We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.
SlllllmsryTo explore mechanisms that prevent autoreactivity in nonautoimmune mice, endogenous immunoglobulin (Ig) light (L) chains that associate with a transgenic anti-DNA heavy chain were analyzed. The antibodies from splenic B cell hybridomas of such mice did not bind doublestranded DNA (dsDNA) and their L chain sequences showed a biased use of V~ and J~ gene segments. The 44 L chains in this survey were coded for by just 18 germline genes. Six of the genes, each belonging to a different V~ group, were used more than once and accounted for three fourths of all sequences. Based on the distribution of V~ genes, the L chain repertoire in this line of transgenic mice was estimated at 37 V~ genes. The most frequently observed gene, a member of the V~ 12/13 group, was identified in 16 hybrids. In addition, the majority of V~ genes used J~5. We interpret the skewed representation of V~ and J~ gene segments to result from negative selection. Based on the data, we suggest that V~ rearrangements giving rise to anti-dsDNA reactivity are removed from the repertoire by a corrective mechanism capable of editing self-reactive Ig.T olerance to self has been studied in Ig transgenic models of autoreactivity. By using Ig transgenes against facultative self-antigens, it has been shown that self-reactive B cells are selected against by anergy or deletion (1, 2). We have constructed mice with transgenes that code for anti-DNA antibodies and obtained similar results. The majority of splenic B lymphocytes from mice with the V,3H9 and V~8 genes express anti-single-stranded DNA (ssDNA) 1 antibodies on their surface, but we do not detect them in the serum (3). By analogy to Goodnow et al. (1), anti-ssDNA B cells in those mice appear to be anergic.Analysis of splenic B cells from mice containing just the V,3H9 transgene suggested a second feature of tolerance to DNA. No anti-double-stranded DNA (dsDNA) activity was detected among hybridomas from these mice. This was surprising, since previous studies have established that the 3H9 H chain can combine with a diverse range of L chains to yield antibodies capable of binding to both ssDNA and dsDNA (4, 5). The absence of hybrids producing anti-dsDNA suggested that dsDNA-spedfic B cells are functionally deleted.Nevertheless, V.3H9 mice have near normal numbers of splenic B cells, and hybridomas can be readily obtained (3). Some of the antibodies produced by these hybridomas bound ssDNA, whereas others did not bind DNA at all. We have now examined the V~ and J~ gene segment use of hybrids obtained from two of these mice. The mAbs were characterized by a sharply reduced repertoire of L chains. L chains that are found among spontaneous anti-DNA antibodies from MLR/lpr (4) or NZB x NZW (6) mice were absent from our sample. In addition, J~5 was overutilized. The data suggested that the major driving force shaping the L chain use in V.3H9 mice is selection against dsDNA-binding B cells. Furthermore, the data are consistent with the notion that B cells can escape deletion if their autoreactive surf...
Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl--L-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of ϳ33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.
The existence of transiently open states in DNA and synthetic polynucleotide double helices as been demonstrated by hydrogen exchange measurements; base pairs reversibly separate and reclose, exposing nucleotide protons to exchange with solvent protons. Recently it has been possible to define the equilibrium kinetic, and activation parameters of the major open state tdat determines base pair hydrogen exchange. However, there is no direct information at the moment about the conformation of the open form. Here we consider the possibility that the low energy and slow opening and closing rates observed reflect a deformation involving several adjacent base pairs. Assuming a mobile open unit capable of diffusing along the double helix, we find that available data are consistent with structures of 10 or so adjacent open pairs. It is further suggested that these structures correspond to thermally induced so on excitations of the double helix, which retain coherence by sharing the energy of a twist deformation among several base tairs. Solitons
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