Long-term effects of peginterferon alfa-2a therapy in Japanese patients with chronic hepatitis B virus infection

Background

There is no information on the long-term effects of peginterferon (PEG-IFN) alfa-2a therapy for chronic hepatitis B (CHB) in Japan. This double-blind, randomized trial investigated the efficacy of PEG-IFN therapy.

Methods

We analyzed 22 Japanese patients with CHB (hepatitis B e antigen [HBeAg]-positive: 17, HBeAg-negative: 5) treated with PEG-IFN alfa-2a and followed-up posttreatment for 5 years. Responders represented patients who showed persistent normalization of alanine transferase (ALT) levels, HBeAg clearance, and low hepatitis B virus (HBV) DNA levels (HBeAg-positive patient; <5 log copies/mL, HBeAg-negative patient; <4.3 log copies/mL) at end of treatment, and at 1, 2, 3, 4 and 5 years posttreatment. In addition, baseline HBeAg-positive patients who showed sustained normalization of ALT level, HBeAg clearance, and low HBV DNA level for more than 6 months until at 1, 2, 3, 4, and 5 years after completion of PEG-IFN were also classified as “triple responders” and the proportion of triple responders relative to all patients was termed the “triple response rate”.

Results

The response rates among HBeAg-positive patients were 13 %, 25 %, 14 %, 21 % and 21 % at end of treatment, and at 1, 2, 3, 4, and 5 years, respectively. The response rate tended to be higher in patients treated for 48 than 24 weeks. The respective response rates among HBeAg-negative patients were 0 %, 20 %, 20 %, 20 % and 25 %. During the treatment period, hepatitis B surface antigen (HBsAg) clearance at 3.5 years was noted in one patient, who was 37-year-old, male, had genotype C and received PEG-IFN alfa-2a at 90 μg for 48 weeks.

Conclusion

At 5 years after completion of PEG-IFN, the triple response rate in HBeAg-positive patients and combined response rate in HBeAg-negative patients were 21 % (3/14) and 25 % (1/4), respectively. The triple response was seen in three patients who had all been treated with PEG-IFN for 48 weeks.

     

Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells

Background

Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.

Methodology/Principal Findings

Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b⁺/CD11c⁺ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.

Conclusions/Significance

The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.     

Improvement in thermal stability and reactivity of pyranose oxidase from Coriolus versicolor by random mutagenesis

A mutant producing a pyranose oxidase, which has a higher thermal stability and lower Km values for d-glucose and 1,5-anhydro-d-glucitol than those of the wild type enzyme, was obtained. A single amino acid substitution, Lys for Glu at position 542, had occurred. This altered enzyme, E542K, was not only stable at 55°C, which was 5°C higher than the wild-type enzyme, but was stable in alkaline solution at pH 8.0–11.0. Km values of E542K for d-glucose and 1,5-anhydro-d-glucitol were 0.7 mM and 14.3 mM, respectively, in contrast with 1.4 mm and 35.3 mM for the wild-type enzyme. A little effect was observed in kcat values, and improvement in reactivity was mainly due to the decreases in Km values. This altered pyranose oxidase is useful for food analysis and diagnosis.     

Evaluation of 11beta-HSD activities in vivo following oral administration of cortisol-13C4,2H1 to a human subject

This study is concerned with an oral administration of 5mg of [1,2,4,19-13C(4),11alpha-2H]cortisol (cortisol-13C(4),2H(1)) to a human subject to reliably evaluate the individual activities of two isozymes of 11beta-HSD. The use of a GC-MS method allowed the simultaneous measurement of the plasma concentrations of cortisol-13C(4),2H(1), cortisone-13C(4), and cortisol-13C(4) together with endogenous cortisol and cortisone. The loss of 11alpha-2H during the conversion of cortisol-13C(4),2H(1) to cortisone-13C(4) by 11beta-HSD2 and the regenerated cortisol-13C(4) from cortisone-13C(4) by 11beta-HSD1 provided a direct and accurate means of distinguishing the activities of the two isozymes. The kinetic analysis associated with the metabolism of orally administered cortisol-13C(4),2H(1) was of great importance in assessing the 11beta-HSD activities. From a viewpoint of the chemical stability and much less pronounced kinetic isotope effect of the 13C-label and the 2H-labeling in the 11alpha-position, cortisol-13C(4),2H(1) used in this study served as an appropriate tracer for elucidating the kinetics of the interconversion of cortisol to cortisone in man.     

High concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome

alpha-Defensins, antimicrobial peptides localized in neutrophils, participate in tissue damage through their cytotoxic effects in neutrophil-mediated pulmonary diseases. Neutrophils play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). We measured alpha-defensins levels in plasma and bronchoalveolar lavage fluid (BALF) of ARDS patients to assess the kinetics of alpha-defensins in ARDS. Plasma alpha-defensins levels were higher in ARDS patients than in control subjects, and BALF levels were also higher in ARDS patients than in control subjects. In ARDS, BALF alpha-defensins levels correlated with those of interleukin (IL)-8, and plasma alpha-defensins levels also correlated with Lung Injury Score. Peripheral neutrophil alpha-defensins contents were higher in ARDS than the control. IL-8 dose-dependently stimulated alpha-defensins release from cultured neutrophils and these levels were higher in ARDS than the control. Reverse-phase high performance liquid chromatography showed high plasma levels of pro-defensins, precursors of alpha-defensins from the bone marrow in ARDS, although alpha-defensins in peripheral and BALF neutrophils were mature type. In conclusion, high plasma alpha-defensins in ARDS patients result from the release of pro-defensins from bone marrow, rather than mature alpha-defensins from neutrophils that accumulate in the alveolar space. The alpha-defensins contents of peripheral neutrophils in ARDS are higher and easier to release than control.     

Simultaneous determination of endogenous and stable isotope-labelled 6beta-hydroxycortisols in human urine by stable isotope dilution mass spectrometry

This study describes a capillary GC-MS method for the simultaneous determination of endogenous 6beta-hydroxycortisol (6beta-OHF) and its stable isotope-labelled analogue, 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6beta-OHF-2H(5)), in human urine. 6beta-Hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (cortisol-13C(4),(2)H(5)) was used as an analytical internal standard. The methoxime trimethylsilyl ether (MO-TMS) derivatization was employed for the GC-MS analysis of 6beta-OHF. Quantitation was carried out by selected-ion monitoring (SIM) of the characteristic fragment ion ([M-31](+.)) of the MO-TMS derivative of 6beta-OHF. The sensitivity limit of the present GC-MS-SIM method was found to be 25 pg per injection for 6beta-OHF (S/N ratio=5.6). The within-day reproducibility in the amounts of unlabelled and labelled 6beta-OHFs determined were in good agreement with the actual amounts added, the relative errors being less than 5.30%. The inter-assay RSDs were less than 4.95% for unlabelled and labelled 6beta-OHFs.     

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3α,11β,17α,21-tetrahydroxy-5β-pregnane-20-one), allo-tetrahydrocortisol (allo-THF, 3α,11β,17α,21-tetrahydroxy-5α-pregnane-20-one) and tetrahydrocortisone (THE, 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione) in human plasma and urine is described. [1,2,3,4,5-²H₅]THF (THF-d₅), allo-[1,2,3,4,5-²H₅]THF (allo-THF-d₅) and [1,2,3,4,5-²H₅]THE (THE-d₅) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M−30]⁺) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.     

Glutamic acid potentiates hepatocyte response to mitogens in primary culture

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L-15 medium. Addition of glutamic acid (1–20 μM) to EGF-containing L-15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF-induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentrations of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose-dependent (0.5–20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5–20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [¹²⁵I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens © 1994 Wiley-Liss, Inc.