Volume 43 (1997) No. 6
Review
The Genetic Basis of Primary, Predominantly Specific Immunodeficiencies
I. ŠTERZL1, J. ŠTERZL2........................211
1Department of Clinical Immunoendocrinology, Institute of Endocrinology, Prague, Czech Republic
2Department of Immunology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Ivan Šterzl, Department of Clinical Immunoendocrinology, Institute of Endocrinology, Národní tr. 8, 116 94 Prague 1, Czech Republic.
Abstract
Articles
Comparative Evaluation of Four Genotyping Methods for Hepatitis C Virus
K. SEME1, M. POLJAK1, P. DOVÈ2, S. KOREN1............................................219
1Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia
2Biotechnical Faculty, Ljubljana, Slovenia
Corresponding author: Katja Seme, Institute of Microbiology and Immunology, Medical Faculty, Zaloška 4, 1105 Ljubljana, Slovenia. Tel (386 61) 316 593; Fax (386 61) 302 895; e-mail: seme@ ibmi.mf.uni-lj.si.
Abstract
Genotoxicity of Purine Acyclic Nucleotide Analogs
B. OTOVÁ1, A. HOLÝ2, I. VOTRUBA2, M. SLADKÁ1, V. BÍLÁ1, B. MEJSNAROVÁ1, V. LEŠKOVÁ1.........................................................................................225
1Department of Biology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic 2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Berta Otová, Department of Biology, 1st Faculty of Medicine, Charles University, Albertov 4, 128 00 6 Prague 2, Czech Republic.
Abstract
Modification of Postradiative Changes of Nucleic Acids by the Bacterial Extract Broncho-Vaxom in Rat Tissues
H. HAKOVÁ , E. MIŠÚROVÁ, K. KROPÁÈOVÁ .............................231
Department of Cellular and Molecular Biology, Faculty of Sciences, P. J. Šafárik University, Košice, Slovakia
Corresponding author: Hedviga Haková, Department of Cellular and Molecular Biology, Faculty of Sciences, P. J. Šafárik University, Moyzesova 11, 041 67 Košice, Slovakia.
Abstract
Testing of Toxic and DNA-Damaging Effects of N-Cyclohexylthiophthalimide (Duslin P) on Hamster V79 Cells
D. SLAMEÒOVÁ, E. HORVÁTHOVÁ, T. FARKAŠOVÁ, ¼. RUŽEKOVÁ, G. BAÈOVÁ, J. KRÈMÁRIKOVÁ ................................................................239
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic
Corresponding author: Darina Slameòová, Department of Mutagenesis, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 812 32 Bratislava, Slovak Republic.
Abstract
Monoclonal Antibody Register A Novel Monoclonal Antibody Specific for Biliary Glycoprotein (CD66a)
L. DRÁBEROVÁ1, C. P. STANNERS2 , P. DRÁBER1,2.....................................243
1Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 2McGill Cancer Centre and Department of Biochemistry, McGill University, Montreal, Quebec, Canada
Corresponding author: Petr Dráber, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeòská 1083, 142 20 Prague 4, Czech Republic [E-mail: draberpe@biomed.cas.cz; Fax: (420-2) 471 3445] Monoclonal Antibody Register A Novel Monoclonal Antibody Specific for Biliary Glycoprotein (CD66a)
Background
Biliary glycoproteins (BGPs), CD66a, are members of the carcinoembryonic antigen (CEA) sugbroup, CD66, of the CEA family which belongs to the immunoglobulin superfamily (Zimmermann and Thompson, 1990; Barnett et al., 1993). The largest BGP isoform, BGPa, is composed of a 108-amino acid N-terminal IgV-like domain, two 178-amino acid IgC2 set domains, the A1 and B1 domains, a 100-amino acid IgC2 set domain, the A2 domain, a transmembrane domain (TM), and a 71-amino acid cytoplasmic tail (Barnett et al., 1989). BGPa and some other BGP splice isoforms are expressed on the surface of several cell types and seem to be involved in cell-cell adhesion and transmembrane signaling (Dráber and Skubitz, 1997). BGPs are also found in human bile and blood (Svenberg et al., 1979). Antibodies prepared by immunization with various CEA family members exhibit cross-reactivity with other members of the family. So far only one monoclonal antibody (mAb) has been prepared which is specific for BGP (Drzeniek et al., 1991). BGP-specific antibodies can be used in research on the expression and function of BGPs in cells (Ilantzis et al., 1997) and body fluids (Svenberg et al., 1979). Increased levels of BGP are found in blood of patients with liver or biliary tract diseases (Svenberg et al., 1979).
Description of the new mAb, TEC-11
Production.
A hybridoma cell line producing a BGP-A2 domain-specific mAb, designated TEC-11, was obtained after immunization of BALB/c mice with a recombinant protein corresponding to the A2 domain of BGP using standard procedures (Dráber et al., 1980; Amoui et al., 1997), except that SP02 myeloma cells were used for fusion. TEC-11 mAb was identified in ELISA by its reactivity with recombinant BGP-A2 fragment but not with isolated CEA. TEC-11 mAb in ascites form showed clear reactivity with BGP-A2 fragment at a dilution greater than 1:200,000 (Fig. 1).
Specificity.
Immunoblotting analysis showed that TEC-11 mAb reacted with cell lines transfected with BGP cDNA in mammalian expression vectors but not with cell lines transfected with cDNA for CEA (CD66e, Fig. 2), NCA (CD66c) and CGM6 (CD66b) (not shown). It also reacted with BGP in extracts from granulocytes and several other cell types and with human bile. ELISA assays with chimeric proteins confirmed that TEC-11 binds only to proteins carrying the BGP-A2 domain (not shown). Properties. TEC-11 (IgG1) binds to the BGP-A2 domain under denaturing conditions; it does not react with native BGP on the cell surface, in cell extracts and bile and cannot therefore be used for immunofluorescent detection of BGP or for its immunoprecipitation.
Acknowledgements
We thank M. Aulická and H. Mrázová for technical assistance. This work was supported by grants from the Ministry of Health of the Czech Republic (No. 3755-3), National Cancer Institute of Canada, and the Medical Research Council of Canada.
References
Amoui, M., Dráberová, L., Tolar, P., Dráber, P. (1997) Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc#epsilon receptors. Eur. J. Immunol. 27, 321-328.
Barnett, T. R., Kretschmer, A., Austen, D. A., Goebel, S. J., Hart, J. T., Elting, J. J., Kamarck, M. E. (1989) Carcinoembryonic antigens: alternative splicing accounts for the multiple mRNAs that code for novel members of the carcinoembryonic antigen family. J. Cell Biol. 108, 267-276.
Barnett, T. R., Drake, L., Pickle II, W. (1993) Human biliary glycoprotein gene: characterization of a family of novel alternatively spliced RNAs and their expressed proteins. Mol. Cell. Biol. 13, 1273-1282.
Dráber, P., Zikán, J., Vojtíšková, M. (1980) Establishment and characterization of permanent murine hybridomas secreting monoclonal anti-Thy-1 antibodies. J. Immunogenet. 7, 455-474.
Dráber, P., Skubitz, K. M. (1997) Signal transduction mediated by the CEA family. In: Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives, ed. Stanners, C. P., Harwood Academic Publishers, Amsterdam, in press.
Drzeniek, Z., Lamerz, R., Fenger, U., Wagener, C., Haubeck, H.-D. (1991) Identification of membrane antigens in granulocytes and colonic carcinoma cells by a monoclonal antibody specific for biliary glycoprotein, a member of the carcinoembryonic antigen family. Cancer Lett. 56, 173-179.
Ilantzis, C., Jothy, S., Alpert, L. C., Dráber, P., Stanners, C. P. (1997) Cell-surface levels of human carcinoembryonic antigen are inversely correlated with colonocyte differentiation in colon carcinogenesis. Lab. Invest., 76, 703-716.
Rojas, M., Fuks, A., Stanners, C. P. (1990) Biliary glycoprotein, a member of the immunoglobulin supergene family, functions in vitro as a Ca2+-dependent intercellular adhesion molecule. Cell Growth Diff. 1, 527-533.
Svenberg, T., Wahren, B., Hammarström, S. (1979) Elevated serum levels of a biliary glycoprotein (BGP I) in patients with liver or biliary tract disease. Clin. Exp. Immunol. 36, 317-325.
Zimmermann, W., Thompson, J. (1990) Recent developments concerning the carcinoembryonic antigen gene family and their clinical implications. Tumor Biol. 11, 1-4.
Monoclonal Antibody Register New Monoclonal Antibodies to Human Leucocyte Surface Molecule CD2
K. DRBAL, I. HILGERT, M. CEBECAUER, P. ANGELISOVÁ, V. HOØEJŠÍ............245
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Václav Hoøejší, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeòská 1083, 142 20 Prague, Czech Republic [E-mail: horejsi@biomed.cas.cz; Fax: (420-2) 472 79 79]
Background
CD2 is an adhesion/signalling receptor of approx. 50 kDa mol. wt. expressed on the surface of T lymphocytes and NK cells (Davis and van der Merwe, 1996). Its extracellular part consists of two immunoglobulin superfamily domains and interacts with a structurally similar ligand, CD58 (LFA-3) (Barclay et al., 1993). Alternative low-affinity ligands may be CD48 (Hahn et al., 1992) and CD59 (Arulanandam et al., 1993). Signalling via CD2 seems to regulate cytokine secretion by T cells (Holter et al., 1996). Monoclonal antibodies (mAbs) to CD2 are useful reagents for research on signalling functions of this receptor and may have a therapeutic potential (Waldmann and Cobbold, 1993).
Description of the new monoclonal antibodies, MEM-65 and MEM-69
Production.
Hybridoma cell lines producing mAbs MEM-65 and MEM-69 were obtained after immunization of BALB/c mice with human peripheral T cells using standard procedures for fusion of the splenocytes with Sp2/0 myeloma cells, selection and cloning.
Specificity.
Cytofluorometric analysis of peripheral blood leucocytes and COS cells transfected with CD2 cDNA revealed that both mAbs gave staining patterns very similar to those yielded by a standard mAb to CD2 (Fig. 1). Both mAbs reacted specifically with COS cells transfected with CD2 cDNA expression vector (Fig. 1). Both mAbs reacted specifically with a CD2-human immunoglobulin fusion protein (H. Stockinger, personal communication) and with the recombinant N-terminal domain of CD2 protein (amino acid residues 1--85; B. Schraven, personal communication). Both mAbs immunoprecipitated from detergent lysates of surface 125I-labelled HPB-ALL cells a protein of approx. 50--55 kDa (not shown). MEM-65 reacted weakly with a 50--55 kDa zone in a sample prepared from HPB-ALL cells under the conditions of Western blotting if a non-reduced, non-boiled sample was used for SDS PAGE. MEM-69 did not recognize the antigen on the blot even under these relatively mild conditions (not shown). The binding of fluorescein-labelled MEM-65 and MEM-69 to peripheral blood T cells was blocked by excess of standard CD2 mAbs OKT11 and RFT11 but not by several other CD2 mAbs tested (not shown). CD2 specificity of MEM-65 was confirmed during the 6th HLDA Workshop (mAb No. T012) (Kishimoto et al., 1997).
Properties. Both mAbs are of the IgG1 isotype; their pI's are 6.4--6.9 (MEM-65) and 7.1--7.5 (MEM-69).
Acknowledgements
We are indebted to Dr. H. Stockinger for CD2 cDNA and testing of the mAbs on CD2-Ig fusion protein, and Dr. B. Schraven for testing the reactivity of the mAbs with recombinant CD2 domains.
References
Arulanandam, A. R. N., Moingeon, P., Concino, M. F., Recny, M. A., Kato, K., Yagita, H., Koyasu, S., Reinherz, E. L. (1993) A soluble multimeric recombinant CD2 protein identifies CD48 as a low affinity ligand for human CD2: divergence of CD2 ligands during the evolution of human and mice. J. Exp. Med. 177, 1439-1450.
Barclay, A. N., Birkeland, M. L., Brown, M. H., Beyers, A. D., Davis, S. J., Somoza, C., Williams, A. F. (1993) The Leucocyte Antigen Facts Book, p. 104, Academic Press, London.
Davis, S. J. and van der Merwe, P. A. (1996) The structure and ligand interactions of CD2: implications for T-cell function. Immunol. Today 17, 177-187.
Hahn, W. C., Menu, E., Bothwell, A. L. M., Sims, P. J., Bierer, B. E. (1992) Overlapping but nonidentical binding sites on CD2 for CD58 and a second ligand CD59. Science 256, 1805-1807. Holter, W., Schwarz, M., Cerwenka, A., Knapp, W. (1996) The role of CD2 as a regulator of human T-cell cytokine production. Immunol. Rev. 153, 325-342.
Kishimoto, T., Goyert, S., Kikutani, H., Mason, D., Miyasaka, M., Moretta, L., Ohno, T., Okumura, K., Shaw, S., Springer, T. A., Sugamura, K., Sugawara, H., von dem Borne, A. E. G. K., Zola, H., eds. (1997) Leukocyte Typing VI. White Cell Differentiation Antigens. Garland Publishing Inc., New York, in press.
Waldmann, H., Cobbold, S. (1993) The use of monoclonal antibodies to achieve immunological tolerance. Immunol. Today, 14, 247-252.
Abstracts
The Genetic Basis of Primary, Predominantly Specific Immunodeficiencies
I. ŠTERZL, J. ŠTERZL
The presented review lists primary immunodeficiencies which essentially involve a mutation in genes coding for functionally important molecules, membrane antigens (e.g., MHC), chains of lymphokine receptors, protein kinases of the signal cascade, transcription factors, and some important regulators of cellular metabolism. Mutations occur as early as during embryogenesis (lymphopoiesis - I) as well as an induction of the immune response by antigen ligand binding to cell receptors, TCR, BCR (immunopoiesis - II). Immunodeficiencies are classified by the stage of development (I) or immune response induction (II) in which they occur most markedly, even in clinical terms. It has been pointed out that the same autoactivation stimuli and mechanisms, allowing differentiation--maturation of cells during embryogenesis (action of stem cell factor (SFC), IL-3, IL-7, and activation cascade), serve even later as a functional prerequisite for an adaptive immune response to antigen. As a result, this attempt to classify primary immunodeficiencies by differentiation periods (when they become evident most markedly in terms of their function) has an inherent logical limitation. Some early mutations turn immediately lethal, some express themselves by blocking embryonic lymphopoiesis while other mutations do not become demonstrable until after cell stimulation by antigens. This explains why the developmental differentiation scheme is bound to turn, in the future, into an immunodeficiency classification by localization of gene mutations and their incidence in time, e.g., increased mutation incidence during proliferation following cell stimulation by antigen stimuli.
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Comparative Evaluation of Four Genotyping Methods for Hepatitis C Virus
K. SEME1, M. POLJAK1, P. DOVÈ2, S. KOREN1
Four most widely accepted genotyping methods for hepatitis C virus (HCV): (i) amplification of the core region with genotype-specific primers; (ii) nested polymerase chain reaction (PCR) in the core region followed by hybridization to HCV type-specific probes; (iii) reverse hybridization with the line probe assay Inno LiPA (Innogenetics, Gent, Belgium) using type-specific probes for the 5' non-coding region (NCR); and (iv) restriction fragment length polymorphism analysis of DNA amplified from the 5' NCR were applied to 40 HCV RNA isolates obtained from Slovenian patients in order to determine the concordance and applicability of various genotyping systems. Additionally, in isolates with discordant results nucleotide sequence analysis of a part of the NS-5 region was performed. Both genotyping methods based on the analysis of the 5' NCR were found more sensitive than those methods based on the analysis of the HCV core region. None of the four genotyping methods correctly classified all Slovenian HCV RNA isolates. PCR with genotype-specific primers was identified as entirely unsuitable for genotyping of Slovenian HCV RNA isolates. The remaining genotyping methods could clearly differentiate between HCV genotypes, but were not entirely reliable for HCV subtyping. The specificity of genotyping methods, which are based on the 5' NCR or core region, was occasionally hampered, due to a lack or excess of sequence variation in their respective target regions.
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