Thrombophilic risk factors in patients with cranial and spinal dural arteriovenous fistulae

OBJECTIVE: Numerous studies have reported the technical aspects and results of surgical and/or endovascular treatment of cranial dural arteriovenous fistulae (cDAVF) and spinal dural arteriovenous fistulae (sDAVF). Only a few of them have addressed the question of thrombophilic conditions, which may be relevant as pathogenetic factors or can increase the risk for venous thromboembolic events. Therefore, the objective of this study is to compare thrombophilic risk factors in patients with cDAVF and sDAVF with no history of trauma. METHODS: A total of 43 patients (25 with cDAVF and 18 with sDAVF) were included in this study. Blood samples were analyzed for G20210A mutation of the prothrombin gene and factor V Leiden mutation. In all patients, prothrombin time, international normalized ratio, fibrinogen, antithrombin, protein C and S activity, von Willebrand factor antigen, ristocetin cofactor activity, D-dimer, coagulation factor VIII activity, and tissue factor pathway inhibitor were determined. Screening was performed for the occurrence of lupus antiphospholipid and cardiolipin antibodies. RESULTS: The prevalence of G20210A mutation of the prothrombin gene was significantly higher in patients with cDAVF (n = 6) compared with patients with sDAVF (n = 0; P < 0.05, Fisher's exact test). A factor V Leiden mutation was found in 3 patients with sDAVF and in 1 patient with cDAVF (P = 0.29, Fisher's exact test). No significant difference was found for other parameters, except for fibrinogen, but decreased protein C activity was more frequent in patients with cDAVF compared with patients with sDAVF (4 versus 1). Decreased protein S activity was encountered in 3 patients (2 with sDAVF and 1 with cDAVF). Cardiolipin antibodies were found in 2 patients with cDAVF but in none with sDAVF, whereas only 1 patient with sDAVF had lupus antiphospholipid antibodies. CONCLUSION: In both groups of patients with dural arteriovenous fistulae, genetic thrombophilic abnormalities occurred in a higher percentage than in the general population. The differences of the genetic abnormalities may be involved in different pathophysiological mechanism(s) in the development of these distinct neurovascular entities.

Antifactor Xa Activity for the Management of Anticoagulation during Cardiac Surgery

Background In patients with autoimmune diseases associated with antiphospholipid antibodies, precise management of anticoagulation during extracorporeal circulation (ECC) is complicated. It was the aim of the present study to determine whether antifactor Xa (aXa) activity is useful in guiding heparin therapy during ECC. Methods In 15 patients undergoing cardiac surgery, anticoagulation with unfractionated heparin (UFH) and its reversal with protamine were guided using activated clotting time (ACT) (>400 second during ECC; ≤100 second for UFH reversal). For each ACT, the corresponding aXa activity levels were measured. Results A total of 144 blood samples were obtained. ACT and aXa activity were significantly correlated (r = 0.771, p< 0.0001, Spearman rank-order correlation). Using receiver operating characteristic curve (ROC) analyses, the cutoffvalues for aXa activity were 1.14 IU/mL (area under the ROC curve [AUC]: 0.89; inaccuracy rate: 9.4%) to predict ACT > 400 seconds and 0.55 IU/mL (AUC: 0.85; inaccuracy rate: 13.3%) for ACT ≤ 100 seconds. Conclusion AXa activity is strongly correlated with ACT, and therefore may be feasible for managing anticoagulation with UFH during ECC.

Double seronegative myasthenia gravis with antiphospholipid syndrome: a case report

INTRODUCTION Myasthenia gravis is an autoimmune disease characterized by fluctuating muscle weakness. It is often associated with other autoimmune disorders, such as thyroid disease, rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome. Many aspects of autoimmune diseases are not completely understood, particularly when they occur in association, which suggests a common pathogenetic mechanism. CASE PRESENTATION We report a case of a 42-year-old Caucasian woman with antiphospholipid syndrome, in whom myasthenia gravis developed years later. She tested negative for both antibodies against the acetylcholine receptor and against muscle-specific receptor tyrosine-kinase, but had typical decremental responses at the repetitive nerve stimulation testing, so that a generalized myasthenia gravis was diagnosed. Her thromboplastin time and activated partial thromboplastin time were high, anticardiolipin and anti-β2 glycoprotein-I antibodies were slightly elevated, as a manifestation of the antiphospholipid syndrome. She had a good clinical response when treated with a combination of pyridostigmine, prednisone and azathioprine. CONCLUSIONS Many patients with myasthenia gravis test positive for a large variety of auto-antibodies, testifying of an immune dysregulation, and some display mild T-cell lymphopenia associated with hypergammaglobulinemia and B-cell hyper-reactivity. Both of these mechanisms could explain the occurrence of another autoimmune condition, such as antiphospholipid syndrome, but further studies are necessary to shed light on this matter.Clinicians should be aware that patients with an autoimmune diagnosis such as antiphospholipid syndrome who develop signs and neurological symptoms suggestive of myasthenia gravis are at risk and should prompt an emergent evaluation by a specialist.

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.

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.

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.

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.

Distinct reactivities of interleukin-4-specific antibodies with recombinant and native canine interleukin-4 in various assays

Interleukin 4 (IL-4) plays a central role in immune responses to parasites and allergens. IL-4 drives the differentiation of naive T cells into Th2 cells and regulates immunoglobulin class switching to IgE.Little is known about the role of IL-4 in canine allergies and parasite infections. Most of the information derives from measurement of IL-4 mRNA expression in dog tissues, but detection of IL-4 protein has been difficult so far, probably due to low sensitivity of available methods. Antibodies (Ab) specific for canine IL-4 are available from various sources, but these Ab have been produced against recombinant Escherichia coli-expressed canine IL-4 and there is only limited information on their reactivities with native canine IL-4. Therefore, in the present study, we tested six available canine IL-4-specific Ab for their reactivities with recombinant canine IL-4 expressed in E. coli (rec.IL-4) or in mammalian cells (mam.IL-4), and with supernatants from stimulated canine peripheral blood mononuclear cells (PBMCs) using several detection methods, including Western blotting, ELISA, cytokine bead assay, and intracellular IL-4 staining. Additionally, we tested a bovine IL-4-specific antibody that has been previously shown to cross-react with canine IL-4. All tested Ab except anti-bovine IL-4 reacted with rec.IL-4, and most of them reacted with mam.IL-4. However, only the cytokine bead assay was sensitive enough to allow the detection of IL-4 in supernatants of canine PBMCs.

Naturally occurring antibodies/autoantibodies in polyclonal immunoglobulin concentrates

It was a long way from the use of hyperimmune animal sera for the treatment of toxin-producing infections to the production of polyclonal, polyspecific human immunoglobulin preparations and the use of NAbs as therapeutic tools for autoimmune and inflammatory diseases. Some highlights of the development of knowledge in blood fractionation techniques, basic science and clinical wisdom are reviewed in this chapter. Proudly we mention the outstanding contribution of Swiss scientists and clinicians in the development of IVIG as clinical tool for some otherwise untreatable diseases or taking advantage of its low adverse event profile in long-term treatment of other chronic autoimmune and inflammatory diseases. This chapter summarizes some of the characteristics and the effects in humans of NAbs which are present in IgG concentrates. We call attention to the fact that the human data remain, at least in part, incomplete, among others because even with the most efficient large-scale techniques available not more than approximately 50% of the total IgG in plasma can be fractionated into an immunoglobulin G concentrate.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.