Primary biliary cirrhosis (PBC) is an autoimmune disease of liver associated with a unique serologic response to mitochondrial autoantigens. Many of the autoantigens recognized by autoantibodies in PBC are members of the 2-oxo-acid dehydrogenase complex. The two major autoantigens are the E2 component of the pyruvate dehydrogenase complex (PDC-E2) and the E2 component of the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2). The autoantibody response to PDC-E2 has been mapped to one immunodominant epitope, which consists of both linear and conformational components. The presence of a single immunodominant epitope in PDC-E2 is unusual when contrasted to the immune response to autoantigens in other human autoimmune diseases. We have mapped the epitope recognized by antimitochondrial autoantibodies (AMA) specific to BCOADC-E2 in patients with PBC by taking advantage of the full-length bovine BCOADC-E2 complementary DNA (cDNA) and a series of expression clones spanning the entire molecule. Reactivity to the various expression clones was studied by immunoblotting, enzyme-linked immunosorbent assay (ELISA), as well as selective absorption of patient sera by expressed protein fragments. Autoantibodies to BCOADC-E2 map within peptides spanning amino acid residues 1 to 227 of the mature protein; our data demonstrate that the epitope is dependent on conformation and includes the lipoic acid binding region. However, only the full-length clone (amino acid residue 1 to 421) is sufficient to remove all detectable BCOADC-E2 reactivity. Moreover, the absence of lipoic acid on the recombinant polypeptides used in this study indicates that antibody binding to BCOADC-E2 is not dependent on the presence of lipoic acid.

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized histologically by nonsuppurative destructive cholangitis. Sera from patients with PBC react with a series of intramitochondrial enzymes with the immunodominant response directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Recently, using tissue sections of late-stage PBC, we showed that there is increased expression in biliary epithelial cells of patients with PDC-E2 or a molecule cross-reactive with PDC-E2. Previous work has shown that biliary epithelial cells of patients with PBC express an increased amount of class II. To address the sequence of events in the evolution of PBC, we have focused our attention in this study on early biliary epithelial lesions. In particular, we have studied the liver of 22 female patients with PBC that was diagnosed as either stage I or stage II using both a mouse monoclonal antibody that has reactivity similar to human autoantibodies as well as a human Fab combinatorial prepared from the lymph node of a PBC patient. Tissues were simultaneously stained using antibodies to PDC-E2, class II, and BB1/B7. As a positive control, tissues from late-stage PBC were studied concurrently. By determining the order of expression among the three molecules, PDC-E2, class II, and BB1/B7, we report that the expression of PDC-E2 or a PDC-E2-like molecule on biliary duct epithelium of patients with PBC precedes the expression of BB1/B7 and major histocompatibility complex (MHC) class II molecules. The alteration of an autoantigen in biliary duct epithelium may be the earliest lesion in PBC.

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other.Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids.Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.

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Although cloud computing has emerged as a promising future computing model, security concerns due to malicious tenants have been deterring its fast adoption. In cloud computing, multiple tenants may share physical systems by using virtualization techniques. In such a virtualized system, a software hypervisor creates virtual machines (VMs) from the physical system, and provides each user with an isolated VM. However, the hypervisor, with a full control over hardware resources, can access the memory pages of guest VMs without any restriction. By compromising the hypervisor, a malicious user can access the memory contents of the VMs used by other users.In this paper, we propose a hardware-based mechanism to protect the memory of guest VMs from unauthorized accesses, even with an untrusted hypervisor. With this mechanism, memory isolation is provided by the secure hardware, which is much less vulnerable than the software hypervisor. The proposed mechanism extends the current hardware support for memory virtualization with a small extra hardware cost. The hypervisor can still flexibly allocate physical memory pages to virtual machines for efficient resource management. However, the hypervisor can update nested page tables only through the secure hardware mechanism, which verifies each mapping change. Using the hardware-oriented mechanism in each system securing guest VMs under a vulnerable hypervisor, this paper also proposes a cloud system architecture, which supports the authenticated launch and migration of guest VMs.

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