Persistence of pandemic influenza H1N1 virus in young patients after oseltamivir therapy in the 2009-2010 season: a comparison with seasonal H1N1 with or without H275Y mutation.

Comparison of the viral persistence of pandemic H1N1 (H1N1pdm) and seasonal H1N1 with or without H275Y mutation after oseltamivir therapy has not been adequately done. Virus was isolated before and on days 4-6 from the start of oseltamivir treatment for 158 cases of seasonal (2007-2008 and 2008-2009 seasons) or pandemic (2009-2010 season) H1N1 influenza. Sequence analysis was done for each season and NA inhibition assay (IC(50)) was done in the 2009-2010 season. H275Y mutation before therapy was 0% in the 2007-2008 and 2009-2010 seasons, but 100% in the 2008-2009 season. Fever and other symptoms were noticeably prolonged after oseltamivir therapy for children with H275Y mutated seasonal H1N1 (2008-2009 season), but not in patients with seasonal H1N1 without mutation (2007-2008) or H1N1pdm (2009-2010). The viral persistence rate was significantly higher for patients 15 years or younger than for those 16 years and older with H275Y mutated seasonal H1N1 (46.2% and 10.5%, respectively) or with H1N1pdm (43.3% and 11.5%, respectively). The H275Y mutation emerged after oseltamivir treatment in 2.4% (2/82) of all patients with H1N1pdm. In two children, the H275Y mutation emerged after therapy and the IC(50) increased more than 200 fold; however, the prolongation of fever was not so prominent. In conclusion, oseltamivir was effective for fever and other clinical symptoms; however, the virus persisted longer than expected after treatment in H1N1pdm influenza-infected children in the 2009-2010 season, similar to seasonal H1N1 with H275Y mutation in the 2008-2009 season.

The relationship between the daily dosage of the carbapenem meropenem (MEPM) and MEPM-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a pathogen which is known to be responsible for nosocomial infection. The appropriate use of antibiotics has become important for preventing the spread of drug-resistant P. aeruginosa. In Hara-doi Hospital, two carbapenem antibiotics, imipenem (IPM) and meropenem (MEPM), are used for patients aged 65 years or older at a daily dosage of 1.0 g and 0.5 g, respectively. Of P. aeruginosa samples isolated in 2003, the sensitivity to IPM was 54% and to MEPM it was 58%. In 2004, the sensitivity to IPM was 55%, i.e., not significantly different from 2003. In 2004, the daily dosage of MEPM was increased to 1.0 g/day, and the sensitivity to MEPM increased to 71%. Based on the Pharmacokinetics/Pharmacodynamics (PK/PD) theory, even though the patients were elderly, a sufficient dosage of antibiotics given over a shorter period of time was effective against MEPM-resistant P. aeruginosa in a hospital ward, and there were no side effects.

[Varicella-zoster virus symptoms and polyneuropathy in a patient with human immunodeficiency virus infection not improved until highly active anti-retroviral therapy added to acyclovir therapy].

In March 2003, a 34-year-old man with left facial palsy, dysphagia, and hoarseness treated with acyclovir suffered worsened dermatological and neurological problems. A routine blood test in early April showed the patient to be HIV-antibody positive, so he was transferred to our hospital. Blood analysis showed serum HIV-RNA at 96,000 copies/mL and a CD 4 count of 170/microL. Brain MRI taken on admission showed a T 2 high lesion in their left medulla. Acyclovir was thought to be ineffective due to reduced cell-mediated immunity because of the HIV infection, and HAART therapy was begun. After two months of HAART, skin lesions and the T 2 high lesion in left medulla improred. HIV-RNA became undetectable and the CD 4 count exceeded 500/microL. Intracellular cytokine analysis by flow cytometry showed a shift from Th 2 to Th 1 dominance. The elimination of VZV may thus have been promoted by the combination of acyclovir and HAART.

A comparison of the antitumor effects of interferon-alpha and beta on human hepatocellular carcinoma cell lines.

The antiviral, antiproliferative and immunomodulatory effects of type I interferons (IFNs) are well documented, however, few studies have been published concerning differences in the antitumor effects of IFN-alpha and beta. In the present study, differences in antitumor effect, including the antiproliferative effect, cell cycle change, apoptosis, and the IFN-stimulated gene (ISG) were examined by flow cytometry between IFN-alpha and beta on three human hepatocellular carcinoma (HCC) cell lines (HepG2, Huh7 and JHH4). The antiproliferative effect of both IFNs on the HCC cell lines was time- and dose-dependent, and IFN-beta was significantly stronger than IFN-alpha. The cell cycle effect by both IFNs was an S-phase accumulation, with IFN-beta having a tendency to increase the S-phase ratio more strongly than IFN-alpha, especially in Huh7. Apoptosis marker expression, Fas antigen and intracellular active caspase-3, was increased after the addition of IFNs, especially of IFN-beta. The expression of human leukocyte antigen-class I molecules, ISG-encoded protein, was increased after the addition of IFNs, especially of IFN-beta. These data suggest that IFN-beta has a greater antitumor effect than IFN-alpha on HCC of a very early stage in patients with chronic hepatitis C.

Interferon-alpha/beta upregulate IL-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis C patients during interferon-alpha/beta treatment.

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8+ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-alpha and -beta dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-beta showed stronger activity in IL-15 production induction in vitro than IFN-alpha. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-alpha/beta induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response.

Kinetic analysis of Mycobacterium tuberculosis-specific cytokine production by PBMC in adults after BCG vaccination.

Although bacille Calmette-Guerin (BCG) has been used worldwide as the only vaccine for tuberculosis, its protective efficacy in human adults is controversial. To investigate human immunological responses to Mycobacterium tuberculosis after BCG vaccination, we analyzed IFN-gamma and IL-10 production by peripheral blood mononuclear cells (PBMC) from health-care workers five times throughout the year after BCG vaccination. Of 449 health-care workers, 36 (8.0%) were negative by the tuberculin skin test, and of these, 20 were vaccinated with BCG. Because all the subjects had received BCG vaccination as infants, the present vaccination was considered to be a revaccination. The cytokine responses of the vaccinated and control tuberculin skin-test-positive subjects (n = 6) were followed at 0, 2, 4, and 8 weeks and at 12 months. The mean IFN-gamma production by PBMC when cultured with purified protein derivative (PPD) gradually increased, reached a peak at week 8, and then declined until 12 months, with four exceptions who showed no IFN-gamma elevation. The IFN-gamma level of the vaccinated group at week 0 was significantly lower than that of the controls. The mean IL-10 production in response to PPD reached a peak at week 2, and then declined to its lowest point at week 8. These results indicate that the BCG vaccine can induce a type I cytokine response to M. tuberculosis in most tuberculin skin-test-negative adults at week 8, suggesting the immunological efficacy of vaccination.

CD27(+) (memory) B cell decrease and apoptosis-resistant CD27(-) (naive) B cell increase in aged humans: implications for age-related peripheral B cell developmental disturbances.

To investigate age-related alterations in human humoral immunity, we analyzed the quantity and quality of peripheral B cell subsets, CD27-negative (CD27(-)) and CD27-positive (CD27(+)) B cells, by flow cytometry analysis in 54 aged individuals (mean age +/- SE, 74.6 +/- 0.7 years) and 30 young individuals (mean age +/- SE, 26.1 +/- 0.5 years). CD27(-) and CD27(+) B cells are regarded as naive and memory B cells, respectively. CD38, Ki-67, CD95 and bcl-2 were used as activation, proliferation and apoptotic markers. Susceptibility to apoptosis was evaluated by cell size and annexin-V binding in culture cells. The percentage of CD27(+) B cells was significantly lower in aged (mean, 19.2%) individuals than that in young individuals (mean, 28.2%). The opposite was true for CD27(-) B cells (mean, 80.8% in aged and 71.8% in young) (P < 0.01). The absolute number of CD27(+) B cells in aged individuals was significantly less than the number of CD27(-) B cells. The CD27(+) B cells from aged individuals showed little susceptibility to apoptosis, although CD95 expression on the CD27(+) B cells was significantly higher in the aged individuals than in the young individuals (P < 0.05). The CD38 and bcl-2 expression on the CD27(-) B cells was significantly higher in the aged individuals than in the young individuals (P < 0.05). In addition, the CD27(-) B cells from the aged individuals showed a decreased susceptibility to apoptosis compared with that of the young individuals. These findings suggested that human aging leads to both quantitative and qualitative alterations in the peripheral B cell developmental system, including memory and naive B cell balance and their surface phenotypes.

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.

Downregulation of CXCR5 in CD27- B cells of HIV-1 infected patients.

The CD27(-) (naive) B cells of HIV-1 infected patients have been shown to be increased in frequency and to be activated, as indicated by high CD38 expression on the cell surface. CXCR5, a B cell chemokine receptor, is expressed on circulating CD27(-) (naive) B cells and plays a pivotal role in peripheral B cell development. To investigate the effect of HIV-1 infection on the expression of this chemokine receptor on naive B cells, the expression level of CXCR5 on CD27(-) B cells was examined in 19 drug-naive HIV-1 infected patients, 27 HAART-treated patients, and 20 controls. CXCR5 expression on CD27(-) B cells was significantly lower in drug-naive patients than in HAART-treated patients and controls (P < 0.01). CD27(-) B cells with high CD38 expression exhibited low CXCR5 expression. The CXCR5 expression level on CD27(-) B cells recovered to within the normal range after effective antiretroviral therapy. These findings suggested that HIV-1 infection induces a remarkable phenotypic alteration of naive B cells and that the activated naive B cells found in HIV-1 infection downregulate CXCR5 on their surface. Impaired homing of naive B cells may contribute to HIV-1 induced immunological deficiencies.

Maturation of monocyte-derived dendritic cells by Hochu-ekki-to, a traditional Japanese herbal medicine.

To investigate the immunological effect of the traditional Japanese herbal medicine (kampo), Hochu-ekki-to (HOT), on dendritic cells (DC), we examined in vitro if HOT would stimulate the maturation process of human monocyte-derived DC as do TNF-alpha and LPS. Monocytes from a healthy volunteer were cultured in the presence of IL-4 and GM-CSF, and the generated immature DC were stimulated with HOT, TNF-alpha, or LPS (HOT-DC, TNF-DC, and LPS-DC, respectively) for 2 days. Flow cytometric analysis showed that HOT stimulated DC to express the surface maturation markers CD80, CD83, and CD86 dose-dependently and that the up-regulation level was identical to TNF-alpha and LPS. The antigen-uptake capacity of HOT-DC was determined by FITC-labeled albumin uptake. HOT-DC lost albumin uptake capacity comparable to LPS-DC, indicating DC maturity. IL-12 (p70) production by HOT-DC and TNF-DC was not increased in comparison with LPS-DC. The antigen-presenting capacity of HOT-DC as analyzed by allogeneic T cell proliferation was significantly increased in comparison with immature DC and was identical to LPS-DC. These results demonstrate that HOT stimulates DC maturation as well as the other known maturation factors, despite low IL-12 production, and suggests the possibility that DC maturation by HOT can play an important role in the improvement of the immunoregulatory function in patients with impaired host defense.

[Evaluation of an immunochromatography test kit for rapid diagnosis of influenza].

We assessed the sensitivity and specificity of the Capilia Flu AB rapid diagnosis kit for influenza that utilizes the immunochromatography method. Tested were 114 influenza like illness patients in the 2001/2002 influenza season. We used Capilia Flu AB and Infu A . B Quick, a rapid diagnosis kit based on enzyme immunoassay. As laboratory confirmation tests, influenza virus isolation and polymerase chain reaction (PCR) were done. Those patients with positive results from virus isolation or PCR were regarded as influenza patients. The sensitivities of nasal swab, pharyngeal swab, and nasal wash specimens were 82.8%, 80.0%, and 75.0%, respectively. The specificities of nasal swab, pharyngeal swab, and nasal wash specimens were 95.3%, 93.9%, and 100%, respectively. A total of 20 patients displayed different results in comparison of their nasal and pharyngeal swabs: 15 patients were positive with the nasal swab but negative with the pharyngeal swab and 5 patients were negative with the nasal swab but positive with the pharyngeal swab. Nasal swab would seem to be preferable in terms of sensitivity. The sensitivity and specificity of Capilia Flu AB were a little higher than those of Influ A . B Quick, but with no significant difference. The one-step operation of Capilia Flu AB is easier than the four steps required by Influ A . B Quick, but the time required to make a diagnosis is the same. No significant age related difference in the effectiveness of the kits was found. The Capilia Flu AB rapid diagnosis kit is useful in clinical practice because it has good sensitivity (about 80%) and specificity (about 90%), and it is easy to use.

Two V(H)5 family genes expressed by human peripheral B cells display differential mutational frequencies in the V(H) region.

The heavy chain variable segment gene (V(H))5 family, one of the seven immunoglobulin (Ig) V(H) families, contains two functional genes, VH251 and VH32. To investigate functional differences between these V(H)5 family genes, V(H) segments expressed by human peripheral B cells were sequenced and analyzed. One hundred fifty-three sequences with unique V(H)DJ(H) recombinations were obtained from 17 adults. The mutational frequency of VH32 derived sequences (6.4%) was higher than that of VH251 derived sequences (4.4%), resulting in a significant difference (P<0.01). Significant differences in mutational frequencies between VH251 and VH32 derived sequences were observed in CDRs and FRs. No significant differences were found in CDR3 length distribution, D segment usage, or J(H) segment usage between VH251 and VH32 derived sequences. These results suggest that mutational frequency is affected, in part, by V(H) gene structure. The difference may occur after recombinational events in B cell development.

Rapamycin blocks tumor progression: unlinking immunosuppression from antitumor efficacy.

BACKGROUND: Malignancy is a dreaded complication of organ transplantation. Immunosuppressive drug therapy-induced impairment of the organ graft recipient's immune surveillance is considered to be the mechanism for the heightened incidence and metastatic progression. We identified a cell-autonomous and host-immunity independent mechanism for cyclosporine-associated tumor progression. In this study, we investigated the effect of rapamycin on tumor progression, in the presence and absence of cyclosporine. METHODS: A spontaneously arising renal adenocarcinoma (renal cancer) of BALB/c origin was used as the model tumor. The effect of rapamycin on renal cancer cell phenotype, molecules (E-cadherin, p27 kip1, cyclin D1) implicated in tumor progression, and the effect of rapamycin on in vivo tumor progression were explored in BALB/c mice and in T-cell, B-cell, and natural killer (NK) cell-deficient severe combined immune deficiency (SCID)-beige mice. In the SCID-beige mice, T24 human bladder transitional cell carcinoma also was used as the tumor inoculum. RESULTS: Rapamycin conditioning of renal cancer cells upregulated E-cadherin expression and induced phenotypic transition from invasive spindle, or dome-shaped cells, with exploratory pseudopodia to noninvasive cuboidal cells that formed cell-to-cell adhesions. Rapamycin increased p27 kip1, reduced cyclinD1, and arrested the growth of renal cancer cells in G1/S phase. In vivo, rapamycin prevented tumor growth and metastatic progression in syngeneic BALB/c or SCID-beige mice, and in BALB/c or SCID-beige mice treated with cyclosporine. Rapamycin treatment alone, or with cyclosporine, prolonged the survival of mice inoculated with renal cancer cells or T24 human bladder cancer cells. CONCLUSIONS: Our findings, in addition to unlinking mechanisms of immunosuppression from that of tumor progression, suggest that rapamycin may be of value for the management of posttransplant malignancy.