Institute of Immunology Co.

Tokyo, Japan

Institute of Immunology Co.

Tokyo, Japan
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Miyazaki-Komine K.,Keio University | Takai Y.,Tohoku University | Huang P.,Keio University | Kusano-Arai O.,Quantitative Medicine | And 11 more authors.
British Journal of Pharmacology | Year: 2016

Background and Purpose Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO-IgG, which recognizes the extracellular domains of the water channel, aquaporin-4. Binding of NMO-IgG to aquaporin-4 expressed in end-feet of astrocytes leads to complement-dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO-IgG to aquaporin-4, using high-avidity, non-pathogenic-chimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies. Experimental Approach cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin-4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Igκ respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies. Key Results Original monoclonal antibodies showed high avidity binding to human aquaporin-4, as determined by ELISA. Live imaging using Alexa-Fluor-555-labelled antibodies revealed that the antibody D15107 more rapidly bound to cells expressing human aquaporin-4 than others and strongly enhanced endocytosis of this water channel, while D12092 also bound rapidly to human aquaporin-4 but enhanced endocytosis to a lesser degree. Chimeric D15107 prevented complement-dependent cytotoxicity induced by NMO-IgG from patient sera in vitro. Conclusions and Implications We have established non-pathogenic, high-avidity, chimeric antibodies against the extracellular domains of human aquaporin-4, which provide a novel therapeutic option for preventing the progress and recurrence of NMO/NMO spectrum disorders. © 2015 The British Pharmacological Society.


Kurtovic T.,University of Zagreb | Lang Balija M.,Institute of Immunology Inc. | Ayvazyan N.,Armenian National Academy of Sciences | Halassy B.,University of Zagreb
Toxicon | Year: 2014

Antivenom raised against the venom of nose-horned viper, Vipera ammodytes (V. a.) ammodytes (European viper venom antiserum, Zagreb antivenom), contains neutralising equine F(ab′)2 fragments that are clinically successful against homologous venom, but also against the venoms of several others medically important European snakes due to its paraspecific action. In this work we demonstrated that Zagreb antivenom is preclinically effective in neutralising lethal toxicity and hemorrhagicity of venoms of Armenian mountain snakes - Montivipera raddei and Macrovipera lebetina obtusa as well. In order to better understand the biochemical basis of the observed paraspecificity, the ability of anti-V. a. ammodytes serum to recognise and neutralise proteinases of the two venoms was also investigated. Anti-V. a. ammodytes serum showed surprisingly low capacity to inhibit metalloproteinases of both venoms included in the study, probably due to weak immunorecognition of their P-I representatives. Also, it completely failed to abolish enzymatic action of serine proteinases from Macrovipera lebetina obtusa venom. Relevance of such finding is yet to be established. © 2014 Elsevier Ltd. All rights reserved.


Kurtovic T.,Institute of Immunology Inc. | Brgles M.,Institute of Immunology Inc. | Leonardi A.,Jozef Stefan Institute | Balija M.L.,Institute of Immunology Inc. | And 4 more authors.
Toxicon | Year: 2011

Ammodytagin, a hemorrhagic Zn 2+-dependent metalloproteinase from Vipera ammodytes ammodytes (Vaa) venom, is a glycosylated heterodimer of 108kDa, as determined by MALDI mass spectrometry. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by Na 2EDTA, Zn 2+ and DTT. It cleaves insulin B-chain, hydrolysing it at positions Gln 4-His 5, His 10-Leu 11 and Tyr 16-Leu 17. Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of a substrate specificity revealed that the hemorrhagic activity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective Vaa specific antivenom should be directed. © 2011 Elsevier Ltd.


Leonardi A.,Jozef Stefan Institute | Sajevic T.,Jozef Stefan Institute | Kovacic L.,Jozef Stefan Institute | Pungercar J.,Jozef Stefan Institute | And 5 more authors.
Toxicon | Year: 2014

In the envenomation caused by a bite of Vipera ammodytes ammodytes, the most venomous snake in Europe, hemorrhage is usually the most severe consequence in man. Identifying and understanding the hemorrhagic components of its venom is therefore particularly important in optimizing medical treatment of patients. We describe a novel high molecular mass hemorrhagin, VaH4. The isolated molecule is a covalent dimer of two homologous subunits, VaH4-A and VaH4-B. Complete structural characterization of A and partial characterization of B revealed that both belong to the P-III class of snake venom metalloproteinases (SVMPs), comprising a metalloproteinase, a disintegrin-like domain and a cysteine-rich domain. However, neither VaH4-A nor VaH4-B possess the Cys174 involved in the inter-subunit disulphide bond of P-III SVMPs. A three-dimensional model of the VaH4 dimer suggests that Cys132 serves this function. This implies that dimers in the P-III class of SVMPs can be formed either between their Cys132 or Cys174 residues. The proteolytic activity and stability of VaH4 depend on Zn2+ and Ca2+ ions and the presence of glycosaminoglycans, which indicates physiological interaction of VaH4 with the latter element of the extracellular matrix (ECM). The molecular mass of VaH4, determined by MALDI/TOF mass spectrometry, is 110.2 kDa. N-deglycosylation reduced the mass of each monomer by 8.7 kDa. The two possible N-glycosylation sites in VaH4-A are located at completely different positions from those in homodimeric P-IIIc VaH3 from the same venom, however, without any evident functional implications. The hemorrhagic activity of this slightly acidic SVMP is ascribed to its hydrolysis of components of the ECM, particularly fibronectin and nidogen, and of some blood coagulation proteins, in particular the α-chain of fibrinogen. VaH4 is also significant medically as we found it cytotoxic against cancer cells and due to its substantial sequence similarity to ADAM/ADAMTS family of physiologically very important human proteins of therapeutic potential. © 2013 Elsevier Ltd. All rights reserved.


Kurtovic T.,Institute of Immunology Inc. | Leonardi A.,Jozef Stefan Institute | Lang Balija M.,Institute of Immunology Inc. | Brgles M.,Institute of Immunology Inc. | And 3 more authors.
Toxicon | Year: 2012

The venom of Vipera ammodytes ammodytes (Vaa), like the venoms of other Viperinae snakes, is largely haemorrhagic and necrotising, and only to a lesser extent neurotoxic to humans. The components most extensively studied so far, and most probably involved in generating the observed pathologies, are haemorrhagins (H), members of the metalloproteinase group of enzymes, and neurotoxic ammodytoxins (Atxs), that belong to the secretory phospholipases A 2. Rabbit antisera were prepared containing functional antibodies specific for each class of pathology-inducing venom constituents and for both classes together. The involvement of these antibodies in neutralising the toxicity of whole Vaa venom was assessed using the ED 50 assay in mice. This assay is the only regulatorily approved assay for estimating anti-venom potency and as such has the task to quantify the active compound neutralising venom-induced pathology of the anti-venom. Fully functional anti-Atx antibodies were shown to be responsible for neutralising the portion of venom toxicity, while anti-H antibodies were not protective in this assay. Thus, the mouse ED 50 assay, intended to measure the active principle of the anti-venom, does not measure antibodies specific for Vaa venom haemorrhagins, and consequently does not fulfil its primary task from the regulatory point of view. © 2012 Elsevier Ltd.


Habjanec L.,Institute of Immunology Inc. | Halassy B.,Institute of Immunology Inc. | Tomasic J.,Institute of Immunology Inc.
International Immunopharmacology | Year: 2010

Structurally related peptidoglycan monomer (PGM) and muramyl dipeptide (MDP) differ in several aspects of biological activity but have in common immunostimulating properties. Comparative study of the effects of these adjuvants on humoral IgG immune response specific for protein antigen ovalbumin (OVA) was carried out in two inbred mouse strains, CBA and NIH/OlaHsd, and their ability to modulate the bias of immune response towards Th1/Th2 was evaluated. MDP had better adjuvant activity at some points than PGM, whereas both adjuvants stimulated Th2-biased immune response specific for OVA. In comparison to Complete Freund's adjuvant (CFA), as a golden standard of adjuvant action, both PGM and MDP exhibited considerably lower activity. Addition of PGM to Incomplete Freund's adjuvant (IFA) on humoral immune response was studied also, and the effect of such adjuvant formulation was compared to the effect of CFA. While CFA induced the switch towards Th1-biased immune response, the addition of PGM into IFA did have no impact on modulating the immune response towards more pronounced Th2-type of immune response, defined by IFA itself. © 2010 Elsevier B.V. All rights reserved.


Santak M.,Institute of Immunology Inc.
Periodicum Biologorum | Year: 2012

Influenza viruses have been with mankind for at least 300 years, with epidemics occurring every few years and pandemics every few decades. They replicate extremely rapidly in the host therefore demanding a fast and effective antiviral response. Despite the availability of seasonal trivalent vaccines and antivirals, which are effective for most recipients, influenza remains serious disease. The reason for that is a grand capacity of the influenza virus to adapt to new environmental conditions and evolutionary pressure. Vaccination remains the main protective measure against influenza for general population. The first vaccine was administered in the 1940s and ever since the influenza vaccine has provided tremendous relief against influenza infections. However, time has revealed the ultimate limit of the vaccine and the call for its reinvention has now come. The purpose of this review is to give a brief but comprehensive overview of the currently used prophylactic and therapeutic approaches against influenza and the new most promising developments in this field.


Forcic D.,Institute of Immunology Inc. | Kosutic-Gulija T.,Institute of Immunology Inc. | Santak M.,Institute of Immunology Inc. | Jug R.,Institute of Immunology Inc. | And 3 more authors.
Vaccine | Year: 2010

The two most commonly used methods for the determination of a virus potency are plaque assay and 50% cell culture infective dose (CCID50) assay, both based on cytopathic effect observation. We compared the potency estimates obtained by plaque and CCID50 assays for nine mumps virus strains that produce different cytopathic effects in Vero cells. The ratios of CCID50 and plaque assay quantification results differed for different strains and were in a range of 0.66-10, indicating that quantification results for some mumps virus strains are almost identical regardless of whether CCID50 or plaque method is used, while the potency estimates of other strains strongly depend on the choice of the assay. © 2009 Elsevier Ltd. All rights reserved.


Patent
University of Tokyo, Institute Of Immunology Co. and Aichi Medical University | Date: 2014-09-03

It is an object of the present invention to provide a monoclonal antibody, which is suitable for the quantitative analysis of asparagine synthetase in a cell. The present invention provides a monoclonal antibody which specifically recognizes asparagine synthetase that is present in a cell.


Patent
University of Tokyo, Aichi Medical University and Institute Of Immunology Co. | Date: 2012-10-03

It is an object of the present invention to provide a monoclonal antibody, which is suitable for the quantitative analysis of asparagine synthetase in a cell. The present invention provides a monoclonal antibody which specifically recognizes asparagine synthetase that is present in a cell.

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