Kouzaburo Yamaji

IL-1 β and TNF-α produced by peripheral blood mononuclear cells before and during interferon therapy in patients with chronic hepatitis C

We investigated the spontaneous and phytohemagglutinin-stimulated production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) by peripheral blood mononuclear cells in patients with chronic hepatitis C during treatment with interferon-α (IFN-α). Spontaneous productions of these were significantly higher in patients with chronic hepatitis C than in healthy subjects. For patients prescribed interferon, stimulated production of TNF-α was significantly higher in complete responders than in partial responders, but the differences were small between the other cytokine levels and outcome of IFN treatment. Spontaneous production of these cytokines was higher in patients with genotype III with complete response than in genotype III patients with a partial response, but this was not the case in patients with genotype II. There was a negative correlation between these cytokines and histological activity index. Spontaneous production of cytokines was decreased only in complete responders after the administration of interferon. These data suggest that the elevated production of cytokines in patients with chronic hepatitis C may be due to host response to the virus, and monitoring cytokines along with alanine aminotransferase and hepatitis C virus RNA during treatment may provide more precise information of the effectiveness of 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.

Age-related accumulation of Ig VH gene somatic mutations in peripheral B cells from aged humans

To investigate age‐related alterations in human humoral immunity, we analysed Ig heavy chain variable region genes expressed by peripheral B cells from young and aged individuals. Three hundred and twenty‐seven cDNA sequences, 163 µ and 164 γ transcripts with VH5 family genes, were analysed for somatic hypermutation and VHDJH recombinational features. Unmutated and mutated µ transcripts were interpreted as being from naive and memory IgM B cells, respectively. In young and aged individuals, the percentages of naive IgM among total µ transcripts were 39% and 42%, respectively. D and JH segment usage in naive IgM from aged individuals was similar to that from young individuals. The mutational frequencies of memory IgM were similar in young and aged individuals. γ transcripts, which are regarded as being from memory IgG B cells, showed a significantly higher mutational frequency (7·6%) in aged than in young individuals (5·8%) (P < 0·01). These findings suggest that VHDJH recombinational diversity was preserved, but that the accumulation of somatic mutations in the IgG VH region was increased in aged humans. The accumulation of somatic mutations in IgG B cells during ageing may imply that an age‐related alteration exists in the selection and/or maintenance of peripheral memory B cells.

Serum levels of soluble interleukin-2 receptors and effects of interferon-α for patients with chronic hepatitis C virus

To characterize the role of serum soluble interleukin-2 receptor (sIL-2R) in hepatitis C virus (HCV) infection, the level of sIL-2R was measured by ELISA in 117 subjects with chronic HCV infection and in 23 healthy controls. HCV RNA was detected by polymerase chain reaction in all subjects with HCV infection. Forty-seven patients with chronic hepatitis and 10 with liver cirrhosis were treated for six months with natural interferon-α. The sIL-2R levels of 40 asymptomatic HCV carriers (632±340 units/ml), 47 patients with chronic hepatitis (547±204 units/ml), 10 with cirrhosis (679±239 units/ml), and 20 with hepatocellular carcinoma (1145±487 units/ml) were significantly higher than those of healthy controls (380±191 units/ml) (P<0.05, respectively). The levels of sIL-2R increased, as did the histological activity index scores (r=0.348,P<0.01). The level of sIL-2R rose after the initial administration of interferon in all 57 patients. In patients in whom HCV RNA was eliminated from the sera within a six-month follow-up after cessation of treatment, the level of sIL-2R reverted to basal values, but in patients in whom HCV RNA was not eliminated the value was significantly higher than that before treatment. These results suggest that monitoring serum sIL-2R in patients with chronic HCV infection treated with interferon may provide information concerning the possibility of the elimination of HCV RNA.

Evidence of B Cell Clonal Expansion in HIV Type 1-Infected Patients

HIV-1 infection results in a gradual decrease in CD4(+) T cell counts and progressive immune deficiency. Increased T cell turnover in HIV-1-infected patients, which can be interpreted as T cell clonal expansion, has been thought to be relevant to its pathogenesis. To investigate whether B cell clonal expansion also occurs in HIV-1-infected patients, we examined the expressed V(H)DJ(H) gene sequences of peripheral B cells in HIV-1-infected patients with hypergammaglobulinemia. Identical V(H)DJ(H) gene rearrangements with additional nucleotide differences in V(H) genes were analyzed as a marker of clonally related B cells. From healthy individuals and HIV-1-uninfected patients with hypergammaglobulinemia, clonally related B cells were detected in none of 10 (0%) and 2 of 10 (20%), respectively. No clonally related B cells were detected in any of the nine HIV-1-infected patients with detectable viral loads and normal Ig levels (0%). In contrast, from 9 of 14 HIV-1-infected patients with hypergammaglobulinemia (64%), clonally related B cells were detected. In addition, no HIV-1-infected patients who exhibited normal Ig levels after antiretroviral therapy had clonally related B cells. These findings suggest that B cell clonal expansion is present in HIV-1-infected patients with hypergammaglobulinemia.

Hepatitis C viral RNA status at two weeks of therapy predicts the eventual response.

We investigated the timing of the disappearance and reappearance of serum hepatitis C viral (HCV) RNA in patients with chronic hepatitis C during interferon treatment and follow-up. Serum samples were tested for HCV RNA by polymerase chain reaction in 62 patients with chronic hepatitis C treated with interferon-alpha for 24 weeks. We found that 17 patients obtained complete response, with absence of serum HCV RNA for 6 months after the treatment. Twenty-nine patients had a partial response, with reappearance of serum HCV RNA within 6 months of follow-up, and 16 patients were nonresponders who were positive for serum HCV RNA throughout the observation period. HCV RNA disappeared within 2 weeks of treatment in 31 patients, including all 17 (100%) complete responders and 14 (48.3%) of the 29 partial responders. The patients remaining positive for HCV RNA at the second week were 15 (51.7%) of the 29 partial responders and the 16 nonresponders. In all of the 29 partial responders, viremia recurred within 1 month after the treatment. These results indicate that the status of HCV RNA at the second week of treatment is a useful predictor of effective treatment, whereas status at the first month after cessation of treatment is useful for assessing the effectiveness of interferon itself.

No Significant Changes in Levels of Hepatitis C Virus (HCV) RNA by HCV Infection Competitive Polymerase Chain Reaction in Blood Samples from Patients with Chronic

To determine if levels of hepatitis C virus(HCV) RNA change over a several-year period, wequantified the amount of HCV RNA by competitivepolymerase chain reaction. The population studiedincluded 44 residents of a rural area with chronic HCVinfection, 39 had chronic hepatitis C and 37 werepatients on hemodialysis. All these Japanese patientshad HCV RNA of genotype II. Blood samples were collected once a year from 1992 to 1995. From 1993 to1995 between the groups, there was no significantdifference in change of HCV RNA levels of 44 residentswith chronic HCV infection, with and without liverdysfunction, nor was there any change in the 31 hemodialysispatients from 1992 to 1995. The HCV RNA levels in the 25with chronic hepatitis who did not respond tointerferon-alpha during 1992-1993 returned topretreatment levels after the cessation of interferontreatment. In two of six hemodialysis patients who wereinfected with HCV during this observation period, HCVRNA was eliminated within one year, and the remaining four became HCV carriers. HCV RNA levels in thelatter rose rapidly after infection and were sustainedat a high level throughout the study period. Thus, HCVRNA level did not change remarkably during a three-year period, a finding which supportsthat it does not correlate with deterioration of liverdamage and aging of HCV carriers.

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.

Hepatitis B Virus Genomes of Chronic Hepatitis Patients Do Not Contain Specific Mutations Related to Acute Exacerbation

To determine the specific viral variants associated with acute exacerbation of chronic hepatitis from hepatitis B virus (HBV) infection, we analyzed the complete nucleotide sequences of the HBV genome in serial serum samples from two chronic active hepatitis patients who seroconverted from HBeAg to anti-HBe. HBV DNA was amplified by polymerase chain reaction (PCR) and sequenced. A 1896 precore stop codon mutant (G to A at nt 1896) coexisting with the wild sequence was found in both patients prior to seroconversion from HBeAg to anti-HBe. Core promoter mutations at nucleotide positions 1762 (A to T) and 1764 (G to A) were found in both patients throughout the observation period. Mutations were observed in the HBV genome of the two patients at different time points, and there was no correlation between the mutations and liver disease or DNA polymerase levels. The nucleotide divergence rate and the composition of quasispecies in the HBV sequence at the time of acute exacerbation were almost the same as were found at other time points. These results suggest that acute exacerbation does not appear to be caused by a characteristic HBV species. The multiple factors that cause generalized HBV replication activation may contribute to acute exacerbation.

Two VH5 family genes expressed by human peripheral B cells display differential mutational frequencies in the VH 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.