Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in <1% of the viral quasispecies may still allow >1000-fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment-naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT-C) in the population, we analyzed HCV genome sequences from 507 treatment-naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication-competent, drug-resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN-191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG-021541; and to the NS4A antagonist ACH-806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug-resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo. Conclusion: Naturally occurring dominant STAT-C resistance mutations are common in treatment-naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin.

SummaryDisease activity in systemic lupus erythematosus is closely associated with the appearance of immunoglobulin (Ig)G antibody to native DNA in both humans and mice. Like normal antibody responses, the anti-DNA autoantibody first appears as IgM and then switches to IgG. Structural studies of IgG anti-DNA suggest that these antibodies are the products of clonally selected, specifically stimulated B cells. The origins of the IgM anti-DNA have been less clear. To determine whether the earlier appearing IgM anti-DNA antibody in autoimmune mice also derives from clonally selected, specifically stimulated B cells or B cells activated by nonselective, polyclonal stimuli, we have analyzed the molecular and serological characteristics of a large number of monoclonal IgM anti-DNA antibodies from autoimmune (NZB x NZW)F1 mice. We have also analyzed IgM and IgG anti-DNA hybridomas obtained from the same individual mice to determine how the later-appearing IgG autoantibody may be related to the earlier-appearing IgM autoantibody within an individual mouse. The results demonstrate that: (a) IgM anti-DNA, like IgG, has the characteristics of a specifically stimulated antibody; (b) IgM and IgG anti-DNA antibodies have similar variable region structures and within individual mice may be produced by B cells derived from the same clonal precursors; (c) recurrent germline and somatically derived VH and VL structures may influence the specificity of anti-DNA monoclonal antibody for denatured vs. native DNA; and (d) the results provide a structural explanation for the selective development of IgG antibody to native DNA as autoimmunity to DNA progresses in (NZB x NZW)F1 mice.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, andXenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes.However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.Numerous recent studies (2,7,8,10,34,49,54) have shown that one of the major heat shock proteins, hsp70, functions in an ATP-dependent manner through transient interactions to mediate folding or unfolding of polypeptide chains. Another major heat shock protein, hsp90, is thought to perhaps also function in some capacity related to folding or protein-protein interactions, but its function(s) remains poorly defined (1). Supporting its potential role in protein folding is a recent demonstration that hsp90 enhances renaturation of some proteins in vitro (52). Perhaps the most widely studied interaction of hsp90 is its identity as a stable component of several unactivated steroid receptor complexes (6, 38, 41). For glucocorticoid receptors, hsp90 binding to receptor is required to maintain high-affinity ligand binding (3,26,40), but other steroid receptors that have been examined do not show this same dependency on hsp90. In all hsp90-nuclear receptor complexes, ligand-dependent activation of the receptor DNA-binding ability is accompanied by dissociation of hsp90 (14,17,31,43), and it appears likely that one hsp90 function is to repress DNA binding by receptor.Steroid receptor-hsp9O interactions provide a model for understanding hsp90 function, but exploiting this model has been hindered by the inability to reversibly assemble receptor-hsp90 complexes in vitro. This drawback was recently overcome by establishing certain physicochemical conditions that permit the use of rabbit reticulocyte...

Apoptotic cells are considered the source of DNA, histones, and nucleoprotein complexes that drive the production of autoantibodies in systemic lupus erythematosus. However, the role of apoptotic cells in the activation of the immune system is not clear. To explore interactions that may initiate or sustain the production of anti-nuclear autoantibodies, we characterized the binding of a large panel of monoclonal autoantibodies to apoptotic cells. Autoantibodies to DNA, individual core histones, histone-DNA complexes, or the native nucleosome core particle revealed a consistent and specific binding pattern in confocal microscopy. Immunoreactive epitopes were detected in the cytoplasm and accumulated along the surface of the fragmenting nucleus in a caspase-dependent manner. Ag-Ab complexes on nuclear fragments that had emerged from the plasma membrane were accessible to anti-isotype-reactive microparticles. Moreover, autoantibodies specific for the nucleosome core or its molecular components selectively precipitated a complex of core histones and DNA from the cytosol at 4 h after induction of apoptosis. These observations identify distinct steps in the release of nucleosomes from the nucleus and their exposure at the cell surface. Furthermore, the results indicate a direct role for nucleosomes in the execution of apoptosis, clearance of apoptotic cells, and regulation of anti-nuclear autoantibody production.

The failure of anti-CD20 antibody (Rituximab) as therapy for lupus may be attributed to the transient and incomplete B cell depletion achieved in clinical trials. Here, using an alternative approach, we report that complete and sustained CD19+ B cell depletion is a highly effective therapy in lupus models. CD8+ T cells expressing CD19-targeted chimeric antigen receptors (CARs) persistently depleted CD19+ B cells, eliminated autoantibody production, reversed disease manifestations in target organs, and extended life spans well beyond normal in the (NZB × NZW) F1 and MRLfas/fas mouse models of lupus. CAR T cells were active for 1 year in vivo and were enriched in the CD44+CD62L+ T cell subset. Adoptively transferred splenic T cells from CAR T cell–treated mice depleted CD19+ B cells and reduced disease in naive autoimmune mice, indicating that disease control was cell-mediated. Sustained B cell depletion with CD19-targeted CAR T cell immunotherapy is a stable and effective strategy to treat murine lupus, and its effectiveness should be explored in clinical trials for lupus.