of fractional concentration of nitric oxide in exhaled air (FeNO) is valuable for the assessment of airway inflammation. Offline measurement of FeNO has been used in some epidemiologic studies. However, the time course of the changes in FeNO after collection has not been fully clarified. In this study, the effects of storage conditions on the stability of FeNO measurement in exhaled air after collection for epidemiologic research were examined.MethodsExhaled air samples were collected from 48 healthy adults (mean age 43.4 ± 12.1 years) in Mylar bags. FeNO levels in the bags were measured immediately after collection. The bags were then stored at 4°C or room temperature to measure FeNO levels repeatedly for up to 168 hours.ResultsIn the bags stored at room temperature after collection, FeNO levels were stable for 9 hours, but increased starting at 24 hours. FeNO levels remained stable for a long time at 4°C, and they were 99.7% ± 7.7% and 101.3% ± 15.0% relative to the baseline values at 24 and 96 hours, respectively. When the samples were stored at 4°C, FeNO levels gradually decreased with time among the subjects with FeNO ≥ 51 ppb immediately after collection, although there were almost no changes among the other subjects. FeNO levels among current smokers increased even at 4°C, although the values among ex-smokers decreased gradually, and those among nonsmokers remained stable. The rate of increase was significantly higher among current smokers than among nonsmokers and ex-smokers from 9 hours after collection onwards.ConclusionsStorage at 4°C could prolong the stability of FeNO levels after collection. This result suggests that valid measurements can be performed within several days if the samples are stored at 4°C. However, the time course of the changes in FeNO levels differed in relation to initial FeNO values and cigarette smoking.

Development of a simplified and convenient assay for cell-mediated immunity to the mumps virus.

Because methods for measuring cell-mediated immunity (CMI) to the mumps virus are expensive, time-consuming, and technically demanding, the role of CMI in mumps virus infection remains unclear. To address this issue, we report here the development of a simplified method for measuring mumps virus-specific CMI that is suitable for use in diverse laboratory and clinical settings. A mumps vaccine was cultured with whole blood, and interferon (IFN)-γ released into the culture supernatant was measured using an enzyme-linked immunosorbent assay. IFN-γ production in blood from vaccinated subjects markedly increased in response to the vaccine and decreased before the antibody titer decreased in some cases, suggesting that this assay may be used as a simple surrogate method for measuring CMI specific for the mumps virus.

Varicella zoster virus antibody detection: A comparison of four commonly used techniques.

BACKGROUND Antibody tests for the varicella zoster virus (VZV) include neutralization, fluorescent antibody to membrane antigen (FAMA), immune adherence hemagglutination (IAHA), enzyme immunoassay (EIA), glycoprotein-based enzyme-linked immunosorbent assay (gpELISA), and complement fixation (CF) tests. Of these, FAMA is considered the most sensitive. However, in Japan, the EIA method is most frequently employed. OBJECTIVE The VZV antibody detection rate of the FAMA, EIA, gpELISA, and IAHA methods was compared. METHODS Four types of antibody tests were conducted with sera collected from 83 college students. The relationships between two antibody tests were examined using Pearsons correlation coefficients. RESULTS All 83 subjects were observed to be VZV antibody-positive using the FAMA method. The Pearson correlation coefficients of gpELISA, EIA, and IAHA relative to FAMA were 0.808, 0.782, and 0.356, respectively. The positive agreement rate of IAHA relative to FAMA was 88.0% (73/83), whereas those of gpELISA and EIA were both 97.6% (81/83). Furthermore, EIA showed 100% positive agreement with gpELISA and a high correlation coefficient of 0.911, whereas these values for IAHA compared to gpELISA were much lower (90.1% and 0.530). The calculated Pearson correlation coefficient for comparison of the EIA and IAHA methods was 0.498, with a positive agreement rate of 90.1% (73/81). CONCLUSIONS The EIA method should be employed in Japan based on the similarity of the positivity between EIA and gpELISA, as it is more available and practical than gpELISA.

Personal and Atmospheric Concentrations of Ozone in Southeastern Hyogo Prefecture, Japan

Twenty-one data sets composed of readings collected by atmospheric ozone monitors worn by individuals on their clothing and installed outside their home or office were collected using Ogawa passive ozone samplers in southeastern Hyogo prefecture, Japan from September 12 to 13, 2011. The concentrations of personal and outdoor ozone ranged from not detectable to 23.2 ppb and from 4.7 to 38.3 ppb, respectively. The mean concentration of personal exposure to ozone was 3.7 ppb and was significantly lower than that of outdoor ozone (18.5 ppb). This suggests that the concentrations of outdoor ozone affect personal ozone exposure. However, in this study, we found no correlation between the concentrations of personal ozone and the total time spent outdoors or the time of day the individual was outside. In contrast, the mean concentrations of outdoor ozone were similar to those of ozone measured at the 12 nearest Ambient Monitoring Stations (AMSs). However, when the AMS was situated near a main road, the regional ozone levels were underestimated.