Monoclonal anti-DNA Antibodies Research Articles

Internalization of Anti-DNA Antibodies by Rat Brain Cells: A Possible Pathogenetic Mechanism of Neuropsychiatric Lupus

Accumulating evidence suggests that DNA-anti-DNA antibody immune complexes are endocytosed by immune cells, where they ligate intracellular nucleic acid sensors. This results in production of type I interferon or other cytokines, which may be a significant pathogenetic mechanism in systemic lupus erythematosus (SLE). Most studies thus far have been performed using cell-lines or peripheral blood mononuclear cells, but it remains unclear whether cells from the brain can take up such immune complexes. Because permeability of the blood-brain interfaces is suggested to be increased and autoantibodies are detected in cerebrospinal fluid of SLE patients with neuropsychiatric manifestations, we hypothesize that DNA-anti-DNA immune complexes may be internalized by cells in the central nervous system and cause dysregulation of neural networks. As a first step to test this hypothesis, here we investigated whether anti-DNA antibodies can be internalized by live brain cells in vitro. Primary culture rat astrocytes were incubated for 1 h at 37°C with or without anti-DNA monoclonal antibodies. After washing, fixation, permeabilization and blocking, internalized anti-DNA antibodies were detected by a fluorescent labeled second antibody. We observed that a dsDNA-specific monoclonal antibody entered the nucleus, while a monoclonal antibody cross-reactive with phospholipid and DNA entered the cytoplasm. Isotype-matched control IgG did not enter the cells, suggesting that they were viable and the cell membrane and nuclear membrane were intact at that time. When the cells were incubated at 4°C, internalization of the anti-DNA antibodies was almost completely abolished. This suggests that antibody internalization was not by passive transfer but is an energy-dependent process. Given that astrocytes play indispensable roles in maintaining the function of neural networks, these results may provide a novel perspective on the pathogenetic mechanisms of the neuropsychiatric manifestations often presented in SLE.

Variable Heavy Chain Domain Derived from a Cell-Penetrating Anti-DNA Monoclonal Antibody for the Intracellular Delivery of Biomolecules

ABSTRACTTargeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA.

Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20-40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ~2.2 × 10-3 s-1, the transition state distance was ~0.94 nm, the apparent on-rate was ~5.26 s-1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies.

An anti-DNA antibody prefers damaged dsDNA over native

DNA–protein interactions, including DNA–antibody complexes, have both fundamental and practical significance. In particular, antibodies against double-stranded DNA play an important role in the pathogenesis of autoimmune diseases. Elucidation of structural mechanisms of an antigen recognition and interaction of anti-DNA antibodies provides a basis for understanding the role of DNA-containing immune complexes in human pathologies and for new treatments. Here we used Molecular Dynamic simulations of bimolecular complexes of a segment of dsDNA with a monoclonal anti-DNA antibody’s Fab-fragment to obtain detailed structural and physical characteristics of the dynamic intermolecular interactions. Using a computationally modified crystal structure of a Fab–DNA complex (PDB: 3VW3), we studied in silico equilibrium Molecular Dynamics of the Fab-fragment associated with two homologous dsDNA fragments, containing or not containing dimerized thymine, a product of DNA photodamage. The Fab-fragment interactions with the thymine dimer-containing DNA was thermodynamically more stable than with the native DNA. The amino acid residues constituting a paratope and the complementary nucleotide epitopes for both Fab–DNA constructs were identified. Stacking and electrostatic interactions were shown to play the main role in the antibody–dsDNA contacts, while hydrogen bonds were less significant. The aggregate of data show that the chemically modified dsDNA (containing a covalent thymine dimer) has a higher affinity toward the antibody and forms a stronger immune complex. These findings provide a mechanistic insight into formation and properties of the pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus, associated with skin photosensibilization and DNA photodamage.

Combining intracellular antibodies to restore function of mutated p53 in cancer.

TP53 is a tumor suppressor gene that is mutated in 50% of cancers, and its function is tightly regulated by the E3 ligase, Mdm2. Both p53 and Mdm2 are localized in the cell nucleus, a site that is impervious to therapeutic regulation by most antibodies. We identified a cell-penetrating lupus monoclonal anti-DNA antibody, mAb 3E10, that targets the nucleus, and we engineered mAb 3E10 to function as an intranuclear transport system to deliver therapeutic antibodies into the nucleus as bispecific single chain Fv (scFv) fragments. Bispecific scFvs composed of 3E10 include PAb421 (3E10-PAb421) that binds p53 and restores the function of mutated p53, and 3G5 (3E10-3G5) that binds Mdm2 and prevents destruction of p53 by Mdm2. We documented the therapeutic efficacy of these bispecific scFvs separately in previous studies. In this study, we show that combination therapy with 3E10-PAb421 and 3E10-3G5 augments growth inhibition of cells with p53 mutations compared to the effect of either antibody alone. By contrast, no enhanced response was observed in cells with wild-type p53 or in cells homozygous null for p53.

The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

Functional Consequences of Complementarity-determining Region Deactivation in a Multifunctional Anti-nucleic Acid Antibody

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering.

Microparticles as autoantigens in human and murine lupus

Systemic lupus erythematosus is a systemic inflammatory disease characterized by anti-DNA production in association with immune complex deposition. These complexes can deposit in the tissue to incite inflammation as well as stimulate cytokine production by plasmacytoid dendritic cells. While the properties of anti-DNA antibodies have been extensively characterized, little is known about the nature of the DNA in the immune complexes although its origin is generally considered to be cell death. Among sources of extracellular DNA, microparticles (MPs) are small membrane-bound vesicles released from activated and dying cells by a blebbing process. As shown by flow cytometry, these particles display DNA as well as other nuclear molecules in an antigenically accessible form as indicated by the binding of monoclonal anti-DNA antibodies as well as plasma from patients with lupus. Furthermore, plasma from patients with lupus contains increased number of particles with bound IgG, suggesting that MPs can form immune complexes found in lupus. To assess whether MPs play a similar role in murine lupus, we used flow cytometry to measure the presence of MPs with bound IgG in the blood of MRL-lpr/lpr and NZB/W mice; in addition, we tested the binding of plasma of these mice to MPs from apoptotic cells. The results of these studies showed important differences in the serological findings of the two strains as reflected by the much higher numbers of MPs with bound IgG in the blood of MRL-lpr/lpr compared with NZB/W mice. These studies also showed that antibodies from MRL-lpr/lpr mice bound much better to MPs from apoptotic cells than those from NZB/W mice. Since particles in NZB/W blood bound to monoclonal anti-nuclear antibodies as well as MRL-lpr/lpr plasma, these findings indicate antigenic activity despite the lack of particle IgG complexes in NZB/W blood. Together, these studies indicate important differences in the serological features of the two strains as reflected by the capacity of antibodies to bind to MPs. These differences may impact on the process of immune complex formation and its consequences as well as the respective role of anti-DNA and other autoantibodies in nephritis in NZB/W mice.

The relevance of antigen binding to the pathogenicity of lupus autoantibodies

Although lupus autoantibodies provide diagnostic value, discordance between serum levels and nephritis poses mechanistic questions. Krishnan and co-workers report that only those that crossreact with basement membrane components produce immune deposits. Thus, other glomerular binding properties probably define where deposits form. Thereafter, Fc- and complement-mediated events influence disease expression. Clearly other factors determine the ultimate phenotype; however, the findings provide insights into the variable disease patterns in lupus nephritis.

IgM anti-DNA antibody and C1q Immune complexes regulate the production of Interferon-α in plasmacytoid dendritic cells (159.26)

Abstract Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the loss of tolerance to autoantigens, and to DNA in particular. Although Immune complexes (ICs) of IgG anti-DNA antibodies are pathogenic, IgM anti-DNA antibodies in SLE are protective. The mechanism responsible for the protective effect of IgM anti-DNA antibody is unclear. We found that a monoclonal IgM anti-DNA antibody cooperated with C1q to suppress interferon (IFN)-α production by plasmacytoid DCs (pDCs). We tested the effects of the antibody on bone marrow-derived pDCs from wild type (WT) and C1q knockout (KO) mice. Interestingly, the absence of C1q significantly enhanced the production of IFN-α induced by IgM anti-DNA antibody. The inhibitory effect of C1q was confirmed using in vivo generated pDCs or in vitro generated pDCs under serum-free conditions. Exogenous C1q suppressed IFN-α transcription in both WT and C1q KO pDCs. Moreover, we identified leukocyte-associated inhibitory receptor (LAIR)-1 as a potential receptor for C1q. Fluorescence labeled C1q strongly bound to pDCs which were highly expressing LAIR-1. LAIR-1 is a collagen receptor containing two ITIMs; its cross-linking with antibody inhibits IFN-a production by pDCs stimulated with DNA containing ICs. In summary, protective IgM anti-DNA antibody/C1q complexes inhibit unwanted IFN-α production through binding to LAIR-1.

Microparticles as antigenic targets in human and murine SLE

Microparticles are small membrane-bound vesicles that are released from activated and dying cells by a blebbing process. These particles circulate in the blood and display potent pro-inflammatory and pro-thrombotic activities. In addition, particles are an important source of extracellular DNA and RNA and may participate in the transfer of informational nucleic acids. Because microparticles contain DNA as well as other nuclear antigens, we have investigated their ability to bind to anti-DNA and other anti-nuclesome antibodies that characterize the prototypic autoimmune disease systemic lupus erythematosus (SLE). For this purpose, we generated microparticles from HL-60, Jurkat and THP-1 cells induced to undergo apoptosis in vitro. Using FACS analysis to assess antibody binding, we showed that particles can bind some but not all monoclonal anti-DNA and anti-nucleosome antibodies from MRL-lpr/lpr and NZB/NZWF1 lupus mice. For the monoclonal anti-DNA, DNase treatment reduced binding. Like the monoclonal antibodies, patient plasma also bound to the particles although this activity was not directly correlated with levels of anti-DNA antibodies as measured by an ELISA. To determine whether particles circulating in the blood of patients can represent immune complexes, FACS analysis was performed on particles isolated from patient plasma. These studies indicated that, while the total levels of microparticles in the blood of patients with SLE did not differ significantly from those of normal controls, the number of IgG-positive particles was significantly elevated using a R-phycoerythrin-labeled anti-human IgG (γ-chain specific) reagent. In this study, the number of IgG-positive particles was correlated with levels of anti-DNA. In similar studies with plasma from MRL-lpr/lpr and NZB/NZWF1 mice, we showed that the total levels of particles were increased compared to those of BALB/c control mice and that the number of particles that stained with an anti-IgG reagent was also increased. Furthermore, plasma of mice could bind to particles generated in vitro from apoptotic cells. Together, these findings indicate that microparticles can express antigenically active DNA in an accessible form, either because of a surface location or particle permeability. Furthermore, they demonstrate that microparticles can form immune complexes and that at least some of the immune complexes in the blood in SLE contain particles. Current studies are characterizing the immune properties of these complexes and their potential role in pathogenicity.

Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus

Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.

Identification of a Lipid Peroxidation Product as the Source of Oxidation-specific Epitopes Recognized by Anti-DNA Autoantibodies*

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including systemic lupus erythematosus (SLE). We have found that bovine serum albumin incubated with peroxidized polyunsaturated fatty acids significantly cross-reacted with the sera from MRL-lpr mice, a representative murine model of SLE. To identify the active substances responsible for the generation of autoantigenic epitopes recognized by the SLE sera, we performed the activity-guiding separation of a principal source from 13-hydroperoxy-9Z,11E-octadecadienoic acid and identified 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as the source of the autoantigenic epitopes. When the age-dependent change in the antibody titer against the ONE-modified protein was measured in the sera from MRL-lpr mice and control MRL-MpJ mice, all of the MRL-lpr mice developed an anti-ONE titer, which was comparable with the anti-DNA titer. Strikingly, a subset of the anti-DNA monoclonal antibodies generated from the SLE mice showing recognition specificity toward DNA cross-reacted with the ONE-specific epitopes. Furthermore, these dual-specific antibodies rapidly bound and internalized into living cells. These findings raised the possibility that the enhanced lipid peroxidation followed by the generation of ONE may be involved in the pathogenesis of autoimmune disorders.

The clinical significance of decreased T cell interleukin-2 production in systemic lupus erythematosus: Connecting historical dots

Although decreased T cell production of interleukin-2 (IL-2) in systemic lupus erythematosus (SLE) was first described more than 25 years ago, questions concerning the significance of this observation and the causative mechanisms remain unresolved. IL-2 is now known to be a critical cytokine for the maintenance of immunologic homeostasis and tolerance to self antigens. Whether the production defect precedes SLE, is secondary to it, or reflects a compensatory attempt to reduce disease activity is not well understood. The article by Song et al, which is published in this issue of Arthritis & Rheumatism, addresses this issue and presents evidence of an acquired T cell defect that is a consequence of the disease (1). These investigators have previously reported that anti–double-stranded DNA (anti-dsDNA) antibodies can penetrate T cells and cause decreased production of IL-2. They now report the mechanism of this effect. Here, I briefly review the principal effects of IL-2 on the immune system, comment on the studies by Song et al in the context of previous studies on IL-2 production in SLE, and conclude with the view that both humoral and cellular mechanisms contribute to an acquired, ineffective, compensatory attempt to control pathologic T cell and B cell hyperactivity in active SLE. IL-2 is a multifunctional cytokine that was first described as a T cell growth factor. Decreased IL-2 production results in various T cell defects, which include impaired cell-mediated immunity associated with enhanced susceptibility to infections, reduced activationinduced cell death, and prolonged survival of autoreactive T cells. Insufficient IL-2 also results in loss of tolerance to self antigens. Tolerance is maintained by regulatory T cells that express the forkhead transcription factor FoxP3. IL-2 is essential for the generation and survival of CD4 FoxP3 regulatory cells that control potentially pathogenic self-reactive T cells (2). Normally, IL-2 produced by activated T cells expands and sustains the Treg cells, which maintain normal immune homeostasis. IL-2 signaling up-regulates STAT-5, two proteins (STAT-5A and STAT-5B) which enhance Treg generation and suppress Th17 differentiation (3), events that are critical in blocking the pathogenesis of lupus nephritis. Thus, it is probably not a coincidence that both decreased IL-2 production and T regulatory dysfunction are observed in SLE (4). Decreased IL-2 production strongly contributes to the generation of T cell– dependent pathogenic antinuclear antibodies and the subsequent development of clinical SLE. The pathogenicity of anti-DNA antibodies is associated with their polyreactivity with diverse antigens. When anti-DNA antibodies bind extracellular antigens directly or in the form of immune complexes, they can induce inflammatory cascades. Convincing evidence now exists that they can also penetrate living cells and alter function. In 1978, Alarcon-Segovia and coworkers reported that anti-DNA and anti-RNP antibodies could enter cells and interact with their intracellular target (5). Although at first, this was a subject of controversy, this work has been confirmed by several groups of investigators who documented that these antibodies can either enhance cell growth and proliferation or induce death and apoptosis, as described in the review by Putterman (6). Sun and colleagues have shown that charged monoclonal anti-DNA antibodies derived from MRL-lpr/lpr mice could penetrate living cells by electrostatic interactions and induce human T cells to produce several proinflammatory cytokines, including IL-1 , tumor necrosis factor , and IL-6 (7). Importantly, these antibodies also inhibited the production of IL-2. In this issue of Arthritis & Rheumatism, the latest contribution from Sun’s group, the report by Song et al, David A. Horwitz, MD: University of Southern California, Los Angeles. Address correspondence and reprint requests to David A. Horwitz, MD, Division of Rheumatology, Department of Medicine, University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, HMR 711, Los Angeles, CA 90033. E-mail: dhorwitz@usc.edu. Submitted for publication April 20, 2010; accepted April 20, 2010.

A New Model of Induced Experimental Systemic Lupus Erythematosus (SLE) in Pigs and Its Amelioration by Treatment with a Tolerogenic Peptide

Systemic lupus erythematosus (SLE) is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, hCDR1, ameliorated lupus manifestations in mice models. The objectives of this study were to induce experimental SLE in pigs and to determine the ability of hCDR1 to immunomodulate the disease manifestations. We report here the successful induction, by a monoclonal anti-DNA antibody, of an SLE-like disease in pigs, manifested by autoantibody production and glomerular immune complex deposits. Treatment of pigs with hCDR1 ameliorated the lupus-related manifestations. Furthermore, the treatment downregulated the gene expression of the pathogenic cytokines, interleukin (IL)-1beta, tumor necrosis factor alpha, interferon gamma, and IL-10, and upregulated the expression of the immunosuppressive cytokine transforming growth factor beta, the antiapoptotic molecule Bcl-xL, and the suppressive master gene, Foxp3, hence restoring the expression of the latter to normal levels. Thus, hCDR1 is capable of ameliorating lupus in large animals and is a potential candidate for the treatment of SLE patients.

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

A human anti-dsDNA monoclonal antibody caused hyaline thrombi formation in kidneys of 'leaky' SCID mice.

There are few studies assessing the pathogenicity of human monoclonal anti-DNA antibodies. The use of SCID mice avoids the problem of rejection of the human hybridoma cells thus allowing in vivo assessment of human immunoglobulins. Using electron microscopy we have shown that the human IgG anti-dsDNA monoclonal antibody, RH14, is nephritogenic in SCID mice, causing morphological changes in the kidney due to immunoglobulin deposition. The problem with using SCID mice is that they have an abnormal immune system; normally they are used at about 2 months of age, at which time they have virtually no functional T or B cells. It is known that older SCID mice become increasingly 'leaky', that is they develop some mature lymphocyte clones. Our aim was to assess if implanting anti-DNA antibodies into older 'leaky' SCID mice would result in pathology which was observable by light microscopy. Eight-month-old SCID mice were implanted with human hybridoma cells secreting either RH14 an anti-dsDNA IgG, CL24, an antiphospholipid antibody or an irrelevant human IgG control. As previously, RH14 deposited in the kidney and caused proteinuria but unexpectedly we also observed hyaline thrombi in the kidney glomeruli and peritubular capillaries. These thrombi occurred only in the case of RH14 implanted mice and were found to stain positively for human IgG and fibrin. However, apart from the interesting thrombi, we did not observe any greater pathological damage resulting from the anti-dsDNA antibody deposition than we had seen in the younger mice; indeed, the electron microscopic findings were more limited.

Detection of DNA-bound immunoglobulins in patients with lupus nephritis, using monoclonal anti-DNA antibody

In order to investigate the relationship between renal histopathology and the characteristics of circulating immune complexes (CICs) in patients with lupus nephritis (LN), we measured the sizes of CICs, DNA-bound immunoglobulins in patients with systemic lupus erythematosus (SLE) and different histopathological forms of nephritis. Sera were obtained from nine patients: four with diffuse proliferative LN (DPLN), four with membranous LN (MLN), and one with mesangial LN, who fulfilled the criteria of the American Rheumatism Association for SLE. The DNA-bound immunoglobulins were measured by ELISA, in which ELISA plates were coated with mouse monoclonal anti-DNA antibodies. The sizes of CICs were analysed by sucrose density gradient ultracentrifugation. Large (larger than 19S), intermediate (19-7S) and small (nearly 7S) sized DNA-bound immunoglobulins (high peaks of IgG and IgA, but low IgM peaks) were found in the patients with DPLN. By contrast, in patients with MLN, the sizes of ICs; DNA-bound IgG, IgA were in general slightly larger than 7S. In one patient with DPLN, at the onset, various sized DNA-bound IgG, IgA and IgM were found. After the methylprednisolone pulse therapy, CICs became smaller and gradually disappeared. We conclude that the characteristics of DNA-anti-DNA IgG, IgA complexes may determine the localization of ICs in the glomeruli and suggest that CICs play an important role in the pathogenesis of LN.

Inhibition of GSK3 Promotes Replication and Survival of Pancreatic Beta Cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Visualization of RecA Filaments and DNA by Fluorescence Microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.