Recombinant ADAMTS13 normalizes von Willebrand factor-cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non-inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme.

Screening for lupus anticoagulant: improving the performance of the lupus-sensitive PTT-LA

Introduction: The aim of the present work was to verify whether calculating a ratio between clotting times obtained with the sensitive PTT-LA and a less sensitive activated partial thromboplastin time (aPTT)-reagent may represent a valuable aPTT-based screening strategy for lupus anticoagulants (LA). Methods: For the pilot study, plasma samples from normal subjects (n = 15) and from patients with LA (n = 10), therapeutic anticoagulation with vitamin K-antagonists (VKA) (n = 15) or unfractionated heparin (n = 15), coagulation factors deficiency (n = 16), and inhibitory antibodies against factor VIII or IX (n = 11) were studied. For the evaluation study, 1553 consecutive plasma samples from nonanticoagulated patients investigated for LA between January 2005 and December 2007 at our institution were studied. Following screening strategies were employed: Pathromtin-SL (aPTT-SL), PTT-LA (aPTT-LA), ratio aPTT-LA/aPTT-SL (aPTT-ratio), and Russell's viper venom (RVV) based LA-Check. LA positive samples were identified by mixing studies and diluted RVV confirmation test (LA-Check/LA-Sure). Results: Pilot study: All screening strategies had a 100% sensitivity, and the aPTT-ratio reached the highest specificity (82%; 95%CI: 74-90%). Within the evaluation study, following sensitivities for LA screening were observed: aPTT-SL 59.0% (95%CI: 57-61%), aPTT-LA 82.1% (95%CI: 80-84%), aPTT-ratio 92.3% (95%CI: 91-94), and LA-Check 83.3% (95%CI: 82-85%). Conclusion: Calculating a ratio between the LA-sensitive PTT-LA and the less sensitive Pathromtin-SL improves the performance of the PTT-LA itself and represents a simple and sensitive aPTT-based integrated strategy for LA screening.

Antisense molecules for targeted cancer therapy

The efficacy of traditional anti-cancer agents is hampered by toxicity to normal tissues, due to the lack of specificity for malignant cells. Recent advances in our understanding of molecular genetics and tumor biology have led to the identification of signaling pathways and their regulators implicated in tumorigenesis and malignant progression. Consequently, novel biological agents were designed which specifically target key regulators of cell survival and proliferation activated in malignant cells and thus are superior to unspecific cytotoxic agents. Antisense molecules comprising conventional single-stranded antisense oligonucleotides (ASO) and small interfering RNA (siRNA) inhibit gene expression on the transcript level. Thus, they specifically target the genetic basis of cancer and are particularly useful for inhibiting the expression of oncogenes the protein products of which are inaccessible to small molecules or inhibitory antibodies. Despite the somewhat disappointing results of recent antisense oncology trials, the identification of new cancer targets and ongoing progress in ASO and siRNA technology together with improvements in tumor targeted delivery have raised new hopes that this fascinating intervention concept will eventually translate into enhanced clinical efficacy.

Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Evidence that anti-muscarinic antibodies in Sjögren's syndrome recognise both M3R and M1R

Inhibitory anti-muscarinic receptor type 3 (M3R) antibodies may contribute to the pathogenesis of Sjögren's syndrome (SS), and putative anti-M3R blocking antibodies in intravenous immunoglobulin (IVIg) have been suggested as a rationale for treatment with IVIg. We investigated the presence of subtype-specific anti-MR autoantibodies in healthy donor and SS sera using MR-transfected whole-cell binding assays as well as M1R and M3R peptide ELISAs. Control antibodies against the second extracellular loop of the M3R, a suggested target epitope, were induced in rabbits and found to be cross-reactive on the peptides M3R and M1R. The rabbit antibodies had neither an agonistic nor an antagonistic effect on M3R-dependent ERK1/2 signalling. Only one primary SS (out of 5 primary SS, 2 secondary SS and 5 control sera) reacted strongly with M3R transfected cells. The same SS serum also reacted strongly with M1R and M2R transfectants, as well as M1R and two different M3R peptides. Strong binding to M1R and low-level activities against M3R peptides were observed both in SS and control sera. IVIg showed a strong reactivity against all three peptides, especially M1R. Our results indicate that certain SS individuals may have antibodies against M1R, M2R and M3R. Our results also suggest that neither the linear M3R peptide nor M3R transfectants represent suitable tools for discrimination of pathogenic from natural autoantibodies in SS.

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS.

IVIG blocks complement deposition mediated by anti-GM1 antibodies in multifocal motor neuropathy

The pathogenesis of multifocal motor neuropathy (MMN) has yet to be established. MMN patients often carry anti-GM1 IgM antibodies, suggesting an autoimmune process involving complement. Intravenous immunoglobulin (IVIG) is the first line treatment, but its action mechanism is unknown.

Influence of anti-infliximab antibodies and residual infliximab concentrations on the occurrence of acquired drug resistance to infliximab in rheumatoid arthritis patients

Infliximab (IFX) can be immunogenic for humans and lead to the formation of antibodies against IFX (anti-IFX Ab), which could induce acquired IFX resistance.

Antibodies recognising sulfated carbohydrates are prevalent in systemic sclerosis and associated with pulmonary vascular disease

Glycosylation represents an important modification that regulates biological processes in tissues relevant for disease pathogenesis in systemic sclerosis (SSc), including the endothelium and extracellular matrix. Whether patients with SSc develop antibodies to carbohydrates is not known.

Does acupuncture needling induce analgesic effects comparable to diffuse noxious inhibitory controls?

Diffuse noxious inhibitory control (DNIC) is described as one possible mechanism of acupuncture analgesia. This study investigated the analgesic effect of acupuncture without stimulation compared to nonpenetrating sham acupuncture (NPSA) and cold-pressor-induced DNIC. Forty-five subjects received each of the three interventions in a randomized order. The analgesic effect was measured using pressure algometry at the second toe before and after each of the interventions. Pressure pain detection threshold (PPDT) rose from 299 kPa (SD 112 kPa) to 364 kPa (SD 144), 353 kPa (SD 135), and 467 kPa (SD 168) after acupuncture, NPSA, and DNIC test, respectively. There was no statistically significant difference between acupuncture and NPSA at any time, but a significantly higher increase of PPDT in the DNIC test compared to acupuncture and NPSA. PPDT decreased after the DNIC test, whereas it remained stable after acupuncture and NPSA. Acupuncture needling at low pain stimulus intensity showed a small analgesic effect which did not significantly differ from placebo response and was significantly less than a DNIC-like effect of a painful noninvasive stimulus.

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.

Favourable prognostic value of antibodies anti-HSP20 in patients with cystic echinococcosis: a differential immunoproteomic approach

Seeking biomarkers reflecting disease development in cystic echinococcosis (CE), we used a proteomic approach linked to immunological characterisation for the identification of respective antigens. Two-dimensional gel electrophoresis (2-DE) of sheep hydatid fluid, followed by immunoblot analysis (IB) with sera from patients with distinct phases of disease, enabled us to identify by mass spectrometry heat shock protein 20 (HSP20) as a potential marker of active CE. Using IB, antibodies specific to the 34 kDa band of HSP20 were detected in sera from 61/95 (64%) patients with CE, but not in sera from healthy subjects. IB revealed anti-HSP20 antibodies in a higher percentage of sera from patients with active disease than in sera from patients with inactive disease (81 vs. 24%; P = 10(-4)). These primary results were confirmed in a long-term follow-up study after pharmacological and surgical treatment. Herewith anti-HSP20 antibody levels significantly decreased over the course of treatment in sera from patients with cured disease, relative to sera from patients with progressive disease (P = 0.017). Thus, during CE, a comprehensive strategy of proteomic identification combined with immunological validation represents a promising approach for the identification of biomarkers useful for the prognostic assessment of treatment of CE patients.

RGD motif of lipoprotein T, involved in adhesion of Mycoplasma conjunctivae to lamb synovial tissue cells

Lipoprotein T (LppT), a membrane-located 105-kDa lipoprotein of Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC) of domestic sheep and wild Caprinae, was characterized. LppT was shown to promote cell attachment to LSM 192 primary lamb joint synovial cells. Adhesion of M. conjunctivae to LSM 192 cells is inhibited by antibodies directed against LppT. The RGD (Arg-Gly-Asp) motif of LppT was found to be a specific site for binding of M. conjunctivae to these eukaryotic host cells. Recombinant LppT fixed to polymethylmethacrylate slides binds LSM 192 cells, whereas LppT lacking the RGD site is deprived of binding capacity to LSM 192, and LppT containing RGE rather than RGD shows reduced binding. Synthetic nonapeptides derived from LppT containing RGD competitively inhibit binding of LSM 192 cells to LppT-coated slides, whereas nonapeptides containing RAD rather than RGD do not inhibit. RGD-containing, LppT-derived nonapeptides are able to directly inhibit binding of M. conjunctivae to LSM 192 cells by competitive inhibition, whereas the analogous nonapeptide containing RAD rather than RGD or the fibronectin-derived RGD hexapeptide has no inhibitory effect. These results reveal LppT as the first candidate of a RGD lectin in Mycoplasma species that is assumed to bind to beta integrins.

Use of an indirect enzyme-linked immunosorbent assay for detection of antibodies in sheep naturally infected with Salmonella Abortusovis

An indirect enzyme-linked immunosorbent assay (ELISA) was modified and validated to detect antibodies against Salmonella Abortusovis in naturally infected sheep. The ELISA was validated with 44 positive and 45 negative control serum samples. Compared with the immunoblot, the sensitivity and specificity of the assay were 98% and 100%, respectively. To follow antibody levels over time, samples from 12 infected ewes were collected at 1, 3, and 10 months after abortion. All animals showed antibody levels above the cutoff value throughout the observation period. One and 3 months after abortion, high antibody levels could be detected in all but one animal, whereas after 10 months, 9 animals had markedly lower but still positive antibody levels. The test characteristics and evidence for the persistence of detectable antibody levels in all infected animals for up to 10 months indicates that the ELISA can be used for herd surveillance testing.