History of Immunology Part 3: START OF IMMUNOLOGY Hans-Martin Jäck

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

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)

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

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

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

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)

PREVENTION OF INFECTION

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

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

IMMUNITY – Timeline 2 Discovery of cells and germs (1683 - 1876)
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)

Immunity - Etymology Late 14c. 1775 1879 "protection from disease“
"exempt from service or obligation" from Latin immunitatem (nom. immunitas) "exemption from performing public service or charge (tax) 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“ Pasteur, Koch, Ehrlich ????? Yufang Shi, Ph.D., Introduction and History of ImmunologyUMDNJ-Robert Wood Johnson Medical School

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

Yufang Shi, Ph.D., UMDNJ-Robert Wood Johnson Medical School
University Professor Department of Molecular Genetics, Microbiology and Immunology UMDNJ-Robert Wood Johnson Medical School The term “immunity” was first used in 1775 by Van Sweiten, a Dutch physician, as“immunitas” to describe the effects induced by an early attempt at variolization. Yufang Shi, Ph.D., UMDNJ-Robert Wood Johnson Medical School

Introduction and History of Immunology
Immunity - Etymology Late 14c. "exempt from service or obligation" from Latin immunitatem (nom. immunitas) "exemption from performing public service or charge (tax) or from Latin “immunis "exempt, free," from in - "not" + munis "performing services“ 1879 "protection from disease“ Pasteur, Koch, Ehrlich ????? The term “immunity” was first used in 1775 by Van Sweiten, a Dutch physician, as“immunitas” 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 Introduction and History of Immunology

IMMUNOLOGY – Timeline Phase I: Phenomenon: Immunity (500 B.C. - 1796)
Phase II: Introduction of Vaccination & Immunochemistry (1860 – 1945) Phase III: Identification of cellular and molecular components (1945 – today)

START OF IMMUNOLOGY

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

Descriptive early period pre-
Early attempts at vaccination: Smallpox (1794 Bacteria cause disease, discovery of bacterial toxins (19th 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

Preventive Vaccination
START OF IMMUNOLOGY Preventive Vaccination

Immunity - Thucydides (430 B.C)
Thucydides reports that people who survived plaque during the Peleponnesian War between Sparta and Athens were protected from disease First description of adaptive immunity !!!!!! 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.”

First description of adaptive immunity
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

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 Variolation, immunization against smallpox, was a common practice before vaccination was common. This worked because the patient was exposed to a weak strain of smallpox, which did not kill, yet provided immunity to the disease. Edward Jenner discovered that cowpox could protect against smallpox, with a much lower incidence of complications than variolation. Pasteur discovered a general method for immunizing people against disease while working on chicken cholera. He coined the term vaccination to describe the technique.

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 , 1st recorded 24 year-long epidemy Worldwide since the 16th century Variolation, immunization against smallpox, was a common practice before vaccination was common. This worked because the patient was exposed to a weak strain of smallpox, which did not kill, yet provided immunity to the disease. Edward Jenner discovered that cowpox could protect against smallpox, with a much lower incidence of complications than variolation. Pasteur discovered a general method for immunizing people against disease while working on chicken cholera. He coined the term vaccination to describe the technique.

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

Small Poxs – Timeline of Variolation
Ca Chinese inoculated children with material from infected people Ca Variolation introduced into Turkish harems 1717 Lady Mary Montagu introduced smallpox inoculation to Europe. 1760 Variolation of the families of Maria Theresia und George III. popularized variolation 1776 Washington began variolating the Continental Army Smallpox was a feared disease throughout human history and justifiably so. It was highly contagious and almost everyone eventually became infected. Mortality rates were as high as 25% in adults and closer to 40% in children. Those who did survive often had scarring due to the blister-like pustules that form on the skin, but they obtained life-long immunity to the disease. As far back at the 11th century in India and China it was realized that 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 virus. Lady Mary Wortley Montgue, wife of ambassador to the Ottoman Empire, introduced variolation to England in 1721 and it became a popular practice throughout Europe. Washington even began variolating the Continental Army in 1776. Variolation had some deleterious side effects. Serious skin lesions inevitably resulted at the site of inoculation, often accompanied by a generalized rash or even a full case of smallpox. The fatality rate from variolation was 1 to 2 %. Today we would find this level of fatality to be unacceptable, but at the time this risk still represented a significant advance. VARIOLATION: The practice of scratching into the skin (usually of children) some matter taken from a part of a person recovering from a mild infection with smallpox. If the amount used was just right this produced only a mild case of smallpox. Wrong treatments could kill or be ineffective. This was practiced by surgeons in Europe during the 18th in an attempt to give immunity to smallpox later in life. Lady Mary Montagu, wife of the British ambassodour to Turkey,

Small Pox – The Jenner Experiment 1796
Country lore (Bauernweisheit)/Obervation “Milkmaids who caught cowpox from their cows could not catch smallpox”. Milk maids infected by cowpox have on their hands scars very similar to smallpox scars. Hypothesis Cowpox infection protects from small pox The experiment (May 14, 1796) Infected a boy with the lymph of a cowpox-infected milkmaid. On 1st July, Jenner infected (variolated) the boy with small pox Result Boy did not get sick Cowpox pustule on the hand of the dairymaid Sarah Nelmes "The use of the term for diseases other than smallpox is due to Pasteur (Trans. 7th Session Internat. Med. Congr. (1881) Jenner infects James Phipps, the son of his gardener who had not yet suffered smallpox with Sarah’s lymph. James became mildly ill

Small Pox – The Jenner Experiment 1796
Conclusion Cow pox infection protects against small pox Problems Human experiment Controls Publication 1797, rejected by Transactions of the Royal Society of London. 1801, published “The Origin of the Vaccine Inoculation” Baxby D Edward Jenner's Inquiry; a bicentenary analysis. Vaccine Jan 28;17(4):301-7. “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)

Small Pox – Jenner‘s Clinic
Roitt p 346

In 1798, a new way to control the spread of a disease (smallpox):
Before Jenner: Disease protection Humoralists Keep the body's humors in balance: perform routine blood-letting administer medicines that cause sweating, urination, bowel evacuation Contagionists Restrict contact: leave the disease-ridden area impose quarantine remove sources of fomites Miasmatists Cleanse the air: remove sources of foul smells flush the air with smoke breathe in aromatic substances In 1798, a new way to control the spread of a disease (smallpox): Vaccinatists Vaccinate: 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

Vaccination – Techniques
Vaccination gun No need for needle replacement and sterilisation. Required too much maintenance Disposable 'bifurcated needle‘ Has a narrow, flattened forked end Draws vaccine by capillary action Was then jabbed repeatedly into the skin

Smallpoxs – The Start of Vaccination
1801 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 1853 Smallpox epidemic duringThe Franco-Prussian War French army was not vaccinated ►23,400 died German army was vaccinated ►only 278 died. 1967The World Health Organization's Smallpox Eradication Campaign Saturation Vaccination In 1967 the World Health Organization (WHO) launched its campaign to eradicate smallpox worldwide. They estimated at that time that there were still up to 15 million cases of smallpox each year. The biggest problem areas were South America, Africa and the Indian subcontinent. Their first approach was to vaccinate every person in the areas at risk. Teams of vaccinators from all over the world journeyed to the remotest of communities. Ring Vaccination The last case of smallpox in South America was reported in As the number of cases in other countries dropped the medical teams were able to change their tactics. They travelled around looking for smallpox outbreaks. They even resorted to putting up posters advertising rewards for people who reported cases of smallpox. Once found, a smallpox sufferer was isolated at home with his family. They and all surrounding families were then vaccinated. The Last Case of Naturally Occurring Smallpox The last case of smallpox in India occurred in 1975, but the disease persisted in Ethiopia and surrounding regions of Africa. In 1977 a hospital worker who had nursed a family in a Somali hospital became ill. Ali Maow Maalim had never himself been vaccinated! WHO officials literally sat on his doorstep, letting no one out or in until the last scab had fallen off his last pock. He recovered. He was the last person on Earth to catch smallpox by natural transmission. Vaccination Techniques Near the beginning of the WHO campaign the invention of a vaccination gun that fired a jet of vaccine using compressed air was heralded as a breakthrough, cutting out the need for needle replacement and sterilisation. It was soon realised however that it required too much maintenance in the desert dusts. The disposable 'bifurcated needle' was adopted instead, its narrow, flattened forked end drawing up just enough vaccine by capillary action. This was then jabbed repeatedly into the skin, to give a painless vaccination. Smallpox is Dead After an anxious period of watching for new cases, in 1980 the WHO formally declared: "Smallpox is Dead!" The most feared disease of all time had been eradicated, fulfilling the prediction that Edward Jenner had made in It has been estimated that the task he started has led to the saving of more human lives than the work of any other person. The last remaining specimens of the smallpox virus are now held in just two laboratories, in Siberia and the USA. The samples, used for research, are afforded higher security than a nuclear bomb. One day they too will be destroyed. Smallpox will have become the first major infectious disease to be wiped from the face of the Earth. Jenner's Legacy Edward Jenner's Inquiry can be identified as the origin of one of the most important branches of modern medicine. All that is known about disease prevention by vaccination, our understanding of allergy, autoimmune diseases (such as rheumatoid arthritis), transplantation and AIDS follows from this fundamental work by Edward Jenner. Jenner is acknowledged as the Father of Immunology - the science of our body's defence against invading bugs and chemicals.

Smallpoxs – Irradication
Up to 15 million cases of smallpox each year. the World Health Organization (WHO) launched its campaign to eradicate smallpox worldwide. 1977 Ali Maow Maalim from Somali was the last person on Earth to catch smallpox by natural transmission 1980 WHO formally declared: "Smallpox is Dead!” 1967The World Health Organization's Smallpox Eradication Campaign Saturation Vaccination In 1967 the World Health Organization (WHO) launched its campaign to eradicate smallpox worldwide. They estimated at that time that there were still up to 15 million cases of smallpox each year. The biggest problem areas were South America, Africa and the Indian subcontinent. Their first approach was to vaccinate every person in the areas at risk. Teams of vaccinators from all over the world journeyed to the remotest of communities. Ring Vaccination The last case of smallpox in South America was reported in As the number of cases in other countries dropped the medical teams were able to change their tactics. They travelled around looking for smallpox outbreaks. They even resorted to putting up posters advertising rewards for people who reported cases of smallpox. Once found, a smallpox sufferer was isolated at home with his family. They and all surrounding families were then vaccinated. The Last Case of Naturally Occurring Smallpox The last case of smallpox in India occurred in 1975, but the disease persisted in Ethiopia and surrounding regions of Africa. In 1977 a hospital worker who had nursed a family in a Somali hospital became ill. Ali Maow Maalim had never himself been vaccinated! WHO officials literally sat on his doorstep, letting no one out or in until the last scab had fallen off his last pock. He recovered. He was the last person on Earth to catch smallpox by natural transmission. Vaccination Techniques Near the beginning of the WHO campaign the invention of a vaccination gun that fired a jet of vaccine using compressed air was heralded as a breakthrough, cutting out the need for needle replacement and sterilisation. It was soon realised however that it required too much maintenance in the desert dusts. The disposable 'bifurcated needle' was adopted instead, its narrow, flattened forked end drawing up just enough vaccine by capillary action. This was then jabbed repeatedly into the skin, to give a painless vaccination. Smallpox is Dead After an anxious period of watching for new cases, in 1980 the WHO formally declared: "Smallpox is Dead!" The most feared disease of all time had been eradicated, fulfilling the prediction that Edward Jenner had made in It has been estimated that the task he started has led to the saving of more human lives than the work of any other person. The last remaining specimens of the smallpox virus are now held in just two laboratories, in Siberia and the USA. The samples, used for research, are afforded higher security than a nuclear bomb. One day they too will be destroyed. Smallpox will have become the first major infectious disease to be wiped from the face of the Earth. Jenner's Legacy Edward Jenner's Inquiry can be identified as the origin of one of the most important branches of modern medicine. All that is known about disease prevention by vaccination, our understanding of allergy, autoimmune diseases (such as rheumatoid arthritis), transplantation and AIDS follows from this fundamental work by Edward Jenner. Jenner is acknowledged as the Father of Immunology - the science of our body's defence against invading bugs and chemicals.

“Jenner is acknowledged as the Father of Vaccination

Develoment of Vaccination
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.

Vaccination against rabies (1885)
Rabies & Cholera - Pasteur Vaccínation against chicken cholera (1878) Vaccination against rabies (1885) (The case of Johann Meister) 1967The World Health Organization's Smallpox Eradication Campaign Saturation Vaccination In 1967 the World Health Organization (WHO) launched its campaign to eradicate smallpox worldwide. They estimated at that time that there were still up to 15 million cases of smallpox each year. The biggest problem areas were South America, Africa and the Indian subcontinent. Their first approach was to vaccinate every person in the areas at risk. Teams of vaccinators from all over the world journeyed to the remotest of communities. Ring Vaccination The last case of smallpox in South America was reported in As the number of cases in other countries dropped the medical teams were able to change their tactics. They travelled around looking for smallpox outbreaks. They even resorted to putting up posters advertising rewards for people who reported cases of smallpox. Once found, a smallpox sufferer was isolated at home with his family. They and all surrounding families were then vaccinated. The Last Case of Naturally Occurring Smallpox The last case of smallpox in India occurred in 1975, but the disease persisted in Ethiopia and surrounding regions of Africa. In 1977 a hospital worker who had nursed a family in a Somali hospital became ill. Ali Maow Maalim had never himself been vaccinated! WHO officials literally sat on his doorstep, letting no one out or in until the last scab had fallen off his last pock. He recovered. He was the last person on Earth to catch smallpox by natural transmission. Vaccination Techniques Near the beginning of the WHO campaign the invention of a vaccination gun that fired a jet of vaccine using compressed air was heralded as a breakthrough, cutting out the need for needle replacement and sterilisation. It was soon realised however that it required too much maintenance in the desert dusts. The disposable 'bifurcated needle' was adopted instead, its narrow, flattened forked end drawing up just enough vaccine by capillary action. This was then jabbed repeatedly into the skin, to give a painless vaccination. Smallpox is Dead After an anxious period of watching for new cases, in 1980 the WHO formally declared: "Smallpox is Dead!" The most feared disease of all time had been eradicated, fulfilling the prediction that Edward Jenner had made in It has been estimated that the task he started has led to the saving of more human lives than the work of any other person. The last remaining specimens of the smallpox virus are now held in just two laboratories, in Siberia and the USA. The samples, used for research, are afforded higher security than a nuclear bomb. One day they too will be destroyed. Smallpox will have become the first major infectious disease to be wiped from the face of the Earth. Jenner's Legacy Edward Jenner's Inquiry can be identified as the origin of one of the most important branches of modern medicine. All that is known about disease prevention by vaccination, our understanding of allergy, autoimmune diseases (such as rheumatoid arthritis), transplantation and AIDS follows from this fundamental work by Edward Jenner. Jenner is acknowledged as the Father of Immunology - the science of our body's defence against invading bugs and chemicals. Though Pasteur knew that the vaccine worked, but no one then in the world of science knew how it worked!

Unterschiede: Aktive Immunisierung vs. passive Immunität
Aktive und passive Immunität unterscheiden sich in einigen wesentlichen Punkten: Protective Vaccination - Summary aktiv passiv Indikation Prophylaxe Prophylaxe, Therapie Gabe von Antigen Antikörper Gabe wie oft wenige Male immer wieder Schutzeintritt spät sofort Schutzdauer lange kurz Gedächtnis ja nein

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)). 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)).

Figure 1-23 Treatment and prevention of disease
Year Event 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. 1721 Lady Mary Wortley Montgue, wife of the ambassador to the Ottoman Empire, introduces variolation to Europe. 1796 Edward Jenner uses cowpox to immunize against smallpox. 1884 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. 1885 Paul Ehrlich proposes that certain chemicals affect bacterial cells and begins a search for one that can treat syphilis. 1886 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. 1891 Ehrlich shows that antibodies are responsible for part of immunity. 1897 Almwroth Wright and David Sample develop an effective vaccine against typhoid fever using killed cells of Salmonella typhi. Waldemar Haffkine develops a vaccine against the plague. 1912 Paul Ehrlich announces the discovery of a cure for syphilis. The cure is the first specific chemotherapeutic agent for a bacterial disease. 1929 Alexander Fleming publishes the first paper describing penicillin. 1935 Gerhard J. Domagk uses Prontosil, a chemically synthesized antimetabolite, to kill Streptococcus in mice. 1938 Max Theiler produces a vaccine against yellow fever by passaging the virus through mice to weaken it. 1940 Howard Florey and Ernest Chain produce an extract of penicillin and show it can kill bacteria in animals. Ernest 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. 1941 Charles Fletcher demonstrates that penicillin is non-toxic to human volunteers, by injecting a police officer suffering from a lethal infection. 1942 Selman Waksman suggests the word "antibiotic" to describe the class of compounds produced by one microorganism that inhibit or kill other microorganisms. 1944 Albert Schatz, E. Bugie, and Selman Waksman discover streptomycin, a very effective drug against tuberculosis. W. H. Feldman and H. C. Hinshaw at the Mayo Clinic successfully treat tuberculosis with streptomycin. 1957 The Soviet delegation to the World Health Organization proposes a vaccination effort to eradicate smallpox. The program finally begins in 1967. 1977 Ali Maow Maalin, age 23 of Somalia, is the last known victim of naturally occurring smallpox. 1979 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.)

START OF IMMUNOLOGY Cellular Immunity

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

START OF IMMUNOLOGY Serotherapy

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

Unterschiede: Aktive Immunisierung vs. passive Immunität
Aktive und passive Immunität unterscheiden sich in einigen wesentlichen Punkten: Passive Vaccination - Summary aktiv passiv Indikation Prophylaxe Prophylaxe, Therapie Gabe von Antigen Antikörper Gabe wie oft wenige Male immer wieder Schutzeintritt spät sofort Schutzdauer lange kurz Gedächtnis ja nein

Vaccination – Passive immunization
Characteritics Transfer of serum, gammaglobulin or monoclonal antibodies from humans or animals Immediate protection Short duration (t/2 = 20 days) Side effects through immune response to foreign proteins (serum disease, anaphylaxy) Contraction of hepatitis or HIV through antibody preparations from human serum Naturally acquired passive immunity placental transport of maternal IgG from mother in the fetus through Transfer if maternal IgA into newborn through milk Artifically induced passive immunization Injection or transfusion of gammaglobulin from other individuals or animals Treatment of an acute infection (diphtheria, tetanus, rabies, FSME, rubella (Röteln) …) Toxins (Insects, snake, scorpions, botulinus) Prophylactic before travel to foreign countries Rhesus factor prophylactic Passive Immunität: Übertragung von Antikörpern Eine Spezifische Immunität kann auch ohne direkten Antigenkontakt erworben werden. Dies wird üblicherweise durch die Übertragung von Serum oder Gammaglobulinen von einem immunisierten Spender auf einen nicht-immunen Empfänger erreicht. Man spricht von einer passiven Immunisierung. Natürlich erworbene passive Immunität : Diese Immunität wird von der Mutter auf den Fetus durch die diaplazentare Übertragung von IgG erreicht. Ein weiteres Beispiel ist die Übertragung von IgA über die Muttermilch auf das Neugeborene. Künstlich herbeigeführte passive Immunität : Dieser Zustand der Immunität kann durch Injektion von Gammaglobulin von anderen Individuen (Hyperimmunglobulin, Standardimmunglobulin) oder von immunen Tieren erreicht werden. Eine derartige Maßnahme wird in einer Vielzahl von akuten Infektionen (Diphtherie, Tetanus, Masern, Tollwut, FSME, Hepatitis A und B, Röteln, Varicella zoster, CMV, Botulismus, Gasbrand etc.), Vergiftungen (Insekten, Schlangen, Skorpion, Botulismus), und prophylaktisch bei Hypogammaglobulinämie und Fernreisen bzw. als Rhesusprophylaxe ergriffen. In den meisten Fällen sind Immunglobuline menschlichen Ursprungs das Mittel der Wahl, in manchen Indikationen stehen nur tierische Präparate zur Verfügung (Schlangenbiss, Diphtherie, Gasgangrän, Botulismus). Die Schutzwirkung tritt bei dieser Art der Immunisierung sofort ein, allerdings sind heterologe Immunglobuline nur relativ kurz wirksam (Halbwertszeit t/2 = 20 Tage) und können auch Nebenreaktionen durch eine Immunantwort gegen das verabreichte fremde Protein mit sich bringen (Serumkrankheit, Anaphylaxie). Homologe Präparate (von anderen Menschen) bergen dagegen wieder die Gefahr der Übertragung von Hepatitis bzw. HIV.

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

SIDE VISIT Bacterial toxins

SIDE VISIT – Bacterial toxins
Exotoxins released by microorganisms act at the surface of host cells, for example by binding to receptors Endotoxins Intrinsic components of microbial structure Trigger e.g., phagocytes to release cytokines that produce local or systemic symptoms exotoxin microbe Janeway, 2011 (8th edition) Toxoid 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.

Endotoxins – Structure and function
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) Immunogenicity Toxicity

Endotoxin – Discovery (LPS)
1894 Richard Pfeiffer Endotoxin - Heat stable toxic material from the membrane of Vibrio Cholerae that is released only after the cells are disintegrated. 1933 Andre 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 Richard Pfeiffer ( ) Germany Otto Westphal ( ) Germany Otto Lüderitz Germany Ref: 1. JOSEPH E. ALOUF (1987). From “Diphtheritic” Poison to Molecular Toxicology ASM News 53, p.547 2. Reimond Beck, A Chronology of Microbiology in Historical Context (paperback)

Exotoxins – (AB toxins)
Diphtheria toxin

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. The toxin catalyses the ADP-ribosylation of (and inactivates) the elongation factor eEF-2. This elongation factor is a protein that is essential to protein synthesis; by inactivating it, the translation portion of protein synthesis is inhibited. The toxin enters the host cell and is hydrolysed by a trypsin-like protease to produce a toxic fragment. The toxin then transfers an ADP-ribose from NAD+ to a diphthamide residue (a modified Histidine amino acid) found within the EF-2 protein. EF-2 is needed for the moving of tRNA from the A-site to the P-site of the ribosome during translation. The ADP-ribosylation is reversible when by giving high doses of nicotinamide (or vitamin B3), one of the reaction's products. Diphtheria toxin is produced by C. diphtheriae only when it is infected with a bacteriophage. The bacteriophage integrates a gene into the bacteria that causes the toxin to be produced. [7] [8] NAD+ + peptide diphthamide ↔ nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide

Exotoxins – More examples AB toxins
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

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. 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. Risk factors include crowded environments, poor hygiene, and lack of immunization.

Diphtheria – Symptoms 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. Those without symptoms who carry diphtheria should be treated with antibiotics. MacGregor RR. Corynebacterium diphtheriae. In: Mandell GL, Bennett JE, Dolan R, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 7th ed. Orlando, FL: Saunders Elsevier; 2009:chap 205.

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 Drooling (suggests airway blockage is about to occur) Fever Hoarseness Painful swallowing Skin lesions (usually seen in tropical areas) Sore 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. Tests used may include: Gram stain or throat culture to identify Corynebacterium diphtheriae Electrocardiogram (ECG)

Treatment If the health care provider thinks you have diphtheria, treatment should be started immediately, even before test results are available. Diphtheria 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. 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. Those without symptoms who carry diphtheria should be treated with antibiotics.

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] 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: Metronidazole Erythromycin (orally or by injection) for 14 days (40 mg/kg per day with a maximum of 2 g/d), or Procaine penicillin G given intramuscularly for 14 days (300,000 U/d for patients weighing <10 kg and 600,000 U/d for those weighing >10 kg). Patients with allergies to penicillin G or erythromycin can use rifampin or clindamycin.

Exotoxins – More examples AB toxins
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

Diphtheria Toxin – Mechanism of action
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. In layman's terms, a bacterium gets infected by a virus. The virus adds DNA into the DNA of the bacteria which causes it to produce Diphtheria toxin. Diphtheria toxin is a single protein with two parts: one that allows entry into host cells and the other prevents the host cell from making proteins. The toxin binds to a cell-surface receptor to gain entry into the cell. Inside the cell, the protein prevents the cell from making new proteins. More technically, Diphtheria toxin consists of a single protein or polypeptide. The protein is broken down (Proteolysis) to yield two fragments (A and B), which are held together by a disulfide bond. Fragment A prevents the host cells from undergoing protein synthesis. Fragment B is a recognition subunit (the host cell basically allows it to enter). Fragment B gains access to the host cell by binding to the plasma membrane at specific sites called the EGF-like domain of Heparin-binding EGF-like growth factor (HB-EGF). The HB-EGF then takes in the fragments through receptor-mediated endocytosis. The low pH of the endosome (sac which transports the HB-EGF and bound fragments A and B) induces fragment B to begin producing pores and catalyses the release of a catalytic fragment A into the cytosol or intracellular fluid. The toxin catalyses the ADP-ribosylation of (and inactivates) the elongation factor eEF-2. This elongation factor is a protein that is essential to protein synthesis; by inactivating it, the translation portion of protein synthesis is inhibited. The toxin enters the host cell and is hydrolysed by a trypsin-like protease to produce a toxic fragment. The toxin then transfers an ADP-ribose from NAD+ to a diphthamide residue (a modified Histidine amino acid) found within the EF-2 protein. EF-2 is needed for the moving of tRNA from the A-site to the P-site of the ribosome during translation. The ADP-ribosylation is reversible when by giving high doses of nicotinamide (or vitamin B3), one of the reaction's products. Diphtheria toxin is produced by C. diphtheriae only when it is infected with a bacteriophage. The bacteriophage integrates a gene into the bacteria that causes the toxin to be produced. [7] [8] Structure Diphtheria toxin is a single polypeptide chain of 535 amino acids consisting of two subunits linked by disulfide bridges. Binding to the cell surface of the less stable of these two subunits allows the more stable part of the protein to penetrate the host cell.[3] [edit] Mechanism Diphthamide This is NAD+-diphthamide ADP-ribosyltransferase (EC ) . It catalyzes the ADP-ribosylation of eukaryotic elongation factor-2 (eEF2), inactivating this protein. It does so by ADP-ribosylating the unusual amino acid diphthamide. In this way, it acts as a RNA translational 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. The exotoxin A of Pseudomonas aeruginosa uses a similar mechanism of action.

(EHEC) – Entero-haemorragic E. coli
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

Diphtheria Exotoxin – Timeline
BC Homer describes Egyptian disease ~1800 Fidele Bretonneau coins “Diphtheria” for the disease (“Häutchen auf Mandeln) and introduces tracheotomy as ultima ratio in treatment 1883 Edwin Klebs (student of Rudolph Virchow) discovers bacteria in diphtheria patients 1884 Friedrich 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 1888 Roux and Yersin - soluble and filterable toxin in diphtheria cultures causes disease 1890 More than 50,000 children/year die in Germany of diphtheria 1890 Behring reports 1. successful vaccination with weakened C. diphtheriae in guinea pigs

Diphtheria toxin - Discovery
Roux 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. 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”. Emile Roux ( ) France Alexandre Yersin ( ) France/Schweiz ALOUF (1987). From “Diphtheritic” Poison to Molecular Toxinology ASM News VOL.53,NO. l0

Infectious Diseases - Online

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. ^ Microbiology: A Human Perspective. Fourth edition. McGraw Hill

Bacterial toxins – Summary

TIMELINE: Serum Therapy
„ANTIGEN“ Deutsch (Detre) 1st Serum therapy (tetanus Mice) Behring & Kitasato anti-toxins 1st Serum therapy in humans (diphtheria) Behring&Ehrlich Ehrlich‘ side chain theory „ANTI-BODY“ Ehrlich Ehrlich Discovery o 1890 1891 1891 1892 1893 1894 1952 1904 1897 Behring 1st serum therapy (diphtheria guinea pgs Industrial production of antisera (diphtheria in sheep (Hoechst) Production of of antisera in US (NYC) Industrial production of antisera (diphtheria in sheep (Hoechst)

Discovery of Inducible Humoral Immunity
Serotherapy -

SEROTHERAPY

Discovery of inducible soluble immunity
1889 Gameleia (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. 229. 1890 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. 1129 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. 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 Pasteur’s 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). In 1899, Gamaleia directed the establishment of a bacteriological institute in Odessa. In 1898 he discovered substances that destroy bacteria—bacteriolysins. 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 “Mechnikov’s 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. 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, 1967. V. N. GUTINA The Great Soviet Encyclopedia, 3rd Edition ( ). © 2010 The Gale Group, Inc. All rights reserved. Nice overview in a „News and Views“ to Behring‘s 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

Discovery of inducible soluble immunity
May 1890 von 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)

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. 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 Pasteur’s 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). In 1899, Gamaleia directed the establishment of a bacteriological institute in Odessa. In 1898 he discovered substances that destroy bacteria—bacteriolysins. 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 “Mechnikov’s 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. 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, 1967. 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.

Discovery of humoral immunity
- Antitoxins and Serum Therapy -

  Tetanus & Diptheria - 1890 Emil von Behring (1890)
Inactivated Tetanus 10 Serum  Pathogenic Tetanus 20  Inactivated Diphteria Serum 10 Emil von Behring (1890) Soluble, inducible and specific immunity trough immunisation with pathogens Individual can be protected by transferring serum from immunized animal → 1st Nobel Price 1901 for Serum Therapie

1st Nobel Price 1901 for Serum Therapy only for Emil von Behring
1. Nobel Price in Medicine Grew pure culture of tetanus bacillus 1890 Discovered Humoral immunity against tetanus together with von Behring Shibasaburo Kitasato Japanese Microbiologist 1st Nobel Price 1901 for Serum Therapy only for Emil von Behring

Immune against pathogenic Tetanus and tetanus toxin
Serum Therapy - Tetanus (December 1890) 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 / 1114. Immune against pathogenic Tetanus and tetanus toxin 10  Control Serum Immune Weakened Tetanus Emil v. Behring ( ) Germany Pathogenic Tetanus 20  No Tetanus Shibasaburo Kitasato ( ) Japan Transferred immune serum protects mice against tetanus Works also therapeutically in sick mice !!!!!! No data of experiments with Diphtheria bacillus were reported !!!!!

  Protection against Diphtheria (December 1890)
Weakened Diphtheria (Jodtrichloride)  BEHRING 1890: “Untersuchungen über das Zustandekommen der Diphtherie-Immunität bei Thieren.” Deut. Med. Wochenschr. 16(50):1145–1147. Pathogenic Diphtheria Emil v. Behring ( ) Germany Non-immunized  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.

Immune against pathogenic diphtheria or diphtheria toxin
Serum Therapy – Diphtheria (1891) Immune against pathogenic diphtheria or diphtheria toxin 10  Wernicke, Frosch and Behring Weakened (JCL3) Diphtheria Immune Serum Pathogenic Diphtheria 20 Behring, Emil “Ueber Desinfection am lebenden Organismus.” Deutsche Medicinische Wochenschrift 17(52):1393– (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) No Diphtheria  Control Serum 10

Serum Therapy – Diphtheria (1891)

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

Zeitschrift für Hygiene und Infectionskrankheiten (1992). 12:10–44.

Behring‘s Goals 1. CAUSATIVE TREATMENT OF INFECTIOUS DISEASES
“ 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, 1113. “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) 2. PROMOTE FIRST IMMUNOLOGICAL PARADIGM „Specific immunity induced by antigens is associated with the formation of soluble antibodies“

Protection against Diphtheria (1892)
Behring, Emil, and Erich Wernicke “Ueber Immunisierung und Heilung von Versuchsthieren bei der Diphtherie.” Zeitschrift für Hygiene und Infectionskrankheiten 12:10–44. Wernicke, Frosch and Behring

Serum Therapy: From Bench to bed side
1890 More than 50,000 children in Germany die every year of diphtheria. Behring and Kitasato discover soluble, inducible and transferable anti-tetanus activity in blood of immunized rabbits 1891 Behring successfully repeats serum therapy of diphtheria- infected guinea pigs Behring apparently treats successfully a diphtheria- diseased child with injection of anti-diphtheria serum produced in horses (report not proven) 1892 Wernicke & Behring improve immunization of rabbits, guinea pigs and goats with weakened diphtheria bacillus The first successful therapeutic serum treatment of a child suffering from diphtheria occurred in Until then more than 50,000 children in Germany died yearly of diphtheria. During the first few years, there was no successful breakthrough for this form of therapy, as the antitoxins were not sufficiently concentrated. Not until the development of enrichment by the bacteriologist Paul Ehrlich ( ) along with a precise quantification and standardization protocol, was an exact determination of quality of the antitoxins presented and successfully developed. Behring subsequently decided to draw up a contract with Ehrlich as the foundation of their future collaboration. They organized a laboratory under a railroad circle (Stadtbahnbogen) in Berlin, where they could then obtain the serum in large amounts by using large animals – first sheep and later horses.

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.

Serum Therapy: Status Quo 1892
1891 – Successful serum therapy of diphtheria in guinea pigs Behring, Emil “Ueber Desinfection am lebenden Organismus.” Deutsche Medicinische Wochenschrift 17(52):1393–1397. 1892 – 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

Diphtheria: D Bench to bed side
In NYC

1892 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 1st clinical trial in Berlin (published by Behring and Ehrlich in 1894) The first successful therapeutic serum treatment of a child suffering from diphtheria occurred in Until then more than 50,000 children in Germany died yearly of diphtheria. During the first few years, there was no successful breakthrough for this form of therapy, as the antitoxins were not sufficiently concentrated. Not until the development of enrichment by the bacteriologist Paul Ehrlich ( ) along with a precise quantification and standardization protocol, was an exact determination of quality of the antitoxins presented and successfully developed. Behring subsequently decided to draw up a contract with Ehrlich as the foundation of their future collaboration. They organized a laboratory under a railroad circle (Stadtbahnbogen) in Berlin, where they could then obtain the serum in large amounts by using large animals – first sheep and later horses.

1892 Roux succesfully produces antisera in horse that protects guinea pigs against diphtheria Samuel Amstrong (1895). The serum tretament of diphtheria. The popular Science Vol XLVI (46), p )

Samuel Amstrong (1895). The serum tretament of diphtheria. The popular Science Vol XLVI (46), p )

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

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 Summary of all treated patients Treatment after onset of infection Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20:

Serum Therapy: Time of application
“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.” 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.

Serum Therapy: Clinical „Trial“ (Paris)

Serum Therapy: Improvements and Quantitation
Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4: Text Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20: Text Ehrlich P, Kossel H (1894): Ueber die Anwendung des Diphtherieantitoxins. Zeitschrift fuer Hygiene und Infektionskrankheiten, medizinische Mikrobiologie, Immunologie und Virologie 17: Text Ehrlich P (1894): Ueber die Behandlung der Diphtherie mit Heilserum. Verhandlungen der Gesellschaft Deutscher Naturforscher und Aerzte : 402. 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

Ehrlich P, Kossel H, Wassermann A (1894): Ueber Gewinnung und Verwendung des Diphterieheilserums. Deutsche medizinische Wochenschrift 20:

1888 - Émile Roux & Alexandre Yersin: Toxinnachweis
Emil Adolf von Behring: Serum Träger der Immunität Behring & Shibasaburo Kitasato: Entdeckung des Antitoxins im Blut kranker Tiere, Antitoxin ist prinzipiell übertragbar William Hallock Park und Anna Wessels Williams entwickeln am New York City Department of Health ein Antitoxin. Behring & Erich Wernicke: Immunität durch Injektion von neutralisiertem Diphtherietoxin Nobelpreis für Physiologie oder Medizin für von Behring von Behring: Toxin-Antitoxinmischung für Immunisierung Gaston Ramon ( ) beha- Gaston Ramon ndelt das Toxin mit Wärme/Formalin für Impfung Émile Roux & Alexandre Yersin: Toxinnachweis Emil Adolf von Behring: Serum Träger der Immunität Behring & Shibasaburo Kitasato: Entdeckung des Antitoxins im Blut kranker Tiere, Antitoxin ist prinzipiell übertragbar William Hallock Park und Anna Wessels Williams entwickeln am New York City Department of Health ein Antitoxin. Behring & Erich Wernicke: Immunität durch Injektion von neutralisiertem Diphtherietoxin Nobelpreis für Physiologie oder Medizin für von Behring von Behring: Toxin-Antitoxinmischung für Immunisierung Gaston Ramon ( ) behandelt das Toxin mit Wärme/Formalin für Impfung

1894 After introduction of serum therapy (Roux serum) mortality of diphtheria in Paris falls from 52% to 25% Production of antitoxins in horses in US (William Hallock Park und Anna Wessels Williams in NYC) 1900 Industrial production of anti-diphtheria sera in US 1895 22 October 1900 One of the first bottles of diphtheria antitoxin produced at the Hygienic State Laboratory which became the NIH in 1930. Advertisement for anti-diphtheria serum by Parke Davis & Company.

By Cristina Luiggi | May 28, 2011
One-Man NIH, 1887 By Cristina Luiggi | May 28, 2011 As epidemics swept across the United States in the 19th century, the US government recognized the pressing need for a national lab dedicated to the study of infectious disease. In 1887, the government set its sights on a small lab located in the Marine Hospital on Staten Island, New York. Its sole member, 27-year-old Joseph James Kinyoun, belonged to a new generation of scientists and physicians who were beginning to understand how microscopic organisms underlay the terrible killers of their day, such as smallpox, yellow fever, and Asiatic cholera. That one-room lab on Staten Island, which Kinyoun originally called “the Laboratory of Hygiene,” ultimately evolved into the 27 institutes and centers that now make up the National Institutes of Health. Kinyoun’s first order of business was to collect blood and stool samples from the sick in order to culture pathogens in the lab. In his first year on the job, he became the first person in the United States to isolate the gram-negative bacterium Vibrio cholerae—providing his American colleagues with their first glimpse of the microorganism responsible for tens of thousands of deaths since it had first reached US shores in the 1830s. This and other successes were duly noted by Congress, which by 1902 had expanded the laboratory to include other divisions, such as chemistry and zoology. Although microorganisms had been visible to the human eye for nearly 400 years thanks to the invention of microscopes in the 1600s, a definitive connection between bacteria and infectious disease wasn’t made until the late 19th century. Instead, filth and poverty were blamed for deadly epidemics, and treatment and prevention strategies were aimed at improving sanitation and welfare. During his time at the Hygienic Laboratory (as it was later called), Kinyoun sailed to Europe for six months to train with the great bacteriologists of his day, including Robert Koch and Louis Pasteur, bringing back laboratory techniques, recipes for effective treatments, and a passionate vision for reforming US health practices, says author and historian Joseph Houts, Kinyoun’s great-grandson. “It was in great part due to him that the ‘germ theory’ made its way back to the United States,” Houts adds. Kinyoun’s tenure as the first director of the NIH lasted 12 years, but his role in shaping how the federal government dealt with the country’s health continued well after his retirement. Following several deaths due to contaminated and shoddily produced vaccines, he pushed hard for the implementation of universal standards for the production of medicines. He was also acutely aware of the need to monitor infectious diseases across the country, as well as the need for an official body that could enforce drastic measures, like quarantines during deadly epidemics. Such efforts, Houts says, eventually led to the creation of federal agencies such as the US Food and Drug Administration and the Centers for Disease Control and Prevention One-Man NIH, 1887 By Cristina Luiggi | May 28, 2011 The Hygienic Laboratory at the Staten Island Marine Hospital Service building.National Cancer Institute

with hand-written labels.
Serum Therapy: From Bench to bed side 1904 Foundation of „Behringwerke" in Marburg (uses two Million Reichsmark from the Nobel prize) → 1. biotech company Photo: Courtesy of Aventis Behring Behring watches diphtheria immunization of horses in Marburg Old vials (1897 and 1906) with hand-written labels.

Roitt p. 321

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“.

Diphtheria – Active Immunizations
1901 Behring uses attenuated diphtheria and successfully immunizes laboratory animals 1913 Behring & Wernicke introduce active vaccination by injection of a safe mixture of diphtheria toxin and antitoxin. Was replaced by active vaccination with inactivated diphtheria toxoid 1924 Gaston Ramon ( ) inactivates tetanus and diphtheria toxins by heat/formalin treatment for active immunizations

Other successful serum therapies
1924 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 1944 Protection against measles Stokes uses globulin fractions of pooled human plasma for passive protection against measles in 891 individuals

SERUM THERAPY – Problem 1
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 Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4:

Serum Therapy: Improvements and Quantitation
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 – Titer (Ehrlich 1894)
Ehrlich P (1894): Über die Gewinnung, Werthbestimmung und Verwerthung des Diphtherieheilserums. Hygienische Rundschau 4:

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:

Anaphylaxis and Related Disorders
START OF IMMUNOLOGY Anaphylaxis and Related Disorders

SERUM THERAPY – Problem 2
1906 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

Hypersensitivity –Injection of Antigens
1902 Anaphylaxis, 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 1913 1903 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!!!!

1902 1903 1906

Hypersensitivity - Today

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

SEROTHERAPY

1st Immunological paradigm:
Serum Therapy: Summary 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) Emil v. Behring ( ) Germany 1st Nobel prize in Medicine for serum therapy (1901) 1st Immunological paradigm: „Specific immunity induced by antigens is associated with the formation of antibodies“

TIMELINE: Serum Therapy of Diphtheria
Safe Active Vacci-nation Ramon Klebs discovers bacteria on material from diceased diphtheria patient Behring& Ehrlich (Berlin) 1st serum therapy in humans Roux and Yersin idenify soluble diphtheria toxin Behring 1st Serum therapy (diphtheria) in guinea pigs Roux develops antisera in horses Behring-werke in Marburg 1883 1884 1888 1890 1891 1892 1893 1894 1904 1924 Löffler identifies C. diphtheriae as the cause of diphtheria Behring & Kitasato 1st serum therapy (tetanus) in mice Hoechst (Behring) Industrial production of antisera in sheep Roux & Chaillon (Paris) Serum therapy in humans Park & Williams (NYC) Production of antisera in

TIMELINE: Serum Therapy of Diphtheria
1st serum therapies in humans Behring& Ehrlich (Berlin) Roux & Chaillon (Paris) Klebs discovers bacteria on material from diceased diphtheria patient Roux and Yersin idenify soluble diphtheria toxin 1st Serum therapy (diphtheria) in guinea pigs (Behring) Roux develops antisera in horses Behring-werke in Marburg 1883 1884 1888 1890 1891 1892 1893 1894 1904 1924 Löffler identifies C. diphtheriae as the cause of diphtheria 1st serum therapy (tetanus) in mice (Behring & Kitasato) Industrial production of antisera in sheep (Hoechst) Production of antisera in US (Park, NYC Safe Active Vacci-nation Ramon

Serum Therapy: Today Llewelyn et al. (1992). Monoclonal antibodies in Medicine. BMJ, 305:1269

IgG und Rhesusunverträglichkeit (Landsteiner 1940)
Hemolytic Disease of the Newborn (HDN) 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 For further explanation, see Kuby, 4th edition, p. 414

Anti-Rh-Antikörper (Rhogam)
Mütterliche Anti-Rh-B-Zelle Fötale Erys Behandlung der Mutter durch Gabe von Rhogam = Anti-D (Rhesusfaktor)-Antikörper vom IgG-Typ) Routinemäßig nach 28. Schwangerschaftswoche und kurz nach Geburt Spätestens 72 Stunden nach Fehlgeburten, Ab- treibungen, Amniocentese oder anderen invasiven Eingriffen Zerstören fötale Erys, die in den mütterlichen Kreislauf übertreten, und verhindern so die Aktivierung von B-Zellen und die Bildung von Gedächtnis-B-Zellen Nur 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. Übertritt größerer fötaler Blutmengen in den mütterlichen Kreislauf Normalerweise nur während der Geburt Während der Schwangerschaft bei Amniocentese oder anderen invasiven Eingriffen Fehlgeburten Abtreibung Schweren inneren Blutungen

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

Antitoxins and cytotoxic antibodies
START OF IMMUNOLOGY Antitoxins and cytotoxic antibodies

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

ANTITOXINS Mechanism of action

Antitoxins: Mechanism of action (Ehrlich, 1897)
Mechanisms of antitoxic effect of Behring‘s 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.“ Seit B ehr in gs Entdeckung der antitoxisclien Functionen hat die Frage nach dem Wesen dieser Erscheinung andauernd das Interesse und die Arbeit fast alles Vertreter der modernen Richtung gefesselt, wie die zahlreichen diesbezüglichen Publilrationen beweisen. Nachdem die ursprüngliche Annahme von einer Zerstörung des Giftes ' durch den Antikörper als unhaltbar sich erwiesen hatte, nachdem sie11 ergeben hatte, dass in den xphysiologi,cjch neutralem Toxin- Antitoxingemischen noch beide Componenten als solche enthalten sind, stehen sich zur Zeit zwei Meinungen schroff gegenüber, Es kann nach der einen Anschauung sich Gift und Gegengift in den Gewebsiiüssi~eiten zii einer Art Doppelverbindung paaren, welche niclit mehr in bestimmten Geweben fiairt wird und welche daher keine Kranldieitser~cheinun~emn ehr auslöst. Im Gegensatz zu dieser chemisclien Auffassung nehmen einige Autoren, insbesondere R o ux und Buchn er, eine meh indirecte Wirkung des Antitoxins an ; dasselbe soll keine active Wirkung auf das Toxin ausüben, sondern in erster Reihe auf die Zellen einwirken und dieselben gewissermassen gegen die Giftwislcung immunisiren. Die S~hwieri~lredietr Entscheidung Probem: In Tierversuchen keine Untertsuchung möglich Abhilfe: Reagenzglasversuche. P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p

Serum and ricin before adition to RBC
Antitoxins: Mechanism of action (Ehrlich, 1897) Mix of anti-ricin Serum and ricin before adition to RBC Experiment: Tubes with blood from un-immunized rabbits Ricin Ricin mediates clumbing of RBC - + + Ricin Oberservation → Anti-ricin toxin prevents ricin (lectin)-mediated clumping of red blood cells in a concentration dependent manner 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 P. Ehrlich (1897). Zur Erkenntniss der Antitoxinwirkung. Fortschritte der Medicin, Bd 15, No 2, p

CYTOTOXIC HUMORAL IMMUNITY
Bacteriolysins (today:antibody + complement)

Blood Plasma and Serum Blood plasma Blood serum
yellow liquid component of blood without blood cells 55% of the total blood volume contains 93% water by volume and dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide Prepared by spinning a tube of fresh blood containing an anti-coagulant Blood serum blood plasma without fibrinogen or the other clotting factors Produced by centrifugation of coagulated blood Blood plasma is the yellow liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid (all body fluid outside of cells). It is mostly water (93% by volume) and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide (plasma being the main medium for excretory product transportation). Blood plasma is prepared by spinning a tube of fresh blood containing an anti-coagulant in a centrifuge until the blood cells fall to the bottom of the tube. The blood plasma is then poured or drawn off.[1] Blood plasma has a density of approximately 1025 kg/m3, or kg/l.[2] Blood serum is blood plasma without fibrinogen or the other clotting factors (i.e., whole blood minus both the cells and the clotting factors).[1]

Bacteriolysins: The beginning
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 1889 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. George Nuttall ( ) San Francisco In 1888, George Henry Falkiner Nuttall showed the blood serum from non-immunized animals contained bactericidal substances, from which he concluded that phagocytes were merely accessory to the protection offered by serum. Buchner‘s Alexin= Ehrlich‘s complement Hans Buchner ( ) Germany

Bacteriolysin: Coining the name
1894 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) Richard Friedrich Johannes Pfeiffer (March 27, 1858 – September 15, 1945) was a German physician and bacteriologist.[1] Pfeiffer was born in Zduny, Province of Posen, and died in Bad Landeck. Pfeiffer is remembered for his many fundamental discoveries in immunology and bacteriology, particularly for the phenomenon of bacteriolysis. In 1894 he found that live cholera bacteria could be injected without ill effects into guinea pigs previously immunised against cholera, and that blood plasma from these animals added to live cholera bacteria caused them to become motionless and to lyse. This could be inhibited by previously heating the blood plasma. He called this bacteriolysis and it became known as the Pfeiffer Phenomenon, or Isayev-Pfeiffer phenomenon. Working with Robert Koch in Berlin he intellectually and experimentally conceived the concept of endotoxin as a heat-stable bacterial poison responsible for the pathophysiological consequences of certain infectious diseases. Endotoxin and anti-endotoxin antibodies have since then fascinated researchers of many disciplines, particularly in the fields of diagnosis, prevention, and therapy of severe Gram-negative infections. Pfeiffer was a pioneer in typhoid vaccination. He discovered the specific bacteria-dissolving immune bodies in cholera and typhus. The British pathologist Almroth Wright is generally credited with the initiation of typhoid vaccination in His claims of priority were challenged as early as 1907 in favour of Richard Pfeiffer. A review of the original literature of the 1890s and the early 1900s revealed that several groups were working on typhoid vaccine at the same time and that the credit for the initiation of typhoid vaccine studies should be shared by these two great researchers. In 1892 he isolated what he thought was the causative agent of influenza. The culprit, according to Pfeiffer, was a small rod-shaped bacterium that he isolated from the noses of flu-infected patients . He dubbed it Bacillus influenzae (or Pfeiffer's bacillus), which was later called Haemophilus influenzae. Few doubted the validity of this discovery, in large part because bacteria had been shown to cause other human diseases, including anthrax, cholera, and plague. When history's deadliest influenza pandemic began in 1918, most scientists believed that Pfeiffer's bacillus caused influenza. With the lethality of this outbreak (which killed an estimated 20 to 100 million worldwide) came urgency—researchers around the world began to search for Pfeiffer's bacillus in patients, hoping to develop antisera and vaccines that would protect against infection. In many patients, but not all, the bacteria were found. Failures to isolate B. influenzae (now known as Haemophilus influenzae) were largely chalked up to inadequate technique, as the bacteria were notoriously difficult to culture. [2] The first blow to Pfeiffer's theory came from Peter Olitsky and Frederick Gates at The Rockefeller Institute. Olitsky and Gates took nasal secretions from patients infected with the 1918 flu and passed them through Berkefeld filters, which exclude bacteria. The infectious agent — which caused lung disease in rabbits — passed through the filter, suggesting that it was not a bacterium.[3][4] Although the duo had perhaps isolated the influenza virus (which they nevertheless referred to as an atypical bacterium called Bacterium pneumosintes), other researchers could not reproduce their results. In 1896 he isolated micrococcus catarrhalis that is the cause of laryngitis. M catarrhalis also causes bronchitis, and pneumonia in children and adults with underlying chronic lung disease. It is occasionally a cause of meningitis. Richard Pfeiffer also invented a universal staining for histological preparations. Pfeiffer studied at the Kaiser-Wilhelms-Akademie in Berlin from 1875 to After completing his studies he was conferred doctor of medicine in 1880 and subsequently served as an army physician and bacteriologist until He was a student of Robert Koch ( ), and from 1887 to 1891 worked as Koch's assistant in the Institute of Hygiene in Berlin. In 1891 he was entrusted with the leadership of the scientific department of the Institute of Infectious Diseases in Berlin. In 1897 Pfeiffer joined the German expedition under Robert Koch to India to investigate the plague. The following year he went to Italy with Koch to do research on Malaria. He moved to Königsberg to enter the chair of hygiene in 1899, succeeding Erwin von Esmarch ( ). He remained in that city until 1909, when he moved on to the same chair in Breslau. Pfeiffer retired there as emeritus in 1925. Richard Friedrich Johannes Pfeiffer Paul Fildes Biographical Memoirs of Fellows of the Royal Society Vol. 2, (Nov., 1956), pp Richard Pfeiffer ( ) Germany Paul Fildes Biographical Memoirs of Fellows of the Royal Society Vol. 2, (Nov., 1956), pp

1901 Complement 1889 1894 1898

Bacteriolysin: Labile component (1895)
Bordet verifies Buchner‘s and Pfeiffer‘s 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 Jules Bordet ( ) Belgium Nobel price Medicine 1919

CYTOTOXIC HUMORAL IMMUNITY
Hemolysins (today:antibody + complement)

Hemolysins Defintion: The activity in serum to clump and lyse erythrocytes 1875 Landois (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. 1898 Carbone & 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, 1898. 1898 Bordet showed that the cytotoxic effect of an immune serum was due to two substances contained in the same fluid, one was Buchner’s alexine (Ehrlich’s complement), the other he name substance sensibilisatrice (Ehrlich‘s 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.

Lysis requires specific Immune factor + heat-labile factor
Hemolysin – The Bordet Experiment (1898) Experimental Set-up Guinea pig blood 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: specific factor no lysis rabbit blood 550C/30 min immune serum Fresh serum from non- immunized animals During his seven years at the Pasteur Institute, Bordet made most of the basic discoveries that led to his Nobel Prize of Soon after his arrival at the Institute, he began work on a problem in immunology. In 1894, Richard Pfeiffer, a German scientist, had discovered that when cholera bacteria was injected into the peritoneum of a guinea pig immunized against the infection, the pig would rapidly die. This bacteriolysis, Bordet discovered, did not occur when the bacteria was injected into a non-immunized guinea pig, but did so when the same animal received the antiserum from an immunized animal. Moreover, the bacteriolysis did not take place when the bacteria and the antiserum were mixed in a test tube unless fresh antiserum was used. However, when Bordet heated the antiserum to 55 degrees centigrade, it lost its power to kill bacteria. Finding that he could restore the bacteriolytic power of the antiserum if he added a little fresh serum from a nonimmunized animal, Bordet concluded that the bacteria-killing phenomenon was due to the combined action of two distinct substances: an antibody in the antiserum, which specifically acted against a particular kind of bacterium; and a non-specific substance, sensitive to heat, found in all animal serums, which Bordet called "alexine" (later named "complement"). In a series of experiments conducted later, Bordet also learned that injecting red blood cells from one animal species (rabbit cells in the initial experiments) into another species (guinea pigs) caused the serum of the second species to quickly destroy the red cells of the first. And although the serum lost its power to kill the red cells when heated to 55 degrees centigrade, its potency was restored when alexine (or complement) was added. It became apparent to Bordet that hemolytic (red cell destroying) serums acted exactly as bacteriolytic serums; thus, he had uncovered the basic mechanism by which animal bodies defend or immunize themselves against the invasion of foreign elements. Eventually, Bordet and his colleagues found a way to implement their discoveries. They determined that alexine was bound or fixed to red blood cells or to bacteria during the immunizing process. When red cells were added to a normal serum mixed with a specific form of bacteria in a test tube, the bacteria remained active while the red cells were destroyed through the fixation of alexine. However, when serum containing the antibody specific to the bacteria was destroyed, the alexine and the solution separated into a layer of clear serum overlaying the intact red cells. Hence, it was possible to visually determine the presence of bacteria in a patient's blood serum. This process became known as a complement fixation test. Bordet and his associates applied these findings to various other infections, like typhoid fever , carbuncle, and hog cholera. August von Wasserman eventually used a form of the test (later known as the Wasserman test ) to determine the presence of syphilis bacteria in the human blood. Already famous by the age of thirty-one, Bordet accepted the directorship of the newly created Anti-rabies and Bacteriological Institute in Brussels in 1901; two years later, the organization was renamed the Pasteur Institute of Brussels. From 1901, Bordet was obliged to divide his time between his research and the administration of the Institute. In 1907, he also began teaching following his appointment as professor of bacteriology in the faculty of medicine at the Free University of Brussels, a position that he held until Despite his other activities, he continued his research in immunology and bacteriology. In 1906, Bordet and Octave Gengou succeeded in isolating the bacillus that causes pertussis (whooping cough) in children and later developed a vaccine against the disease. Between 1901 and 1920, Bordet conducted important studies on the coagulation of blood. When research became impossible because of the German occupation of Belgium during World War I, Bordet devoted himself to the writing of Traité de l'immunité dans les maladies infectieuses (1920), a classic book in the field of immunology. He was in the United States to raise money for new medical facilities for the wardamaged Free University of Brussels when he received word that he had been awarded the Nobel Prize. After 1920, he became interested in bacteriophage , the family of viruses that kill many types of bacteria, publishing several articles on the subject. In 1940, Bordet retired from the directorship of the Pasteur Institute of Brussels and was succeeded by his son, Paul. Bordet himself continued to take an active interest in the work of the Institute despite his failing eyesight and a second German occupation of Belgium during World War II. Many scientists, friends, and former students gathered in a celebration of his eightieth birthday at the great hall of the Free University of Brussels in He died in Brussels in 1961. unspecifc heat-labile factor  lysis Non- immune serum No lysis lysis inducible factor no lysis Lysis requires specific Immune factor + heat-labile factor

↑ BELFANTI, S. AND CARBONE, T
↑ 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, 1898. ↑ 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:

Hemolysin – The Bordet Experiment (1898)
1898 Bordet 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. Bordet‘s 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). Bordet’s Observation (at Pasteur) Hemolytic activity of serum was lost after heating to 56oC Activity was restored upon addition of fresh non-immune serum from rabbit or guinea pig

Bordet: Alexin „helps“ inducible factor (1898)
Explanation Hemolysin activity of immune serum was due to two substances contained in the same serum: a heat-labile unspecific factor present in blood of non-immunized and immunized animals (Buchner’s/Bordet’s alexin, today Ehrlich’s complement) a heat-stable species-specific factor only present in the immune serum (Bordet’s substance of sensibilisatrice, today Ehrlich‘s antibodies) Bordet’s mechanism of action During his seven years at the Pasteur Institute, Bordet made most of the basic discoveries that led to his Nobel Prize of Soon after his arrival at the Institute, he began work on a problem in immunology. In 1894, Richard Pfeiffer, a German scientist, had discovered that when cholera bacteria was injected into the peritoneum of a guinea pig immunized against the infection, the pig would rapidly die. This bacteriolysis, Bordet discovered, did not occur when the bacteria was injected into a non-immunized guinea pig, but did so when the same animal received the antiserum from an immunized animal. Moreover, the bacteriolysis did not take place when the bacteria and the antiserum were mixed in a test tube unless fresh antiserum was used. However, when Bordet heated the antiserum to 55 degrees centigrade, it lost its power to kill bacteria. Finding that he could restore the bacteriolytic power of the antiserum if he added a little fresh serum from a nonimmunized animal, Bordet concluded that the bacteria-killing phenomenon was due to the combined action of two distinct substances: an antibody in the antiserum, which specifically acted against a particular kind of bacterium; and a non-specific substance, sensitive to heat, found in all animal serums, which Bordet called "alexine" (later named "complement"). In a series of experiments conducted later, Bordet also learned that injecting red blood cells from one animal species (rabbit cells in the initial experiments) into another species (guinea pigs) caused the serum of the second species to quickly destroy the red cells of the first. And although the serum lost its power to kill the red cells when heated to 55 degrees centigrade, its potency was restored when alexine (or complement) was added. It became apparent to Bordet that hemolytic (red cell destroying) serums acted exactly as bacteriolytic serums; thus, he had uncovered the basic mechanism by which animal bodies defend or immunize themselves against the invasion of foreign elements. Eventually, Bordet and his colleagues found a way to implement their discoveries. They determined that alexine was bound or fixed to red blood cells or to bacteria during the immunizing process. When red cells were added to a normal serum mixed with a specific form of bacteria in a test tube, the bacteria remained active while the red cells were destroyed through the fixation of alexine. However, when serum containing the antibody specific to the bacteria was destroyed, the alexine and the solution separated into a layer of clear serum overlaying the intact red cells. Hence, it was possible to visually determine the presence of bacteria in a patient's blood serum. This process became known as a complement fixation test. Bordet and his associates applied these findings to various other infections, like typhoid fever , carbuncle, and hog cholera. August von Wasserman eventually used a form of the test (later known as the Wasserman test ) to determine the presence of syphilis bacteria in the human blood. Already famous by the age of thirty-one, Bordet accepted the directorship of the newly created Anti-rabies and Bacteriological Institute in Brussels in 1901; two years later, the organization was renamed the Pasteur Institute of Brussels. From 1901, Bordet was obliged to divide his time between his research and the administration of the Institute. In 1907, he also began teaching following his appointment as professor of bacteriology in the faculty of medicine at the Free University of Brussels, a position that he held until Despite his other activities, he continued his research in immunology and bacteriology. In 1906, Bordet and Octave Gengou succeeded in isolating the bacillus that causes pertussis (whooping cough) in children and later developed a vaccine against the disease. Between 1901 and 1920, Bordet conducted important studies on the coagulation of blood. When research became impossible because of the German occupation of Belgium during World War I, Bordet devoted himself to the writing of Traité de l'immunité dans les maladies infectieuses (1920), a classic book in the field of immunology. He was in the United States to raise money for new medical facilities for the wardamaged Free University of Brussels when he received word that he had been awarded the Nobel Prize. After 1920, he became interested in bacteriophage , the family of viruses that kill many types of bacteria, publishing several articles on the subject. In 1940, Bordet retired from the directorship of the Pasteur Institute of Brussels and was succeeded by his son, Paul. Bordet himself continued to take an active interest in the work of the Institute despite his failing eyesight and a second German occupation of Belgium during World War II. Many scientists, friends, and former students gathered in a celebration of his eightieth birthday at the great hall of the Free University of Brussels in He died in Brussels in 1961. 1898 Bordet 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). Jules Bordet ( ) Belgium Nobel price Medicine 1919 senibilsation Substance Sensibilisatrice (specificity and sensibilsation) Buchner‘s Alexin (toxic) RBC Cleary separeted haemolysin activity into an innate and an adaptive humoral component

Paul Ehrlich: Alexin → Complement (1899)
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 termed “amboreceptors” (today: antibodies). and the heat-labile component was termed “complement” due to the fact that it “complemented” the activity of the amboreceptors. Paul Ehrlich ( ) Germany Nobel price Medicine 1908 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,

Paul Ehrlich Morgenroth: Lysins
Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 36, 6–9 (1899) Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 36, 481–486 (1899) Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 37, 453–458 (1900) Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr –687 (1900) Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 38, 251–257 (1901) Ehrlich, P. & Morgenroth, J. Berlin klin.Wochenschr. 38, 569–574 (1901) 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).

Paul Ehrlich: Alexin → Complement (1899)
Paul 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) The term "complement" was introduced by Paul Ehrlich in the late 1890s, as part of his larger theory of the immune system. According to this theory, the immune system consists of cells that have specific receptors on their surface to recognize antigens. Upon immunization with an antigen, more of these receptors are formed, and they are then shed from the cells to circulate in the blood. These receptors, which we now call "antibodies," were called by Ehrlich "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. Ehrlich, therefore, named this heat-labile component "complement," because it is something in the blood that "complements" the cells of the immune system. In the early half of the 1930s, a team led by the renowned Irish researcher, Jackie Stanley, stumbled upon the all-important opsonization-mediated effect of C3b. Building off Ehrlich's work, Stanley's team proved the role of complement in both the innate as well as the cell-mediated immune response. Ehrlich believed that each antigen-specific amboceptor has its own specific complement, whereas Bordet believed that there is only one type of complement. In the early 20th century, this controversy was resolved when it became understood that complement can act in combination with specific antibodies, or on its own in a non-specific way. Ehrlich & Morgenroth (1899). Über Haemolysie – zweite Mitteilung. Berl. Klin. Wochenschr. Bd. 22, p („Komplement „ mentioned on page 482, left column)

Bordet: Alexin „helps“ inducible factor (1898)
Explanation Hemolysin activity of immune serum was due to two substances contained in the same serum: a heat-labile unspecific factor present in blood of non-immunized and immunized animals (Buchner’s/Bordet’s alexin, today Ehrlich’s complement) a heat-stable species-specific factor only present in the immune serum (Bordet’s substance of sensibilisatrice, today Ehrlich‘s antibodies) Bordet’s mechanism of action During his seven years at the Pasteur Institute, Bordet made most of the basic discoveries that led to his Nobel Prize of Soon after his arrival at the Institute, he began work on a problem in immunology. In 1894, Richard Pfeiffer, a German scientist, had discovered that when cholera bacteria was injected into the peritoneum of a guinea pig immunized against the infection, the pig would rapidly die. This bacteriolysis, Bordet discovered, did not occur when the bacteria was injected into a non-immunized guinea pig, but did so when the same animal received the antiserum from an immunized animal. Moreover, the bacteriolysis did not take place when the bacteria and the antiserum were mixed in a test tube unless fresh antiserum was used. However, when Bordet heated the antiserum to 55 degrees centigrade, it lost its power to kill bacteria. Finding that he could restore the bacteriolytic power of the antiserum if he added a little fresh serum from a nonimmunized animal, Bordet concluded that the bacteria-killing phenomenon was due to the combined action of two distinct substances: an antibody in the antiserum, which specifically acted against a particular kind of bacterium; and a non-specific substance, sensitive to heat, found in all animal serums, which Bordet called "alexine" (later named "complement"). In a series of experiments conducted later, Bordet also learned that injecting red blood cells from one animal species (rabbit cells in the initial experiments) into another species (guinea pigs) caused the serum of the second species to quickly destroy the red cells of the first. And although the serum lost its power to kill the red cells when heated to 55 degrees centigrade, its potency was restored when alexine (or complement) was added. It became apparent to Bordet that hemolytic (red cell destroying) serums acted exactly as bacteriolytic serums; thus, he had uncovered the basic mechanism by which animal bodies defend or immunize themselves against the invasion of foreign elements. Eventually, Bordet and his colleagues found a way to implement their discoveries. They determined that alexine was bound or fixed to red blood cells or to bacteria during the immunizing process. When red cells were added to a normal serum mixed with a specific form of bacteria in a test tube, the bacteria remained active while the red cells were destroyed through the fixation of alexine. However, when serum containing the antibody specific to the bacteria was destroyed, the alexine and the solution separated into a layer of clear serum overlaying the intact red cells. Hence, it was possible to visually determine the presence of bacteria in a patient's blood serum. This process became known as a complement fixation test. Bordet and his associates applied these findings to various other infections, like typhoid fever , carbuncle, and hog cholera. August von Wasserman eventually used a form of the test (later known as the Wasserman test ) to determine the presence of syphilis bacteria in the human blood. Already famous by the age of thirty-one, Bordet accepted the directorship of the newly created Anti-rabies and Bacteriological Institute in Brussels in 1901; two years later, the organization was renamed the Pasteur Institute of Brussels. From 1901, Bordet was obliged to divide his time between his research and the administration of the Institute. In 1907, he also began teaching following his appointment as professor of bacteriology in the faculty of medicine at the Free University of Brussels, a position that he held until Despite his other activities, he continued his research in immunology and bacteriology. In 1906, Bordet and Octave Gengou succeeded in isolating the bacillus that causes pertussis (whooping cough) in children and later developed a vaccine against the disease. Between 1901 and 1920, Bordet conducted important studies on the coagulation of blood. When research became impossible because of the German occupation of Belgium during World War I, Bordet devoted himself to the writing of Traité de l'immunité dans les maladies infectieuses (1920), a classic book in the field of immunology. He was in the United States to raise money for new medical facilities for the wardamaged Free University of Brussels when he received word that he had been awarded the Nobel Prize. After 1920, he became interested in bacteriophage , the family of viruses that kill many types of bacteria, publishing several articles on the subject. In 1940, Bordet retired from the directorship of the Pasteur Institute of Brussels and was succeeded by his son, Paul. Bordet himself continued to take an active interest in the work of the Institute despite his failing eyesight and a second German occupation of Belgium during World War II. Many scientists, friends, and former students gathered in a celebration of his eightieth birthday at the great hall of the Free University of Brussels in He died in Brussels in 1961. 1898 Bordet 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). Jules Bordet ( ) Belgium Nobel price Medicine 1919 senibilsation Substance Sensibilisatrice (specificity and sensibilsation) Buchner‘s Alexin (toxic) RBC Cleary separeted haemolysin activity into an innate and an adaptive humoral component

Paul Ehrlich: Hemolysin – Mode of Action
Anti-Toxin toxophore Complement toxophore haptophore Zwischenkörper Immunkörper Amboreceptor Haptophore 2 haptophore Haptophore 1 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 Mechanism of Action Complement Amboreceptor Receptor auf RBC 2. lysis 1. recognition Ehrlich & Morgenroth (1900). Über Haemolysie – vierte Mitteilung. Berl. Klin. Wochenschr. Juli p. 681.

Mechanism of Hemolysis: Textbook 1903
Deutsch, L. & Feistmantel, C. Die Impfstoffe und Sera. Thieme, Leipzig, 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

FINDS NEW REMEDY FOR PNEUMONIA; Dr. L. B
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

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 ( ) 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, 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,

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 Bordet‘s landmark experiment in 1895)

Complement: Mechanims of Action (1899)
Ehrlich Active haemolysin (or bacteriolysin) consists (in analogy to a toxin) of two parts: Immunkörper (or amboreceptor) und „groups“: senibilsation Buchner‘s alexin Substance sensibilisatrice Blood cell Source:Ehrlich & Morgenroth (1900). Über Haemolysie – vierte Mitteilung. Berl. Klin. Wochenschr. Juli p. 681. Studies by Bordet and Ehrlich clearly separated the haemolysin activity into an innate (complement) and a adaptive (antibody) component

Complement - Today Lysis - rupturing membranes of foreign cells
Opsonization - enhancing phagocytosis of antigens Chemotaxis - attracting macrophages and neutrophils Clumping of antigen-bearing agents

Complement - Today Opsonization - enhancing phagocytosis of antigens
Chemotaxis - attracting macrophages and neutrophils Lysis - rupturing membranes of foreign cells Clumping of antigen- bearing agents

SUMMARY – Bordet‘s and Ehrlich‘s Impact
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)

HUMORAL IMMUNITY Other activities

Properties of Antibodies (Opsonins)
1903 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 Wright’s vaccine. Sir Almroth Edward Wright (1861–1947) 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: Typhusepidemien waren in der britischen Armee ein großes Problem zu einer Zeit, als Infektionskrankheiten regelmäßig in der britischen Armee mehr Soldaten töteten als die direkte Feindeinwirkung. Wright entwickelte einen Impfstoff gegen Typhus mit abgetöteten Bakterien, den er an Truppen (3000 Soldaten) in Indien testete. Er schlug 1899 eine Massenimpfung britischer Truppen im Burenkrieg mit dem Impfstoff vor, stieß aber auf erheblichen Widerstand und konnte nur Freiwillige impfen. Als Ergebnis kam es im Burenkrieg zu verheerenden Typhusepidemien unter den britischen Truppen mit Erkrankten und 9000 Toten. Auch unter britischen Medizinern war die Impfung damals umstritten und Wright lieferte sich mit dem berühmten Statistiker Karl Pearson im British Medical Journal eine heftige Auseinandersetzung über den Wert der Impfung, bevor dann ein groß angelegter Versuch im Burenkrieg den Erfolg deutlich machte. Im Ersten Weltkrieg wurde in der britischen Armee in großem Umfang gegen Typhus geimpft als einzige der beteiligten Armeen. Wright selbst war während des Ersten Weltkriegs bei den britischen Truppen in Frankreich um Wundinfektionen zu studieren. Diggins F. (2002). Who was... Almroth Wright? Biologist (London). 49(6):280-2. See also Roitt p. 322

Another Paradigm in Immunology
„Infections are cleared by cellular and humoral immunity“ 1908 Paul Ehrlich: Ehrlich (1908). Über Antigene und Antikörper. Einleitung in „Handbuch der Immunitätsforschung“. P.1 -10 Very nice overview about the knowledge of antibody and antigen in 1908.

Agglutinins 1869 Adolf Creite (Göttingen) reports accumulation (“Anhäufung”) of red blood cells when adding sera from one animal to the blood of another animal 1875 Leonard Landois (Göttingen) reports disolving (“Auflösung”) und ball formation (“Zusammen-ballung”) when he mixed blood and serum from diferent animals Creite, A. (1869a) Versuche über die Wirkung des Serumeiweisses nach Injection in das Blut. Zeitschrift für Rationelle Medicin, 36, 90–108. Germany Two bacteriologists, Herbert Edward Durham (-1945) and Max von Gruber (1853–1927), discovered specific agglutination in The clumping became known as Gruber-Durham reaction. Gruber introduced the term agglutinin (from the Latin) for any substance that caused agglutination of cells. French physician Fernand Widal (1862–1929) put Gruber and Durham's discovery to practical use later in 1896, using the reaction as the basis for a test for typhoid fever. Widal found that blood serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. This Widal test was the first example of serum diagnosis. Austrian physician Karl Landsteiner found another important practical application of the agglutination reaction in Landsteiner's agglutination tests and his discovery of ABO blood groups was the start of the science of blood transfusion and serology which had made transfusion possible and safe after a while. Landois, L. (1875) Die Transfusion des Blutes, Leipzig. – 1902 Germany 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.

Agglutinins 1896 Herbert E. Durham and Max von Gruber introduce the term Agglutinin for any substance that caused agglutination of cells → Gruber-Durham reaction 1896 Fernand Widal uses the Gruber-Durham reaction to develop a test for typhoid fever. Obseravtion: Serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. Widal test was the first example of serum diagnosis. 1853 – 1927 Ausria 1862–1929 France

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

Karl Landsteiner – Discoveries
1901 Discovered that blood types of donors and recipients need to be matched before transfusions made blood transfusions a routine saved countless lives Nobel Prize for Medicine1930. 1920 Introduces the term hapten 1909 discovered (with biologist Erwin Popper) the infectious nature of poliomyelitis (the polio virus). 1927 discovered with pathologist Philip Levine,the M and N agglutinogens. 1940 discovered (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

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

http://www. biologieunterricht. info/pool/pdf/ab_landsteinerversuch

Precipitins PRECIPITINS
1897 Rudolf 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 PRECIPITINS Kraus, R. Wien. Klini Wochenzeitschrift 10:736 (1897)

Summary: Humoral immunity (1905)
SOLUBLE, INDUCIBLE & SPECIFIC IMMUNITY (Antitoxins, Immunkörper, Amboceptor, Zwischenkörper, Immunisin, substance sensibilisatrice, copula, Desmon, philocytase, fixateur) Antitoxins Behring (1890) Bacteriolysins Pfeiffer (1895) Agglutinins von Gruber (1896) Precipitins Kraus (1897) Hemolysins Belfanti & Crabone (1898) Opsonins Wright (1903) The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different".[67] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.[67] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper.[67] 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 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,

Humoral immunity (1905) SOLUBLE, INDUCIBLE, & SPECIFIC IMMUNINITY (Antitoxins, Immunkörper, Amboceptor, Zwischenkörper, Immunisin, substance sensibilisatrice, copula, Desmon, philocytase, fixateur) Antitoxins Behring (1890) Bacteriolysins Pfeiffer (1895) Agglutinins von Gruber (1896) Precipitins Kraus (1897) Hemolysins Belfanti & Crabone (1898) Opsonins Wright (1903) The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different".[67] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.[67] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper.[67] 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 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,

Major discoveries in the study of humoral immunity[4]
Major discoveries in the study of humoral immunity[4] Substance Activity Discovery Alexin(s) Complement Soluble components in the serum that are capable of killing microorganisms Buchner (1890), Ehrlich (1892)[3] Antitoxins Substances in the serum that can neutralize the activity of toxins, enabling passive immunization von Behring and Kitasato (1890)[5] Bacteriolysins Serum substances that work with the complement proteins to induce bacterial lysis Richard Pfeiffer (1895)[6] Bacterial agglutinins & precipitins Serum substances that agglutinate bacteria and precipitate bacterial toxins von Gruber and Durham (1896),[7] Kraus (1897)[8] Hemolysins Serum substances that work with complement to lyse red blood cells Belfanti and Carbone (1898)[9] Jules Bordet (1899)[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] Antibody formation (1900), antigen-antibody binding hypothesis (1938), produced by B cells (1948), structure (1972), immunoglobulin genes (1976) Founder: P Ehrlich[3]

Origin: Terms „Antitoxin“ & „Antikörper“
1890 Behring never uses the noun „Antitoxin“ his his two 1890 publications (Only used adj. „antitoxisch“ in a footnote in the 1890 paper with Kitasato) 1891 Tizzioni 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, 1891. 1891 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, 1897 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 The first use of the term "antibody" occurred in a text by Paul Ehrlich. The term Antikörper (the German word for antibody) appears in the conclusion of his article "Experimental Studies on Immunity", published in October 1891, which states that "if two substances give rise to two different antikörper, then they themselves must be different".[67] However, the term was not accepted immediately and several other terms for antibody were proposed; these included Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.[67] The word antibody has formal analogy to the word antitoxin and a similar concept to Immunkörper.[67]

Coining Term „Antikörper“ – Ehrlich
1891 1897 Deutsche Med. Wochenzeitschr. (1891) Nr. 32, p 976

Term Antikörper – Lindemann 1984
JEAN LINDENMANN (1984). Origin of the Terms 'Antibody' and 'Antigen‘ Scand. J. Immunol., 19, Ehrlich’s 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 body—the anti- abrin—which completely paralyses the effects of abrin—probably 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 !!!????

START OF IMMUNOLOGY Serology

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

Widal Test (Typhoid fever)
1896 Herbert E. Durham and Max von Gruber introduce the term Agglutinin for any substance that caused agglutination of cells → Gruber-Durham reaction 1896 Fernand Widal uses the Gruber-Durham reaction to develop a test for typhoid fever. Obseravtion: Serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. Widal test was the first example of serum diagnosis. 1853 – 1927 Ausria 1862–1929 France

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. Principle of complement fixation test The complement is able to bind to the antigen antibody complex : this phenomenon is called the reaction of complement fixation. Once the complement is bound, it attacks the antigen : the bacterium is destroyed by bacteriolysis. Later, Bordet discovered that when red blood cells are added to the blood serum (where bacteria can be found), those are also destroyed if the complement is in the serum : this is called hemolysis. However, if red blood cells (= erythrocytes) are added after the bacteriolysis, they remain intact since the free complement is already bound to the antigen antibody complexes. This is how a specific bacteria can be detected in a sample of blood serum. This discovery allowed him to develop blood tests (serodiagnosis technique) that indicate whether a person has been in contact with any infectious agent. If the red blood cells remain intact when 1) the bacterium's specific antibody, 2) the complement's specific antibody and 3) the red blood cells' specific antibody are added to the serum, that means that the bacterium is present. This technique, called "Bordet-Wassermann's reaction", allows to detect the presence of some bacteria that cause diseases like typhoid, tuberculosis, syphilis... 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:

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

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

August Paul von Wassermann
Wassermann Test (Syphillis) 1904 Fritz R. Schaudinn and Paul E. Hoffmann isolate the causative organism of syphilis. 1905 Wassermann 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 A complement-fixation test as a diagnostic of syphilis using blood serum or cerebrospinal fluid. It is a modification of the complement-fixation reaction by Jules Jean Baptiste Vincent Bordet ( ) and Octave Gengou ( ) in 1901. The "antigen" used is an alcoholic muscle extract, usually from a bull's heart. The active substance is e lipid called cardiolipin, which has nothing to do with syphilis. However, this antigen reacts with certain antibodies, so-called reagines, that commonly occur in the blood of syphilis patients. Because the antibodies are not specific for syphilis, they may occasionally occur in other diseases, like malaria and lepra. The results are designated as 1, 2, 3, and 4 plus, the intensity of the reaction usually corresponding to the severity of the infection. The disease may still exist with a negative reaction. Several negative Wassermann reactions a few years after treatment indicate the absence of syphilis. Wassermann described his test one year after Fritz Richard Schaudinn ( ) and Paul Erich Hoffmann's ( ) discovered the causative organism of syphilis. Many modifications have since been made of this test, such as the Kahn, Kolmer, etc, but the general principled applied by Wassermann continue to guide the procedures. The term Wasserman's reaction is therefore applied to almost any serological test for syphilis. Bordet and Gengou will be entered later. August Paul von Wassermann ( ) Bamberg, Germany A Wassermann, A. Neisser and C. Bruck. Eine serodiagnostische Reaktion bei Syphilis. Deutsche medicinische Wochenschrift, Berlin, 1906, 32:

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 the bacterium's specific antibody, complement and the red blood cells' specific antibody

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

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) Königliches Institut für experimentelle Therapie, Frankfurt 1899 Königliches Institut für experimentelle Therapie + Georg-Speyer-Haus 1922. Frankfurt Ab 1947 Paul-Ehrlich-Institut für Exp. Therapie Paul-Ehrlich-Institut im Jahre 1990 als Bundesamt für Sera und Impfstoffe in Langen bei Frankfurt/Main Das Paul-Ehrlich-Institut für Serumprüfung und Serumforschung Jahre1896 in Steglitz bei Berlin Georg-Speyer-Haus, 1906, Frankfurt

IMMUNOLOGY: Own Discipline
JOURNALS Zeitschrift für Immunitätsforschung (1908) J. of Immunology (1913) Eur. J. Immunology (1970) PROFESSIONAL SOCIETIES American Associaten of Immunologist (1913) Deutsche Gesellschaft für Immunologie = DGfI (1953)

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)

IMMUNOCHEMISTRY The Antibody Problem ( )

History of Immunology Part 3: START OF IMMUNOLOGY Hans-Martin Jäck

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History of Immunology Part 3: START OF IMMUNOLOGY Hans-Martin Jäck

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