A peptide DNA surrogate that binds and inhibits anti-dsDNA antibodies

Autoantibodies to dsDNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Although the anti-dsDNA antibodies present in SLE are indicative of an antigen-driven response, the antigen has not been conclusively identified. The specific SLE anti-dsDNA antibodies were obtained by affinity purification using a dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide display library, we demonstrated that purified polyclonal anti-dsDNA antibodies and a monoclonal anti-dsDNA antibody specifically bind a 15 mer peptide ASPVTARVLWKASHV. This chemically synthesized peptide could be recognized by anti-dsDNA antibodies in ELISA and Dot blot. This 15 mer peptide can inhibit anti-dsDNA antibodies binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay while a control peptide did not inhibit anti-dsDNA antibodies. This study demonstrates the potential usefulness of the peptide DNA surrogate in diagnostic tests of SLE and in the investigation of the origin of anti-dsDNA antibodies. It may also be used in studies of the DNA-anti-DNA antibody interaction.     

Quantitative determination of anti-dsDNA antibodies and antibody/dsDNA stoichiometries in prepared,soluble complement-fixing antibody/dsDNA immune complexes

We have investigated quantitatively the complement-mediated binding of prepared, soluble ¹²⁵I-7S IgG antibody/³H-dsDNA immune complexes to human red blood cells (RBCs). We have performed these studies by using a detailed modification of the RBC-CF assay [Pedersen et al., J. Immun. Meth. 38, 2692–2280 (1980)] which now allows for the simultaneous measurement of both ³H-DNA and ¹²⁵I-binding to the cells. Our results indicate that, in the case of three SLE patients, their anti-dsDNA antibody titers are sufficiently high that a small fraction of their ¹²⁵I-7S IgG antibodies (ca 0.1–0.2%) can be identified as specifically anti-dsDNA. We have also used an indirect method (with ¹²⁵I-labelled rabbit anti-human IgG) for the determination of IgG anti-dsDNA antibodies in complement-fixing antibody/dsDNA immune complexes that bind to RBCs, and the results of these measurements are in reasonable agreement with the direct binding experiments. These studies have also allowed us to estimate the antibody/DNA stoichiometries in complement-fixing immune complexes. The results of these experiments may provide a useful standard for the analysis of monoclonal anti-dsDNA antibodies.     

The Trypanosoma lewisi immunofluorescence test: A new simple technique for simultaneous determination of total antinuclear antibodies and the detection of antibodies to double-stranded DNA

An indirect immunofluorescence technique was developed for the detection of antibodies to dsDNA and the simultaneous assessment of antinuclear antibodies ‘in toto’ (ANA). This assay was based upon the use as substrate of smears of peripheral blood derived from rats infected with Trypanosoma lewisi. T. lewisi possesses a giant kinetoplast posteriorly to the nucleus. Enzyme digestion and absorption experiments provided strong evidence that T. lewisi kinetoplast contains dsDNA uncontaminated by other nuclear antigens. The T. lewisi immunofluorescent test was evaluated on a total of 130 sera (30 from patients with SLE) and compared with radioimmunoassays for antibodies to dsDNA ([¹²⁵I]dsDNA-RIA) and antibodies to ssDNA ([¹²⁵I]ssDNA-RIA). Excellent correlation was found between kinetoplast immunofluorescence and [¹²⁵I]dsDNA-RIA, whereas no non-SLE sera showing significant ssDNA binding activity gave kinetoplast staining. With a single exception, only SLE sera reacted with T. lewisi kinetoplast. Sera containing auto-antibodies other than ANA did not induce fluorescene of any part of the parasite, including the flagellum and its base. These results indicated that the T. lewisi immunofluorescence test is specific and reliablem and combines the advantages of Crithidia luciliae with those of Trypanosoma gambiense. It may be used routinely for evaluating of total ANA and simultaneous detection of antibodies against dsDNA.     

Systemic Lupus Erythematosus and Related Diseases

NZB (H-2^d) mice are well known for the production of IgM autoantibodies to ssDNA. However, an FI cross between NZB and either NZW or SWR mice is required to produce IgG nephritogenic antibodies to dsDNA and glomerulonephritis. The contribution of parental class II loci in the hybrid mice is clearly important to the development of anti-dsDNA antibodies, In contrast, NZB mice congenic with the la^bm12 mutation develop IgG autoantibodies to dsDNA despite being homozygous for Ia. As a part of our effort to examine the mechanisms of disease development in NZB.H-2^bm12 mice, we have generated a panel of monoclonal antibodies against nucleic acids. A subgroup of these antibodies exhibited strong electrostatic interaction with nucleic acids as evidenced by inhibition of their binding by a moderate increase in ionic strength. Interestingly, the effect of salt was either all or none; e.g., antibodies were either markedly inhibited or virtually unaffected. The importance of this ionic interaction was highlighted by analysis of DNA binding of antibodies from serum and nephritic kidneys of NZB.H-2^bm12 mice. Antibodies specific for ssDNA, which are common in NZB mice and not associated with nephritic lupus, are largely unaffected by salt. However, serum and kidney eluted IgG antibodies specific for dsDNA were markedly inhibited by salt. We postulate that B cell clones whose antibodies exhibit electrostatic interaction with DNA are preferentially expanded during the course of lupus in NZB.H-2^bm12 mice and that such antibodies contribute significantly to glomerulonephritis.     

Epstein–Barr virus antibodies mark systemic lupus erythematosus and scleroderma patients negative for anti‐DNA

Systemic lupus erythematosus (SLE) is an autoimmune disease that can attack many different body organs; the triggering event is unknown. SLE has been associated with more than 100 different autoantibody reactivities – anti‐dsDNA is prominent. Nevertheless, autoantibodies to dsDNA occur in only two‐thirds of SLE patients. We previously reported the use of an antigen microarray to characterize SLE serology. We now report the results of an expanded study of serology in SLE patients and scleroderma (SSc) patients compared with healthy controls. The analysis validated and extended previous findings: two‐thirds of SLE patients reacted to a large spectrum of self‐molecules that overlapped with their reactivity to dsDNA; moreover, some SLE patients manifested a deficiency of natural IgM autoantibodies. Most significant was the finding that many SLE patients who were negative for autoantibodies to dsDNA manifested abnormal antibody responses to Epstein–Barr virus (EBV): these subjects made IgG antibodies to EBV antigens to which healthy subjects did not respond or they failed to make antibodies to EBV antigens to which healthy subjects did respond. This observation suggests that SLE may be associated with a defective immune response to EBV. The SSc patients shared many of these serological abnormalities with SLE patients, but differed from them in increased IgG autoantibodies to topoisomerase and centromere B; 84% of SLE patients and 58% of SSc patients could be detected by their abnormal antibodies to EBV. Hence an aberrant immune response to a ubiquitous viral infection such as EBV might set the stage for an autoimmune disease.     

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.     

联合检测对类风湿性关节炎的诊断及临床意义

目的探讨类风湿因子（RF）、抗环瓜氨酸多肽抗体（Anti—CCP）、抗核抗体（ANA）、抗双链DNA抗体（抗ds—DNA）、抗ENA抗体联合检测对类风湿性关节炎（RA）患者的诊断及临床意义。方法分别检测20例RA患者（RA组）;20例确诊为非RA自身免疫痛患者（非RA患者组）和20例健康体检者（对照组）的RF、Anti—CCP、ANA、抗ds—DNA、抗ENA抗体。RF采用速率散射比浊法、Anti—CCP采用化学发光微粒予免疫检测法,ANA,ds—DNA抗体检测采用间接免疫荧光法,抗ENA抗体检测采用免疫印迹法。结果1．RA组患者的RF的阳性率为55％、And—CCP的阳性率为80％、ANA阳性率分别为65．0％、抗ds—DNA的阳性率为5．0％、抗ENA抗体阳性率为5．0％,五者联合检测的阳性率为95．0％,明显高于对照组（P〈0．01）。2．非RA组患者的RF的阳性率为20．0％、Anti—CCP的阳性率为5．0％、ANA阳性率分别为60．0％、抗ds—DNA的阳性率为5．0％、抗ENA抗体阳性率为30．0％,五者联合检测的阳性率为85．0％,明显高于对照组（P〈0．01）、结论RA患者中RF、AInj—CCP阳性率较高．五者的联合捡测可提高捡出率．对RA病情的鉴别诊断及发展和疗效的观察均有重要的意义。     

Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome

The use of small interfering RNAs (siRNAs) has been under investigation for the treatment of several unmet medical needs, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) wherein siRNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the mRNA level. The properties such as clear anatomy, accessibility, and relatively low enzyme activity make the lung a good target for local siRNA therapy. However, the translation of siRNA is restricted by the inefficient delivery of siRNA therapeutics to the target cells due to the properties of naked siRNA. Thus, this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable siRNA formulations for human use before siRNA therapeutics for ALI/ARDS become available in the clinic.