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Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg History of Immunology.

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Presentation on theme: "Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg History of Immunology."— Presentation transcript:

1 Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg History of Immunology Part 3: START OF IMMUNOLOGY Core Module Immunology Doctoral Training Group GK1660 Erlangen 2011

2 Doctoral Training Group GK University of Erlangen-Nürnberg 2 TOPICS - History of Immunology Repeat: Discovery of cells and germs ( ) Repeat: Prevention of Infection (1840 – today) Start of Immunology ( ) Immunochemistry - The antibody problem ( ) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

3 DISCOVERY OF CELLS & GERMS (brief repeat from last lecture) ( )

4 Doctoral Training Group GK University of Erlangen-Nürnberg 4 Germ Theory 1683Leeuwenhoek described in a letter to the Royal Society very likely bacteria (types of animalcules) in the saliva and tooth scrapings from his mouth. 1859Louis Pasteur – disproves spontaneous generation of cells from decayed organic matter Germ Theory: Infectious disease are caused by germs ~1860Louis Pasteur - Fermentation process and souring of wine is caused by the growth of different microorganisms 1876Robert Koch isolates the first disease-causing pathogen (anthrax) and provides definitive proof of the germ theory 1884Kochs postulates to classify infectious agent

5 Doctoral Training Group GK University of Erlangen-Nürnberg 5 Germ Theory 1683Leeuwenhoek described in letter to the Royal Society very likely bacteria (types of animalcules) in the saliva and tooth scrapings from his mouth. 1854John Snow - Traces the source of the 1854 cholera outbreak in Soho, London to the drinking water and not to the bad smell (miasma theory) 1859Louis Pasteur – Germ Theory: Infectious disease are caused by germs 1859Louis Pasteur - Fermentation process is caused by the growth of microorganisms 1876Robert Koch isolates the first disease-causing pathogen (anthrax) and provides definitive proof of the germ theory 1882Koch isolates tubercle bacillus 1884Kochs postulates to classify infectious agent

6 Doctoral Training Group GK University of Erlangen-Nürnberg 6 TOPICS - History of Immunology Discovery of cells and germs ( ) Prevention of Infection (1840 – today) Start of Immunology ( ) Immunochemistry - The antibody problem ( ) Self-/non-self discrimination (1940 – today) Generation of antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

7 PREVENTION OF INFECTION

8 Doctoral Training Group GK University of Erlangen-Nürnberg 8 Preventions of Infections DESINFECTION – External 1840s Ignaz Semmelweis - hand washing prevents childbirth fever 1867Joseph Lister - carbol to treat wounds for surgery DESINFECTION – Internal (antimicrobial compounds) 1881E. v. Behring - unsuccessful attempts to treat infections with chemicals 1909P. Ehrlich - First organic compound to treat syphilis (Salvarsan) 1929Fleming - Penicillin 1935Domagk - Sulfonamides PREVENTIVE VACCINATION 1796Jenner - Cow pox vaccination 1880Pasteur - cholera vaccination in chicken – generalization of Jenners use of cow pox vaccine. Pasteur introduces general term vaccination 1886Pasteur - rabies vaccination

9 Doctoral Training Group GK University of Erlangen-Nürnberg 9 Summary: Preventions of Infections DESINFECTION – External 1840s Ignaz Semmelweis - hand washing prevents childbirth fever 1867Joseph Lister - carbol to treat wounds for surgery DESINFECTION – Internal (antimicrobial compounds) 1881E. v. Behring - unsuccessful attempts to treat infections with chemicals 1909P. Ehrlich - First organic compound to treat syphilis (Salvarsan) 1929Fleming - Penicillin 1935Domagk - Sulfonamides PREVENTIVE VACCINATION 1796Jenner - Cow pox vaccination 1880Pasteur - cholera vaccination in chicken – generalization of Jenners use of cow pox vaccine. Pasteur introduces general term vaccination 1886Pasteur - rabies vaccination

10 Doctoral Training Group GK University of Erlangen-Nürnberg 10 IMMUNITY – Timeline 2 Discovery of cells and germs ( ) Prevention of Infection (1840 – today) Start of Immunology (1796 or 1890?? -1910) Immunochemistry - The antibody problem ( ) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

11 Doctoral Training Group GK University of Erlangen-Nürnberg 11 Immunity - Etymology Late 14c. "exempt from service or obligation" o from Latin immunitatem (nom. immunitas) "exemption from performing public service or charge (tax) o or from Latin immunis "exempt, free," from in - "not" + munis "performing services 1775 The term Immunitas was first used by Van Sweiten, a Dutch physician, to describe the effects induced by an early attempt at variolization. 1879"protection from disease o Pasteur, Koch, Ehrlich ????? Yufang Shi, Ph.D., Introduction and History of ImmunologyUMDNJ-Robert Wood Johnson Medical School

12 Doctoral Training Group GK University of Erlangen-Nürnberg 12 Immunity - Etymology Late 14c."exempt from service or obligation" o from Latin immunitatem (nom. immunitas) "exemption from performing public service or charge (tax) o or from Latin immunis "exempt, free," from in - "not" + munis "performing services 1879"protection from disease o Pasteur, Koch, Ehrlich ????? The term immunity was first used in 1775 by Van Sweiten, a Dutch physician, asimmunitas to describe the effects induced by an early attempt at variolization. Introduction and History of Immunology Yufang Shi, Ph.D. University Professor Department of Molecular Genetics, Microbiology and Immunology UMDNJ-Robert Wood Johnson Medical School Yufang Shi, Ph.D., UMDNJ-Robert Wood Johnson Medical School

13 Doctoral Training Group GK University of Erlangen-Nürnberg 13 Yufang Shi, Ph.D., UMDNJ-Robert Wood Johnson Medical School The term immunity was first used in 1775 by Van Sweiten, a Dutch physician, asimmunitas to describe the effects induced by an early attempt at variolization. Yufang Shi, Ph.D. University Professor Department of Molecular Genetics, Microbiology and Immunology UMDNJ-Robert Wood Johnson Medical School

14 Doctoral Training Group GK University of Erlangen-Nürnberg 14 Immunity - Etymology Late 14c."exempt from service or obligation" o from Latin immunitatem (nom. immunitas) "exemption from performing public service or charge (tax) o or from Latin immunis "exempt, free," from in - "not" + munis "performing services 1879"protection from disease o Pasteur, Koch, Ehrlich ????? The term immunity was first used in 1775 by Van Sweiten, a Dutch physician, asimmunitas to describe the effects induced by an early attempt at variolization. Introduction and History of Immunology Yufang Shi, Ph.D. University Professor Department of Molecular Genetics, Microbiology and Immunology UMDNJ-Robert Wood Johnson Medical School

15 Doctoral Training Group GK University of Erlangen-Nürnberg 15 Phase I: Phenomenon: Immunity ( 500 B.C ) Phase II: Introduction of Vaccination & Immunochemistry (1860 – 1945) Phase III: Identification of cellular and molecular components (1945 – today) IMMUNOLOGY – Timeline

16 START OF IMMUNOLOGY

17 Doctoral Training Group GK University of Erlangen-Nürnberg 17 1.Preventive Immunization o Jenner (1789) - 1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic antibodies und complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Bordet (1901) - Complement fixation test o Wassermann (1905) - Syphilis-Nachweis o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma TOPCIS: Start of Immunology Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930

18 Doctoral Training Group GK University of Erlangen-Nürnberg 18 Descriptive early period pre- Early attempts at vaccination: Smallpox (1794 Bacteria cause disease, discovery of bacterial toxins (19 th century) Discovery of antibodies ( until 1975) Theories of antibody formation (1897 and 1960s) Discovery of B and T cells (60s) The molecular revolution (1974 -present

19 START OF IMMUNOLOGY Preventive Vaccination

20 Doctoral Training Group GK University of Erlangen-Nürnberg 20 Thucydides reports that people who survived plaque during the Peleponnesian War between Sparta and Athens were protected from disease From Thucydides History of the Peloponnesian War Yet it was with those who had recovered from the disease that the sick and the dying found most compassion. These knew what it was from experience, and had now no fear for themselves; for the same man was never attacked twice- never at least fatally. Immunity - Thucydides (430 B.C) First description of adaptive immunity !!!!!!

21 Doctoral Training Group GK University of Erlangen-Nürnberg 21 Immunity – First written reports 430 B.C.Thucydides reported that people who survived plaque during the Peleponnesian War between Sparta and Athens were protected from disease Yet it was with those who had recovered from the disease that the sick and the dying found most compassion. These knew what it was from experience, and had now no fear for themselves; for the same man was never attacked twice- never at least fatally. From Thucydides History of the Peloponnesian War 65 B.C.Roman poet Marcus Annaeus Lucanus uses in the epic poem Pharsalia the term immunes to describe his obervations that African tripe placed poison snake glands under skin to protect themselves against snake bites Silverstein, Arthur M. (1989) History of Immunology, Academic Press. First description of adaptive immunity

22 Doctoral Training Group GK University of Erlangen-Nürnberg 22 Small Pox - Disease Dt., Pocken, Blattern; engl. Small pox; lat., Variola The German word Pocken originated from Germanic poccas and means Beutel, Tasche, Blase (= Blatter) und is related to the Engl. pocket/pox/pocks Variola from lat. varius = dt. bunt, scheckig, fleckig Highly contagious viral and very often fetal disease 10% of all children before the age of 10 died of small pox

23 Doctoral Training Group GK University of Erlangen-Nürnberg 23 Small Pox - Disease The disease killed as many as 30% of those infected Queen Mary II of England, Emperor Joseph I of Austria, King Luis I of Spain, Tsar Peter II of Russia, and King Louis XV of France) Between 65–80% of survivors were pox-marked Very old disease Originated in China and India 3000 years ago Symptoms are already mentioned in Old Testament Mumy of Pharao Ramses II. of Egypt showed pox scars Reached Europe around 165 through Roman legions returning from Irak Antoninische Pest, 1 st recorded 24 year-long epidemy Worldwide since the 16th century

24 Doctoral Training Group GK University of Erlangen-Nürnberg 24 Small Poxs – Variolation & Vaccination Variolation o Oriental habit to protect children through intentional infection with lymph or pustules from a person that recovered from a mild infection of small pox. o Common practice before vaccination o Worked if exposed to a weak strain of smallpox o Wrong treatments could kill or be ineffective. o Strains of small pox differ in mortality rate (1-20%) Vaccination o Edward Jenner discovered that cowpox could protect against smallpox with less complications than variolation. o Louis Pasteur coined the term vaccination (from lat, vacca = cow) as a general procedure o immunize people against other disease

25 Doctoral Training Group GK University of Erlangen-Nürnberg 25 Ca Chinese inoculated children with material from infected people Ca. 1500Variolation introduced into Turkish harems 1717Lady Mary Montagu introduced smallpox inoculation to Europe. 1760Variolation of the families of Maria Theresia und George III. popularized variolation 1776Washington began variolating the Continental Army Small Poxs – Timeline of Variolation Lady Mary Montagu, wife of the British ambassodour to Turkey,

26 Doctoral Training Group GK University of Erlangen-Nürnberg 26 Country lore (Bauernweisheit)/Obervation o Milkmaids who caught cowpox from their cows could not catch smallpox. o Milk maids infected by cowpox have on their hands scars very similar to smallpox scars. Hypothesis o Cowpox infection protects from small pox The experiment (May 14, 1796) o Infected a boy with the lymph of a cowpox- infected milkmaid. o On 1st July, Jenner infected (variolated) the boy with small pox Result o Boy did not get sick Cowpox pustule on the hand of the dairymaid Sarah Nelmes Jenner infects James Phipps, the son of his gardener who had not yet suffered smallpox with Sarahs lymph. James became mildly ill Small Pox – The Jenner Experiment 1796

27 Doctoral Training Group GK University of Erlangen-Nürnberg 27 Pasteur introduced VACCINATION (from lt. vacca = cow) as a general term for the procedure to protect individuals with weaken pathogens (Pasteur: Trans. 7th Session Internat. Med. Congr. (1881) Conclusion o Cow pox infection protects against small pox Problems o Human experiment o Controls Publication o 1797, rejected by Transactions of the Royal Society of London. o 1801, published The Origin of the Vaccine Inoculation Baxby D Edward Jenner's Inquiry; a bicentenary analysis. Vaccine Jan 28;17(4): Small Pox – The Jenner Experiment 1796

28 Doctoral Training Group GK University of Erlangen-Nürnberg 28 Roitt p 346 Small Pox – Jenners Clinic

29 Doctoral Training Group GK University of Erlangen-Nürnberg 29 Humoralists Keep the body's humors in balance: o perform routine blood-letting o administer medicines that cause sweating, urination, bowel evacuation Contagionists Restrict contact: o leave the disease-ridden area o impose quarantine o remove sources of fomites Miasmatists Cleanse the air: o remove sources of foul smells o flush the air with smoke o breathe in aromatic substances In 1798, a new way to control the spread of a disease (smallpox): Vaccinatists Vaccinate: o purposefully infect the population with another, less virulent disease Modified after B. J. Becker: Infectious and Epidemic Disease in History, Department of History, University of California, Irvine Before Jenner: Disease protection

30 Doctoral Training Group GK University of Erlangen-Nürnberg 30 Humoralists Keep the body's humors in balance: o perform routine blood-letting o administer medicines that cause sweating, urination, bowel evacuation Contagionists Restrict contact: o leave the disease-ridden area o impose quarantine o remove sources of fomites Miasmatists Cleanse the air: o remove sources of foul smells o flush the air with smoke o breathe in aromatic substances In 1798, a new way to control the spread of a disease (smallpox): Vaccinatists Vaccinate: o purposefully infect the population with another, less virulent disease Modified after B. J. Becker: Infectious and Epidemic Disease in History, Department of History, University of California, Irvine Before Jenner: Disease protection

31 Doctoral Training Group GK University of Erlangen-Nürnberg 31 Vaccination gun o No need for needle replacement and sterilisation. o Required too much maintenance Disposable 'bifurcated needle o Has a narrow, flattened forked end o Draws vaccine by capillary action o Was then jabbed repeatedly into the skin Vaccination – Techniques

32 Doctoral Training Group GK University of Erlangen-Nürnberg Jenner: The annihilation of the Small Pox, the most dreadful scourge of the human species, must be the final result of this practice' Compulsory vaccinations in Bavaria (1807), Denmark (1810), Prussia (1835), Germany (1874) and Britain in Smallpox epidemic duringThe Franco-Prussian War French army was not vaccinated 23,400 died German army was vaccinated only 278 died. Smallpoxs – The Start of Vaccination

33 Doctoral Training Group GK University of Erlangen-Nürnberg Up to 15 million cases of smallpox each year. 1967the World Health Organization (WHO) launched its campaign to eradicate smallpox worldwide. 1977Ali Maow Maalim from Somali was the last person on Earth to catch smallpox by natural transmission 1980WHO formally declared: "Smallpox is Dead! Smallpoxs – Irradication

34 Jenner is acknowledged as the Father of Vaccination

35 Doctoral Training Group GK University of Erlangen-Nürnberg 35 Berühmte Wissenschaftler setzen die Forschung fort Seine Visionen wurden von großen Forschern zielstrebig weiter verfolgt: In Paris entwickelte Louis Pasteur eine Impfung gegen die Tollwut, Robert Koch wies auf den Zusammenhang von Mikroorganismen und Infektionen hin und entdeckte den Tuberkelbazillus, Paul Ehrlich begründete die moderne Chemotherapie, und Emil von Behring entdeckte das Serum gegen Diphtherie und Wundstarrkrampf. Impfstoffproduktion beginnt Inzwischen werden viele der nach und nach entwickelten Schutzimpfungen in vielen Ländern empfohlen. Nachdem die beiden Amerikaner Frederick Robbins und Thomas Weller gezeigt hatten, dass sich lebende Zellen in Kulturlösungen im Reagenzglas züchten lassen (Nobelpreis 1954), war auch der Weg für die Produktion von Impfstoffen geebnet. So lässt sich heute mit den abgetöteten oder abgeschwächten Erregern von Viren, Bakterien oder den von diesen gebildeten Giften eine lang bestehende Immunität erreichen. Develoment of Vaccination

36 Doctoral Training Group GK University of Erlangen-Nürnberg 36 Rabies & Cholera - Pasteur Vaccination against rabies (1885) (The case of Johann Meister) Vaccínation against chicken cholera (1878) Though Pasteur knew that the vaccine worked, but no one then in the world of science knew how it worked!

37 Doctoral Training Group GK University of Erlangen-Nürnberg 37 aktivpassiv IndikationProphylaxeProphylaxe, Therapie Gabe vonAntigenAntikörper Gabe wie oftwenige Maleimmer wieder Schutzeintrittspätsofort Schutzdauerlangekurz Gedächtnisjanein Unterschiede: Aktive Immunisierung vs. passive Immunität Aktive und passive Immunität unterscheiden sich in einigen wesentlichen Punkten: Protective Vaccination - Summary

38 Doctoral Training Group GK University of Erlangen-Nürnberg 38 Anforderungen: Was macht einen Impfstoff zu einem guten Impfstoff? Ein Impfstoff zur aktiven Immunisierung muss folgende Anforderungen erfüllen: Das Antigen soll definiert sein. Der Impfstoff darf nicht selbst zur Erkrankung führen und muss vor Krankheit durch lebende Erreger schützen. Der Schutz soll lange anhalten. Die Impfung soll möglichst geringe Kosten verursachen und wenige Nebenwirkungen haben. Der Impfstoff muss biologisch stabil und leicht zu verabreichen sein. Auch der Proband muss gewisse Anforderungen erfüllen: Er soll möglichst früh impfen und den Schutz durch Auffrischungsimpfungen aufrechterhalten. Optimal wäre eine Erfolgskontrolle durch Antikörpertiter-Kontrolle. Besondere Vorsicht gilt bei Immundefekten bei Lebendimpfungen (angeboren oder erworben (AIDS, iatrogen)).

39 Doctoral Training Group GK University of Erlangen-Nürnberg 39 YearEvent 1100 Physicians in India and China realize that the liquid from the pustules of a smallpox victim, when scratched on the skin of a healthy patient, would most often cause mild disease. This intentional infection, termed variolation, would also give life-long protection against the illness. 1721Lady Mary Wortley Montgue, wife of the ambassador to the Ottoman Empire, introduces variolation to Europe. 1796Edward Jenner uses cowpox to immunize against smallpox Ilya Ilich Metchnikoff demonstrates that certain body cells move to damaged areas of the body where they consume bacteria and other foreign particles. He calls the process phagocytosis. This is the beginning of the science of immunology, the study of the immune system. 1885Paul Ehrlich proposes that certain chemicals affect bacterial cells and begins a search for one that can treat syphilis Theobald Smith and D. E. Salmon develop a treatment for hog cholera by injecting killed hog cholera microorganisms into pigeons and demonstrate immunity to subsequent administration of a live microbial culture of cholera. 1891Ehrlich shows that antibodies are responsible for part of immunity. 1897Almwroth Wright and David Sample develop an effective vaccine against typhoid fever using killed cells of Salmonella typhi. 1897Waldemar Haffkine develops a vaccine against the plague. 1912Paul Ehrlich announces the discovery of a cure for syphilis. The cure is the first specific chemotherapeutic agent for a bacterial disease. 1929Alexander Fleming publishes the first paper describing penicillin. 1935Gerhard J. Domagk uses Prontosil, a chemically synthesized antimetabolite, to kill Streptococcus in mice. 1938Max Theiler produces a vaccine against yellow fever by passaging the virus through mice to weaken it. 1940Howard Florey and Ernest Chain produce an extract of penicillin and show it can kill bacteria in animals. 1940Ernest Chain and E.P. Abraham describe a substance from E. coli that can inactivate penicillin. This demonstrates how rapidly bacteria can become resistant to antibiotics Selman Waksman and H. Boyd Woodruff discover actinomycin, the first antibiotic obtained pure from a group of soil organisms, the actinomycetes. In subsequent years many antibiotics are isolated from this group including tetracycline and streptomycin. 1941Charles Fletcher demonstrates that penicillin is non-toxic to human volunteers, by injecting a police officer suffering from a lethal infection. 1942Selman Waksman suggests the word "antibiotic" to describe the class of compounds produced by one microorganism that inhibit or kill other microorganisms. 1944Albert Schatz, E. Bugie, and Selman Waksman discover streptomycin, a very effective drug against tuberculosis. 1944W. H. Feldman and H. C. Hinshaw at the Mayo Clinic successfully treat tuberculosis with streptomycin. 1957The Soviet delegation to the World Health Organization proposes a vaccination effort to eradicate smallpox. The program finally begins in Ali Maow Maalin, age 23 of Somalia, is the last known victim of naturally occurring smallpox Smallpox is declared to be eliminated. This is the only example of a microbial disease that has been wiped from the face of the Earth. (However, the recent specter of bioterrorism and the smallpox stocks kept by several governments make new epidemics of smallpox still possible.) Figure 1-23 Treatment and prevention of disease

40 START OF IMMUNOLOGY Cellular Immunity

41 Doctoral Training Group GK University of Erlangen-Nürnberg 41 1.Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic antibodies und complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Bordet (1901) - Complement fixation test o Wassermann (1905) - Syphilis-Nachweis o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 Will be covered by C. Bogdan TOPCIS: Start of Immunology

42 START OF IMMUNOLOGY Serotherapy

43 1.Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic antibodies und complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Bordet (1901) - Complement fixation test o Wassermann (1905) - Syphilis-Nachweis o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 TOPCIS: Start of Immunology

44 Doctoral Training Group GK University of Erlangen-Nürnberg 44 aktivpassiv IndikationProphylaxeProphylaxe, Therapie Gabe vonAntigenAntikörper Gabe wie oftwenige Maleimmer wieder Schutzeintrittspätsofort Schutzdauerlangekurz Gedächtnisjanein Unterschiede: Aktive Immunisierung vs. passive Immunität Aktive und passive Immunität unterscheiden sich in einigen wesentlichen Punkten: Passive Vaccination - Summary

45 Doctoral Training Group GK University of Erlangen-Nürnberg 45 Characteritics o Transfer of serum, gammaglobulin or monoclonal antibodies from humans or animals o Immediate protection o Short duration (t/2 = 20 days) o Side effects through immune response to foreign proteins (serum disease, anaphylaxy) o Contraction of hepatitis or HIV through antibody preparations from human serum Naturally acquired passive immunity o placental transport of maternal IgG from mother in the fetus through o Transfer if maternal IgA into newborn through milk Artifically induced passive immunization o Injection or transfusion of gammaglobulin from other individuals or animals o Treatment of an acute infection (diphtheria, tetanus, rabies, FSME, rubella (Röteln) …) o Toxins (Insects, snake, scorpions, botulinus) o Prophylactic before travel to foreign countries o Rhesus factor prophylactic Vaccination – Passive immunization

46 Doctoral Training Group GK University of Erlangen-Nürnberg 46 1.Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic antibodies und complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Bordet (1901) - Complement fixation test o Wassermann (1905) - Syphilis-Nachweis o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 TOPCIS: Start of Immunology

47 SIDE VISIT Bacterial toxins

48 Doctoral Training Group GK University of Erlangen-Nürnberg 48 Exotoxins o released by microorganisms o act at the surface of host cells, for example by binding to receptors Endotoxins o Intrinsic components of microbial structure o Trigger e.g., phagocytes to release cytokines that produce local or systemic symptoms SIDE VISIT – Bacterial toxins Janeway, 2011 (8th edition) microbe exotoxin Toxoid o Coined by Ehrlich (1908) to describe a derivate of a toxin that still bound to immunoreceptor but has lost its toxic group (toxophore) Ehrlich P (1908): Über Antigene und Antikörper. Handbuch der Technik und Methodik der Immunitätsforschung: 1-10.

49 Doctoral Training Group GK University of Erlangen-Nürnberg 49 Endotoxins – Structure and function Immunogenicity Toxicity Endotoxin is a lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens (Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemo-philus influenzae, Bordetella pertussis and Vibrio cholerae) LPS induces proliferation of mouse B cells via TLR4 (TI1 antigen) mitogen LPS activates/induces Phagocytosis IL1 (fever) TNFa (endotoxin-induced shock in patients severely infected by gram- negative bacteria)

50 Doctoral Training Group GK University of Erlangen-Nürnberg Richard Pfeiffer Endotoxin - Heat stable toxic material from the membrane of Vibrio Cholerae that is released only after the cells are disintegrated. 1933Andre Boivin and Lydia Mesrobeanu (Pasteur) Re-discover endotoxin and show that the partially purified toxic fraction contains polysaccharides, lipids, and proteins. 1950s Otto Westphal und Otto Lüderitz (MPI Freiburg) Prepare protein-free endotoxin Ref:1. JOSEPH E. ALOUF (1987). From Diphtheritic Poison to Molecular Toxicology ASM News 53, p Reimond Beck, A Chronology of Microbiology in Historical Context (paperback) Endotoxin – Discovery (LPS) Richard Pfeiffer ( ) Germany Otto Westphal ( ) Germany Otto Lüderitz Germany

51 Doctoral Training Group GK University of Erlangen-Nürnberg 51 Exotoxins – (AB toxins) Diphtheria toxin

52 Doctoral Training Group GK University of Erlangen-Nürnberg 52 Diphteria Toxin – Mechanism of Action Diphtheria toxin (about 530 aa) is cleaved by proteolysis in disulfide-linked A and B fragment Fragment B facilitates entry into the cell via Heparin-binding EGF-like growth factor (through receptor-mediated endocytosis) as well as the transport of A fragment into the cytosol. Fragment A prevents protein synthesis by inactivting eEF2 through transfer of a ADP ribosyl moiety form NAD+ onto the unusual amino acid diphthamide in the eEF2. NAD + + peptide diphthamide nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide

53 Doctoral Training Group GK University of Erlangen-Nürnberg 53 Diphtheria- ADP ribosylates translational elongation factor 2 cells dies Pseudomonas- ADP ribosylates translational elongation factor 2 cells dies Pertussis- ADP ribosylates adenylate cyclase Gi regulatory protein (blocks e.g., chemokine receptors) Cholera toxin- ADP ribosylates eucaryotic adenylate cyclase Gs regulatory protein - increased level of intracellular cAMP, which promotes secretion of fluid and electrolytes in intestinal epithelium leading to diarrhea Botulinus- Zn ++ dependent protease acts on synaptobrevin at motor neuron - Inhibits acetylycholine release from peripheral cholinergic neurons resulting in flaccid paralysis Tetanus- Zn ++ dependent protease acts on synaptobrevin in CNS - Inhibits neurotransmitter release from inhibitory neurons in the CNS resulting in spastic paralysis Exotoxins – More examples AB toxins

54 Doctoral Training Group GK University of Erlangen-Nürnberg 54 Causes, incidence, and risk factors Diphtheria spreads through respiratory droplets (such as those produced by a cough or sneeze) of an infected person or someone who carries the bacteria but has no symptoms. Diphtheria can also be spread by contaminated objects or foods (such as contaminated milk). The bacteria most commonly infects the nose and throat. The throat infection causes a gray to black, tough, fiber-like covering, which can block the airways. In some cases, diphtheria may first infect the skin, producing skin lesions.throat infectionskin lesions Once infected, dangerous substances called toxins, produced by the bacteria, can spread through your bloodstream to other organs, such as the heart, and cause significant damage. Because of widespread and routine childhood DPT immunizations, diphtheria is now rare in many parts of the world. There are fewer than five cases of diphtheria a year in the United States.DPT immunizations Risk factors include crowded environments, poor hygiene, and lack of immunization.

55 Doctoral Training Group GK University of Erlangen-Nürnberg 55 MacGregor RR. Corynebacterium diphtheriae. In: Mandell GL, Bennett JE, Dolan R, eds. Mandell, Douglas, and Bennetts Principles and Practice of Infectious Diseases. 7th ed. Orlando, FL: Saunders Elsevier; 2009:chap 205. Expectations (prognosis) - today Diphtheria may be mild or severe. Some people may not have symptoms. In others, the disease can slowly get worse. The death rate is 10%. Recovery from the illness is slow. Protection Anyone who has come into contact with the infected person should receive an immunization or booster shots against diphtheria. Protective immunity lasts only 10 years from the time of vaccination, so it is important for adults to get a booster of tetanus-diphtheria (Td) vaccine every 10 years.booster shotsimmunity Those without symptoms who carry diphtheria should be treated with antibiotics. Diphtheria – Symptoms

56 Doctoral Training Group GK University of Erlangen-Nürnberg 56 Symptoms Symptoms usually occur 2 to 5 days after you have come in contact with the bacteria. Bluish coloration of the skin Bloody, watery drainage from nose Breathing problems Difficulty breathing Rapid breathing Stridor Chills Croup-like (barking) cough DroolingDrooling (suggests airway blockage is about to occur) Fever Hoarseness Painful swallowing Skin lesions (usually seen in tropical areas) Sore throatSore throat (may range from mild to severe) Note: There may be no symptoms. Signs and tests The health care provider will perform a physical exam and look inside your mouth. This may reveal a gray to black covering (pseudomembrane) in the throat, enlarged lymph glands, and swelling of the neck or larynx.health care providerenlarged lymph glands Tests used may include: Gram stain or throat culture to identify Corynebacterium diphtheriaethroat cultureCorynebacterium diphtheriae Electrocardiogram (ECG)ECG

57 Doctoral Training Group GK University of Erlangen-Nürnberg 57 Treatment If the health care provider thinks you have diphtheria, treatment should be started immediately, even before test results are available.health care provider Diphtheria antitoxinDiphtheria antitoxin is given as a shot into a muscle or through an IV (intravenous line). The infection is then treated with antibiotics, such as penicillin and erythromycin.intravenouserythromycin People with diphtheria may need to stay in the hospital while the antitoxin is being received. Other treatments may include: Fluids by IV Oxygen Bed rest Heart monitoring Insertion of a breathing tube Correction of airway blockages Anyone who has come into contact with the infected person should receive an immunization or booster shots against diphtheria. Protective immunity lasts only 10 years from the time of vaccination, so it is important for adults to get a booster of tetanus- diphtheria (Td) vaccine every 10 years.booster shotsimmunity Those without symptoms who carry diphtheria should be treated with antibiotics.

58 Doctoral Training Group GK University of Erlangen-Nürnberg 58 Patients with severe cases will be put in a hospital intensive care unit (ICU) and be given a diphtheria anti-toxin. Since antitoxin does not neutralize toxin that is already bound to tissues, delaying its administration is associated with an increase in mortality risk. Therefore, the decision to administer diphtheria antitoxin is based on clinical diagnosis, and should not await laboratory confirmation. [6]intensive care unitdiphtheria anti-toxinantitoxin [6] Antibiotics have not been demonstrated to affect healing of local infection in diphtheria patients treated with antitoxin. Antibiotics are used in patients or carriers to eradicate C. diphtheriae and prevent its transmission to others. The CDC recommends [9] either:antitoxin [9] Metronidazole ErythromycinErythromycin (orally or by injection) for 14 days (40 mg/kg per day with a maximum of 2 g/d), or Procaine penicillin GProcaine penicillin G given intramuscularly for 14 days (300,000 U/d for patients weighing 10 kg). Patients with allergies to penicillin G or erythromycin can use rifampin or clindamycin.rifampinclindamycin

59 Doctoral Training Group GK University of Erlangen-Nürnberg 59

60 Doctoral Training Group GK University of Erlangen-Nürnberg 60 Diphtheria- ADP ribosylates translational elongation factor 2 cells dies Pseudomonas- ADP ribosylates elongation factor 2 cells dies Pertussis- ADP ribosylates adenylate cyclase Gi regulatory protein (blocks e.g., chemokine receptors) Cholera toxin- ADP ribosylates eucaryotic adenylate cyclase Gs regulatory protein - increased level of intracellular cAMP, which promotes secretion of fluid and electrolytes in intestinal epithelium leading to diarrhea Botulinus- Zn ++ dependent protease acts on synaptobrevin at motor neuron - Inhibits acetylycholine release from peripheral cholinergic neurons resulting in flaccid paralysis Tetanus- Zn ++ dependent protease acts on synaptobrevin in CNS - Inhibits neurotransmitter release from inhibitory neurons in the CNS resulting in spastic paralysis Exotoxins – More examples AB toxins

61 Doctoral Training Group GK University of Erlangen-Nürnberg 61 Diphtheria Toxin – Mechanism of action Mechanism Diphtheria toxin is a NAD+-diphthamide ADP-ribosyltransferase. It catalyzes the ADP-ribosylation of the unusual amino acid diphthamide in the eukaryotic elongation factor-2 (eEF2 eEF is inactivetd and mRNA translationalis inhibieted inhibitor. The catalysed reaction is as follows: NAD + + peptide diphthamide nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide The exotoxin A of Pseudomonas aeruginosa uses a similar mechanism of action.exotoxin APseudomonas aeruginosa The exotoxin A of Pseudomonas aeruginosa uses a similar mechanism of action.exotoxin APseudomonas aeruginosa

62 Doctoral Training Group GK University of Erlangen-Nürnberg 62 Gehört zu einem genetisch veränderten Stamm der harmlosen E. coli-Bakterien Der Keim kommt vor allem im Darm von Wiederkäuern wie Rindern, Schafen oder Ziegen vor. Produziert Toxine, die zu wässrigem Durchfall und bis zu blutiger Diarrhoe mit Bauchkrämpfen Spezielles Hüllenprotein (Adhäsin), das sich an die Epithelzellen der Darmwand anheftet. Ein über Phageninfektion eingeschleustes Gen für das neurotoxische und nekrotisierende Shiga-Toxin oder auch Vero-Toxin (zerstört Vero-Zellen = Affennierenzellen) Hemmt Proteinsynthese Plasmidkodiertes Hämolysin blutzellenzerstörendes Toxin (EHEC) – Entero-haemorragic E. coli

63 Doctoral Training Group GK University of Erlangen-Nürnberg 63 BCHomer describes Egyptian disease ~1800Fidele Bretonneau coins Diphtheria for the disease (Häutchen auf Mandeln) and introduces tracheotomy as ultima ratio in treatment 1883Edwin Klebs (student of Rudolph Virchow) discovers bacteria in diphtheria patients 1884Friedrich Löffler Cultivates and identifies C. diphtheriae as the agent of the disease hypothesize that infected people die of a toxic bacterial product since the bacillus does not grow well in infected patients 1888Roux and Yersin - soluble and filterable toxin in diphtheria cultures causes disease 1890More than 50,000 children/year die in Germany of diphtheria 1890Behring reports 1. successful vaccination with weakened C. diphtheriae in guinea pigs Diphtheria Exotoxin – Timeline

64 Doctoral Training Group GK University of Erlangen-Nürnberg 64 Major Observations The filtrates of from old alkaline diphtheria cultures when injected into animals mimicked the symptoms of the natural disease. Germ-free urine of infected children contained sufficient toxin to kill guinea pigs. The discovery was serendipity since high calcium tap water led to precipitation of calcium phosphate and, with it, to the lowering of free iron ions in the medium, which, as we know today, is required for optimal toxinogenesis. 1888Roux and Yersin discover at the newly founded Pasteur institute the first bacterial protein toxin from diphtheria, explains, for the first time, the mechanism of pathogenicity of a microorganism for humans in terms of a soluble toxic substance. Diphtheria toxin - Discovery Emile Roux ( ) France Alexandre Yersin ( ) France/Schweiz ALOUF (1987). From Diphtheritic Poison to Molecular Toxinology ASM News VOL.53,NO. l0

65 Doctoral Training Group GK University of Erlangen-Nürnberg s_immunology.html#section2 Infectious Diseases - Online

66 Doctoral Training Group GK University of Erlangen-Nürnberg 66 _diphtheriae A low concentration of iron is required in the medium for toxin production. At high iron concentrations, iron molecules bind to an aporepressor on the beta bacteriophage, which carries the Tox gene. When bound to iron, the aporepressor shuts down toxin production [1]. Elek's test for toxogenecity is used to determine whether the organism is able to produce the diphtheria toxin or not.iron aporepressorbacteriophagerepressor [1]Elek's testdiphtheria toxin ^^ Microbiology: A Human Perspective. Fourth edition. McGraw Hill

67 Doctoral Training Group GK University of Erlangen-Nürnberg 67 Bacterial toxins – Summary gy.net/endotoxin.htm

68 Doctoral Training Group GK University of Erlangen-Nürnberg 68 1 st Serum therapy (tetanus Mice) Behring & Kitasato anti-toxins Ehrlich Discovery o ANTIGEN Deutsch (Detre) st Serum therapy in humans (diphtheria) Behring&Ehrlich 1 st Serum therapy in humans (diphtheria) Behring&Ehrlich Ehrlich side chain theory TIMELINE: Serum Therapy Behring 1 st serum therapy (diphtheria guinea pgs ANTI- BODY Ehrlich 1891 Industrial production of antisera (diphtheria in sheep (Hoechst) Production of of antisera in US (NYC)

69 Discovery of Inducible Humoral Immunity - Serotherapy -

70 SEROTHERAPY

71 Doctoral Training Group GK University of Erlangen-Nürnberg 71 Discovery of inducible soluble immunity 1889Gameleia (Gamaleia) describes inducible humoral immunity against anthrax Serum from sheep immunized with attenuated anthrax kills anthrax in vitro Activity in blood vanishes after one month But sheep remains immune for much longer time (memory !!!!???) soluble activity with short in vitro half-live and Memory Gamelai (1889). Sur la Destruction des Microbes dans les Corps des Animaux. Febricitants. Ann. Inst. Pasteur, p Bouchard shows that bacteria-killing power is greater in blood serum from immunized than from naive animals Inducible soluble activity M. BOUCHARD (1890). ACTIONS DES PRODUITS SCREnTtS PAR LBS MICROBES PATHOGiNEsBy. Paris: Gauthier, Villars et File. (Summary published in Nov The British Medical JOURNAL. P Nice overview in a News and Views to Behrings Dec 1890 article about the serum therapy in animals by Hankin, EH (1890). A cure for Tetatus and Diphthria, Nature No 1101, Vol. 43, p. 121

72 Doctoral Training Group GK University of Erlangen-Nürnberg 72 May 1890von Behring and Nissen show that serum from animals immunized with anthrax killed anthrax in vitro but not bacillus pyocyaneus Inducibilty and Specificity von Behring and Nissen (May 1890), Ueber den bakterienfeindlichen Einfluss von verschiedenen Serumarten, Z. für Hygine vol. viii, p. 412) Discovery of inducible soluble immunity

73 Doctoral Training Group GK University of Erlangen-Nürnberg 73 Warning! The following article is from The Great Soviet Encyclopedia (1979). It might be outdated or ideologically biased. Gamaleia, Nikolai Fedorovich Born Feb. 5 (17), 1859, in Odessa; died Mar. 29, 1949, in Moscow. Soviet microbiologist. Honorary academician of the Academy of Sciences of the USSR (1940; corresponding member, 1939). Academician of the Academy of Medical Sciences of the USSR (1945). Member of the CPSU from 1948.Academy of Medical Sciences Gamaleia graduated from Novorossiiskii University in Odessa in After graduating from the Academy of Military Medicine in St. Petersburg in 1883, he began to study the bacteriology of tuberculosis and anthrax in Odessa. In 1886 he worked in Paris with L. Pasteur. Studying vaccination against rabies, he improved on Pasteurs method of prophylactic inoculations. In the same year he and I. I. Mechnikov organized a bacteriological station in Odessa. He discovered that cattle plague is caused by a filterable virus. From 1887 to 1891 he did research on rabies, tuberculosis, cholera, and inflammatory processes. In 1892 he defended his doctoral dissertation, Etiology of Cholera From the Standpoint of Experimental Pathology (published in 1893).cattle plague In 1899, Gamaleia directed the establishment of a bacteriological institute in Odessa. In 1898 he discovered substances that destroy bacteriabacteriolysins. He introduced many new ideas into the study of microbial toxins. In the years he was in charge of efforts to control the plague epidemic raging in Odessa. During the ensuing years he fought against cholera in southern Russia. Gamaleia discovered Mechnikovs vibrio, the causative agent of a cholera-like disease of birds. He proposed a vaccine against cholera in man and devised various sanitary and hygienic measures to control the disease. In 1908, Gamaleia was the first to demonstrate that typhus is transmitted by lice. He worked hard to prevent typhus and relapsing fever, cholera, smallpox, and other infectious diseases. In 1910 he was the first to prove the value of disinsection (destruction of insects) in eradicating typhus and relapsing fever. From 1910 to 1913 he edited and published the journal Gigiena i sanitariia (Hygiene and Sanitation) that he founded. From 1912 to 1928 he was the scientific director of the Institute of Smallpox Vaccination in Leningrad, and from 1930 to 1938 he occupied the same post in the Central Institute of Epidemiology and Bacteriology in Moscow. From 1938 until the end of his life Gamaleia was a professor in the Microbiology subdepartment of the Second Moscow Medical Institute and from 1939, chief of the laboratory of the Institute of Epidemiology and Microbiology of the Academy of Medical Sciences of the USSR. From 1939 he was president and then honorary president of the all-Union Society of Microbiologists, Epidemiologists, and Specialists in Infectious Diseases. In his works he was a consistent materialist and a supporter of the theory of evolution. He trained numerous Soviet microbiologists. Gamaleia received the State Prize of the USSR in 1943 and was awarded two Orders of Lenin, the Order of the Red Banner of Labor, and medals.relapsing feverMicrobiologistsmicrobiologists WORKS Sobr. soch., vols Moscow, REFERENCES Nikolai Fedorovich Gamaleia. (AN SSSR: Matly k bibliografii uchenykh SSSR: Seriia biologicheskikh nauk, vol. 1.) Moscow-Leningrad, Milenushkin, Iu. I. N. F. Gamaleia. Moscow, V. N. GUTINA The Great Soviet Encyclopedia, 3rd Edition ( ). © 2010 The Gale Group, Inc. All rights reserved. How to thank TFD for its existence? Tell a friend about us, add a link to this page, add the site to iGoogle, or visit webmaster's page for free fun content. Tell a friend about usadd the site to iGooglewebmaster's page for free fun content

74 Discovery of humoral immunity - Antitoxins and Serum Therapy -

75 Doctoral Training Group GK University of Erlangen-Nürnberg 75 Pathogenic Tetanus 2020 Inactivated Tetanus 1010 Serum Inactivated Diphteria Serum 1010 Emil von Behring (1890) Soluble, inducible and specific immunity trough immunisation with pathogens Individual can be protected by transferring serum from immunized animal 1 st Nobel Price 1901 for Serum Therapie Tetanus & Diptheria

76 Doctoral Training Group GK University of Erlangen-Nürnberg 76 Shibasaburo Kitasato Japanese Microbiologist 1889Grew pure culture of tetanus bacillus 1890Discovered Humoral immunity against tetanus together with von Behring 1. Nobel Price in Medicine st Nobel Price 1901 for Serum Therapy only for Emil von Behring

77 Doctoral Training Group GK University of Erlangen-Nürnberg 77 Weakened Tetanus Weakened Tetanus Transferred immune serum protects mice against tetanus Works also therapeutically in sick mice !!!!!! No data of experiments with Diphtheria bacillus were reported !!!!! Shibasaburo Kitasato ( ) Japan Emil v. Behring ( ) Germany Immune against pathogenic Tetanus and tetanus toxin Control Serum Control Serum Immune Serum Immune Serum No Tetanus No Tetanus Pathogenic Tetanus Pathogenic Tetanus BEHRING und KITASATO, 1890: Ueber das Zustandekom- men der Diphtherie-Immunität und der Tetanus-Immunität bei Thieren. Deutsche Medicinische Wochenschrift, 16. Jahrgang, Nr. 49, 4. December, S / Serum Therapy - Tetanus (December 1890)

78 Doctoral Training Group GK University of Erlangen-Nürnberg 78 Pathogenic Diphtheria Pathogenic Diphtheria Weakened Diphtheria (Jodtrichloride) Weakened Diphtheria (Jodtrichloride) Induced immunity with chemically weakened germs. Did not transfer serum of immunized animals (as cited in many text books) However, tried internal desinfection (chemotherapy) using Jodtrichloride or Peroxide (not convincing) 1891 – Successful serum therapy of diphtheria in guinea pigs Behring, Emil Ueber Desinfection am lebenden Organismus. Deutsche Medicinische Wochenschrift 17(52):1393–1397. Emil v. Behring ( ) Germany Non- immunized BEHRING 1890: Untersuchungen über das Zustandekommen der Diphtherie- Immunität bei Thieren. Deut. Med. Wochenschr. 16(50):1145–1147. Protection against Diphtheria ( December 1890)

79 Doctoral Training Group GK University of Erlangen-Nürnberg 79 Behring, Emil Ueber Desinfection am lebenden Organismus. Deutsche Medicinische Wochenschrift 17(52):1393–1397. (Summary of experiment) Behring, Emil, and Erich Wernicke Ueber Immunisierung und Heilung von Versuchsthieren bei der Diphtherie. Zeitschrift für Hygiene und Infections- krankheiten 12:10–44. (Description of experiments) Weakened (JCL 3 ) Diphtheria Weakened (JCL 3 ) Diphtheria Immune against pathogenic diphtheria or diphtheria toxin Control Serum Control Serum Immune Serum Immune Serum No Diphtheria No Diphtheria Wernicke, Frosch and Behring Pathogenic Diphtheria Pathogenic Diphtheria Serum Therapy – Diphtheria ( 1891)

80 Doctoral Training Group GK University of Erlangen-Nürnberg 80 p Serum Therapy – Diphtheria ( 1891)

81 Doctoral Training Group GK University of Erlangen-Nürnberg 81 Zeitschrift für Hygiene und Infectionskrankheiten (1992). 12:10–44. p.16 p.12 p.13 p.10 Wernicke, Frosch and Behring Visibilty of serum therapy in humans Inducible immunity Weakening of diphtheria toxin Sucessful serum therapy of diphtheria Serum Therapy – Diphtheria ( 1892)

82 Doctoral Training Group GK University of Erlangen-Nürnberg 82 Zeitschrift für Hygiene und Infectionskrankheiten (1992). 12:10–44.

83 Doctoral Training Group GK University of Erlangen-Nürnberg 83 Behrings Goals... may also be of use for the treatment of humans suffering from diphtheria or tetanus. … Das Blut ist ein ganz besonderer Saft (Goethe) Behring (1890): Behring E. A. and Kitasato S. (1890) Uber das Zustandekommen der Diphtherie- Immunitat und der Tetanus-Immunitlt bei Thieren. Dtsch. med. Wochenschr. 49, The final aim of our experiments remains the production of the substance in such amounts and with such effectivity that humans, too, may be treated for diphtheria with it Behring (1893): Behring E. A. (1893). Die Geschichte der Diphtherie, p Thieme, Leipzig. Behring E. A. (1894) 1. CAUSATIVE TREATMENT OF INFECTIOUS DISEASES 2. PROMOTE FIRST IMMUNOLOGICAL PARADIGM Specific immunity induced by antigens is associated with the formation of soluble antibodies

84 Doctoral Training Group GK University of Erlangen-Nürnberg 84 Behring, Emil, and Erich Wernicke Ueber Immunisierung und Heilung von Versuchsthieren bei der Diphtherie. Zeitschrift für Hygiene und Infectionskrankheiten 12:10–44. p2Res=.25&printThumbnails=no Protection against Diphtheria (1892) Wernicke, Frosch and Behring

85 Doctoral Training Group GK University of Erlangen-Nürnberg More than 50,000 children in Germany die every year of diphtheria. 1890Behring and Kitasato discover soluble, inducible and transferable anti-tetanus activity in blood of immunized rabbits 1891Behring successfully repeats serum therapy of diphtheria- infected guinea pigs 1891Behring apparently treats successfully a diphtheria- diseased child with injection of anti-diphtheria serum produced in horses (report not proven) 1892Wernicke & Behring improve immunization of rabbits, guinea pigs and goats with weakened diphtheria bacillus Serum Therapy: From Bench to bed side

86 Doctoral Training Group GK University of Erlangen-Nürnberg 86 Wernicke, Frosch and Behring Behring, Emil, and Erich Wernicke Ueber Immunisierung und Heilung von Versuchsthieren bei der Diphtherie. Zeitschrift für Hygiene und Infectionskrankheiten 12:10–44.

87 Doctoral Training Group GK University of Erlangen-Nürnberg 87 Serum Therapy: Status Quo – Successful serum therapy of diphtheria in guinea pigs Behring, Emil Ueber Desinfection am lebenden Organismus. Deutsche Medicinische Wochenschrift 17(52):1393– – Successful serum therapy in other animals but not yet tested in humans Behring, Emil, and Erich Wernicke Ueber Immunisierung und Heilung von Versuchsthieren bei der Diphtherie. Zeitschrift für Hygiene und Infectionskrankheiten 12:10–44

88 Doctoral Training Group GK University of Erlangen-Nürnberg 88 In NYC Diphtheria: D Bench to bed side

89 Doctoral Training Group GK University of Erlangen-Nürnberg Ehrlich & Behring develop procedures to enrich and standardize antitoxins from large animals (goats) 1992 Behring and Ehrlich set-up a lab in Berlin-Steglitz (Stadtbahnbogen, now MPI), where they could obtain large amounts of serum by using large animals – first sheep and later horses Industrial production by Hoechst provides anti- diphtheria sera from 20 sheep for 1 st clinical trial in Berlin (published by Behring and Ehrlich in 1894) Serum Therapy: From Bench to bed side

90 Doctoral Training Group GK University of Erlangen-Nürnberg 90 me_46.djvu Samuel Amstrong (1895). The serum tretament of diphtheria. The popular Science Vol XLVI (46), p ) Serum Therapy: From Bench to bed side 1892 Roux succesfully produces antisera in horse that protects guinea pigs against diphtheria

91 Doctoral Training Group GK University of Erlangen-Nürnberg 91 cience_Monthly_Volume_46.djvu Samuel Amstrong (1895). The serum tretament of diphtheria. The popular Science Vol XLVI (46), p ) MBAJ&pg=PA512&lpg=PA512&dq=frist+seru m+therapy+diphtheria+france&source=bl&ot s=RFyFBiOaIw&sig=AeveGTdw6FkxbHtqUlkT UC33J20&hl=de&ei=1VjiTdXkMIrIsgaoo6XwB Q&sa=X&oi=book_result&ct=result&resnum= 2&ved=0CCEQ6AEwAQ#v=onepage&q&f=fal se

92 START OF TRANSLATIONAL IMMUNOLOGY Use results obtained in experiments with animals to treat disease in humans

93 Doctoral Training Group GK University of Erlangen-Nürnberg 93 Serum Therapy: 1st Clinical Trial (Berlin) Six hospitals treated 220 patients under der supervision of Behring and Ehrlich with a serum that was standardized by Ehrlich (1893) Therefore, all patients received the same effective dose of antiserum Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20: Summary of all treated patients Treatment after onset of infection

94 Doctoral Training Group GK University of Erlangen-Nürnberg 94 Since antitoxin does not neutralize toxin that is already bound to tissues, delaying its administration is associated with an increase in mortality risk. Therefore, the decision to administer diphtheria antitoxin is based on clinical diagnosis, and should not await laboratory confirmation. Serum Therapy: Time of application Atkinson W, Hamborsky J, McIntyre L, Wolfe S, eds. (2007). Diphtheria. in: Epidemiology and Prevention of Vaccine- Preventable Diseases (The Pink Book) (10 ed.). Washington DC: Public Health Foundation. pp. 59–70.

95 Doctoral Training Group GK University of Erlangen-Nürnberg 95 p. 520 Serum Therapy: Clinical Trial (Paris)

96 Doctoral Training Group GK University of Erlangen-Nürnberg 96 Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4: Text Text Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20: Text Text Ehrlich P, Kossel H (1894): Ueber die Anwendung des Diphtherieantitoxins. Zeitschrift fuer Hygiene und Infektionskrankheiten, medizinische Mikrobiologie, Immunologie und Virologie 17: Text Text Ehrlich P (1894): Ueber die Behandlung der Diphtherie mit Heilserum. Verhandlungen der Gesellschaft Deutscher Naturforscher und Aerzte : 402. Text Text Ehrlich P, Wassermann A (1894): Ueber die Gewinnung der Diphterie-Antitoxine aus Blutserum und Milch immunisirter Thiere. Zeitschrift fuer Hygiene und Infektionskrankheiten, medizinische Mikrobiologie, Immunologie und Virologie 18: Text Text Serum Therapy: Improvements and Quantitation

97 Doctoral Training Group GK University of Erlangen-Nürnberg 97

98 Doctoral Training Group GK University of Erlangen-Nürnberg 98 Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20:

99 Doctoral Training Group GK University of Erlangen-Nürnberg 99

100 Doctoral Training Group GK University of Erlangen-Nürnberg Émile Roux & Alexandre Yersin: ToxinnachweisÉmile RouxAlexandre Yersin Emil Adolf von Behring: Serum Träger der ImmunitätEmil Adolf von BehringImmunität Behring & Shibasaburo Kitasato: Entdeckung des Antitoxins im Blut kranker Tiere, Antitoxin ist prinzipiell übertragbarShibasaburo Kitasato William Hallock Park und Anna Wessels Williams entwickeln am New York City Department of Health ein Antitoxin.William Hallock ParkAnna Wessels Williams Behring & Erich Wernicke: Immunität durch Injektion von neutralisiertem DiphtherietoxinErich Wernicke Nobelpreis für Physiologie oder Medizin für von BehringNobelpreis für Physiologie oder Medizin von Behring: Toxin-Antitoxinmischung für Immunisierung Gaston Ramon ( ) beha - Gaston Ramon ndelt das Toxin mit Wärme/Formalin für ImpfungGaston Ramon Impfung

101 Doctoral Training Group GK University of Erlangen-Nürnberg After introduction of serum therapy (Roux serum) mortality of diphtheria in Paris falls from 52% to 25% 1894Production of antitoxins in horses in US (William Hallock Park und Anna Wessels Williams in NYC) 1900Industrial production of anti-diphtheria sera in US g/wiki/File:AmDruggist1900N oOtherSerum.jpg 22 October 1900 Advertisement for anti-diphtheria serum by Parke Davis & Company. One of the first bottles of diphtheria antitoxin produced at the Hygienic State Laboratory which became the NIH in /history/docs/page_03.html Serum Therapy: From Bench to bed side

102 Doctoral Training Group GK University of Erlangen-Nürnberg 102 The Hygienic Laboratory at the Staten Island Marine Hospital Service building.National Cancer InstituteMarine Hospital Service One-Man NIH, 1887 By Cristina Luiggi | May 28, nih-1887/

103 Doctoral Training Group GK University of Erlangen-Nürnberg Foundation of Behringwerke" in Marburg (uses two Million Reichsmark from the Nobel prize) 1. biotech company Serum Therapy: From Bench to bed side Photo: Courtesy of Aventis Behring Old vials (1897 and 1906) with hand-written labels. Behring watches diphtheria immunization of horses in Marburg

104 Doctoral Training Group GK University of Erlangen-Nürnberg 104 Roitt p. 321 Serum Therapy: From Bench to bed side

105 Doctoral Training Group GK University of Erlangen-Nürnberg 105 Abb. 10 und 11: Nicht anders erging es Emil von Behring der bei seinen Forschungen beginnend mit Meerschweinchen, später erfolgreich das Pferd und in Fortsetzung auch Rinder und Schafe zur Gewinnung des Diphtherieantitoxins heranzog – er wurde als Pferdedoktor belächelt. Behring als Dompteur der Kühe mit Peitsche und Spritze und der Titel Heilserum vom Pferd, beides aus den Lustigen Blättern 1894.

106 Doctoral Training Group GK University of Erlangen-Nürnberg Behring uses attenuated diphtheria and successfully immunizes laboratory animals 1913Behring & Wernicke introduce active vaccination by injection of a safe mixture of diphtheria toxin and antitoxin. Was replaced by active vaccination with inactivated diphtheria toxoid 1924Gaston Ramon ( ) inactivates tetanus and diphtheria toxins by heat/formalin treatment for active immunizations Diphtheria – Active Immunizations

107 Doctoral Training Group GK University of Erlangen-Nürnberg Felton serum (Felton antibodies) 29 of 30 pneumonia patients were successfully treated with partially purified antibodies from anti- pneumococcal horse sera Felton, L. D Bull. Johns Hopkins Hosp. 38:33-60 PARK, William H. (1924). Use of Vaccines and Pneumonia Antibody in the Treatment and Prevention of Pneumonia and the Use of Convalescent Serum in the Prevention of Measles. Proceedings. Int. Conf. Health Probl. Trop. Amer., Kingston, Jamaica, July 22-August 1, 1924, pp Protection against measles Stokes uses globulin fractions of pooled human plasma for passive protection against measles in 891 individuals Other successful serum therapies

108 Doctoral Training Group GK University of Erlangen-Nürnberg 108 Problem Antisera were not sufficiently concentrated No Standardization to compare different anti-toxin preparations Solution Concentration e.g., by ammonium sulfate precipation (Behring para-albumin, Morawitz pseudo-albumin, today gamma-globulin) Higher titers by improving immunization procedures Ehrlich (1892 – 1894) Immunitätseinheiten - Develops a method to standarize anti-toxin preparations by comparig activity of antiserum against a standard anti-toxin serum in an in vivo toxin neutralization test Coins the term Titer: Dilution of anti-toxin that just neutralizes completely a given amount of toxin SERUM THERAPY – Problem 1 Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4:

109 Doctoral Training Group GK University of Erlangen-Nürnberg 109 Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4: Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20: Ehrlich P, Kossel H (1894): Ueber die Anwendung des Diphtherieantitoxins. Zeitschrift fuer Hygiene und Infektionskrankheiten, medizinische Mikrobiologie, Immunologie und Virologie 17: Ehrlich P (1894): Ueber die Behandlung der Diphtherie mit Heilserum. Verhandlungen der Gesellschaft Deutscher Naturforscher und Aerzte : 402. Ehrlich P, Wassermann A (1894): Ueber die Gewinnung der Diphterie-Antitoxine aus Blutserum und Milch immunisirter Thiere. Zeitschrift fuer Hygiene und Infektionskrankheiten, medizinische Mikrobiologie, Immunologie und Virologie 18: Serum Therapy: Improvements and Quantitation

110 Doctoral Training Group GK University of Erlangen-Nürnberg 110 SERUM THERAPY – Titer (Ehrlich 1894) Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4:

111 Doctoral Training Group GK University of Erlangen-Nürnberg 111 Ehrlich P, Wassermann A (1894): Ueber die Gewinnung der Diphterie-Antitoxine aus Blutserum und Milch immunisirter Thiere. Zeitschrift fuer Hygiene und Infektionskrankheiten, medizinische Mikrobiologie, Immunologie und Virologie 18:

112 Doctoral Training Group GK University of Erlangen-Nürnberg 112

113 Doctoral Training Group GK University of Erlangen-Nürnberg 113

114 Doctoral Training Group GK University of Erlangen-Nürnberg 114

115 START OF IMMUNOLOGY Anaphylaxis and Related Disorders

116 Doctoral Training Group GK University of Erlangen-Nürnberg 116 SERUM THERAPY – Problem Inflammation (immune repones can cause disease !!! Freiherr Clemens von Pirquet (Wiener Kinderarzt) beobachtete bei einigen Diphteriepatienten nach wiederholter Injektion mit Antiseren gegen Diphterie eine Entzündungsreaktion erkannte, dass Antikörper nicht nur schützende Immunantworten vermitteln, sondern auch Überempfindlichkeitsreaktionen auslösen können. Pirquet führte für diese Serumkrankheit den Begriff Allergie (aus dem Griechischen die Fremdreaktion) ein Unter Allergie versteht man überschießende Reaktion des Immunsystems auf normaler-weise harmlose, fremde Stoffe

117 Doctoral Training Group GK University of Erlangen-Nürnberg 117 Hypersensitivity –Injection of Antigens 1902Anaphylaxis, an acute and serious hypersensitivity reaction Paul Portier and Charles Richet (France) report that some dogs that had received a sublethal dose of sea anemone die after second subcutaneous injection Richet receives Nobel price for Medicine Arthus reaction Nicolas Maurice Arthus (a Swiss physician) observes serve local inflammation in rabbits that had received several injections of harmless substances such as milk and horse serum Occurs also with repeated expose of airborne antigens such as fungi Cautioned that the same could happen during a serum therapy!!!!

118 Doctoral Training Group GK University of Erlangen-Nürnberg

119 Doctoral Training Group GK University of Erlangen-Nürnberg 119 Hypersensitivity - Today

120 Doctoral Training Group GK University of Erlangen-Nürnberg Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic antibodies und complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Bordet (1901) - Complement fixation test o Wassermann (1905) - Syphilis-Nachweis o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 Will be covered by S. Finotto TOPCIS: Start of Immunology

121 SEROTHERAPY

122 Doctoral Training Group GK University of Erlangen-Nürnberg 122 Naive individual can be protected and cured !!!! from diphtheria and tetanus by transfering serum from an immunized animal Retter der Kinder (Diphtherie) Retter der Soldaten (Tetanus) 1 st Nobel prize in Medicine for serum therapy (1901) Emil v. Behring ( ) Germany Serum Therapy: Summary 1 st Immunological paradigm: Specific immunity induced by antigens is associated with the formation of antibodies

123 Doctoral Training Group GK University of Erlangen-Nürnberg 123 Klebs discovers bacteria on material from diceased diphtheria patient Behring- werke in Marburg Behring& Ehrlich (Berlin) 1 st serum therapy in humans Behring& Ehrlich (Berlin) 1 st serum therapy in humans TIMELINE: Serum Therapy of Diphtheria Löffler identifies C. diphtheriae as the cause of diphtheria Roux and Yersin idenify soluble diphtheria toxin Hoechst (Behring) Industrial production of antisera in sheep Roux develops antisera in horses Behring 1st Serum therapy (diphtheria) in guinea pigs Behring & Kitasato 1 st serum therapy (tetanus) in mice Roux & Chaillon (Paris) Serum therapy in humans Park & Williams (NYC) Production of antisera in 1924 Safe Active Vacci- nation Ramon

124 Doctoral Training Group GK University of Erlangen-Nürnberg 124 Klebs discovers bacteria on material from diceased diphtheria patient Behring- werke in Marburg 1 st serum therapies in humans Behring& Ehrlich (Berlin) Roux & Chaillon (Paris) 1 st serum therapies in humans Behring& Ehrlich (Berlin) Roux & Chaillon (Paris) TIMELINE: Serum Therapy of Diphtheria Löffler identifies C. diphtheriae as the cause of diphtheria Roux and Yersin idenify soluble diphtheria toxin Industrial production of antisera in sheep (Hoechst) Roux develops antisera in horses 1st Serum therapy (diphtheria) in guinea pigs (Behring) Production of antisera in US (Park, NYC 1 st serum therapy (tetanus) in mice (Behring & Kitasato) 1924 Safe Active Vacci- nation Ramon

125 Doctoral Training Group GK University of Erlangen-Nürnberg 125 Serum Therapy: Today Llewelyn et al. (1992). Monoclonal antibodies in Medicine. BMJ, 305:1269

126 Doctoral Training Group GK University of Erlangen-Nürnberg 126 IgG und Rhesusunverträglichkeit (Landsteiner 1940) For further explanation, see Kuby, 4th edition, p. 414 Mutter ist Rh- Vater ist Rh+ Baby ist Rh+ Während 1. Schwanger- schaft werden nur geringe Mengen an anti-Rh-AK produziert (IgM). Jedoch entstehen Gedächtnis-B- Zellen Während 2. Schwanger- schaft werden Gedächt- nis-B aktiviert und schnell hochaffine IgG-anti-Rh-AK produziert Anti-Rhesusfaktor-AK sind vom IgG-Typ und passie- ren die Plazenta Lyse von Erys Erythroblastosis Fetalis Hemolytic Disease of the Newborn (HDN)

127 Doctoral Training Group GK University of Erlangen-Nürnberg 127 Übertritt größerer fötaler Blutmengen in den mütterlichen Kreislauf oNormalerweise nur während der Geburt oWährend der Schwangerschaft bei Amniocentese oder anderen invasiven Eingriffen Fehlgeburten Abtreibung Schweren inneren Blutungen Behandlung der Mutter durch Gabe von Rhogam = Anti-D (Rhesusfaktor)-Antikörper vom IgG-Typ) oRoutinemäßig nach 28. Schwangerschaftswoche und kurz nach Geburt oSpätestens 72 Stunden nach Fehlgeburten, Ab- treibungen, Amniocentese oder anderen invasi- ven Eingriffen oZerstören fötale Erys, die in den mütterlichen Kreislauf übertreten, und verhindern so die Aktivierung von B-Zellen und die Bildung von Gedächtnis-B-Zellen oNur kleine Mengen an anti-Rh-AK werden verab- reicht, diese werden im Blut der Mutter verdünnt, treten deshalbb nur langsam und in sehr geringen Mengen den fötalen Kreislauf über, zerstören aber effizient fötale Eyrs im Blut der Mutter. Rhogam Fötale Erys Mütterliche Anti-Rh-B-Zelle Anti-Rh-Antikörper (Rhogam)

128 Discovery of a inducible, soluble and specific activity in the blood (later termed antibodies) in 1890 The first paradigm in immunology Specific immunity induced by antigens is associated with the formation of antibodies The first paradigm in immunology Specific immunity induced by antigens is associated with the formation of antibodies START OF IMMUNOLOGY ??

129 START OF IMMUNOLOGY Antitoxins and cytotoxic antibodies

130 Doctoral Training Group GK University of Erlangen-Nürnberg Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic humoral immunity and complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Widal (1896) – Widal agglutination test for typhoid fever o Bordet (1901) - Complement fixation test o Wassermann (1905) – Syphilis test o Landsteiner (1901) – Blood goups in human 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 TOPCIS: Start of Immunology

131 ANTITOXINS Mechanism of action

132 Doctoral Training Group GK University of Erlangen-Nürnberg 132 Mechanisms of antitoxic effect of Behrings serum therapy? Hypothesis 1 : Antitoxins destroys toxin. Disproved since toxins could be detected in toxin/anti-toxin mixtures Hypothesis 2 (e.g., Roux und Buchner): Antitoxin soll keine aktive Wirkung auf das Toxin ausüben, sondern in erster Reihe auf die Zellen einwirken und dieselben gewissermassen gegen die Giftwirkung immunisieren. Hypothesis 3 (Ehrlich): Gift und Gegengift paaren in den Gewebsflüssigkeiten zu einer Art Doppelverbindung, welche nicht mehr in bestimmten Geweben fixiert wird und welche daher keine Krankheitserscheinungen mehr auslöst. P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p Antitoxins: Mechanism of action (Ehrlich, 1897)

133 Doctoral Training Group GK University of Erlangen-Nürnberg 133 Experiment: Antitoxins: Mechanism of action (Ehrlich, 1897) P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p Conclusion Cellular explanation of Roux and Buchner (hypothesis 2) disproved First evidence for direct (from mix in vitro) and chemical interference (from titration) of antitoxin with toxin Ricin Mix of anti-ricin Serum and ricin before adition to RBC Tubes with blood from un-immunized rabbits - + Oberservation Anti-ricin toxin prevents ricin (lectin)-mediated clumping of red blood cells in a concentration dependent manner Ricin mediates clumbing of RBC + Ricin + + +

134 CYTOTOXIC HUMORAL IMMUNITY Bacteriolysins (today:antibody + complement)

135 Doctoral Training Group GK University of Erlangen-Nürnberg 135 Blood plasma o yellow liquid component of blood without blood cells o 55% of the total blood volume o contains 93% water by volume and dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide o Prepared by spinning a tube of fresh blood containing an anti-coagulant Blood serum o blood plasma without fibrinogen or the other clotting factors o Produced by centrifugation of coagulated blood Blood Plasma and Serum

136 Doctoral Training Group GK University of Erlangen-Nürnberg George Nuttall (US, working in Breslau) reports for the frist that time the bactericidal power of the blood serum from non-immunized animals in the absence of cells (macrophages) Nuttal, G. (1888) Experimente über die bakterienfeindlichen Einflüsse des thierischen Körpers, Z. Hygiene vol iv, p On the Formation of Specific Anti-Bodies in the Blood, Following Upon Treatment with the Sera of Different Animals George H. F. Nuttall The American Naturalist. Vol. 35, No. 419 (Nov., 1901), pp Hans Buchner (1850 – 1902, Germany) was the first to demonstrate the presence of a soluble bacteria-killing, heat-labile substance (alexin, from the Greek alexô defend) in normal serum Buchner, H. E. (1889). "Über die bakterientödtende Wirkung des zellenfreien Blutserums (Concerning the Bacteriological Effects of Cell- free Blood Serum), Zbl. Bakt. 5: 817) Co-discovererd with his younger brother Eduard Buchner (1907 Nobel Prize in Chemistry for cell-free fermentation) the yeast enzyme zymase. Bacteriolysins: The beginning Hans Buchner ( ) Germany George Nuttall ( ) San Francisco Buchners Alexin= Ehrlichs complement

137 Doctoral Training Group GK University of Erlangen-Nürnberg Describes a heat labile antibactericidal activity in the blood of immunized animals Guinea pigs were immunized with cholera bacteria Blood was collected and added to live cholera cultures Bacteria became motionless Heating the plasma abolished the effect (destroyed complement) Bacteriolysis or Pfeiffer Phenomenon Other Discoveries Vaccination of guinea pigs against cholera or typhus (1894) Initiated (in parallel with Almoth Wright) the first successful typhus vaccination (1996) Discovered endotoxin as a heat-stabile toxic activity of bacteria Isolated in 1892 Haemophilus influenzae, which he thought was the causative agent of influenza. (Peter Olitsky and Frederick Gates, Rockefeller, could not isolate bacteria in influenza patients during the 1918 pandemic) Bacteriolysin: Coining the name Richard Pfeiffer ( ) Germany Paul Fildes Biographical Memoirs of Fellows of the Royal Society Vol. 2, (Nov., 1956), pp

138 Doctoral Training Group GK University of Erlangen-Nürnberg 138 Complement

139 Doctoral Training Group GK University of Erlangen-Nürnberg 139 Jules Bordet ( ) Belgium Nobel price Medicine 1919 Bacteriolysin: Labile component (1895) Bordet verifies Buchners and Pfeiffers obervations of the prescence of a heat- labile factor required for bacterilysis in vitro Fresh serum from animals immunized with bacteria lysis bacteria in vitro However, if serum ages, bacteriolytic activity is lost

140 CYTOTOXIC HUMORAL IMMUNITY Hemolysins (today:antibody + complement)

141 Doctoral Training Group GK University of Erlangen-Nürnberg 141 Hemolysins 1875Landois (Greifswald), in his work on transfusion, demonstrates the hemolytic action of animal sera on heterologous erythrocytes, i.e., the ability of a serum from one animal to dissolve red cells from an animal of another species. 1898Carbone & Belfanti (Italy) show for the first time that red cells could act as antigens and that the serum of an animal species 1 treated with injections of blood from animals of another species had a high toxicity for animal species 1 BELFANTI, S. AND CARBONE, T.: Produzione di sostanze tossiche mmcl siero di animale inoculati con sangue eterogeneo. Gior. d.r. Accad. di. med. di Torino, Series 4, 46: 321, Bordet showed that the cytotoxic effect of an immune serum was due to two substances contained in the same fluid, one was Buchners alexine (Ehrlichs complement), the other he name substance sensibilisatrice (Ehrlichs amboceptor). Bordet, J Sur l'agglutination et la dissolution des globules rouges par le serum d'animaux injectes de sang defibrine. Ann. De l'Inst. Pasteur. xii: John M. POLK (1904). A CLINICAL STUDY OF THE HEMOLYTIC ACTION OF HUMAN BLOOD SERUM. Journal of Medical Research. (NEW SERIES, VOLUME VII.) VOL. XII. OCTOBER, No. 3. Defintion: The activity in serum to clump and lyse erythrocytes

142 Doctoral Training Group GK University of Erlangen-Nürnberg 142 immune serum Lysis requires specific Immune factor + heat-labile factor Experimental Set-up Hemolysin – The Bordet Experiment (1898) Fresh serum from non- immunized animals rabbit blood no lysis Non- immune serum Guinea pig blood no lysis 55 0 C/30 min specific factor lysis inducible factor unspecifc heat-labile factor No lysis Bordet, J Sur l'agglutination et la dissolution des globules rouges par le serum d'animaux injectes de sang defibrine. Ann. De l'Inst. Pasteur. xii:

143 Doctoral Training Group GK University of Erlangen-Nürnberg 143 BELFANTI, S. AND CARBONE, T.: Produzione di sostanze tossiche mmcl siero di animale inoculati con sangue eterogeneo. Gior. d.r. Accad. di. med. di Torino, Series 4, 46: 321, Bordet, J Sur l'agglutination et la dissolution des globules rouges par le serum d'animaux injectes de sang defibrine. Ann. De l'Inst. Pasteur. xii:

144 Doctoral Training Group GK University of Erlangen-Nürnberg 144 Bordets Observation (at Pasteur) Hemolytic activity of serum was lost after heating to 56 o C Activity was restored upon addition of fresh non-immune serum from rabbit or guinea pig Bordets Conclusion This action of the serum was due to two substances contained in the Same fluid, which he named alexine (Ehrlich: complement) and substance sensibilisatrice (Ehrlich: amboceptor). 1898Bordet demonstrate that the toxic effect of the serum was due to its ability to injure the red cells in such a way that they yielded their hernoglobin to the suspending fluid. This action of the serum he showed was due to two substances contained in the Same fluid, which he named alexine (Ehrlich: complement) and substance sensibilisatrice (Ehrlich: amboceptor). John M. POLK (1904). A CLINICAL STUDY OF THE HEMOLYTIC ACTION OF HUMAN BLOOD SERUM. Journal of Medical Research. (NEW SERIES, VOLUME VII.) VOL. XII. OCTOBER, No. 3. Hemolysin – The Bordet Experiment (1898)

145 Doctoral Training Group GK University of Erlangen-Nürnberg 145 RBC Bordet: Alexin helps inducible factor (1898) Explanation Hemolysin activity of immune serum was due to two substances contained in the same serum: 1.a heat-labile unspecific factor present in blood of non-immunized and immunized animals (Buchners/Bordets alexin, today Ehrlichs complement) 2.a heat-stable species-specific factor only present in the immune serum (Bordets substance of sensibilisatrice, today Ehrlichs antibodies) Bordets mechanism of action Jules Bordet ( ) Belgium Nobel price Medicine 1919 Substance Sensibilisatrice (specificity and sensibilsation) senibilsation Buchners Alexin (toxic) Cleary separeted haemolysin activity into an innate and an adaptive humoral component

146 Doctoral Training Group GK University of Erlangen-Nürnberg 146 Paul Ehrlich ( ) Germany Nobel price Medicine 1908 Together with J. Morgenroth, Ehrlich verified the presence of the two factors in the immune serum (of a goat) required to lysis red blood cells from a mutton (German: Hammel). The thermostable and inducible immunebodies were termedamboreceptors (today: antibodies). and the heat-labile component was termed complement due to the fact that it complemented the activity of the amboreceptors. Paul Ehrlich: Alexin Complement (1899) Ehrlich & Morgenroth (1899). Über Haemolysie – zweite Mitteilung. Berl. Klin. Wochenschr. Bd. 22, p (Komplement mentioned on page 482, left column) Ehrlich (1899). Zur Theorie der Lysinwirkung. Berl. Klein. Wochenzeitschr. No. 1, p. 6 (very nice summary of the first two publications) Ehrlich, P. & Morgenroth, J. (1900). Ueber Hämolysine – 3. Mitteilung. Berl. Klin. Wochenchr. 37,

147 Doctoral Training Group GK University of Erlangen-Nürnberg Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 36, 6–9 (1899) 2.Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 36, 481–486 (1899) 3.Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 37, 453–458 (1900) 4.Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr –687 (1900) 5.Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 38, 251–257 (1901) 6.Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 38, 569–574 (1901) Paul Ehrlich Morgenroth: Lysins Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 36, 6–9 & 481–486 (1899); 37, 453–458 & 681–687, (1900); 38, 251–257 & 569–574 (1901).

148 Doctoral Training Group GK University of Erlangen-Nürnberg 148 Paul Ehrlich: Alexin Complement (1899) 1899Paul Ehrlich & Morgenroth verfied the presence of two blood factors that are part of the anti haemolysine activity required to lyse red blood cells in vitro rename alexin in complement and show that amboreceptor (an antitoxin with only the haptophore part) remediates complement Ehrlich & Morgenroth (1899). Über Haemolysie – zweite Mitteilung. Berl. Klin. Wochenschr. Bd. 22, p (Komplement mentioned on page 482, left column)

149 Doctoral Training Group GK University of Erlangen-Nürnberg 149 RBC Bordet: Alexin helps inducible factor (1898) Explanation Hemolysin activity of immune serum was due to two substances contained in the same serum: 1.a heat-labile unspecific factor present in blood of non-immunized and immunized animals (Buchners/Bordets alexin, today Ehrlichs complement) 2.a heat-stable species-specific factor only present in the immune serum (Bordets substance of sensibilisatrice, today Ehrlichs antibodies) Bordets mechanism of action Jules Bordet ( ) Belgium Nobel price Medicine 1919 Substance Sensibilisatrice (specificity and sensibilsation) senibilsation Buchners Alexin (toxic) Cleary separeted haemolysin activity into an innate and an adaptive humoral component

150 Doctoral Training Group GK University of Erlangen-Nürnberg 150 Mechanism of Action Ehrlich & Morgenroth (1900). Über Haemolysie – vierte Mitteilung. Berl. Klin. Wochenschr. Juli p a.Complement b.Amboreceptor c.Receptor auf RBC toxophore haptophore Anti-Toxin Hemolysin Complement Zwischenkörper Immunkörper Amboreceptor toxophore Haptophore 1 Haptophore 2 haptophore 2. lysis 1. recognition Amboceptors have bifunctional binding capacity: recognizes the specific antigen AND binds to the heat-labile antimicrobial component of fresh serum. So complement gives the amboreceptor the potential to kill In contrast, antitoxins posseses both binding (via haptophore grouop) and killing (via taxophore) activity Paul Ehrlich: Hemolysin – Mode of Action

151 Doctoral Training Group GK University of Erlangen-Nürnberg 151 Deutsch, L. & Feistmantel, C. Die Impfstoffe und Sera. Thieme, Leipzig, Mechanism of Hemolysis: Textbook 1903 Although Ehrlich and Bordet did not agree on the mechanism of action, their work clearly separated the haemolysin activity into an unspecific innate (complement) and an specific adaptive (antibody) component

152 FINDS NEW REMEDY FOR PNEUMONIA; Dr. L.B. Felton, Harvard, After 5 Years' Research, Isolates Antibody That Kills Germ. USED IN 120 HOSPITAL CASES Doctors, Here and in Boston, Say It Will Cut Death Rate 25 to 50 Per Cent. FINDS NEW REMEDY FOR PNEUMONIA

153 Doctoral Training Group GK University of Erlangen-Nürnberg 153 Paul Ehrlich ( ) verified the presence of the two factors in the immune serum was required to produce cell lysis. He termed the thermostable form as amboreceptors or immune bodies (presently known as antibodies). On the other hand, the heat- labile component was termed complement due to the fact that it complemented the activity of the amboreceptors. Ehrlich (Ehrlich & Morgenroth, 1899, 1900) demonstrated that this same factor was involved in the lysis of erythrocytes by immune serum. Ehrlich coined the term 'complement' to indicate his belief that this factor augmented or 'complemented' the bacterolytic and haemolytic activity inherent in antibody. Ehrlich, P. & Morgenroth, J. (1899) Berl. Klin. Wochenchr. 36, 6-9 Ehrlich, P. & Morgenroth, J. (1900) Berl. Klin. Wochenchr. 37, verified the presence of the two factors in the immune serum was required to produce cell lysis. He termed the thermostable form as amboreceptors or immune bodies (presently known as antibodies). On the other hand, the heat-labile component was termed complement due to the fact that it complemented the activity of the amboreceptors. Paul Ehrlich: Hemolysin – Mode of Action

154 Doctoral Training Group GK University of Erlangen-Nürnberg 154 Ehrlich call the soluble, inducible and specific Immunkörper "amboceptors" to emphasize their bifunctional binding capacity: They recognize and bind to a specific antigen, but they also recognize and bind to the heat-labile antimicrobial component of fresh serum. So complement gives the amboreceptor the potential to kill In contrast, antitoxins posses both binding (via haptophore grouop) and killing (via taxophore) Source:Ehrlich & Morgenroth (1900). Über Haemolysie – vierte Mitteilung. Berl. Klin. Wochenschr. Juli p (Ehrlich summarizes also Bordets landmark experiment in 1895) Paul Ehrlich: Hemolysin – Mode of Action

155 Doctoral Training Group GK University of Erlangen-Nürnberg 155 Ehrlich Source:Ehrlich & Morgenroth (1900). Über Haemolysie – vierte Mitteilung. Berl. Klin. Wochenschr. Juli p Studies by Bordet and Ehrlich clearly separated the haemolysin activity into an innate (complement) and a adaptive (antibody) component Active haemolysin (or bacteriolysin) consists (in analogy to a toxin) of two parts: Immunkörper (or amboreceptor) und groups: Substance sensibilisatrice senibilsation Blood cell Buchners alexin Complement: Mechanims of Action (1899)

156 Doctoral Training Group GK University of Erlangen-Nürnberg 156 Lysis - rupturing membranes of foreign cells Opsonization - enhancing phagocytosis of antigens Chemotaxis - attracting macrophages and neutrophils Clumping of antigen-bearing agents hway.png Complement - Today

157 Doctoral Training Group GK University of Erlangen-Nürnberg 157 Opsonization - enhancing phagocytosis of antigens Chemotaxis - attracting macrophages and neutrophils Lysis - rupturing membranes of foreign cells Clumping of antigen- bearing agents Complement - Today ent.jpg

158 Doctoral Training Group GK University of Erlangen-Nürnberg 158

159 Doctoral Training Group GK University of Erlangen-Nürnberg 159 Studies by Bordet and Ehrlich clearly separated the haemolysin activity into an innate (complement) and an adaptive (antibody) component Established that humoral immune bodies (i.e., antibodies) can be cytotoxic Allowed to establish sensitive tests to detect the presence of antibodies against a germ in the blood of patients – Start of serology (e.g., complement fixation test) SUMMARY – Bordets and Ehrlichs Impact

160 HUMORAL IMMUNITY Other activities

161 Doctoral Training Group GK University of Erlangen-Nürnberg Almroth Wright (nickname Almost Right) and Douglas communicate to the Royal Society that humoral, inducible and specific substances (i.e., antibodies) in the body fluids reinforce he action of phacytosis named these factors opsonins (today antibodies) 189?Develops anti-typhoid vaccine with weakened bugs (all British solders were immunized during the 1st WW) with Wrights vaccine. Properties of Antibodies (Opsonins) Sir Almroth Edward Wright (1861–1947) Diggins F. (2002). Who was... Almroth Wright? Biologist (London). 49(6): Wright, A. E., and S. R. Douglas An experimental investigation of the role of the body fluids in connection with phagocytosis. Proc. R. Soc. London 72: See also Roitt p. 322

162 Doctoral Training Group GK University of Erlangen-Nürnberg

163 1908 Paul Ehrlich: Ehrlich (1908). Über Antigene und Antikörper. Einleitung in Handbuch der Immunitätsforschung. P Very nice overview about the knowledge of antibody and antigen in Another Paradigm in Immunology Infections are cleared by cellular and humoral immunity Another Paradigm in Immunology Infections are cleared by cellular and humoral immunity

164 Doctoral Training Group GK University of Erlangen-Nürnberg Adolf Creite (Göttingen) reports accumulation (Anhäufung) of red blood cells when adding sera from one animal to the blood of another animal 1875Leonard Landois (Göttingen) reports disolving (Auflösung) und ball formation (Zusammen- ballung) when he mixed blood and serum from diferent animals Agglutinins Germany – 1902 Germany Creite, A. (1869a) Versuche über die Wirkung des Serumeiweisses nach Injection in das Blut. Zeitschrift für Rationelle Medicin, 36, 90–108. Landois, L. (1875) Die Transfusion des Blutes, Leipzig. N. C. Hughes-Jones & Brigitte Gardner (2002). Historical Review: RED CELL AGGLUTINATION: THE FIRST DESCRIPTION BY CREITE (1869) AND FURTHER OBSERVATIONS MADE BY LANDOIS (1875) AND LANDSTEINER (1901). British Journal of Haematology, 2002, 119, 889–893.

165 Doctoral Training Group GK University of Erlangen-Nürnberg Herbert E. Durham and Max von Gruber introduce the term Agglutinin for any substance that caused agglutination of cells Gruber-Durham reaction 1896Fernand Widal uses the Gruber-Durham reaction to develop a test for typhoid fever. o Obseravtion: Serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. o Widal test was the first example of serum diagnosis – 1927 Ausria 1862–1929 France Agglutinins

166 Doctoral Training Group GK University of Erlangen-Nürnberg Landsteiner's first agglutination test with blood 1901Landsteiner discovers two types of blood antigens (blood groups) Agglutinins – Discovery of Blood Groups Austria Nobelpreis 1930 Landsteiner (1900). Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe.Centralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten, vol. 27,pp ool/pdf/ab_landsteinerversuch.pdf Landsteiner, K. (1901) Über Agglutinationserscheinungen normal menschlichen Blutes. Wiener Klinische Wochenschrift, 14, 1132–1134.

167 Doctoral Training Group GK University of Erlangen-Nürnberg Discovered that blood types of donors and recipients need to be matched before transfusions o made blood transfusions a routine o saved countless lives o Nobel Prize for Medicine Introduces the term hapten 1909discovered (with biologist Erwin Popper) the infectious nature of poliomyelitis (the polio virus). 1927discovered with pathologist Philip Levine,the M and N agglutinogens. 1940discovered (with pathologist Alexander Wiener the rhesus (Rh) factor in blood. While working in his laboratory on 24 June 1943, Landsteiner suffered a heart attack, and died from its after-effects two days later Austria(US) Nobel Prize 1930 Karl Landsteiner – Discoveries

168 Doctoral Training Group GK University of Erlangen-Nürnberg 168 ^^ Landsteiner K, Wiener AS. An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proc Soc Exp Biol Med 1940;43: immune sera ^^ Landsteiner K. Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe. Zentralblatt Bakteriologie 1900;27: ^^ Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and "incomplete" Rh agglutinins. Brit J Exp Path 1945;26:

169 Doctoral Training Group GK University of Erlangen-Nürnberg 169

170 Doctoral Training Group GK University of Erlangen-Nürnberg 170 Precipitins 1897Rudolf Kraus ( ; Austria) observes precipitates between immune sera and soluble lystes of bacteria Experiment Filter lysates from cholera, plaque and typhus through a bacteria-removing filter Add sera from patients or animals Obeservation Precipitates are visible Conclusion Not only bateria but also their soluble compounds react with corresponding antibodies Kraus, R. Wien. Klini Wochenzeitschrift 10:736 (1897) PRECIPITINS

171 Doctoral Training Group GK University of Erlangen-Nürnberg 171 Summary: Humoral immunity (1905) Eichmann, Klaus (2000). The network collective: rise and fall of a scientific paradigm JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen Scand. J. Immunol., 19, In 1905 it was clear that all these humoral activities can be traced back to the same class of inducible compounds (i.e., the antibody molecule) Today, Antikörper (Antibody) is a neutral term for the common component in all the different biological activities of immune sera

172 Doctoral Training Group GK University of Erlangen-Nürnberg 172 Summary: Humoral immunity (1905) Eichmann, Klaus (2000). The network collective: rise and fall of a scientific paradigm JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen Scand. J. Immunol., 19, In 1905 it was clear that all these humoral activities can be traced back to the same class of inducible compounds (i.e., the antibody molecule) Today, Antikörper (Antibody) is a neutral term for the common component in all the different biological activities of immune sera

173 Doctoral Training Group GK University of Erlangen-Nürnberg 173 Humoral immunity (1905) Eichmann, Klaus (2000). The network collective: rise and fall of a scientific paradigm JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen Scand. J. Immunol., 19, In 1905 it was clear whether all these activities are caused by the same inducible class of compounds Today, Antikörper (Antibody) is a neutral term for the various properties of soluble, inducible and specific immunity

174 Doctoral Training Group GK University of Erlangen-Nürnberg 174 SubstanceActivityDiscovery Alexin(s) Complement Complement Soluble components in the serum that are capable of killing microorganisms Buchner (1890), Ehrlich (1892) [3] Ehrlich [3] Antitoxins Substances in the serum that can neutralize the activity of toxins, enabling passive immunizationpassive immunization von Behring and Kitasato (1890) [5] [5] Bacteriolysins Serum substances that work with the complement proteins to induce bacterial lysis complement proteins Richard PfeifferRichard Pfeiffer (1895) [6] [6] Bacterial agglutinins & precipitinsagglutininsprecipitins Serum substances that agglutinate bacteria and precipitate bacterial toxins von Grubervon Gruber and Durham (1896), [7] Kraus (1897) [8] [7] [8] Hemolysins Serum substances that work with complement to lyse red blood cellsred blood cells Belfanti and Carbone (1898) [9] Jules Bordet (1899) [10] [9] Jules Bordet [10] Opsonins serum substances that coat the outer membrane of foreign substances and enhance the rate of phagocytosis by macrophages Wright and Douglas (1903) [11] [11] Antibody formation (1900), antigen-antibody binding hypothesis (1938), produced by B cells (1948), structure (1972), immunoglobulin genes (1976)immunoglobulin Founder: P Ehrlich [3] [3] Major discoveries in the study of humoral immunity [4] [4] y#cite_note-G.E-3

175 Doctoral Training Group GK University of Erlangen-Nürnberg Behring never uses the noun Antitoxin his his two 1890 publications (Only used adj. antitoxisch in a footnote in the 1890 paper with Kitasato) 1891Tizzioni and Cattani who were looking rather for a substance than an activity introduced for the first time the noun antitoxin Tizzoni, G. & Cattani, G. Ueber die Eigenschaften des Tetanus-Antitoxins. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 9, 685, Ehrlich uses for the first time the term Antikörper in his second 1891 paper on page 1219 at the end of the 6. paragraph; If two substances give rise to two different 'Antikörper', then they themselves must be different. Ehrlich, P. Experimentelle Untersuchungen über Immunität. II. Ueber Abrin. Dtsch. med. Wochenschr. 17, 1218, Ehrlich uses again the term Antikörper in his 1897 paper: Nachdem die ursprüngliche Annahme von einer Zerstörung des Giftes durch den Antikörper als unhaltbar sich erwiesen hatte, …. P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p Other terms:Immunkörper, Amboceptor, Zwischenkörper,, Immunisin, substance sensibilisatrice, copula, Desmon, philocytase, fixateur Origin: TermsAntitoxin & Antikörper

176 Doctoral Training Group GK University of Erlangen-Nürnberg Deutsche Med. Wochenzeitschr. (1891) Nr. 32, p 976 Coining Term Antikörper – Ehrlich

177 Doctoral Training Group GK University of Erlangen-Nürnberg 177 Ehrlichs term was not accepted immediately and several other terms were used: Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin. Antibody was obviously constructed in formal analogy to 'Antitoxin', but in conceptual analogy to 'Immunkörper'. The antitoxin is something directed against a toxin; the antibody is a body directed against something (a body ???) This imbroglio (Verwirrung) is apparent in Ehrlich's paper (Ehrlich, P. Experimentelle Untersuchungen er Immunitat. II. Ueber Abrin. Dtsch. med. Wochenschr. 17, 1218, 1891): All these phenomena result from the presence, in the blood, of a bodythe anti- abrinwhich completely paralyses the effects of abrinprobably by destruction of this body Body number 1 in this sentence is antibody; body number 2 is antigen. Antibody is actually confusing since it is itself a body that reacts against another body. Therefore, Immunkörper might have been a better term !!!???? Term Antikörper – Lindemann 1984 JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen Scand. J. Immunol., 19,

178 START OF IMMUNOLOGY Serology

179 Doctoral Training Group GK University of Erlangen-Nürnberg Preventive Immunization o Jenner (1789)-1. designed immunization (1798) o Pasteur (1880) – chicken cholera generalized Jenners small pox approach 2.Cellular Immunity o Methnikoff (1884) - discovers phagocytic activity 3.Humoral Immunity & Serotheraphy o Bering (1890/91) – Tetanus/Diphtheria o Ehrlichs Sidechain Theory (1897) 4.Cytotoxic humoral immunity and complement o Bordet (1899): substance sensibilisatrice + Buchners Alexin o Ehrlich (1899): Amboreceptor + Komplement 5.Serodiagnostic (Start of Serology) o Widal (1896) – Widal agglutination test for typhoid fever o Landsteiner (1901) – Blood goups in human o Bordet (1901) - Complement fixation test o Wassermann (1905) – Syphilis test 6.Anaphylaxis and Related Disorders (harmless antigens make us sick) o Portier & Richet (1902) - Anaphylaxis o Arthus reaction (1903) o Von Pirquet (1906) - Serum sickness – Allergie o Wolff_Eisner (1906) - Heufieber o Meltzer (1910) - Asthma Nobel 1908 Nobel 1901 Nobel 1908 Nobel 1919 Nobel 1913 Nobel 1930 TOPCIS: Start of Immunology

180 Doctoral Training Group GK University of Erlangen-Nürnberg Herbert E. Durham and Max von Gruber introduce the term Agglutinin for any substance that caused agglutination of cells Gruber-Durham reaction 1896Fernand Widal uses the Gruber-Durham reaction to develop a test for typhoid fever. o Obseravtion: Serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. o Widal test was the first example of serum diagnosis – 1927 Ausria 1862–1929 France Widal Test (Typhoid fever)

181 Doctoral Training Group GK University of Erlangen-Nürnberg 181 Bordet/Gengou: Complement fixation (1901) Observation (Jules Bordet and Octave Gengou 1898, Pasteur Institute) Antigen-antibody reaction leads to the binding (fixation) of complement to the target antigen This discovery allowed him to develop blood tests (serodiagnosis technique) that indicate whether a person has been in contact with any infectious agent. Jules Bordet ( ) Belgium Nobel price Medicine 1919 Bordet, J. and O. Gengou Sur l'existencede substances sensibilisatrices dans la plupart des serums antimicrobiens. Ann. De l'Inst. Pasteur. xv:

182 Doctoral Training Group GK University of Erlangen-Nürnberg Serum is isolated from the patient (has antibodies) and control (no antibodies). 2.Complement proteins in the patients serum are destroyed by heating (antibody survives) 3.Known amount of standardized complement (guinea pig serum) and antigen are added 4.Sheep red blood cells (sRBCs) which have been pre-bound to anti-sRBC antibodies are added to the serum. 5.The test is considered negative if the solution turns pink at this point and positive otherwise. Nice Animation: Complement fixation test

183 Doctoral Training Group GK University of Erlangen-Nürnberg 183 Complement in patient (has anti-germ) and ctrl serum (no anti-germ) is destroyed by heating Sheep red blood cells (sRBCs) and anti-sRBC are added Equal amounts of fresh C (guinea pig serum) and germ- Ag are added Antigen Fixed Compl. Anti- germ Anti- SRBC Free Compl. Agglutinated sRBC Lysed SRBC Complement fixation test Nice Animation: Patient Control Anti-SRBC

184 Doctoral Training Group GK University of Erlangen-Nürnberg A Wassermann, A. Neisser and C. Bruck. Eine serodiagnostische Reaktion bei Syphilis. Deutsche medicinische Wochenschrift, Berlin, 1906, 32: http://plumbot.com/Wassermann_test 3.http://www.whonamedit.com/synd.cfm/2962.html Wassermann Test (Syphillis) August Paul von Wassermann ( ) Bamberg, Germany 1904Fritz R. Schaudinn and Paul E. Hoffmann isolate the causative organism of syphilis. 1905Wassermann introduces complement-fixation test with blood serum or cerebro-spinal fluid to detect a syphilis infection Procedure Used lipid called cardiolipin from heart as "antigen", which has nothing to do with syphilis. However, this antigen reacts with certain antibodies that are commonly present in the blood of syphilis patients Antibodies are also present in malaria, lepra and autoimmune disorders Not specific for syphilis Also called Bordet-Wassermann

185 Doctoral Training Group GK University of Erlangen-Nürnberg 185 Complement Fixation Test - Summary The bacterium is present, if the red blood cells (reporter) added to the blood of infected patients remain intact in the presence of 1.the bacterium's specific antibody, 2.complement and 3.the red blood cells' specific antibody

186 Doctoral Training Group GK University of Erlangen-Nürnberg 186 Bacterio- & Hemolysins: Summary 1888George Nuttal reports that bloodserum from non-immunzed animals kills bacteria in the absence of cells 1889Hans Buchner describes for the frist time a heat-labile substance in blood serum that was capable of destroying bacteria (alexin) 1894Richard Pfeiffer shows for the frist time that blood from immunized animals can lyse bacteria (bacteriolysis) in the absence of cells 1898Jules Bordet shows that two different bodies (today antibody and complement) are required for lysis of erythrocytes (immune hemolysis) 1899Paul Ehrlich & Morgenroth rename alexin in complement and show that amboreceptor (today antibody) mediates complement 1901Bordet introduces the complement-fixation test 1905August v. Wassermann invents antibody test for syphyilis

187 Doctoral Training Group GK University of Erlangen-Nürnberg 187 IMMUNOLOGY: Own Discipline PAUL-EHRLICH-INSTITUT Paul-Ehrlich Institute für Serum-forschung und Serumprüfung (1896) bis Paul-Ehrlich Institute Bundesamt für Sera und Impfstoffe (1990) Das Paul-Ehrlich- Institut für Serumprüfung und Serumforschung Jahre1896 in Steglitz bei Berlin Paul-Ehrlich-Institut im Jahre 1990 als Bundesamt für Sera und Impfstoffe in Langen bei Frankfurt/Main Königliches Institut für experimentelle Therapie + Georg- Speyer-Haus Frankfurt Ab 1947 Paul- Ehrlich-Institut für Exp. Therapie Georg-Speyer-Haus, 1906, Frankfurt Königliches Institut für experimentelle Therapie, Frankfurt 1899

188 Doctoral Training Group GK University of Erlangen-Nürnberg 188 JOURNALS o Zeitschrift für Immunitätsforschung (1908) o J. of Immunology (1913) o Eur. J. Immunology (1970) PROFESSIONAL SOCIETIES o American Associaten of Immunologist (1913) o Deutsche Gesellschaft für Immunologie = DGfI (1953) IMMUNOLOGY: Own Discipline

189 Doctoral Training Group GK University of Erlangen-Nürnberg 189 TIME LINE - History of Immunology Discovery of cells and germs ( ) Prevention of Infection (1840 – today) Start of Immunology ( ) Immunochemistry - The antibody problem ( ) Self-/non-self discrimination (1940 – today) Models to explain antibody diversity (1897 and 1950s) Discovery of B and T cells (1960s) The molecular revolution (1974 – today)

190 IMMUNOCHEMISTRY The Antibody Problem ( )


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