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.

Cupiennin 1a exhibits a remarkably broad, non-stereospecific cytolytic activity on bacteria, protozoan parasites, insects, and human cancer cells

Cupiennin 1a, a cytolytic peptide isolated from the venom of the spider Cupiennius salei, exhibits broad membranolytic activity towards bacteria, trypanosomes, and plasmodia, as well as human blood and cancer cells. In analysing the cytolytic activity of synthesised all-d- and all-l-cupiennin 1a towards pro- and eukaryotic cells, a stereospecific mode of membrane destruction could be excluded. The importance of negatively charged sialic acids on the outer leaflet of erythrocytes for the binding and haemolytic activity of l-cupiennin 1a was demonstrated. Reducing the overall negative charges of erythrocytes by partially removing their sialic acids or by protecting them with tri- or pentalysine results in reduced haemolytic activity of the peptide.

Seropositivity and Risk Factors Associated with Toxoplasma gondii Infection in Wild Birds from Spain

Toxoplasma gondii is a zoonotic intracellular protozoan parasite of worldwide distribution that infects many species of warm-blooded animals, including birds. To date, there is scant information about the seropositivity of T. gondii and the risk factors associated with T. gondii infection in wild bird populations. In the present study, T. gondii infection was evaluated on sera obtained from 1079 wild birds belonging to 56 species (including Falconiformes (n = 610), Strigiformes (n = 260), Ciconiiformes (n = 156), Gruiformes (n = 21), and other orders (n = 32), from different areas of Spain. Antibodies to T. gondii (modified agglutination test, MAT titer ≥1:25) were found in 282 (26.1%, IC95%:23.5–28.7) of the 1079 birds. This study constitute the first extensive survey in wild birds species in Spain and reports for the first time T. gondii antibodies in the griffon vulture (Gyps fulvus), short-toed snake-eagle (Circaetus gallicus), Bonelli's eagle (Aquila fasciata), golden eagle (Aquila chrysaetos), bearded vulture (Gypaetus barbatus), osprey (Pandion haliaetus), Montagu's harrier (Circus pygargus), Western marsh-harrier (Circus aeruginosus), peregrine falcon (Falco peregrinus), long-eared owl (Asio otus), common scops owl (Otus scops), Eurasian spoonbill (Platalea leucorodia), white stork (Ciconia ciconia), grey heron (Ardea cinerea), common moorhen (Gallinula chloropus); in the International Union for Conservation of Nature (IUCN) “vulnerable” Spanish imperial eagle (Aquila adalberti), lesser kestrel (Falco naumanni) and great bustard (Otis tarda); and in the IUCN “near threatened” red kite (Milvus milvus). The highest seropositivity by species was observed in the Eurasian eagle owl (Bubo bubo) (68.1%, 98 of 144). The main risk factors associated with T. gondii seropositivity in wild birds were age and diet, with the highest exposure in older animals and in carnivorous wild birds. The results showed that T. gondii infection is widespread and can be at a high level in many wild birds in Spain, most likely related to their feeding behaviour.

Toxoplasma gondii in Switzerland: a serosurvey based on meat juice analysis of slaughtered pigs, wild boar, sheep and cattle

Toxoplasmosis is one of the most important zoonotic diseases worldwide and is caused by the protozoan Toxoplasma gondii. Besides vertical infection during pregnancy, humans can get infected post-natally either by peroral uptake of sporulated Toxoplasma oocysts or by ingestion of tissue cysts upon consumption of raw or undercooked meat. The aim of this study was to approximate the risk of human infection via meat consumption by estimating the seroprevalence of T. gondii in slaughtered animals in Switzerland and to compare data with prevalences assessed 10 years ago. The study included pigs, cattle, sheep and wild boar of different age groups and housing conditions whenever possible and applicable. A P-30-ELISA was used to detect T. gondii-specific antibodies and to determine seroprevalences in meat juice of slaughtered animals. A total of 270 domestic pigs (120 adults, 50 finishing, 100 free-ranging animals), 150 wild boars, 250 sheep (150 adults, 100 lambs) and 406 cattle (47 calves, 129 heifers, 100 bulls, 130 adult cows) were tested. Seropositivity increased with the age of the assessed animals. Independent of the age-group, the overall seroprevalence was lowest in wild boars (6.7%), followed by pigs (23.3%), cattle (45.6%) and sheep (61.6%), respectively. Conventional fattening pigs and free-ranging pigs surprisingly had comparable seroprevalences (14.0% and 13.0%, respectively). Unlike in other European countries, where generally a decrease in the number of seropositive animals had been observed, we found that the prevalence of seropositive animals, when compared with that of 10 years ago, had increased for most species/age groups. Conclusively, the results demonstrated a high seroprevalence of T. gondii in animals slaughtered for meat production and revealed that increasing age of the animals is a more important risk factor than housing conditions in Switzerland.

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.

Drug target identification in intracellular and extracellular protozoan parasites

The increasing demand for novel anti-parasitic drugs due to resistance formation to well-established chemotherapeutically important compounds has increased the demands for a better understanding of the mechanism(s) of action of existing drugs and of drugs in development. While different approaches have been developed to identify the targets and thus mode of action of anti-parasitic compounds, it has become clear that many drugs act not only on one, but possibly several parasite molecules or even pathways. Ideally, these targets are not present in any cells of the host. In the case of apicomplexan parasites, the unique apicoplast, provides a suitable target for compounds binding to DNA or ribosomal RNA of prokaryotic origin. In the case of intracellular pathogens, a given drug might not only affect the pathogen by directly acting on parasite-associated targets, but also indirectly, by altering the host cell physiology. This in turn could affect the parasite development and lead to parasite death. In this review, we provide an overview of strategies for target identification, and present examples of selected drug targets, ranging from proteins to nucleic acids to intermediary metabolism.

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.