The Impact of HBV-DNA Fluctuations on Virus-Specific CD8+ T Cells i

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The Impact of HBV-DNA Fluctuations on Virus-Specific CD8+ T Cells in HBeAg+ Chronic Hepatitis B Patients Treated With a Steroid and Lamivudine

Posted 06/07/2006

J. Gotto; G. J. M. Webster; D. Brown; J. Jenkins; G. M. Dusheiko; A. Bertoletti


Summary

Restoration of anti-viral immune response may be a requisite for sustained virological response to treatment in chronic hepatitis B patients. Over a 13-month period, we examined the dynamics of hepatitis B virus (HBV)-specific CD8+ cells in six human leucocyte antigen (HLA)-A2+ hepatitis B e antigen (HBeAg)+ 'immunotolerant' chronic hepatitis B patients treated sequentially with corticosteroid and lamivudine. Our results show that the combination treatment did not result in a sustained restoration of anti-viral specific CD8+ cells in five of the six patients studied. However, HBV-specific CD8+ cells, despite being severely compromised, were not totally deleted. Paradoxically, steroid treatment was not associated with inhibition but with a minimal increase of the HBV-specific CD8 response, and we observed that nucleocapsid-specific CD8 responses were not rescued by stable and prolonged inhibition but became detectable after rapid rebounds of HBV replication. In most patients, the transient and minimal restoration of HBV-specific immunity was not associated with clinical benefits. Our results describe a dynamic relationship between HBV-specific CD8+ cells and HBV-DNA values, that could potentially be used for a better design of HBV treatment in HBeAg+ 'immunotolerant' chronic hepatitis B patients.
Introduction

The treatment of chronic hepatitis B virus (HBV) infection, which affects 300 million people worldwide, has advanced considerably through the development of nucleoside analogues and the pegylated form of interferon (IFN)-alpha, but the overall results are not completely satisfactory. Lamivudine, for example, reduces levels of plasma HBV-DNA in >90% of cases[1] and induces hepatitis B e antigen (HBeAg) seroconversion in up to 60% of patients with a pre-treatment alanine aminotransferase (ALT) >200 IU/L, but in only 6% of patients with an ALT of <80 IU/L.[2] This latter group, defined as 'immunotolerant', is characterized by a high level of HBV replication (HBV-DNA >107 genomes/mL of serum), the presence of circulating HBeAg and normal or only slightly elevated ALT levels and is extremely frequent in Asia, where 70% of the world population of HBV-infected subjects are concentrated.[3] It is possible to hypothesize that the low efficacy of treatment in this patient population is related to the absence of HBV-specific adaptive immunity, which is essential for the control of infection.[4-8] A recent longitudinal analysis of HBV-specific CD8+ responses performed in a heterogeneous population of chronic hepatitis B patients has shown that quantitative and qualitative defects of HBV-specific CD8+ cells are particularly manifest in HBeAg+ 'immunotolerant' patients.[9] HBV-specific CD8+ cells can hardly be detected in the blood and liver of these patients, and when CD8+ cells are detected, they are characterized by distinct functional defects.[10] Thus attempts have been made to manipulate the pre-treatment conditions of 'HBeAg+ immunotolerant patients', using short courses of glucocorticoids as immunomodulators.[11] The rationale for this unconventional use of glucocorticoids in persistent viral disease is derived from the historical observations that withdrawal of prednisolone in patients with HBeAg+ chronic hepatitis could result in transient ALT elevations.[12] These ALT elevations have always been interpreted as a sign of recovery of HBV-specific T-cell response after the immune suppressor effects of steroids.[11] Studies by Liaw et al. showed the recovery of HBV-specific Th1 responses after cortisone treatment,[13] but whether this Th1 recovery was associated with cortisone or lamivudine treatment was not clear.

We report here the results of a study designed to analyse the profile of HBV-specific CD8+ T cells in 'immunotolerant' HBeAg+ patients during sequential therapy with prednisolone and lamivudine.

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Materials and Methods
Patients

Six human leucocyte antigen (HLA)-A2+ patients chronically infected with HBV were studied. The selection criteria were HBsAg and HBeAg positivity for at least 6 months, a liver biopsy within a year confirming chronic hepatitis with no evidence of cirrhosis, stable ALT values less than twice the upper limit of normal (i.e. <80 U/L) at least on two occasions 1 month before therapy and normal liver function (i.e. normal prothrombin time, serum albumin and bilirubin) ( Table 1 ). Screening for HLA-A2 positivity was performed by staining peripheral blood mononuclear cells (PBMC) of patients with fluorescent conjugated anti-HLA-A0201 antibody (Serotec, Kidlington, UK). All patients were negative for antibodies to hepatitis C virus, hepatitis D virus and human immunodeficiency virus (HIV)-1, 2. The longitudinal analysis of anti-viral T-cell immunity in the periphery was approved by the local ethics committee and all patients provided written consent.
Treatment

The six patients were treated with prednisolone, 30 mg/day for 2 weeks, tapering to 15 mg/day for a further 2 weeks, followed by 2 weeks without treatment. Lamivudine 100 mg/day was given from week 6 for 12 months (Fig. 1). Clinical, virological and immunological data were collected on two to three occasions preceding therapy, and over a period of 13 months (weeks −8, −4, 0, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 and 56).

Click to zoom       
Figure 1.  (click image to zoom)

Longitudinal analysis of HBV-DNA levels, ALT and HBV-specific CD8+ T-cell responses in a treated representative patient. (a) Serum ALT levels (white bars) and HBV-DNA (black squares) are represented on the y-axis and measured at the indicated time points (x-axis). (b) Percentages of tetramer+ CD8+ T-cells are represented on the y-axis and the x-axis shows time in weeks prior to and during therapy. PBMC were tested directly ex vivo with the tetramers for core18-27, env183-91 and env348-57 specific CD8+ T-cells. Positive results were detected only using tetramer core18-27 (Tc18-27 •). The background level of direct tetramer staining with Tc18-27 (0.05%, indicated by a horizontal line) was calculated in HLA-A2 + non-HBV-infected subjects and in HLA-A2-negative HBV-infected patients.[9,29] (c) Percentage of peptide-specific IFN-γ-producing CD8+ T-cells after in vitro expansion (bars). PBMC were stimulated with the indicated peptides. After 10 days of in vitro expansion, the frequency of peptide-specific IFN-γ-producing CD8+ T-cells was calculated. The indicated percentages of IFN-γ-producing CD8+ T-cells were calculated after subtraction of double positive cells present in non-peptide stimulated cells.
     

Virological Assessment

Hepatitis B surface antigen, anti-HBs, total and IgM anti-HBc, HBeAg, anti-HBe, were determined by commercial enzyme immunoassay kits (Abbot Laboratories, North Chicago, IL, USA; Ortho Diagnostic System, Raritan, NJ, USA; Sanofi Diagnostic Pasteur, Marnes-la Coquette, France). Serum HBV-DNA was quantified using the Roche Amplicor Monitor assay (Roche Pharmaceuticals Ltd., Branchburg, NJ, USA), with a DNA detection limit of 400 copies/mL (0.0014 pg/mL).
PCR and HBV-DNA Sequencing

DNA was extracted from serum samples using the QIAamp DNA Blood mini kit (QIAGEN, Crawley, UK). The HBV-DNA was amplified with primers specific for the HBV core and envelope genes, as described previously.[24] The amplicons were purified and the core/envelope regions were sequenced directly using an ABI 377 Automated Sequencer (Applied Biosystems, Foster City, CA, USA). The HBV genotype infecting these patients was determined by analysis of sequenced portions of the core and surface antigens.[14]
Synthetic Peptides

Peptides corresponding to different HBV-specific HLA-A2 restricted CD8 epitopes were purchased from Primm (Milan, Italy). The purity of peptides was greater than 90% by high-performance liquid chromatography (HPLC) analysis. The peptides were based on HBV genotype D (serotype ayw) and the degree of homology among different genotypes is shown in Table 2 .
Isolation of PBMCs and Production of T-cell Lines

Peripheral blood mononuclear cells were isolated from fresh heparinized blood by Ficoll-Hypaque density-gradient centrifugation and suspended at a concentration 1.5-2 × 106 cells/mL in RPMI1640 (Invitrogen Ltd., Paisley, UK), 10% foetal calf serum (FCS). Cells were stimulated with 1 μm of various peptides in a 96-well plate. Recombinant interleukin 2 (IL-2) (R&D systems, Abingdon, UK) was added on day 4 of culture and cells were analysed after 10-12 days of culture. PBMC stimulation was performed with a panel of 11 peptides ( Table 2 ) corresponding to HBV sequences previously identified as HLA-A2-restricted CTL epitopes.[15]
Intracellular IFN-γ Production

Ex vivo-purified PBMC or short-term T-cell lines were stimulated at 2-3 × 106 cells/mL in RPMI1640, 10% FCS, with HBV peptides (1 μm) for 6 h at 37 °C in the presence of Brefeldin A (Sigma-Aldrich, Poole, Dorset, UK) at 10 μg/mL. Cells were washed, stained with Cy-chrome-conjugated anti-CD8 antibodies, then permeabilized and fixed using Cytofix/Cytoperm (Pharmingen, San Diego, CA, USA) according to the manufacturer's instructions. Flourescein isothiocyanate (FITC)-conjugated anti-cytokine antibodies or isotype-matched controls were added (30 min, 22 °C), washed twice and analysed by flow cytometry.
Staining With HLA-tetrameric Complexes

Human leucocyte antigen-class I tetramers were purchased commercially (Proimmune, Oxford, UK). Tetramer staining was performed as previously described[6] and cells were analysed on FACSort® (Becton Dickinson: BD Biosciences, San Diego, CA, USA) using CELL Quest™ (BD Biosciences, San Diego, CA, USA) software immediately or after addition of 1% paraformaldehyde.

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Results

Six HLA-A2+ chronic hepatitis B patients (HBeAg+, ALT < 80 IU/L; Table 1 ) were treated with lamivudine and prednisolone. Sequencing of the envelope 183-91, 348-57 and core 18-27 regions was performed to exclude possible variations between the peptides utilized and the virus infecting the patients. No mutations within epitopes were detected before or after treatment (not shown).

Of the six patients treated, only one (patient 1) seroconverted to anti-HBe and showed a sustained response to therapy with normal ALT levels 3 months after the end of therapy. Patient 2 developed a YMDD mutation, while the compliance with therapy of patient 6 was questionable.

The HBV-specific CD8+ response was analysed longitudinally in all treated patients using two different methods: the direct ex vivo frequencies of CD8+ cells specific for core 18-27, env 183-91 and env348-57 epitopes were measured using specific HLA tetramers; the functional capabilities of CD8+ cells specific for 11 different HLA-A2-restricted HBV epitopes ( Table 2 ) to proliferate and produce IFN-γ were tested with intracellular cytokine staining after in vitro expansion. An example of the analysis is shown in Fig. 1. Overall, the treatment did not result in a sustained and significant restoration of HBV-specific CD8 response. Other than for patient 1, who seroconverted to anti-HBe (Fig. 1), direct ex vivo tetramer staining was negative in all patients analysed before and during lamivudine treatment (not shown). In addition, as we have observed the presence of tetramer-negative cells in chronic HBV patients,[10] we performed direct ex vivo intracellular cytokine staining in patients who demonstrated the presence of IFN-γ producing HBV-specific CD8+ cells after in vitro expansion, but we were consistently unable to detect directly ex vivo envelope and polymerase-specific CD8+ T cells. However, HBV-specific CD8+ cells were not totally deleted as they were demonstrated after in vitro expansion.[9]

The analysis of the HBV-specific CD8 response after in vitro expansion with HBV peptides showed a transient increase of the detection frequency of CD8+ T cells specific for core, envelope and polymerase epitopes during prednisolone therapy (Figs 2 and 3). The detection frequency of envelope and polymerase-specific CD8+ T cells was instead similar before and during lamivudine treatment (Figs 2 and 4), while core-specific CD8+ T cells which were undetectable before therapy in all patients, became detectable in three of the six patients during lamivudine treatment (Figs 2 and 5).

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Figure 2.  (click image to zoom)

Changes of HBV-specific CD8+ responses before and during treatment. (a) Frequency of HBV-specific CD8+ T-cell responses before and during treatment. Bars indicate the frequency of positive core, envelope and polymerase-specific CD8+ responses found in the treated patients. Numbers above bars indicate the number of positive responses out of the number of assays performed. A response was positive when the % of IFN-γ-producing CD8+ cells found in peptide stimulated cell lines minus the % of IFN-γ-producing CD8+ cells found in nonstimulated cell lines was more than 0.05. (b) Frequency of core, envelope and polymerase-specific CD8+ T-cell positive responses within the indicated periods of treatment (before, during prednisolone, week 6-24, week 28-56).
     

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Figure 3.  (click image to zoom)

Prednisolone treatment and pattern of HBV-specific CD8+ T-cell responses. Black bars indicate the % of IFN-γ-producing CD8+ T-cells specific for the different HBV epitopes after 10 days of in vitro expansion with peptides. HBV-DNA levels are given in copies/mL. ALT levels are given in units per litre (U/L). Patients 1, 2, 3 and 4 displayed an elevation of ALT levels after prednisolone treatment. ALT levels did not change in patients 5 and 6.
     

Click to zoom       
Figure 4.  (click image to zoom)

Changes in envelope and polymerase-specific CD8+ T-cell frequencies in the six treated patients. For each patient the figures show: the time course analysis of HBV-DNA and ALT levels (upper graph); the percentage of CD8+ T-cells producing IFN-γ after stimulation with envelope or polymerase peptides (lower graph) and the serological profile of HBeAg (horizontal bar). Upper graph: serum ALT levels (units per litre; U/L white bars); HBV-DNA (copies per millilitre, black squares) are represented on the y-axis and were measured at the indicated time points (x-axis). Lower graph: the percentages of CD8+ T-cells producing IFN-γ after stimulation with envelope peptides (env183-91, env335-43, env338-47, env348-57: white square) or with polymerase peptides (pol455-63, pol502-10, pol575-83, pol655-63, pol816-24; black square) are represented on the y-axes and the x-axes show the number of weeks before and during treatment.

[ 本帖最后由 FORRESTFU 于 2008-5-24 19:47 编辑 ]

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Figure 5.  (click image to zoom)

Changes in Core18-27-specific CD8+ T-cell frequencies in the 6 treated patients. For each patient the figures show: the time course analysis of HBV-DNA and ALT levels (upper graph); the percentages of CD8+ T-cells producing IFN-γ after stimulation with core18-27 peptide (lower graph) and the serological profile of HBeAg (horizontal bar). Upper graph: Serum ALT levels (units per litre; U/L white bars); HBV-DNA (copies per millilitre, black squares) are represented on the y-axis and were measured at the indicated time points (x-axis). Lower graph: the percentages of CD8+ T-cells producing IFN-γ after stimulation with core18-27 peptide (•) are represented on the y-axes and the x-axes show the number of weeks before and during treatment. For clarity, grey boxes are used to indicate the presence of core18-27-specific CD8+ T-cell responses.
     

Steroid Treatment and HBV-specific CD8+ Cell Response

Despite the already high level of HBV-DNA detectable in all patients, prednisolone treatment increased serum HBV-DNA values in four of six patients (patients 1, 3, 4, 6; Fig. 3). Elevation of serum ALT, 4-6 weeks after steroid withdrawal, was detectable in four of the six patients studied and the higher values of ALT elevation (>100 U/L) were detected in the patients with a more consistent increase of their HBV-DNA values (patients 1, 3, 4; Fig. 3).

All four of the patients with elevated ALT levels after prednisolone therapy displayed a recovery of HBV-specific immunity that was temporally associated with steroid therapy (Fig. 3) and not, as hypothesized, with its withdrawal. ALT flares were not associated with an increase of the peripheral HBV-specific CD8+ response, which with the exception of patient 3 reached a maximum during prednisolone therapy (Fig. 3). HBV-specific CD8 cells were also detected during prednisolone in one of the two patients who did not show any subsequent ALT elevation. The HBV-specific CD8+ response during prednisolone treatment was quantitatively minimal. It could be demonstrated only after in vitro expansion in all patients except patient 1. In this patient, an increased ex vivo frequency of core 18-27-specific CD8+ responses was detectable by tetramer staining (Fig. 1). In all other patients, not only tetramer but also intracellular cytokine staining methods failed to detect envelope or polymerase-specific CD8+ cells directly ex vivo (not shown).
HBV-specific CD8+ Response During Lamivudine Treatment: Envelope and Polymerase-Specific CD8+ T-cells

The quantity of envelope and polymerase-specific CD8+ cells observed after in vitro expansion fluctuated during the study with no clear association with HBV-DNA and ALT levels or lamivudine treatment (Fig. 4).

Although the envelope-specific CD8+ T-cell response increased during prednisolone (seven of 24 positive tests, 29%), detection of envelope-specific CD8+ cells during lamivudine treatment, in both the first (weeks 6-24; 12.5% positive tests) and the second (weeks 28-56; 13% positive tests) periods of treatment were identical to the pre-treatment level (12.5% positive tests) (Fig. 2b). A similar pattern was detectable for polymerase-specific CD8+ cells. Not only lamivudine did not restore polymerase-specific CD8+ T-cell response, but detection frequency modestly decreased during treatment (positive tests 5% before therapy vs 1.8% during therapy; Fig. 2b). Although some peptide-specific CD8 + T-cell responses were strong (see, e.g. patients 3, 2 and 5), direct ex vivo analysis of peptide-specific CD8+ T cells displayed negligible frequency (not shown).
Core-specific CD8+ T-cells are Associated With Dynamic Fluctuations of HBV-DNA

A pattern of association between core18-27-specific CD8+ responses and HBV-DNA levels was observed (Figs 2 and 5). Core18-27-specific CD8+ cells were not detectable in any of the patients participating in this study before therapy, either directly ex vivo or after in vitro expansion (0 of 14 tests; Fig. 2b). They were instead detected in three of six patients (15%; 12 positive tests of 80) during treatment (Fig. 2a). The recovery of core18-27-specific CD8+ responses was not associated with stable suppression of HBV replication but rather with a rapid rebound of HBV-DNA levels (Fig. 5). Core18-27-specific CD8+ cells were absent throughout the study in patients 3, 4 and 6 who displayed stable inhibition of HBV-DNA (<105 copies/mL) during lamivudine treatment. In contrast, core18-27-specific CD8+ response recovery was detectable: (i) in patient 1 during prednisolone therapy, immediately after a rapid rise of HBV-DNA levels (from 107 copies/mL at week −4 to 109 copies/mL at week 4, patient 1); (ii) in patient 2 during the rapid rise of HBV-DNA levels because of the selection of a lamivudine-resistant variant (from 105 copies/mL at week 40 to 5 × 106 copies/mL, at week 48); (iii) in patient 5, in a temporal relationship with the rapid fluctuations of HBV-DNA which occurred because of the poor compliance with treatment of this patient. The core18-27-specific CD8+ response became consistently detectable only in patient 1, where HBV-DNA levels decreased to <105 copies/ml and the patient seroconverted to anti-HBe (Fig. 5, patient 1).

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Discussion

Hepatitis B e antigen-positive immunotolerant chronic hepatitis B patients are difficult to treat[2] and their HBV-specific T-cell response is severely compromised.[9] The combination treatment with steroid and lamivudine examined in this study displays a very limited efficacy in inducing HBeAg to anti-HBe seroconversion. Five of the six patients examined returned 1 month after the end of treatment to the profile of HBV infection present at the start (HBeAg positive, with ALT < 80 U/L). In line with the poor clinical response, inhibition of viral replication by lamivudine was not associated in these patients with a sustained restoration of HBV-specific CD8+ responses. These results are apparently in contrast with previous work performed in patients treated with lamivudine alone,[16,17] where inhibition of HBV replication was associated with a recovery of HBV-specific CD8+ T-cell response. However, the patient population analysed here is different from the patients with chronic active hepatitis analysed by Boni et al..[16,17] Chronic active hepatitis B patients have before therapy a higher frequency of HBV-specific CD8+ cells than HBeAg+ 'immunotolerant patients'.[9] Thus the results reported here show that the prolonged and stable inhibition caused by lamivudine is not sufficient to rescue the profound defects of HBV-specific CD8+ T-cell responses displayed by 'immunotolerant' HBeAg+ patients. Only re-activation of HBV replication was temporally linked with a recovery of a virus-specific CD8+ T-cell response and we also unexpectedly observed that prednisolone was associated with a transitory increase of anti-viral specific CD8+ response. As steroid and lamivudine treatments have different mechanisms of action we will discuss the implications of these findings separately.
Prednisolone Treatment: Effect on HBV-specific CD8+ T Cells

Combinations of corticosteroid with a specific anti-HBV agent have historically demonstrated equal or better virological responses than treatment with single antiviral agents.[18] The rationale of this approach, however, was based on very little scientific evidence and on the speculation that the ALT flares occurring afterwithdrawal of steroid were the consequence of a rebound of the HBV-specific T-cell response after suppression by steroids.[11]

Our findings did not support this hypothesis. Although our HBV-specific CD8+ T-cell analysis was limited to only a few HLA-A2 restricted HBV epitopes, we found an increased circulating HBV-specific CD8+ response during and not after prednisolone treatment in all 4 of the patients where ALT elevation was triggered.

The increased frequency of HBV-specific CD8+ T-cells in the circulation (detected directly ex vivo in a single patient - patient 1) could be the result of a simple cell redistribution from lymph nodes to blood and this possibility is supported by the fact that leucocyte counts increased during steroid treatment. However, simple redistribution cannot explain the efficient in vitro expansion and IFN-γ production shown by HBV-specific CD8+ cells during prednisolone treatment. In addition, the most convincing reconstitution of an HBV-specific CD8+ response during prednisolone treatment was associated with clinical and virological efficacy (patient 1). Taken together, these data do not support the idea that prednisolone has a suppressive effect on the HBV-specific T-cell response.

Corticosteroids have a complex interaction with the immune system. Recent investigations have shown that the effect of glucocorticoids on T-cell function may depend on the state of activation and or differentiation of T cells.[19,20] For example, glucocorticoids can suppress apoptosis of effector CD8+ cells[21] and thus increase and not only decrease anti-viral immunity. A further, and not mutually exclusive possibility is that the increased rate of HBV replication directly stimulated by glucocorticoids can result in an HBV-specific CD8+ boost. Increased levels of HBV-DNA were detectable in most of the patients treated.

Irrespective of the causes responsible for the increased detection of HBV-specific CD8+ T-cells during prednisolone, these results might explain the reported clinical efficacy of combination treatment with steroids and IFN-alpha or lamivudine in chronic active hepatitis B patients[13] and of the clinical observations that steroid-free chemotherapy in HBV-carriers with lymphoma decreases the risk of HBV reactivation.[22]
Lamivudine Treatment: Effect of Viral Inhibition on HBV-specific CD8+ Responses

Lamivudine treatment was not associated with a stable restoration of the HBV-specific CD8+ response, which was inferior to that detected during steroid treatment. The frequency of detection and the magnitude of envelope and polymerase-specific CD8+ cell responses was not influenced by treatment, while we observed that the restoration of core18-27 CD8+ T-cells was associated with poor compliance with treatment, selection of lamivudine resistant mutants or steroid treatment. All these events resulted in a rapid rebound of HBV-DNA levels. It can be suggested that core18-27 CD8+ T-cells, which are activated in vitro by a low concentration of peptide (10 pm),[9,23] are the first to perceive the increase in HBV replication and respond to it.

The need for active viral replication in order to sustain the CD8+ T-cell response present in chronic viral infection has been well documented in a recent animal study[24] and also in chronic active hepatitis B patients where a clear decay of the HBV-specific CD8+ response after 5-6 months of lamivudine therapy was observed.[17] The relationship between viral replication and CD8+ responses has recently been mathematically modelled and has revealed that the magnitude of CD8+ expansion is dependent on the rate of viral replication: slowly replicating viruses are particularly inefficient at expanding a virus-specific CD8+ cell population.[25]

The temporal association between the HBV CD8+ T-cell recovery and increasing HBV-DNA levels might suggest that structured, interrupted regimes of anti-viral therapy might rescue in HBeAg+ immune-tolerant patients the HBV immunity, more than stable and prolonged viral inhibition. A therapeutic strategy designed to boost the anti-viral T-cell response by allowing the virus to replicate after active suppression was initially proposed for HIV infection[26] and tested in chronic hepatitis B patients.[27] Recent data by Sarin et al. demonstrate a beneficial effect of an interrupted treatment scheme in HBeAg+ patients with normal ALT levels.[28] This study provides immunological data to support such approaches, even though the minimal degree of restoration of HBV-specific CD8+ immunity that we observed in patients after HBV-DNA reactivation is a warning that these schemes may have a limited efficacy.

特深沉

我记得hiv宿主是CD4 T Cell

日月同辉81

能不能翻译一下
看不懂

FORRESTFU

这篇文章虽然是06年的，但今天看到后还是立即贴上来了。

本来不想说什么，反正每个人从不同的角度去看就会有不同的感触。

这里做个小提醒，即目前医学界对血清HBVDNA的认知程度仍保持着惊人的浅薄和愚蠢记录，他们对持续和强力抑制HBVDNA的热情不但没有因为免疫生物方面研究的推进而有丝毫消退，反而越来越表现出临床与研究成果的脱离。

对持续和强力抑制血清HBVDNA的空前高涨的热情，几乎可以与当年滥用抗生素的热情媲美。

jjwwhbv3

ni你好可怜，你完全\不懂医学

走遍四方

原帖由 jjwwhbv3 于 2008-5-25 11:53 发表
ni你好可怜，你完全\不懂医学

please解释得清楚一点