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2	Reflections on the development and 46 years work in immunology Morten Harboe Norwegian Society of Immunology, 8 March 2005
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5	Polymorphisms in human serum proteins published as of 1959 1) Haptoglobin. Hp 1-1, Hp 2-1, and Hp 2-2 types detected by electrophoresis in starch gel. O. Smithies, 1955. 2) Gc-globulin. (Now called vitamin D-binding protein) Gc 1-1, Gc 2-1, and Gc 2-2 types detected by immunoelectro- phoresis. J. Hirschfeld, 1959. 3) Transferrin. Several forms with different electrophoretic mobility. O. Smithies, 1957; E. R. Giblett et al., 1959. It was only at the beginning. The field was entirely open. TODAY we find genetic variation in genes coding for proteins almost whereever we look for it.
6	References to previous Gm papers in our first paper on Gm^x (1959) Demonstration and properties of Gm^a Grubb, 1956 Grubb and Laurell, 1956 Two alternatives: Gm^a and Gm, the allele Confirmation of Gm^a Moullec et al., 1956 Linnet Jepsen, 1957 There were four papers in the field … Today, beginning work on a Thesis …
7	Anti-Gm^x 1) First, we established a basic hemagglutination inhibition assay for typing of Gm^a. The anti-Gm^a antibody was a selected rheumatoid factor, i.e. a serum from a patient with RA. Indicator cells were group O Rh(D) positive red cells coated with anti-D to fix one individual’s IgG on to the red cell surface. To find new factors was trial and error with a need of good luck. 2) Two of 20 RA sera showed a different reaction pattern which defined a new factor, called Gm^x. 3) Studies of 32 families with 91 children showed that Gm^x was determined as an autosomal dominant character. 4) It was related to Gm^a, a bit strange, so we called it Gm^x.
8	Anti-Gm 1) Further trial and error: Red cells coated with one of 8 anti-D antibodies were agglutinated by one of 65 sera containing strong rheumatoid factor activity. 2) A hemagglutination inhibition system worked well with this combination of anti-D S.V. and RA serum J.K. 3) Of sera from 119 individuals, 97 (81.5 %) inhibited the agglutination, i.e. they contained the reactive factor. 4) Sera from 25 familes with 66 children were studied. The pattern indicated that we had found the allele of Gm^a. 5) We were stunned and did not dare to call the factor anything else than suggested by Grubb. The manuscript was sent to Nature and accepted without any comment.
9	Comments on the anti-Gm paper Dear Dr. Harboe, Congratulations with your Nature paper! You are entitled to, and perhaps even expected to give the factor a name. Yours sincerely, A. E. Mourant M.D., M.R.C.P. etc.
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11	To Henry G. Kunkel’s laboratory, July 1961 1) He studied antibody structure, specificity and function in human disease, in particular in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple myeloma and macroglobulinemia. 2) The stage: He had a laboratory, a ward for inpatients in The Rockefeller University Hospital, and a weekly outpatient clinic. 3) Sentral subjects: Rheumatoid factors, anti-nuclear antibodies in SLE. 4) The driving concept: Monoclonal myeloma proteins and macroglobulins were isolated and studied as model proteins for antibodies. A central point was immunization of rabbits to produce antibodies to individual myeloma proteins to obtain information on their antigenic determinants and structural relationship. This proved to be highly informative and productive, at the cutting edge of the time. 5) In addition studies of complement, basic structure and involvement in human disease, H. J. Müller-Eberhard.
12	Understanding antibodies in 1961 Antibody behaviour and function 1) Antibody mediated immunity was well known. Anti-toxin in diphteria, first Nobel prize in Medicine or Physiology, Emil von Behring 1901. 2) Antibodies were extensively used in therapy. Antibody formation after vaccination was known to be responsible for its effect. 3) The specificity of serological reactions, K. Landsteiner 1936. 4) Interaction with complement and its effect well known. 5) Large array of immunochemical techniques, important application for diagnosis of infectious diseases. 6) Produced by plasma cells. In culture of single plasma cells, one cell produced antibodies of a single specificity. 7) Burnet’s clonal selection theory had been developed, 1957-1959.
13	Understanding cells in 1961 Lymphocytes TODAY: At the center of immunology. THEN: Known to be present almost everywhere, in particular in the gut. Associated with chronic infection. Present in increased amounts in 2nd set skin grafts after accelerated rejection in rabbits. Delayed type hypersensitivity with pronounced local lymphocyte infiltration was transferable by cells, not by serum (antibodies). No one understood their function. JL Gowans was on the track studying their recirculation. JFAP Miller (Lancet, Sept. 1961): Neonatal thymectomy in mice induced lymphopenia and delayed transplant rejection.
14	JEM FROM THE ARCHIVE The first entry in a new series, 3 January 2005 Discovering lymphocyte subsets Heather L. Van Epps JEM News Editor At a scientific meeting (8 years later) in 1968, Jacques Miller was accused of complicating immunology. He and others suggested that there was not one but two kinds of lymphocytes – one from the thymus and one from the bone marrow. In a pair of groundbreaking articles published in the Journal of Experimental Medicine in 1968, Miller and his student Graham Mitchell proved that two subsets of lymphocytes did exist and identified which subset mediated antibody responses.
15	Understanding antibodies in 1961 Antibody structure, very limited information 1) Known to be proteins. 2) Electrophoresis: They were γ-globulins, but also located in the β₂ area. 3) Ultracentrifugation: 7S and 19S. Kabat and Kunkel had been in Uppsala to study antibodies! 4) The γG molecule was shown to consist of two kinds of chains, light and heavy, held together by disulfide bonds (Edelman & Poulik 1961). 5) γG was split in two different kinds of fragments by papain, only one of them reacting with antigen (Porter 1959). 6) γG: the F fragment distinct for γG, the S fragment shared (antigenic) properties with β₂M and β₂A. Edward C. Franklin, JEM October 1961.
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17	Understanding antibodies in 1961 Antibody structure, very limited information 1) Proteins. 2) Electrophoresis: They were γ-globulins, but also located in the β₂ area. 3) Ultracentrifugation: 7S and 19S. Kabat and Kunkel went to Uppsala to study antibodies! 4) The γG molecule was shown to consist of two kinds of chains, light and heavy, held together by disulfide bonds (Edelman & Poulik 1961). 5) γG was split in two different kinds of fragments by papain, only one of them reacting with antigen (Porter 1959). 6) γG: the F fragment distinct for γG, the S fragment shared (antigenic) properties with β₂M and β₂A. Edward C. Franklin, JEM October 1961.
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24	The strange Gm(b+) myeloma protein 1) Gm(a+) 60 % of American whites, Gm(b+) 91 % of American whites Gm(a+) myeloma proteins: 15/47, 32 % Gm(b+) myeloma proteins: 2/47, 4.2 % 2) Markedly increased inhibiting capacity/mg Gm(b+) mye- loma protein compared with polyclonal normal IgG. 3) Hypothesis which would explain the observations as stated in the Discussion part of the JEM paper: ”The Gm(b) character may be present only on a small percentage of the γ-globulin molecules in a normal Gm(b+) individual.”
25	Subclasses of human IgG 1) Kunkel’s antibodies to myeloma proteins could separate them into four distinct groups, corresponding protein being present in normal IgG in different concentrations. 2) Gm^a occurred in only one of these groups, now called IgG1, Gm^b in another, IgG3. Jacob B. Natvig had a central role in this further work. 3) After we were back in Oslo all gradually fell into place: The Gm factors were markers of different genes coding for the four subclasses of IgG. Gm^a and Gm^b behaved as alleles in the initial family studies. They are not, but polymorphic characters present in closely linked genes. In today’s language: This is haplotype inheritance and linkage disequilibrium, matters intensely studied at IMMI.
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27	Subclasses of human IgG 1) Kunkel’s antibodies to myeloma proteins could separate them into four distinct groups, corresponding protein being present in normal IgG in different concentrations. 2) Gm^a occurred in only one of these groups, now called IgG1, Gm^b in another, IgG3. Jacob B. Natvig had a central role in this further work. 3) After we were back in Oslo all gradually fell into place: The Gm factors were markers of different genes coding for the four subclasses of IgG. Gm^a and Gm^b behaved as alleles in the initial family studies. They are not, but polymorphic characters present in closely linked genes. In today’s language: This is haplotype inheritance and linkage disequilibrium, matters intensely studied at IMMI.
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31	Theses from work connected to the Institute of Experimental Medical Research (subject area) Hemolytic anemia O.J.Mellbye (1969) Transplantation E.Thorsby (1969) Connective tissue diseases R.A.Larsen (1972) Eye diseases B.Berger (1972), H.O.Sandberg (1980) Immunoglobulins B.G.Solheim (1972), K.Hannestad (1973), T.Eskeland (1975), E.Saltvedt (1977) Mycobacterial infection B.Myrvang (1975), O.Closs (1975), G.Bjune (1980), R.N.Mshana (1983), R.Melsom (1983), L.J.Reitan (1985), M.Løvik (1986) Tumour immunology J.L.Svennevig (1982) Surgery and trauma E.Fosse (1987)
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35	Mechanisms of nerve damage during reversal reactions 1 Reversal reactions are associated with an abrupt increase in cell mediated immune reactions to mycobacterial antigenic determinants. Godal T et al., Acta Pat Microbiol Scand 1973;236:45-53 Bjune G et al., Clin Exp Immunol 1976;25:85-94 Mshana RN et al., Clin Exp Immunol 1983;52:441-448
36	Mechanisms of nerve damage during reversal reactions 2 Delayed type hypersensitivity (DTH) reactions against M. leprae antigenic determinants released from Schwann cells damage the nerve as an innocent bystander. This view influenced introduction of new therapy, immunosuppression combined with continued anti-mycobacterial chemo- therapy. It still remains valid today.
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