Shigeki Nabeshima

Selective CD27+ (memory) B cell reduction and characteristic B cell alteration in drug-naive and HAART-treated HIV type 1-infected patients

To investigate HIV-1-related B cell disorders, the quantity of CD27 positive (CD27+) B cells and their CD38, CD95, and bcl-2 intensities were examined by flow cytometry analysis in 16 drug-naive patients, 27 highly active antiretroviral therapy (HAART)-treated patients, and 20 uninfected controls. CD27+ B cells have been recognized as memory B cells. The mean percentage of CD27+ B cells was significantly lower in drug-naive patients (11.9%) and in HAART-treated patients (16.1%) than in controls (31.4%) (p < 0.01). The intensities of CD38 and CD95 on CD27+ B cells were higher in drug-naive patients than in controls (p < 0.01 in CD95). The intensity of CD95 on CD27+ B cells in HAART-treated patients was lower than that of drug-naive patients, but significantly higher than that of controls (p < 0.01). The intensity of bcl-2 on CD27+ B cells was equivalent among the three groups. These findings suggest that disturbance of peripheral B cell composition, exemplified by CD27+ B cell reduction, exists in both drug-naive and HAART-treated HIV-1-infected patients. In addition, the augmented apoptotic state of CD27+ B cells found in HAART-treated patients with undetectable viral loads, indicated by CD95 elevation, suggests that some HIV-1-related B cell disorders last for years after effective antiviral therapy.

Increased frequency of CD27- (naive) B cells and their phenotypic alteration in HIV type 1-infected patients

To investigate HIV-1-related B cell disorders, the quantity of peripheral CD27 negative (CD27-) B cells, their CD38, CD95, and bcl-2 intensities, and their apoptosis susceptibility were examined by flow cytometry analysis in 16 drug-naive patients, 27 HAART-treated patients, and 20 uninfected controls. CD27- B cells have been recognized as naive B cells. The mean percentage of CD27- B cells was significantly higher in drugnaive patients (88.1%) and in HAART-treated patients (83.9%) than in controls (68.6%) (p < 0.01). The intensities of CD38 and CD95 on CD27- B cells were significantly higher in drug-naive patients than in controls (p < 0.01). The intensity of CD95 on CD27- B cells in HAART-treated patients was lower than that of drug-naive patients, but significantly higher than that of controls (p < 0.01). The intensity of bcl-2 on CD27- B cells in drug-naive patients was lower than that of controls. In drug-naive patients, CD27-B cells with high CD38 expression represented low bcl-2 expression. The CD27- B cells of drug-naive patients showed an increased susceptibility to apoptosis, characterized by diminished cell size and a high frequency of annexin-V binding, compared with controls and HAART-treated patients. These findings suggested that HIV-1 infection affects peripheral CD27- (naive) B cells as well as CD27+ (memory) B cells and that CD27- B cells might be activated and rendered highly susceptible to apoptosis by HIV-1 infection. Some phenotypic alterations in CD27- B cells may continue after the reduction of HIV-1 loads by effective antiviral therapy.

Increased frequency of interferon-γ-producing peripheral blood CD4+ T cells in chronic hepatitis C virus infection

Increased frequency of interferon-γ-producing peripheral blood CD4 + T cells in chronic hepatitis C virus infection

The role of γδ T cells in induction of bacterial antigen‐specific protective CD8+ cytotoxic T cells in immune response against the intracellular bacteria Listeria monocytogenes

The role of T‐cell receptor (TCR) γδ T cells in the induction of protective TCR αβ T cells against infection by the intracellular bacteria Listeria monocytogenes was analysed. We found that depletion of γδ T cells by anti‐TCR δ monoclonal antibody treatment before intravenous immunization of mice with a sublethal dose of viable L. monocytogenes resulted in reduction of protection against secondary challenge infection in the immunized mice. The γδ T‐cell depletion also reduced induction of protective αβ T cells capable of transferring the protection against challenge infection of L. monocytogenes into naive mice. Furthermore, the protective T cells that were affected by the γδ T‐cell depletion were suggested to be CD8+ cytotoxic T cells rather than CD4+ T cells by the following observations. First, induction of cytotoxic T lymphocytes specific to a L. monocytogenes‐derived H‐2Kd‐restricted peptide (listeriolysin O 91–99) was significantly suppressed by γδ T‐cell depletion before immunization. Second, γδ T‐cell depletion did not affect cytokine production and proliferation of T cells from immunized mice in response to in vitro stimulation with heat‐killed Listeria which preferentially stimulates CD4+ T cells. Third, CD8+αβ T cells from control immunized mice transferred protection against infection of L. monocytogenes into naive mice but only a limited degree of protection was transferred by CD8+ T cells from the γδ T‐cell‐depleted immunized mice; and fourth, CD4+αβ T cells from the γδ T‐cell‐depleted mice transferred a similar level of protection as those from the control immunized mice. All these results suggest that γδ T cells participate in establishment of protective immunity against intracellular bacteria by supporting priming of bacterial antigen‐specific CD8+ cytotoxic T cells.

A reduction in the number of peripheral CD28+CD8+T cells in the acute phase of influenza

Influenza patients show a high incidence of T lymphocytopenia in the acute phase of the illness. Since CD8+ T cells play an important role in influenza virus infection, we investigated which subset of CD8+ T cells was involved in this lymphocytopenia. CD8+ T cells from eight patients with influenza A were studied for lymphocyte count, surface marker, and intracellular IFN‐γ production in the acute (days 1–3) and recovery phases (days 9–12). Total and T lymphocyte counts in the acute phase were approximately three times less than in the recovery phase; however, the CD4/8 ratio was the same in both phases. The cell count reduction in the acute phase was attributed predominantly to the CD28+ CD8+ subset, compared with the CD28− CD8+ subset. The memory/activation marker CD45RO on the CD8+ T cells was assessed. The CD28+ CD45RO− subset, a naive phenotype, was reduced significantly in number in the acute phase compared with the recovery phase. The CD28+ CD45RO+ subset, a memory phenotype, was also reduced in the acute phase, but the reduction was not statistically significant. Intracellular IFN‐γ in the CD8+ subset after mitogenic stimulation was measured by flow cytometry; the percentage of CD28+ IFN‐γ−/CD8+ subset in the acute phase was significantly less than in the recovery phase. These results indicated that the predominant reduction of peripheral CD8+ T cells in the acute phase of influenza was from naive‐type lymphocytes, suggesting that these quantitative and qualitative changes of CD8+ T cells in influenza are important for understanding the immunological pathogenesis.

Increased frequency of interferon-gamma-producing peripheral blood CD4+ T cells in chronic hepatitis C virus infection.

OBJECTIVES:To determine the profile of cytokine secretion by CD4+ T helper (Th) cells in chronic hepatitis C virus (HCV) infection, we used flow cytometry to determine the percentage of interferon (IFN)-γ and interleukin (IL)-4 producing cells from CD4+ T lymphocytes in peripheral blood obtained from patients chronically infected with HCV.METHODS:Peripheral blood mononuclear cells isolated from 89 HCV infected subjects (22 asymptomatic carriers, 56 patients with chronic hepatitis, and 11 patients with liver cirrhosis) and 24 healthy controls were stained with surface CD4 and intracellular IFN-γ and IL-4. Serum soluble IL-2 receptor (sIL-2R) levels were analyzed by ELISA.RESULTS:The frequency of IFN-γ producing CD4+ cells in asymptomatic HCV carriers, patients with chronic hepatitis, and patients with liver cirrhosis were significantly higher than those of healthy controls (p < 0.01, respectively). In contrast, the percentages of IL-4-producing CD4+ cells were very low, and there were no significant correlations with disease progression. A significant elevation in serum sIL-2R levels was found in chronic HCV infection compared to healthy controls, and serum sIL-2R levels significantly correlated with the frequency of IFN-γ-producing cells.CONCLUSIONS:In HCV infected subjects, both serum sIL-2R and IFN-γ are increased in chronic HCV infection no matter the stage of disease, meaning they are no different in asymptomatic carriers, patients with chronic hepatitis, and patients with liver cirrhosis, and that Th1 cytokine or Th1 cells may participate in the pathogenesis of liver damage in chronic HCV infection.

Hepatitis C virus (HCV) RNA level determined by second-generation branched-DNA probe assay as predictor of response to interferon treatment in patients with chronic HCV viremia

Using first- and second-generation branched-DNA probe assays (1st- and 2nd-bDNA), we investigated the predictors of favorable clinical response to interferon (IFN) treatment in patients with chronic HCV viremia. A total of 122 patients (85 genotype 1b and 37 genotype 2a) with chronic HCV viremia received 24-week IFN-α treatment. Patients with sustained clearance of serum HCV RNA by polymerase chain reaction at six months after IFN treatment were defined as having a sustained response (SR). HCV RNA level was determined by 1st- and 2nd-bDNA assays prior to treatment. Mean HCV RNA level by 1st-bDNA was significantly higher in genotype 1b patients [5.4 × 106 HCV genome equivalent (Meq)/ml] than in genotype 2a patients (0.9 Meq/ml) (P < 0.05). There was no significant difference between patients with these genotypes in the level by 2nd-bDNA (1b: 5.2 Meq/ml and 2a: 3.1 Meq/ml). SR was achieved by 43 (35.2%) of 122 patients. Mean HCV RNA levels by both the 1st- and 2nd-bDNA of SR patients (1.0 and 1.9 Meq/ml) were significantly lower than those of non-SR patients (5.3 and 6.0 Meq/ml) (both P < 0.05). The SR rate in genotype 2a patients (59.5%) was significant higher than in genotype 1b patients (24.7%) (P < 0.05). Stepwise logistic regression analysis showed that HCV RNA level ≦1.0 Meq/ml by 2nd-bDNA (odds ratio = 7.6, compared to level > 1.0 Meq/ml, P < 0.05) was a significant predictive cutoff for SR. Using 2nd-bDNA, a significantly higher rate of SR was found in genotype 1b patients with level ≤1.0 Meq/ml (57.6%) than in those with level >1.0 Meq/ml (3.8%) (P < 0.05). The SR rate of genotype 2a patients with level >1.0 Meq/ml (68.6%) was somewhat higher than for those with level ≤1.0 Meq/ml (52.4%). These findings suggested that, using 2nd-bDNA, a low HCV RNA level of ≦1.0 Meq/ml was the most favorable marker of successful IFN treatment and that patients with genotype 2a, even those with level >1.0 Meq/ml, had a high rate of SR to IFN treatment.

Identifying interacting predictors of falling among hospitalized elderly in Japan : A signal detection approach

Falling is a complex phenomenon that involves interaction of multiple risk factors. The authors analyzed factors related to falls in a geriatric hospital to elucidate interaction of multiple risk factors for falls in elderly inpatients. Subjects were 364 patients (mean age, 81.7; women 76.7%) who were aged 60 years and over and had been hospitalized for more than 6 months between April 2000 and March 2001. A signal detection model was used to identify baseline variables that best divided the sample into subgroups using incidence of falling as an outcome variable. During a follow‐up period, 91 patients (25%) had at least one incident of fall. Out of 14 independent variables, a higher‐order interaction consisting of six significant variables was identified. Consequently, the subjects were categorized into seven subgroups whose fall rate varied 5.7–80.9%. We found that the combination of non‐bedridden state, dementia, and medication of tranquilizers or sleeping drugs was the highest fall rate (80.9%). Signal detection analysis is useful to identify the combination of multiple risk factors of falling, and applicable to develop prevention programs for each subgroups.

HIV infection with concomitant cerebral toxoplasmosis and disseminated histoplasmosis in a 45-year-old man

Although disseminated histoplasmosis is a common opportunistic infection in HIV patients in endemic areas, it is not widely known in Japan. We report a rare case of a man from Ghana infected with HIV who was hospitalized in Japan and who suffered from coinfection with cerebral toxoplasmosis and disseminated histoplasmosis. The diagnosis of cerebral toxoplasmosis was confirmed by a brain biopsy, and the therapy for the disease resulted in almost complete resolution of the brain lesion. However, fever of unknown origin continued for 2 weeks, and disseminated histoplasmosis was diagnosed by examination of a blood smear and by the detection of the histoplasma genome in the peripheral blood by means of polymerase chain reaction. The isolate was confirmed to be Histoplasma capsulatum var. duboisii. Therapy with amphotericin B was initiated, and no histoplasma genome in the peripheral blood was detected 3 days later. Unfortunately, the patient died after 10 days from acute respiratory syndrome. This case highlights that histoplasmosis should be included in the differential diagnosis of opportunistic infections in AIDS patients when patients have a history of travel to or arrival from endemic areas.

Deconstructing the traditional Japanese medicine “Kampo”: compounds, metabolites and pharmacological profile of maoto, a remedy for flu-like symptoms

Pharmacological activities of the traditional Japanese herbal medicine (Kampo) are putatively mediated by complex interactions between multiple herbal compounds and host factors, which are difficult to characterize via the reductive approach of purifying major bioactive compounds and elucidating their mechanisms by conventional pharmacology. Here, we performed comprehensive compound, pharmacological and metabolomic analyses of maoto, a pharmaceutical-grade Kampo prescribed for flu-like symptoms, in normal and polyI:C-injected rats, the latter suffering from acute inflammation via Toll-like receptor 3 activation. In total, 352 chemical composition-determined compounds (CCDs) were detected in maoto extract by mass spectrometric analysis. After maoto treatment, 113 CCDs were newly detected in rat plasma. Of these CCDs, 19 were present in maoto extract, while 94 were presumed to be metabolites generated from maoto compounds or endogenous substances such as phospholipids. At the phenotypic level, maoto ameliorated the polyI:C-induced decrease in locomotor activity and body weight; however, body weight was not affected by individual maoto components in isolation. In accordance with symptom relief, maoto suppressed TNF-α and IL-1β, increased IL-10, and altered endogenous metabolites related to sympathetic activation and energy expenditure. Furthermore, maoto decreased inflammatory prostaglandins and leukotrienes, and increased anti-inflammatory eicosapentaenoic acid and hydroxyl-eicosapentaenoic acids, suggesting that it has differential effects on eicosanoid metabolic pathways involving cyclooxygenases, lipoxygenases and cytochrome P450s. Collectively, these data indicate that extensive profiling of compounds, metabolites and pharmacological phenotypes is essential for elucidating the mechanisms of herbal medicines, whose vast array of constituents induce a wide range of changes in xenobiotic and endogenous metabolism.Systems biology of traditional medicine: comprehensive analysis of a traditional Japanese medicine, maotoPharmacological activities of Kampo, or traditional Japanese herbal medicine, are putatively mediated by complex interactions between the plant-derived compounds and endogenous molecules. To elucidate these properties, we performed comprehensive phytochemical profiling, and pharmacological and metabolomic analyses of maoto, an herbal remedy for flu-like symptoms. In the plasma of maoto-treated rats, we detected maoto-derived compounds, metabolites produced from the chemical transformation of maoto compounds by host metabolism and gut microbes, and endogenous metabolites that were appeared following maoto administration. In an acute inflammatory rat model, maoto ameliorated symptoms of sickness behavior, suppressed inflammatory cytokines, and extensively affected common metabolites and lipid mediators. These data suggest that the diverse chemical composition of Kampo broadly affects the host’s endogenous metabolism and exerts specific pharmacological effects.