Immunology Letters 164 (2015) 40–43

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Anti-Gal IgM, IgA and IgG natural antibodies in childhood Marketa Hamanova a , Magda Chmelikova a , Ivo Nentwich b , Vojtech Thon a,∗ , Jindrich Lokaj a a b

Department of Clinical Immunology and Allergy, Faculty of Medicine, Masaryk University, Brno, Czech Republic Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway

a r t i c l e

i n f o

Article history: Received 24 September 2014 Received in revised form 27 January 2015 Accepted 3 February 2015 Available online 12 February 2015 Keywords: Natural antibodies Childhood Anti-Gal Kinetics Immunodeficiency

a b s t r a c t The target for the most abundant xenoreactive natural antibodies in humans is the ␣-Gal epitope. Anti-Gal could provide natural immune defense against pathogens that express the ␣-Gal epitope. Anti-Gal natural antibodies are usually studied in adult individuals. Data demonstrating the incidence and concentration of anti-Gal natural antibodies in childhood are in short supply and incomplete. In the present study we prospectively quantified anti-Gal IgM, IgA and IgG levels in different age groups of children from delivery to 24 months of age and compared these levels to the level of these antibodies in their respective mothers. Measurement of anti-Gal antibodies may broaden the spectrum of specific antibodies that are available for determination of specific antibody responses in physiological and pathological conditions in children. Plasma was collected from umbilical cord blood of full term newborn, from blood of infants at age 6, 12 and 24 months and from their respective mothers at time of delivery. Quantitative determination of anti-Gal antibodies IgM, IgA and IgG were made with the enzyme immunoassays Human Anti-Alpha Galactosyl IgM ELISA, IgG ELISA and IgA ELISA. Hemagglutination activity was titrated against rabbit erythrocytes. The kinetic processes for the formation of natural antibodies in the first two years of life, in general, compared with the kinetics for the formation of total immunoglobulins IgM, IgA and IgG. There were no detectable anti-Gal IgM and IgA in the cord blood, whereas anti-Gal IgG were found at similar levels in both neonate cord blood and peripheral blood of their respective mothers. When comparing the percentage of natural antibodies in the plasma of children, the level of natural antibodies in children at the age of two years was approximately 37% for IgM, 25% for IgG and 15% for IgA. The titration of antibodies required for agglutination of rabbit red blood cells over the 24 month period followed the same trend observed for the formation of natural antibodies. Our study demonstrates the kinetics of formation of anti-Gal IgM, IgA and IgG natural antibodies in the first two years of life. The relative lack of these antibodies in this period should be taken into account when assessing for humoral immunodeficiencies, particularly with regards to the potential for children to mount an anti-carbohydrate response. © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction The presence of natural antibodies in the sera of normal, non-immunized humans and animals has been known since the beginning of immunological research [1]. The repertoire of these antibodies includes both autoantibodies [2,3] as well as xenoantibodies [4].

∗ Corresponding author at: Department of Clinical Immunology and Allergy, Faculty of Medicine, Masaryk University, St. Anne’s University Hospital, Pekarska 53, CZ-656 91 Brno, Czech Republic. Tel.: +420 543183128; fax: +420 543183143. E-mail address: [email protected] (V. Thon).

The target for the most abundant xenoreactive natural antibodies in humans is the ␣-Gal epitope (Gal␣1–3Gal␤1–4GlcNAc-R) which is expressed on the terminal sugar units of oligosaccharides conjugated to protein or lipid molecules in several microorganisms including protozoa, bacteria, enveloped viruses and non-primate mammals, but not in humans [5]. Their expression on rabbit and pig erythrocytes is of practical diagnostic importance. The presence of anti-Gal throughout a human’s life implies a constant antigenic stimulation which, as is the case for the ABO isohaemagglutinins, may originate in the intestinal microbiota [5–7]. There is no definitive explanation for the abundance of these natural antibodies with this particular specificity, presumably above that of any other natural antibodies. Anti-Gal could provide natural immune defense against pathogens that express

http://dx.doi.org/10.1016/j.imlet.2015.02.001 0165-2478/© 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

M. Hamanova et al. / Immunology Letters 164 (2015) 40–43

␣-Gal epitope and thus play a physiological role in the recognition and removal of senescent cells and other self-antigens [8–10]. From a clinical laboratory immunology point of view the titration of anti-Gal in serum may serve as a useful method for the assessment of humoral immunodeficiency disorders [6,11].

2. Patients and methods 2.1. Peripheral blood and cord blood samples Blood samples from mothers and their children in the research project – immune factors of maternal milk and atopic reactivity in breast-feed infants – were collected with the parent’s informed consent and with the permission of the ethical commission of the Masaryk University in Brno, Czech Republic. Plasma was collected from umbilical cord blood of 41 full term newborn, from the blood of infants at age 6 (n = 26), 12 (n = 16) and 24 (n = 23) months and from their respective mothers at time of delivery. Plasma was aliquoted, frozen, stored at −70 ◦ C and freshly thawed prior to use. Concentration of total IgM, IgA and IgG in plasma was measured nephelometrically using the analyzers IMMAGE 800 (Beckman, Brea, CA, USA) and BNII (Dade Behring, Marburg, Germany). The results are stated in g/l.

2.2. Determination of anti-Gal antibodies and total immunoglobulins Quantitative determination of anti-Gal IgM, IgA and IgG was made with the new standardized enzyme immunoassays (Human Anti-Alpha-Galactosyl IgM ELISA, IgG ELISA and IgA ELISA, all BioVendor, Brno, Czech Republic) that quantifies anti-Gal binding to a chemically synthesized molecule displaying the terminal beta disaccharide Gal␣1–3Gal epitope. Plasma samples were diluted from 1:20 to 1:2560 with dilution buffer. The absorbance at 450 nm was determined using an EL 808 Bio Kinetics Reader (BioTek Instruments, Winooski, VT) with the reference wavelength set to 630 nm. Sample concentrations were calculated from the

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calibration curve and multiplied by their respective dilution factors. The results are presented in concentrations (U/ml). 2.3. Hemagglutination assay Hemagglutination activity was titrated by mixing two-fold serial dilutions (diluent: saline) of the plasma samples with equal volumes of 0.5% rabbit erythrocytes in polystyrene tubes (Turbox Acrylic Cuvettes, Orion Diagnostica, Espoo, Finland). Agglutination was evaluated after incubation for 2 h at 37 ◦ C and 4 ◦ C overnight. The titre was defined as the highest serum dilution causing agglutination. 2.4. Statistical analysis Statistical analysis was performed using Statistica software (StatSoft CR, version 10, Prague, Czech Republic). The Mann–Whitney U-test was used for analysis in independent groups and paired Wilcoxon test was applied to dependent variables. 3. Results The kinetics for the formation of natural antibodies in the first two years of life, in general compares with the kinetics for the formation of total immunoglobulins IgM, IgA and IgG (Fig. 1a–c). There were no detectable anti-Gal IgM and IgA in the cord blood, whereas anti-Gal IgG were found at similar levels in both neonate cord blood and peripheral blood of their respective mothers. The ratio of anti-Gal to total serum immunoglobulins was calculated for each isotype (IgM, IgA and IgG). The concentration increase towards the normal adult levels in IgM isotype was markedly quicker than for IgG and IgA (Table 1). When comparing the percentage of natural antibodies in the plasma of children (setting concentrations in mothers = 100%), we see that the level of natural antibodies in children at the age of two years was approximately 37% for IgM, 25% for IgG and 15% for IgA (Fig. 2). The titration of antibodies required for agglutination of rabbit red blood cells over the 24 month period

Fig. 1. Dynamics of production of anti-Gal and immunoglobulins in infants. (A) Anti-Gal IgM antibodies and IgM immunoglobulins. (B) Anti-Gal IgA antibodies and IgA immunoglobulins. (C) Anti-Gal IgG antibodies and IgG immunoglobulins. CB denotes cord blood; 6, 12, 24 means age in months; “mo” stands for blood of mothers.

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M. Hamanova et al. / Immunology Letters 164 (2015) 40–43

Table 1 Relationship of anti-Gal antibodies and total serum immunoglobulins. The ratio (expressed as median) was calculated as anti-Gal to total serum immunoglobulins for each isotype (IgM, IgA, IgG). Ratio of anti-Gal antibodies to total serum immunoglobulins (U/mg)

Cord blood 6 months 12 months 24 months Mothers

IgM

IgA

IgG

0.8 2.6 20.1 40.3 28.5

0 4.4 16.6 25.1 27.1

3.1 0.9 1.0 1.9 4.6

Fig. 3. Natural agglutinins to rabbit erythrocytes in infants. CB denotes cord blood; 6, 12, 24 means age in months; “mo” stands for blood of mothers.

Fig. 2. Concentration changes of anti-Gal antibodies and total immunoglobulins in infants expressed as a percentage of the maternal value (100%).

followed the same trend observed for the formation of natural antibodies (Fig. 3). 4. Discussion Anti-Gal are usually measured in adult individuals. They have traditionally been detected by agglutination test with rabbit or porcine erythrocytes [12,13] with the epitope against which they are raised identified as Gal␣1–3Gal␤1–4GlcNAc-R [5,14]. Data on the incidence and levels of anti-rabbit erythrocytes and/or anti-Gal in childhood are lacking or incomplete [6,17,21,22]. In the present

study we prospectively quantified anti-Gal IgM, IgA and IgG levels in different age groups of children from delivery to 24 months of life and compared these levels to the levels of these antibodies in their respective mothers. In clinical medicine, anti-Gal are a major immune barrier to proficient xenotransplantation, particularly in the transplantation of organs from pigs to humans [5,15–17]. In addition, anti-Gal may be exploited for eliciting a protective immune response against micro-metastases by enhancing the immunogenicity of tumourassociated antigens on autologous tumour cells [5,18,19] and may also be harnessed for acceleration of wound healing and possibly in the regeneration of ischaemic myocardium and injured nerve tissue [5,20]. The titration of anti-Gal in serum may serve as a useful method for the assessment of humoral immunodeficiency disorders [6,11] and was originally suggested in the 1960s using agglutination of rabbit erythrocytes [12]. Measurement of anti-Gal antibodies may broaden the spectrum of specific antibodies that are available for the determination of specific antibody responses in physiological and pathological conditions in children. The period from birth to two years of age is characterized by a steeper increase of IgM compared to IgG and IgA, which could be due to failure in switching of the isotype antibody response. In the cord blood of newborns, anti-␣-Gal IgG were found at similar concentrations to that present in the peripheral blood of their respective mothers which reflects the transplacental transfer of maternal IgG antibodies. Consequently, specific anti-Gal IgG were of low concentration at six months of age and started to slowly increase with increasing age. Even at 12 months of age the concentrations were significantly lower than the concentrations in their mothers as calculated with the ratio of specific anti-␣-Gal antibodies to total immunoglobulins. To our knowledge, our study is the first demonstrating the kinetics for the formation of anti-Gal natural antibodies (IgM, IgA, IgG) in the first two years of life. The relative lack of these antibodies in this period should be taken into account when assessing for humoral immunodeficiencies, particularly with regards to the potential for children to mount an anti-carbohydrate response.

Conflict of interest The authors declare that they have no conflict of interest.

M. Hamanova et al. / Immunology Letters 164 (2015) 40–43

Acknowledgment This work was supported by the European Community’s Seventh Framework Programme FP7/2007–2013 under grant agreement no. 201549 (EURO-PADnet HEALTH-F2-2008-201549). References ˇ [1] Boyden S. The occurrence and significance of natural antibodies. In: Sterzl J, editor. Molecular and cellular basis of antibody formation. Prague: Czech Acad. Sci.; 1965. p. 329–40. [2] Coutinho A, Kazatchkine MD, Avrameas S. Natural autoantibodies. Curr Opin Immunol 1995;7:812–8. [3] Madi A, Bransburg-Zabary S, Kenett DY, Ben-Jacob E, Cohen IR. The natural autoantibody repertoire in newborns and adults: a current overview. Adv Exp Med Biol 2012;750:198–212. [4] Parker W, Bruno D, Platt JL. Xenoreactive natural antibodies in the world of natural antibodies: typical or unique. Transpl Immunol 1995;3:181–91. [5] Galili U. Anti-Gal: an abundant human natural antibody of multiple pathogeneses and clinical benefits. Immunology 2013;140:1–11. [6] Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med 1984;160: 1519–31. [7] Galili U, Mandrell RE, Hamadeh RM, Shohet SB, Griffiss JM. Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun 1988;56:1730–7. [8] Ochsenbein AF, Zinkernagel RM. Natural antibodies and complement link innate and acquired immunity. Immunol Today 2000;21:624–30. [9] Grabar P. Autoantibodies and the physiological role of immunoglobulins. Immunol Today 1983;4:337–40.

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[10] Avrameas S. Natural autoantibodies: from ‘horror autotoxicus’ to ‘gnothi seauton’. Immunol Today 1991;12:154–9. [11] Bernth-Jensen JM, Møller BK, Jensenius JC, Thiel S. Biological variation of anti-␣Gal-antibodies studied by a novel Time-Resolved ImmunoFluorometric Assay. J Immunol Methods 2011;373:26–35. [12] Tönder O, Natvig JB, Matre R. Antibodies in human sera to rabbit erythrocytes. Immunology 1967;12:629–37. [13] Parker W, Bruno D, Holzknecht ZE, Platt JL. Characterization and affinity isolation of xenoreactive human natural antibodies. J Immunol 1994;153:3791–803. [14] Galili U, Macher BA, Buehler J, Shohet SB. Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha (1–3)-linked galactose residues. J Exp Med 1985;162:573–82. [15] Galili U. Interaction of the natural anti-Gal antibody with alpha-galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today 1993;14:480–2. [16] Cramer DV. Natural antibodies and the host immune responses to xenografts. Xenotransplantation 2000;7:83–92. [17] Minanov OP, Itescu S, Neethling FA, Morgenthau AS, Kwiatkowski P, Cooper DK, et al. Anti-GaL IgG antibodies in sera of newborn humans and baboons and its significance in pig xenotransplantation. Transplantation 1997;63:182–6. [18] Galili U, Wigglesworth K, Abdel-Motal UM. Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines. J Immunol 2007;178:4676–87. [19] Whalen GF, Sullivan M, Piperdi B, Wasseff W, Galili U. Cancer immunotherapy by intratumoral injection of ␣-gal glycolipids. Anticancer Res 2012;32:3861–8. [20] Galili U. Discovery of the natural anti-Gal antibody and its past and future relevance to medicine. Xenotransplantation 2013;20:138–47. [21] Tönder O, Larsen B, Aarskog D, Haneberg B. Natural and immune antibodies to rabbit erythrocyte antigens. Scand J Immunol 1978;7:245–9. [22] Doenz U, Nydegger UE, Kueng A, Carrel T, Mohacsi P. Anti-Galalpha1-3Gal IgM/IgG antibody levels in infants: do they have a clinical relevance in pediatric xenotransplantation. J Heart Lung Transplant 2000;19:1108–13.

Anti-Gal IgM, IgA and IgG natural antibodies in childhood.

The target for the most abundant xenoreactive natural antibodies in humans is the α-Gal epitope. Anti-Gal could provide natural immune defense against...
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