Characterization of palytoxin binding to HaCaT cells using a monoclonal anti-palytoxin antibody.

Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a K(d) of 3 × 10-10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3-1.0 × 10-7 M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1-3.0 × 10-9 M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10-5 M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

Highly sensitive electrochemiluminescent nanobiosensor for the detection of palytoxin.

Marine toxins appear to be increasing in many areas of the world. An emerging problem in the Mediterranean Sea is represented by palytoxin (PlTX), one of the most potent marine toxins, frequently detected in seafood. Due to the high potential for human toxicity of PlTX, there is a strong and urgent need for sensitive methods toward its detection and quantification. We have developed an ultrasensitive electrochemiluminescence-based sensor for the detection of PlTX, taking advantage of the specificity provided by anti-PlTX antibodies, the good conductive properties of carbon nanotubes, and the excellent sensitivity achieved by a luminescence-based transducer. The sensor was able to produce a concentration-dependent light signal, allowing PlTX quantification in mussels, with a limit of quantification (LOQ = 2.2 µg/kg of mussel meat) more than 2 orders of magnitude more sensitive than that of the commonly used detection techniques, such as LC-MS/MS.

Simple scale-up of recombinant antibody production using an UCOE containing vector.

Recombinant proteins, in particular antibodies, have become fundamental in biomedical research where they are used in numerous therapeutic and diagnostic applications. For this reason there is an increasing demand for quick and economical production systems for recombinant proteins in mammalian cells.

Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies.

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.

B7h triggering inhibits umbilical vascular endothelial cell adhesiveness to tumor cell lines and polymorphonuclear cells.

Vascular endothelial cells (ECs) are key players in leukocyte recruitment into tissues and metastatic dissemination of tumor cells. ECs express B7h, which is the ligand of the ICOS T cell costimulatory molecule. The aim of this work was to assess the effect of B7h triggering by a soluble form of ICOS (ICOS-Fc) on the adhesion of colon carcinoma cell lines to HUVECs. We found that B7h triggering inhibited HUVEC adhesiveness to HT29 and DLD1 cells (by 50 and 35%, respectively) but not to HCT116 cells. The effect was dependent on the ICOS-Fc dose and was detectable as early as 30 min after treatment and was still present after 24 h. It was inhibited by soluble anti-ICOS reagents (mAb and B7h-Fc) and silencing of B7h on HUVECs, and it was not displayed by an F119S mutated form of ICOS-Fc that does not bind B7h. HUVEC treatment with ICOS-Fc did not modulate expression of adhesion molecules and cytokines, but it substantially downmodulated ERK phosphorylation induced by E-selectin triggering or osteopontin, which may influence HUVEC adhesiveness. Moreover, HUVEC treatment with ICOS-Fc also inhibited adhesion of polymorphonuclear cells and several tumor cell lines from different origins. Therefore, the B7h-ICOS interaction may modulate spreading of cancer metastases and recruitment of polymorphonuclear cells in inflammatory sites, which opens a view on the use of ICOS-Fc as an immunomodulatory drug.

Anti transglutaminase antibodies cause ataxia in mice.

BACKGROUND: Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one. METHODS: We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice. CONCLUSION: The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia.

Gluten sensitivity: from gut to brain.

Gluten sensitivity is a systemic autoimmune disease with diverse manifestations. This disorder is characterised by abnormal immunological responsiveness to ingested gluten in genetically susceptible individuals. Coeliac disease, or gluten-sensitive enteropathy, is only one aspect of a range of possible manifestations of gluten sensitivity. Although neurological manifestations in patients with established coeliac disease have been reported since 1966, it was not until 30 years later that, in some individuals, gluten sensitivity was shown to manifest solely with neurological dysfunction. Furthermore, the concept of extraintestinal presentations without enteropathy has only recently become accepted. In this Personal View, we review the range of neurological manifestations of gluten sensitivity and discuss recent advances in the diagnosis and understanding of the pathophysiological mechanisms underlying neurological dysfunction related to gluten sensitivity.

Cerebellar ataxia as a possible organ-specific autoimmune disease.

The purpose of this study was to investigate the possibility that autoimmunity is responsible for some cases of sporadic idiopathic ataxia. We prospectively investigated 400 patients with progressive ataxia and identified a group of patients with idiopathic sporadic ataxia. A comparison of the prevalence of autoimmune diseases, the autoimmunity linked HLA DQ2, and serum anticerebellar antibodies was made between patients with idiopathic sporadic and those with genetically characterized ataxia. Ninety-one of 400 (23%) patients with progressive ataxia had idiopathic sporadic ataxia. The prevalence of autoimmune diseases in this group was 47% as compared with 6% in the group of patients with genetic ataxias (P < 0.0001). The HLA DQ2 was found in 71% of patients with sporadic ataxia, in 34% in patients with genetic ataxia, and in 36% of healthy local population (P = 0.0005 by Chi squared test). Anticerebellar antibodies were detected in 12 out of 20 patients with idiopathic sporadic as opposed to one of 20 patients with genetic ataxia. The significantly higher prevalence of autoimmune diseases, HLA DQ2 and anti-cerebellar antibodies in patients with idiopathic sporadic ataxia compared to genetic ataxia supports the notion that autoimmunity may account for some cases of idiopathic sporadic cerebellar ataxia.

Gluten ataxia: passive transfer in a mouse model.

Gluten sensitivity is an autoimmune disease that usually causes intestinal atrophy resulting in a malabsorption syndrome known as celiac disease. However, gluten sensitivity may involve several organs and is often associated with extraintestinal manifestations. Typically, patients with celiac disease have circulating anti-tissue transglutaminase and anti-gliadin antibodies. When patients with gluten sensitivity are affected by other autoimmune diseases, other autoantibodies may arise like anti-epidermal transglutaminase in dermatitis herpetiformis, anti-thyroid peroxidase antibodies in thyroiditis, and anti-islet cells antibodies in type 1 diabetes. The most common neurological manifestation of gluten sensitivity is ataxia, the so-called gluten ataxia (GA). In patients with GA we have demonstrated that anti-gliadin and anti-tissue transglutaminase antibodies cross-react with neurons but that additional anti-neural antibodies are present. The aim of the present article is to review the knowledge on animal models of gluten sensitivity, as well as reviewing the role of anti-neural antibodies in GA.

Quantification of antineural antibodies in autoimmune neurological disorders.

More than 50 different neurological pathologies have a confirmed or suspected autoimmune etiology affecting an estimated number of 75 million people worldwide. Autoantibodies are a useful diagnostic marker for most autoimmune diseases even though their pathological role is not evident, and several tests for their detection are commercially available. However, for autoimmune diseases involving the nervous system, lack of clear information on the identity of antineural antibody targets and the presence of many rare diseases have hampered the development of specific diagnostic assays. This review focuses on the actual knowledge on confirmed and suspected autoimmune diseases that target the CNS and the diagnostic relevance of corresponding antineural autoantibodies.

Detection of anti-brain serum antibodies using a semi-quantitative immunohistological method.

The number of autoimmune disorders that may involve the nervous system is increasing. The diagnosis of neurological involvement in the context of systemic diseases may be helped by the detection of autoantibodies reacting against neural autoantigens. If the autoantigen is not known but the target tissue is suspected, immunohistochemistry is one of the main techniques used to certify the presence of autoantibodies. Autoreactive antibodies are also present in the healthy population but in low quantity compared to patients with such diseases. Quantification of such autoantibodies could help to discriminate between disease and healthy states. We have developed a densitometric immunohistological method for the evaluation of human serum anti-neural reactivity. Using a densitometric analysis of rat brain sections incubated with the serum from 107 healthy subjects, we have defined the baseline of natural anti-neural autoreactivity, and the cut-off for subsequent quantification of anti-neural reactivity in patients with neurological involvement in the context of autoimmune diseases, including systemic lupus erythematosus, paraneoplastic cerebellar degeneration, and stiff person syndrome. The test sensitivity was 81% with a positive predictive value of 52%, a specificity of 89% with a negative predictive value as high as 97%. In conclusion, this standardised semi-quantitative procedure makes immunohistochemistry a reliable diagnostic test for autoimmune neuropathologies and represents an excellent exclusion test for anti-neural autoimmunity.