The brain and the immune system, or the “supersystems”, a term coined by Tada (1997), are the two major adaptive systems of the body.
Although the immune system has been often regarded as autonomous, the evidence accumulated over several decades indicates that the central nervous system (CNS) receives messages from the immune system and vice versa messages from the brain regulate immune functions.
Two pathways link the brain and the immune system: the neuroendocrine humoral outflow, via the pituitary, and the autonomic nervous system, via direct neuronal influences. Thus, circulating hormones or locally released neurotransmitters and neuropeptides regulate major immune functions, such as antigen presentation, secretion of antibodies and cytokines, selection of T helper (Th)1, Th2 and Th17 responses, lymphocyte activity, proliferation and traffic. On the other hand, messages from the immune system such as proinflammatory cytokines influence CNS components of the stress system and alter neurotransmitter networks’ activity, which, in turn affects thermoregulation, sleep, physical activity, appetite, mood, cognitive behavior, sex drive etc. Thus, during an immune response the brain and the immune system “talk to each other” and this process is essential for maintaining the stability and integrity of the internal milieu or homeostasis. Neurosciences and immunology developed independently for many years, and thus, the question of how the brain communicates with the immune system remained enigmatic until fairly recently. This overview is an attempt to provide some historical perspectives on major figures, events, concepts and ideas that eventually brought together the fields of neuroscience, endocrinology and immunology. The reader will find more detailed information on some of these issues in the history section on this site.
Claudius Galen was a second century Greek physician, philosopher and writer who is often considered the most significant contributor to ancient medicine following Hippocrates. He was born in Pergamum (modern-day Bergama, Turkey) in 129 A.D., an ancient center of civilization, where he received extensive training in philosophy and medicine, and later trained in Smyrna (modern Izmir), Corinth and Alexandria. From 162 A.D., he lived in Rome where he gained reputation as an experienced physician, served under five Roman emperors, and spent the rest of his time writing and experimenting. He was a teacher, philosopher, pharmacist and leading scientist of his day. Galen was the originator of the experimental method in medical investigation. He proved that urine was formed in the kidney; identified seven of the twelve cranial nerves, discovered the valves of the heart, recognized the contagiousness of tuberculosis, and the possible spread of rabies via dogs. Galen''s most important discovery was that arteries carry blood not air. He also argued that the mind was in the brain, not in the heart, as Aristotle had claimed. Galen''s most lasting technique is the palpation of the pulse – still done by doctors to this day.
Galen mentioned that ''the best doctor is also a philosopher''. The profit motive, said Galen, was incompatible with a serious devotion to the art. Galen developed further on Hippocrates'' idea about ''Humoralism'' or the doctrine of the Four Temperaments as a medical theory, which retained its popularity for centuries largely though the influence of his writings. The imbalance of four humours (blood, phlegm, yellow bile and black bile), or ''dyscrasia'' was thought to be the direct cause of all diseases. In his work De temperamentis, Galen developed probably the first typology of four temperaments, and searched for physiologic reasons for different behaviors in humans – throughout history they had different names, but sanguine, choleric, melancholic and phlegmatic eventually became the most popular. While the term ''temperament'', came to refer just to psychological dispositions, Galen used it to refer to bodily dispositions, which determined a person''s susceptibility to particular diseases as well as behavioral and emotional inclinations. Thus, it was probably Galen who first integrated the ''immune system'' or ''cancer'' with the person''s emotional and physiologic processes – circa 200 A.D. he wrote that melancholic women were more prone to develop ''swellings'' of the breasts than were sanguine women. Galen''s medical theory of the Four Temperaments was widely accepted up to 1858, until it was ''displaced'' by Rudolf Virchow''s newly published premise of cellular pathology.
Late Middle Ages - Early Modern Times
Galen''s influence dominated medicine right until the 16th century. He taught that nerves were hollow tubes distributing ''animal spirits'' in the body, thereby fostering concerted action, or ''sympathy'' of the organs. In 1552, Bartolomeo Eustachius first depicted the sympathetic nerves and the adrenal glands. Only many years later (see below) will it become evident that the adrenal gland secreted the two major ''stress'' hormones – cortisol and epinephrine (adrenaline). Winslow reintroduced the sympathetic nervous system in 1732 to describe the chains of ganglia and nerves connected to the thoracic and lumbar spinal cord. The functions of these structures remained unknown until the 19th century, when Bernard and others first reported the effects of sympathetic nerve stimulation and epinephrine was isolated .
The 19th Century
Caleb Parry – stress and hyperthyroidism
The relationship between stressful life events and the onset of Grave''s disease (the most common cause of hyperthyroidism, known as Basedow''s disease in the European Continent) was initially documented by Caleb Parry in 1825, Robert Graves in 1935, and later by Basedow and others. In 1786, Caleb Hillier Parry treated a 37-year-old woman a few months after childbirth that had palpitations, tachycardia, goiter, and prominent eyes. Parry''s second case was precipitated by acute stress. In August 1803 Caleb Parry''s patient, 21-year-old Elizabeth S., was pushed down the stairs in an out-of control wheelchair. From that time, it was said, she was ''subject to palpitations of the heart and nervous troubles''. Two weeks later, she developed swelling of the thyroid gland which was variable; at times almost disappearing. The right lobe was larger than the left, was quite painless and accompanied by marked pulsation of the carotid arteries and a ''small, hard, regular pulse of 96'' . Parry''s report of eight patients, however was not published until 1825, when Parry''s son, also a physician published his father''s case records, 3 years after Parry''s death, but still ten years ahead of Graves.
Caleb Parry had a lifelong friendship with Edward Jenner. When Jenner in 1798 published An Enquiry into the Causes and Effects of the Variolae Vaccinae, he dedicated his historic work to Parry. In 1778 Parry commenced general practice in Bath, where he spent the rest of his life. As a physician he kept meticulous records of his patients, and excelled as physiologist and skilled experimentor. Parry wrote that it is more important to know what sort of patient has the disease than what kind of disease the patient has. He was the first to suggest the correct mechanism of angina, although his explanation was ignored for more than 100 years. He expounded the concept that ischemic heart disease resulted from energy demands of the myocardium, which the vascular system was unable to supply (a collaborative ''study'' with his friend Edward Jenner). Parry was very willing to collaborate with his colleagues. His willingness for collaboration is further illustrated by Parry''s involvement in the Fleece Medical Society, which he and Edward Jenner formed so that its members could meet regularly to present papers to each other. This club was one of the earliest English provincial medical societies.
Claude Bernard – the birth of physiology
Laboratory sciences, and in particular, experimental physiology, had made little progress before the middle of the 19th century. Up to that time, at best, treatment of the sick was based upon the practitioner''s experience. At worst, it was based upon his theories and philosophic imaging. Among the great contributions to the advance of clinical and experimental medicine of the 19th century, was the brilliant information that came welling out of the laboratories of Claude Bernard, a French physiologist, considered by many as the father of physiology and experimental medicine. Alexis Carel said of Bernard: "Before him, medicine was purely empirical. He is responsible for the introduction of the scientific method in the art of healing".
Bernard started his career as a pharmacist''s assistant in a suburb of Lyon. He had the occasion to observe the rude empiricism of the pharmacotherapy and healing practices of his day, of which opium was probably the most important. Shortly he pursued a career as a play writer – he wrote a five act drama and setting out in 1834 for Paris. Here, however he followed the advice of a famous literary critic to study medicine. Thus, France lost a potential play writer, but she gained her greatest 19th century physiologist. Bernard achieved no great success as a student as he eventually passed out of the medical school 26th out of 29; some of his instructors even regarded him as lazy.
In 1839, he was appointed interne to professor Magendie, at the Collège de France, the most famous French physiologist of the time, and later became his préparateur, or lecture assistant. Later, in 1855 Bernard succeeded Magendie as professor at the Collège de France. Towards the end of the 1840s, Bernard began a series of remarkable discoveries. As his pupil Paul Bert said ''No one ever made discoveries more simply, more naïve. He discovered as others breathed.'' He began by showing that the pancreas, secreting its juice into the duodenum, was capable of digesting foodstuffs, that both pancreatic juice and bile were necessary for the absorption of fat from the gut and that the main processes of digestion occur in the small intestine and not, as was previously thought, in the stomach. He was the first scientist to appreciate the importance of internal glandular secretions, and to understand interrelations of organic function. In 1844 Bernard began his work on the physiological effects of poisons, particularly of curare and carbon monoxide. He found that the former, an arrow poison used by South American Indians rendered the motor nerves inactive, while the sensory and central nervous system remained intact.
This was followed by the discovery of the glycogenic function of the liver, and was perhaps his most noteworthy achievement. In 1856 he discovered glycogen, whose role is to build up a reserve of carbohydrate, which can be broken down to sugars as required. The digestive system, he found, is not just catabolic, but anabolic, producing complex molecules (such as glycogen) from simple ones (such as sugars). Another of Bernard''s brilliant discoveries was that of existence and function of vasomotor nerves. He established that two sets of nerves affected blood vessels. One set constricted the vessels; the other set dilated them, and thus able to formulate the statement: "the sympathetic nerve is the constrictor of the blood vessels; the chorda tympani is their dilator"; in hot weather blood vessels of the skin expand, releasing surplus heat, contracting during cold to conserve heat. The body is therefore able to maintain a constant environment separate from outside influences. In 1859, his lectures at the Collège de France were published and one of these contained perhaps his most fertile idea. He saw the animal''s external environment (''le milieu extérieur'') as constantly changing: but the composition of the fluids within the body (''le milieu intérieur'') was kept remarkably constant, so protecting the cells of the body from the vicissitudes of the external environment.
In 1865 he published his masterpiece – the phenomenal book An Introduction to the Study of Experimental Medicine. The only sure way forward in experimental medicine was to design experiments in which every variable was controlled. Furthermore, every experiment should be based on a hypothesis. Bernard''s younger contemporary and good personal friend, Louis Pasteur, commented thus on the Introduction: "Nothing so complete, nothing so profound and so luminous has ever been written on the true principles of the difficult art of experimentation. This book will exert an immense influence on medical science, its teaching, its progress…." Bernard and Pasteur not only were good friends, but at times collaborated. Bernard was chairman of the committee of Academie des Sciences, and in 1860 presented the prize for experimental physiology to Pasteur for his work on fermentation.
In the Introduction Bernard brought the concept of the constancy of the internal environment (milieu intérieur) and wrote that "La fixité du milieu intérieur est la condition d''une vie libre et indépendante." ("The constancy of the internal environment is the condition required for a free and independent life"). He discussed that the body possesses control systems, which adjust their interactions and exchanges with their surroundings in such a way that the physical state and chemical composition of the internal environment remains essentially constant. This new concept entailed the overcoming of the concept of physiology as ''anatomy in motion'' and a radical shift in perspective [as Denis Noble recently stated ''Bernard can be regarded as the first systems biologist – higher-level systems biology is classical physiology by another name. The new vogue for systems biology today is an important development, since it is time to complement reductionist molecular biology by integrative approaches'' . The concept of the constancy of the internal environment was further developed 50-60 years later by the American physiologist Walter Cannon, who introduced and coined the term homeostasis (see below).
Some terms and concepts introduced at the end of 19th century
Microbiology/Immunology: In 1876 Heinrich Hermann Robert Koch provided first demonstration that microbes can cause disease – he discovered that bovine anthrax is caused by a specific bacillus, and thus developed an experimental procedure to prove the microbic etiology of infectious diseases. In 1877 Paul Ehrlich described mast cells. In 1878 Louis Pasteur developed the germ theory of disease, and the next year introduced attenuated chicken cholera vaccine. Between 1880-1881 he developed the concept that bacterial virulence could be attenuated by culture in vitro and used as vaccines. In 1882 and 1883, respectively Koch discovered tuberculosis bacillus and the vibrio cholera. In 1885, Pasteur developed the concept of a "therapeutic vaccination" and introduced the live "attenuated" vaccine for rabies. In 1888, Pierre Roux and Alexandre Yersin identified bacterial toxins (diphtheria bacillus).
Between 1883 and 1905 Ilya Ilyich Mechnikov (also known as Elie Metchnikoff) developed the cellular theory of immunity via phagocytosis by macrophages and microphages (polymorhonuclear leukocytes). From Edward Jenner''s discovery of smallpox vaccination in 1798 to Louis Pasteur''s discovery of immunization with attenuated pathogens in 1880, nothing was known of the factors that might mediate the resulting protection. The idea of cells being directly involved in the defense of the body was first suggested by Mechnikov in 1884. He was well aware that single cell organisms took in food by phagocytosis and released debris by exocytosis. He observed that starfish and other invertebrates were able to mobilize phagocytic cells in response to insult. He first proposed that the phagocyte is crucial to an understanding of the general pathology of inflammation and then extended his theory to assign to this ubiquitous cell a central role in both natural and acquired immunity in vertebrates . Interestingly, in 1888 Pasteur helped Mechnikov to get an appointment at the Pasteur Institute, where he remained for the rest of his life.
In 1890 Robert Koch published the famous Koch''s postulates, the four criteria designed to establish a causal relationship between a causative microbe and a disease. The postulates were formulated by Koch and Friedrich Loeffler in 1884, and refined by Koch in 1890. Koch applied the postulates to establish the etiology of anthrax and tuberculosis, but they have been generalized to other diseases. In 1891, Koch was the first to demonstrate a delayed type hypersensitivity reaction, but it wasn''t until the 1940s that Landsteiner and Chase proved that the reaction was mediated by the cellular and not the humoral arm of the immune system (see below). Koch attempted to use his killed tuberculin preparation as a prophylactic and therapeutic vaccine. Unfortunately, the antigen did not confer protection to naive patients, and when injected intravenously in infected patients, caused reactivation of the disease and in some cases death. Nevertheless, when the antigen was injected intradermally, the delayed inflammatory response (tuberculin reaction) could indicate whether or not an asymptomatic person had been exposed to Mycobacterium tuberculosis. The "Koch phenomenon" later became known as "delayed type hypersensitivity". Understanding hypersensitivity would be the key to recognition of a cellular immune system. In 1905, Koch was awarded the Nobel Prize in Physiology and Medicine for his tuberculosis findings.
In 1890 Emil von Behring and Shibasaburo Kitasato, of Koch''s Institute in Berlin reported that mice immunized with diphtheria and tetanus toxins develop something in their blood that seemed to mediate antitoxic activity, because the protection could be transferred passively to normal animals with immune serum. Shortly thereafter, Paul Ehrlich, soon to become associated with Koch, showed similar humoral responses to the plant toxins ricin and abrin, and then Koch''s student Richard Pfeiffer showed that humoral substances could lyse typhoid and cholera organisms. All this initiating the development of the humoral theory of immunity – all immunity is mediated by humoral factors, or anti-bodies, specific for the disease-causing toxin or pathogenic organism. Some of the findings, mentioned above provoked one of the most original and ''dramatic'' debate in immunology about the role of cell mediated and humoral immunity that began in the 19th century between the French "cellularists," led by Mechnikov and the German "humoralists", led by Ehrlich .
In 1892 Dmitri Iwanowski described the first "filterable" infectious agent - tobacco mosaic virus (TMV) - smaller than any known bacteria. Iwanowski was the first person to discriminate between viruses and other infectious agents, although he was not fully aware of the significance of this finding. In 1893 William Coley used live bacteria and bacterial lysates to treat tumors, the "Coley''s Toxins". In 1895, Jules Bordet discovered complement and the antibody activity in bacteriolysis. In 1898 Martinus Willem Beijerinck, a Dutch microbiologist and botanist extended Iwanowski''s work with TMV and formed the first clear concept of the virus "contagium vivum fluidum" - soluble living germ. Beijerinck confirmed and extended Iwanowski''s work and was the person who developed the concept of the virus as a distinct entity. He named that new pathogen virus and is considered the founder of virology. Beijerinck maintained that viruses were liquid in nature, a theory discredited in the 1930s by Wendell Meredith Stanley, an American biochemist, virologist and Nobel Prize laureate, who proved they were particulate. In 1900, Paul Ehrlich developed the antibody formation theory.
Neuroscience/Physiology: In 1875, Richard Caton was first to record electrical activity from the brain. In 1877, Jean-Martin Charcot published Lectures on the Diseases of the Nervous System. In 1883, Emil Kraepelin coined the terms neuroses and psychoses. In 1885, Paul Ehrlich noted that intravenous dye does not stain brain tissue. In 1889, Santiago Ramon y Cajal argued that nerve cells are independent elements. In 1889, William His coined the term dendrite, and two years later Wilhelm von Waldeyer coined the term neuron. In 1895, William His first used the term hypothalamus. In 1896, Rudolph Albert von Kolliker coined the term axon. In 1897, Ivan Petrovich Pavlov published work on physiology of digestion, while Sir Charles Scott Sherrington coined the term synapse. In 1900, M. Lewandowsky coined the term blood-brain barrier, while Sigmund Freud published The Interpretation of Dreams.
Pasteur and cold water stress
In 1878, Louis Pasteur observed that chickens normally resistant to anthrax became susceptible following immersion in cold water. He concluded that lower temperature of the host was sufficient to increase growth or virulence of the bacteria. But it seemed more likely that Pasteur also discovered the effect of ''cold stress'' on the humoral and/or cellular branches of the host immune system . As noted by H Selye, many years later, in 1955 ''Pasteur failed to recognize the importance of the ''terrain'', because he was too preoccupied with the pathogen (microorganism) itself. In fact, Pasteur and Bernard, two of the giants of nineteenth-century biology, argued all their lives whether the most important factor in disease was the "terrain" – the human body" – or the germ. At the end of his life, however, Pasteur admitted that Bernard had been right, declaring, "It is the terrain", and he allegedly said, ''Le microbe n''est rien, le terrain est tout''. Regardless of the way that Pasteur''s experiment is interpreted, these data with a cold-stressed chicken inoculated with B. anthracis provided the first documented evidence, conducted by the father of the germ theory of disease, that environment of the host affects susceptibility to disease . In 1890 R Charrin extended these observations by reporting that forced physical exercise (a physical stressor) was associated with an increase in the mortality of rats infected with B. anthracis.
Receptor theory and magic bullets
The two founders of the receptor concept, the Cambridge physiologist John Newport Langley and the Berlin immunologist Paul Ehrlich mentioned the idea first in 1878, and both returned to their former approaches only several years later – at the turn of the 19th to the 20th century [6,7]. In 1878 Langley wrote: "there is some substance… in the nerve endings or gland cells with which atropine and pilocarpine are capable of forming compounds ... according to some law of which their relative mass and chemical affinity for the substances are factors". The same year Ehrlich speculated that there must be a specific chemical character of the cell that is responsible for the selective binding of dyes. Later he turned to immunological problems, especially to the interactions between bacterial toxins and antitoxins or antibodies, which are formed in the blood. In 1897 Ehrlich proposed the ''side-chain theory'' for the binding of toxins to cells. Ehrlich postulated the existence of many side chains, and, in 1900, he introduced the term ''receptor'' as a replacement for the term ''side chain [6,7].
In 1905, Langley examined the effect of nicotine on muscles in which nerves had been cut and allowed to degenerate. The animal model that was used in these trials was the anaesthetized fowl, in which injection of nicotine produces a characteristic tonic contraction of certain muscles of the leg. This contraction could also be induced in the denervated leg muscle, which indicated that nicotine might act directly on the muscle cells. By injecting curare into the animal, the contraction could be abolished. Langley realized that this was a parallel case to the antagonism between pilocarpine and atropine that he had described 27 years earlier. From this observation, Langley drew the critical conclusion that the poisons did not act directly on the contractile substance, but rather on some accessory substance of the muscle cell. Thus, Langley proposed that nicotine and curare act on the ''receptive substance'' of muscle cells. Ehrlich accepted Langley''s point that receptors existed not only for toxins but also for drugs. In 1907, on the basis of his experiments with dyes on trypanosomes, Ehrlich assumed the existence of ''chemoreceptors''. It was also Ehrlich who envisioned the creation of ''magic bullets'', compounds that would specifically target disease-causing microorganisms. These magic bullets would seek out these microorganisms and destroy them, and having no harmful effects on the bodies of patients. Ehrlich''s first magic bullet was Salvarsan, developed in 1909, together with his student Sahachiro Hata, which provided the only cure for syphilis until it was later superseded and replaced by penicillin .
Autonomic nervous system and the discovery of adrenaline (epinephrine)
Between 1880 and 1920, J. N. Langley, in conjunction with H. K. Anderson, defined the major functional features of the sympathetic and parasympathetic systems, showing how different effector tissues were affected by segmental ventral root stimulation. In 1898 Langley coined the term autonomic nervous system (ANS) to denote which section of the nervous system was responsible for involuntary, unconscious functions of internal organs, in contrast with the portion responsible for voluntary, conscious functions of skeletal muscle. Later, in 1905 Langley also introduced the term parasympathetic nervous system to denote the cranial and sacral portions of the ANS, in contrast with the sympathetic nervous systems, which originates from thoracolumbar ganglia . In 1899, Tonkoff showed that nerves, independent of blood vessels enter lymph nodes, thus providing the first evidence that lymphoid organs are innervated.
In 1895, George Oliver, a rural medical practitioner, paid a visit to Professor Edward Schaefer at the Physiological Laboratory, University College London. Oliver''s own experiments had yielded a pressor effect of adrenal extracts on various animals, and Oliver wanted to verify his findings. After Schaefer agreed to the collaboration, the two men conducted a series of experiments to examine the effects of graded intravenous injection of extracts of adrenal medulla on the blood pressure (recorded kymographically on a smoked drum) of the anaesthetized dog. The effects were dramatic: ''Invariably there is a rise of blood pressure…The rise is usually very rapid; sometimes the pen of the kymograph rises almost vertically...indeed, even although we have employed an exceptionally long manometer, in more than one instance the mercury has been entirely driven out from the open end of the tube''. Thus, Oliver and Schaefer discovered the potent cardiovascular effects of adrenal medulla''s extracts, and concluded that ''the suprarenal capsules are to be regarded although ductless, as strictly secreting glands. The material which they form…in the medulla of the gland produces striking physiological effects upon muscle tissue generally, especially upon that of the heart and arteries'' [8,9].
In 1899, Lewandowsky first reported that the effects of stimulating sympathetic nerves resembled influences produced by injecting animals with extracts f the adrenal gland. There is a controversy over who first isolated the active component adrenaline (epinephrine) – Napoleon Cybulski, in 1895, John Jacob Abel of Johns Hopkins University, together with A. C. Crawford, in 1897, or Jokichi Takamine and Thomas Aldrich in 1900 – Aldrich found the correct formula (C9H13NO3), and independently, they isolated the responsible component in crystalline form. Abel also named the hormone "epinephrin" (no "e", now called epinephrine in the United States), while Takamine arranged for Parke, Davis & Company to market the pure crystalline substance as "adrenaline." Thus, about 110 years ago, epinephrine (adrenaline) was the first hormone to be isolated from tissue, and the first intercellular messenger to be chemically identified and synthesized.
In 1898, after Jules Bordet''s demonstration of the phenomenon of immune hemolysis, Paul Ehrlich assigned his assistant Julius Morgenroth to extend these studies . They described the hemolytic antibodies that result when animals are injected with the blood of unrelated species. They followed this by attempting to immunize animals with the blood of their own species, and even with the animal''s own blood. Whereas they obtained iso-antibodies in many instances, they failed in every attempt to elicit the formation of auto-antibodies. This led Ehrlich to postulate, in 1901 the existence of what he termed horror autotoxicus, the unwillingness of the organism to endanger itself by the formation of toxic autoantibodies, or the "extremely dysteleological" situation that might result from the formation of "autotoxins", noxious for the individual himself.
When it was called to Ehrlich''s attention that Metalnikoff and others, in 1900 had demonstrated in animals the formation of antibodies against their own sperm, Ehrlich did not recant. In 1904, Julius Donath, and Karl Landsteiner reported the first known autoimmune disease in humans, paroxysmal cold hemoglobinuria. They showed that an antibody in this disease reacted in the cold with the patient''s own erythrocytes, a phenomenon then frequently seen in syphilis patients. By definition, the Donath–Landsteiner antibody was an autoantibody. Over the ensuing years, other more-or-less convincing demonstrations suggested that autoimmune reactions might be responsible for sympathetic ophthalmia, for ocular inflammation due to lens antigens, for some hemolytic anemias and for certain encephalitides. However, for many years Paul Ehrlich''s theory of horror autotoxicus dominated 20th Century thinking. Ehrlich''s absolute dictum that autoimmune disease cannot occur would influence over decades many investigators to disregard data that argued otherwise. This prevented full acceptance of a growing reality, and the full acceptance of autoimmunity concept had to await another 50–60 years. This may also support the statement by Ludwik Fleck (still valid nowadays) that "the acceptance of a fact in science may depend less upon its truth than upon the willingness of the leaders in the field to acknowledge it!" (cf.).
The 20th Century
Early 20th century – the birth of modern immunology and neurosciences
Near the end of the 19th century and the beginning of the 20th century, Ilya Mechnikov and Paul Ehrlich, respectively, developed the concepts of cellular and humoral immunity. Even though Mechnikov and Ehrlich shared the 1908 Nobel Prize in Physiology and Medicine, during this period the humoralists had all but won the day, and the cellular theory for the immune system had to wait until the 1940s for revival of interest and ultimate discovery. In 1903, Ivan Pavlov coined the term conditioned reflex, while in 1906, Sir Charles Scott Sherrington published The Integrative Action of the Nervous system that described the synapse, and his theory that the nervous system acts as the coordinator of various parts of the body, and that the reflexes are the simplest expressions of the interactive action of the nervous system. At approximately the same time, Thomas Elliott, and later Sir Henry Dale and Otto Loewi introduced the concept of chemical neurotransmission, basic principles in neuroscience.
In 1902 Charles Richet and Paul Portier discovered anaphylaxis. About 1900, while cruising in tropical waters, they studied the poison of the tropical jellyfish. Richet and Portier found that injection of a glycerol solution of the poison produced the symptoms of poisoning by the jellyfish. On their return to France they studied the toxins of local jellyfish. They determined the minimum dose that was fatal for dogs several days after its injection. Smaller doses than this produced only transient effects. But if a dog that had been injected with a small dose received a similar small dose after an interval of several weeks, a violent reaction killed the dog. By 1902 Richet had studied this phenomenon in different animals. Reactions produced by the injection of antitoxins or minute doses of toxins had already been called prophylactic, or protective. Richet realized that in this new phenomenon the first dose sensitized the animal, so that the second injection produced a violent reaction. The first dose was the opposite of prophylactic, and he therefore called the phenomenon anaphylaxis. He also established that, to produce the violent reaction, there must be an interval of several weeks between the injections.
The genesis of the concept of chemical synaptic transmission has been attributed to John Newport Langley, but actually his research student, Thomas Renton Elliott was able to formulate first this concept [8,9]. In 1904, Elliott produced a number of additional and striking examples of the correspondence between the effects of adrenaline and of sympathetic nerve stimulation. As was then the custom, Elliott published separately from Langley, and in a famously prophetic Physiological Society communication from May 21 1904 (On the action of adrenalin) he hypothesized ''….sympathetic axons cannot excite the peripheral tissue except in the presence, and perhaps through the agency, of the adrenalin or perhaps its immediate precursor ….Adrenalin might then be the chemical stimulant liberated on each occasion when the impulse arrives at the periphery''. Elliott''s proposal was extraordinarily prescient. Elliott''s colleagues were skeptical, including his mentor Professor Langley, known as an individual who disapproved of speculative theories, and epinephrine did not perfectly reproduce the effects of sympathetic nerve stimulation (see below the discovery in 1946 of norepinephrine as the principal neurotransmitter of the sympathetic nervous system). In this talk, however young Elliott dared to propose a hypothesis in which nerves signal by releasing a specified chemical substance, and the target cells possess sites which are specialized to recognize that particular signal – i.e., the two fundamental elements in chemical neurotransmission. It was only later that Dale realized that Elliott had been correct in principle and erroneous only with regard to the actual identification of the mediator. Elliott became a close friend of Langley''s immediately previous graduate student Henry Dale, who always gave Elliott much credit for originating the ideas whose pursuit ultimately lead Dale to the 1936 Nobel Prize for neurotransmission.
In 1904, Loeper and Crouzon were the first to describe a pronounced leukocytosis (2- to 3 fold increase) after subcutaneous injection of the adrenomedullary hormone epinephrine (adrenaline) in humans. During experiments in 1907, a by-product in the synthesis of adrenaline was identified. This substance, which became commercially available as "Arterenol" in 1908, was in fact norepinephrine (noradrenaline), which would formally be discovered and isolated from tissue 40 years later (see below). In 1914 W Frey reported that the response upon adrenaline injection was biphasic in both animals and humans: a quick (<30 min) phase characterized by an increase in lymphocytes numbers, followed by a phase with an increase in granulocyte numbers .
In 1906, Clemens Peter Freiherr von Pirquet, an Austrian scientist and pediatrician coined the term allergy. Pirquet worked as pediatrician at the Viennese St.-Anna-Kinderspital (children''s hospital). When treating children contracted with diphtheria he was surprised by the side effects of vaccinations. That''s why his research and the allergy definition were at the beginning focused on the so called serum disease. He noticed that patients who had previously received injections of horse serum or smallpox vaccine had quicker, more severe reactions to a second injection. He, along with Bela Schick, coined the word allergy (from the Greek allos meaning ''other'', ''changed'' or ''altered state'' and ergon meaning ''reaction'') to describe this hypersensitivity reaction. Pirquet was aware that not only these vaccination reactions were allergic reactions but that there was an even more diverse range of allergies. Soon after, the observation with smallpox led Pirquet to realize that tuberculin, which Robert Koch isolated from the bacteria that caused tuberculosis in 1890, might lead to a similar type of reaction. Charles Mantoux expanded upon Pirquet''s ideas and the Mantoux test, in which tuberculin is injected under the skin, became a diagnostic test for tuberculosis in 1907.
With the term allergy Pirquet wanted to describe in general a change in reactivity of the organism, namely in time, quality and quantity. In contrast to the widely accepted use of the word "allergy" today, where it is restricted to specific immunologic hypersensitivity reactions against harmless foreign antigens, allergy in Pirquet''s sense comprised as general term likewise increases and decreases of the reactivity and so both "hyper-" and "hyposensitivity reactions". In the context with the expansion of allergy to the human predisposition Pirquet emphasized, that the change of reactivity does not only depend on exogenous substances (so called allergens), but also on endogenous factors of the organism itself . The orientation towards the organism and his reactivity is the central idea, which can be found in the complete works of Pirquet from 1903 to 1929. It is the true essence of his theory of allergy. At that point in time, however, von Pirquet had no means of scientifically proving that these immunological changes actually occurred in the body. It was not until the mid-1920''s that a second significant event occurred. Researchers found that, by injecting a minute quantity of purified allergen under the skin, certain individuals would develop a clear skin response; a "wheal," with or without itching and redness, could be provoked. This positive skin test for allergies would show itself most prominently in patients with hay fever, asthma, chronic rhinitis, hives and eczema. The "prick test" became a method of demonstrating the involvement of the immune system in allergic reactions. It was not until the 1960s, when an important discovery occurred which provided long-awaited scientific support for the classical allergy theory and removed any doubts about the relationship of the immune system with allergies. This breakthrough came about with the scientific discovery of immunoglobulin E (IgE) by a Japanese couple named Ishizaka.
In 1908, Karl Landsteiner, an Austrian physician proved that poliomyelitis was caused by a virus. One day in 1908 the body of a young polio victim was brought in for autopsy. Landsteiner took a portion of the boy''s spinal column and injected it into the spinal canal of several species of experimental animals, including rabbits, guinea-pigs, mice and monkeys. Only the monkeys contracted the disease. Landsteiner reported the results of the experiment, conducted with Erwin Popper, an assistant at the Wilhelmina Hospital in Vienna. Landsteiner and Popper were the first to prove that viruses could infect humans as well as animals. In 1900, Landsteiner also wrote a paper in which he described the agglutination of blood that occurs when one person''s blood is brought into contact with that of another. In 1901, Landsteiner demonstrated that the blood serum of some people could clump the blood of others. From his observations he devised the idea of mutually incompatible blood groups. He placed blood types into three groups: A, B, and C (later referred to as O). Two of his colleagues subsequently added a fourth group, AB. In 1907 the first successful transfusions were achieved by Dr. Reuben Ottenberg of Mt. Sinai Hospital, New York, guided by Landsteiner''s work. Landsteiner''s accomplishment saved many lives on the battlefields of World War I. In 1909 Landsteiner classified the bloods of human beings into the now well-known A, B, AB, and O groups, for which he was given the Nobel Prize for Physiology and Medicine in 1930.
In 1910 Barger and Dale, working on an ergot extract, discovered that a substance in it, later called histamine, had a direct stimulant effect on plain (smooth) muscle, especially that of the uterus and bronchioles. (Histamine had previously been synthesized, but it was not known to occur naturally, in the animal body or elsewhere.) They also showed that it caused a general fall in blood pressure and that its injection produced most of the features of anaphylactic shock. In 1911 they were the first to show that it could be present in animal tissues, as they had isolated it from the wall of the intestine.
In 1913 Dale noticed the extreme sensitivity of the isolated uterus of a particular guinea pig when treated with a normally quite innocuous dose of horse serum. He later discovered that this particular guinea pig had already been used for the assay of diphtheria antitoxin and was therefore already sensitized to horse serum. By following up this chance observation Dale was able to produce (in guinea pig plain muscle) all the essential features of anaphylaxis, thus greatly advancing knowledge of the cause of this condition. Later, in 1922 Dale and Charles Kellaway showed that anaphylactic phenomena are probably due to the location of the antibody in the cell surface. Ten years later other workers showed that in anaphylaxis histamine is actually released by the injured cells.
In 1913 Dale found unusual activities in a batch of ergot extract, and the active principle responsible for these unusual effects was isolated by Dale''s chemical coworker, Arthur James Ewins. It proved to be acetylcholine (first synthesized in 1867), and a ''lucky accident'' that Dale was fond of claiming in later life. This work led to an important paper by Dale in 1914, in which he showed that the action of acetylcholine on plain muscle and glands was very similar to the action of parasympathetic fibers, and that acetylcholine reproduces those effects of autonomic nerves that are absent from the action of adrenaline. These observations had no direct sequel at that time, because there was then no evidence that acetylcholine was normally present in the animal body. Nevertheless, this paper foreshadowed an understanding of the chemical transmission of the nerve impulse [8,9,13].
The fight-or-flight response
In 1902, Walter Bradford Cannon became an assistant professor of physiology, at Harvard and in 1906 he succeeded Bowditch as the George Higginson Professor of Physiology and became the chair of the department, a position he would hold until 1942. Early in his career, Cannon reasoned that 1) it was known that the adrenal medulla is linked to the sympathetic nervous system; 2) many physiological changes known to accompany emotional arousal, such as dilation of the pupils, fast pulse, piloerection, and inhibition of gastrointestinal function, etc., are all signs of increased sympathetic nervous system activity. Cannon had been perhaps predisposed to think along these original lines as a result of the deep impression made upon him, in his early experience in 1909, by noting the marked sensitivity of the stomach and intestines to psychological stimuli. In 1911 Cannon and de la Paz reported that when a cat, placed in a holder, was frightened by a barking dog, detectable amounts of "adrenalin"—measured by an intestinal-strip bioassay—appeared in inferior venal caval blood. The same year Cannon, Shohl, and Wright also reported that under similar experimental conditions cats showed glycosuria in about an hour, while adrenalectomized cats failed to show this response. Thus, in 1911 Cannon and de la Paz concluded that the general nature of the cat''s responses when frightened were "all signs of nervous discharges along sympathetic paths" . In 1914 evidence for "emotional glycosuria" in 4 of 9 medical students taking a difficult scholastic examination and in 12 of 25 members of the Harvard University football squad (including players on the bench) during the most exciting contest of the season also was reported from Cannon''s laboratory. These and other findings led Cannon in 1914 to propound the "emergency function" theory of adrenal-medulla function based upon the view that the many physiological or metabolic consequences of "adrenalin" release are each "directly serviceable in making the organism more efficient in the struggle, which fear or rage or pain may involve. He then championed the view that "…the absolutely essential organs—the ''tripod of life''—the heart, lungs and brain (as well as the skeletal muscles)—are, in times of excitement, when the adrenal glands discharge, abundantly supplied with blood taken from organs of less importance in critical moments. "
All this prompted Cannon to write his famous book Bodily Changes in Pain, Hunger, Fear and Rage: An Account of Recent Researches into the Function of Emotional Excitement, published in 1915, where he coined the term fight-or-flight response to describe an animal''s response to threats. His theory stated that animals react to threats with a general discharge of the sympathetic nervous system, priming the animal for fighting or fleeing, and that "it is the sympathetic division of the autonomic system which is the primary agency in mobilizing the bodily forces in times of great fear or rage". This served as a basis for the concepts of homeostasis and stress, developed later by him and Hans Selye, respectively (see below). Langley did not include the adrenal medulla in the autonomic nervous system. In the 1920s, however Walter Cannon considered the sympathetic nerves and adrenal medulla as a functional unit – the ''sympathico-adrenal'' system.
Stress and infections
In 1910 A Abbott reported in the University of Pennsylvania Medical Bulletin that forced exercise in a revolving drum increased the mortality due to Staphylococcus aureus in rabbits, but this effect was observed only when exercise was initiated at the time of infection. Although exercise before infection with Staphylococcus aureus had no impact on mortality, rabbits were susceptible to Streptococcus pyogenes when they were exercised before being inoculated with this organism .
In 1919, Dr. Tohru Ishigami of Japan published an article ''The influence of psychic acts on the progress of pulmonary tuberculosis'', which appeared in the American Review of Tuberculosis. Ishigami was probably the first to indicate the role of the stress-immune system interaction in tuberculosis. While studying subjects suffering from chronic tuberculosis, he observed a decrease in the phagocytic activity of leukocytes (white blood cells) during the periods of greatest psychological stress. Some stable patients often deteriorated and died after learning of the loss of a loved one. In other, more severe cases, a surprisingly complete recovery came about, despite the fact that no specific therapy was available. "These patients are found to be optimistic and not easily worried", he wrote. He therefore suggested that stress conditions induced immunodepression and consequently an increase in susceptibility to pulmonary tuberculosis. Ishigami concluded that the key to progression of this disease lay in the "emotional life of the patient." Causal factors cited included business failures, family discord, jealousy, nervousness or the death of a loved one. Interestingly, according Sir William Osler (the first professor of medicine at Johns Hopkins University Medical School and later Regius Chair of Medicine at the University of Oxford) "Tuberculosis is a social disease with a medical aspect". In 1909, he also stated that: "The care of tuberculosis depends more on what the patient has in his head than what he has in his chest."
In 1922 G Oppenheimer, E Nicholls & R Spaeth provided probably the first evidence that stress is not always immunosuppressive – they demonstrated that Streptococcus pneumoniae is among the few infections in which stress, under certain conditions may actually protect the animals from the bacterial pathology – mortality was reduced in rats and guinea pigs when exercise preceded Streptococcus pneumoniae infection. In 1925 G Bailey extended these observations in studies with type I Streptococcus pneumoniae, where forced exercise decreased mortality of rabbits when the exercise regimen was initiated before bacterial challenge; however, he demonstrated that an increased mortality was observed if exercise commenced at the time of inoculation with S. pneumoniae. Thus, these studies also underscored the potential importance of the timing of the stressor applied [15,16].
1920s and 1930s
In 1920, the classic demonstration of chemical neurotransmission was finally achieved by a simple, yet ingenious experiment carried out by the German-born Austrian-American physician and pharmacologist Otto Loewi. Working at that time in Vienna, Austria, the night before Easter Sunday, in 1920, after awaking from a sound sleep, Loewi formulated an idea for testing the hypothesis of chemical transmission and scribbled a few notes on a pad before going back to sleep. Early the next morning the idea returned to him; so he went to his laboratory and performed the now-classic experiment that was to revolutionize concepts of nerve function. After Loewi placed two frog hearts into a single bath, the vagus nerve of one heart was stimulated, thereby slowing it, while causing the rate of the second heart to also diminish. From this experiment, Loewi reached the obvious conclusion that a substance liberated from the first heart was responsible for causing inhibition of the second heart. He termed the unknown substance vagusstoff. He suspected that the parasympathetic substance might be acetylcholine, but he cautiously called it the "vagus substance" because even then acetylcholine was not known to be present in the animal body. Indeed, it was not until 1933 that two of Dale''s coworkers proved that Loewi''s vagus substance was acetylcholine. In 1929 Dale and Dudley found acetylcholine in the spleens of horses and oxen - the first occasion on which it had ever been found in the animal body - and the experiments of Dale and John Gaddum (1930) strongly suggested that the effects produced by stimulation of Para-sympathetic nerves were due to the liberation of acetylcholine. In 1934 Dale proposed a chemical classification of the two divisions of the autonomic nervous system: parasympathetic fibres should be referred to as ''cholinergic'' and sympathetic fibres as ''adrenergic''. Between 1933 and 1937 Dale and his co-workers George Brown, Marthe Vogt, John Gaddum, Frank MacIntosh and Wilhelm Feldberg provided first evidence for the role of acetylcholine in gangloinic transmission, at parasympathetic post-ganglionic junction and the neuromuscular junction. Thus, acetylcholine (in parasympathetic or somatomotor nerves) turned out to be the first identified mammalian neurotransmitter. In recognition of their extraordinary achievements, Dale and Loewi shared the Nobel Prize in 1936 for their work on chemical transmission of nerve impulses [8,9,13].
In 1926 Serguei Metal''nikov and Victor Chorine at the Pasteur Institute showed that immune reactions could be conditioned by classical Pavlovian means. They demonstrated that rabbits that had received intra-peritoneal injections of a culture broth of killed microbes and that received external stimuli - heating or grating of the skin where the broth was injected - reacted later by increasing the production of humoral antibodies following the sole repetition of the stimulation of the skin, without injection of an antigen. Guinea pigs were similarly ''conditioned'' to produce haemolysins when their skin was stimulated. Similar results were obtained when the reaction to stimulation of the skin in conditioned animals (either guinea pigs or rabbits) was measured by the migration of monocytes to the area. In addition, the smells of camphor as well as visual cues have been used in association with agents that modulated immune parameters. Later, in 1933 G Smith and R Salinger observed that asthmatic attacks were provoked in some patients with visual stimuli in the absence of the allergen.
In 1927 Dr. Israel Bram from Philadelphia, extended the observations of Caleb Parry, made 100 years earlier when reported that a clear history of traumatic stress was found in 85% of more than 3000 cases of thyrotoxicosis. The precipitating conditions largely involved severe life-threatening crises, now commonly referred to as traumatic stress, such as fires, shipwrecks, earthquakes, combat experiences, and narrow escapes from accidents, as well as various types of object loss. The most striking common feature associated with these stressful experiences seems to be extreme fear concerning biological survival. Later, in 1936 Bram extended his observations in his report ''Psychic trauma in the etiology of Grave''s disease: a survey of 5000 case histories'' published in The American Journal of Psychiatry.
In 1935 J Hammar first detected the presence of silver-stained neural profiles in the developing human thymus, branching into the parenchyma and forming a plexus in the medulla by 16-20 GW. However, at this time, the thymus was regarded as a rudimentary organ, whose function as a primary lymphoid organ of the immune system would be discovered about 30 years later.
Living organisms survive by maintaining an immensely complex dynamic steady state of the internal milieu or homeostasis, terms coined by Walter Cannon, a kind of a brilliant follow-up and extension of Claude Bernard''s work and concepts. Homeostasis is constantly challenged or outright threatened by intrinsic or extrinsic, real or perceived disturbing forces or stressors. Cannon extended the homeostatic concept to emotional as well as physical parameters and to the ''fight or flight reaction'', and linked the adaptive response to stress with catecholamine (CA) secretion and actions. Indeed, Cannon in 1924 had already implicated adrenal secretions as mediators of stress: Evidence points to the sympatho-adrenal system as the chief agency in resisting alterations of our internal environment for when that system is not functioning the same stresses - cold, lack of oxygen, low blood sugar, loss of blood - which had no considerable influence on normal animals, become ominous for continued existence.
He developed the concept of homeostasis, and popularized it in his book The Wisdom of the Body, published in 1932. Cannon presented four tentative propositions to describe the general features of homeostasis: 1) Constancy in an open system, such as our bodies represent, requires mechanisms that act to maintain this constancy. Cannon based this proposition on insights into the ways by which steady states such as glucose concentrations, body temperature and acid-base balance were regulated. 2) Steady-state conditions require that any tendency toward change automatically meets with factors that resist change. An increase in blood sugar results in thirst as the body attempts to dilute the concentration of sugar in the extracellular fluid. 3) The regulating system that determines the homeostatic state consists of a number of cooperating mechanisms acting simultaneously or successively. Blood sugar is regulated by insulin, glucagons, and other hormones that control its release from the liver or its uptake by the tissues. 4) Homeostasis does not occur by chance, but is the result of organized self-government. Interestingly, in 1935, Cannon was probably the first to introduce stress into experimental biology in the publication "Stresses and strains of homeostasis", a summary that preceded Selye''s syndrome of nocuous agents by a year.
The concept of stress
In the 1930s Hans Hugo Bruno Selye, a Canadian endocrinologist of Austro-Hungarian origin developed the concept of stress – extending the term stress from physics and set it to mean the mutual actions of forces that take place across any section of the body. He referred to this state as the ''general adaptation or stress syndrome''. According Selye ''stress is reflected by the sum of the nonspecific changes as they develop throughout time during continued exposure to a stressor, the general adaptation syndrome encompasses all nonspecific changes as they occur during continued exposure to a stressor. One is a snapshot, the other a motion picture of the response to demands. It might be compared to other general defense actions such as inflammation or the formation of immune bodies''. The modern term stress is defined as a state of threatened or perceived threatened homeostasis .
Selye received his medical degree in 1931 from the German University in Prague, he moved on to do postdoctoral work at Johns Hopkins and then migrated to Montreal, where endocrinology was in its heyday, beginning at McGill and then at the University of Montreal, where, later his Institute of Experimental Medicine became a major center of experimental science. The discovery of the stress syndrome was somehow accidental during Selye''s attempts, while at Mc Gill to isolate some hormones from the placenta. At some point it occurred to Selye that his observations, in fact, could be a nonspecific response to nocuous agents. In 1936, he published in Nature the short report ''A syndrome produced by diverse nocuous''. During the same year, in The British Journal of Experimental Pathology he described in more detail this syndrome consisting of involution of the thymus, pleural transudate, adrenal enlargement with loss of cortical (adrenocortical) lipoids and rapid loss of body weight in the rat by certain operative injuries, drugs (atropine, morphine, formaldehyde and adrenaline) and exposure to low temperature. Selye concluded that the involution of the thymus was in fact mediated by the adrenal gland as it was absent in adrenalectomized animals if stressed, and that the syndrome represent an ''alarm reaction'', which enables the organism to meet critical situations more efficiently.
Selye had used "stress" in his initial letter to the Editor of Nature in 1936, who suggested that it be deleted since this implied nervous strain and substituted alarm reaction. In fact, Selye later admitted that had his knowledge of physics and English been more precise, he would have introduced the "strain" concept (in 1822 the French mathematician, Augustin Cauchy, coined the terms "stress" and "strain," defining stress as the pressure per unit area and strain as the ratio of the increase or decrease in the length of an object to its original length). According Selye in his first papers he ''….tried to demonstrate that stress is not a vague concept, but rather that it is clearly a definable biological and medical phenomenon whose mechanisms can be objectively identified and with which we can cope much better once we know how to handle it''.
It all started in 1929 when Dr. Phillip Hench, who was Mayo Clinic''s first rheumatologist, noted a clinical remission in one of his patients, a 65-year-old doctor with rheumatoid arthritis who suffered an intercurrent episode of jaundice. By 1938 he had collected a further 31 cases of improvement with jaundice. He observed that other conditions, including pregnancy and the postsurgical state, could also lead to temporary remission. Convinced that this was no coincidence he decided to devote himself to the discovery of the nature of "antirheumatic substance X" in remissions associated with these conditions. Because remissions associated with jaundice occurred as frequently in women as in men, Hench concluded that factor X, if a hormone, must be present in both sexes. Knowledge that the postsurgical state led to an adrenal response and the observation that the fatigue in Addison''s disease was similar to that seen in rheumatoid arthritis may have led Hench to turn his attention to the adrenals as the possible source of substance X. Fortuitously, he was able to cooperate with Edward Kendall, PhD, Professor of Physiological Chemistry at the Mayo Clinic, who in 1914, aged 28, had been the first to isolate thyroxine in its crystalline form. During the early 1930''s Kendall had isolated six hormones from the adrenal cortex; these were named compounds A through E. Hench asked if Kendall thought any of the isolated hormones might help in treating rheumatoid arthritis. Over the course of many conferences, the two physicians decided that Substance X was most likely an essential hormone, possibly a steroid. In January 1941, Dr. Hench jotted in his red notebook: "Try Compound E in rheumatoid arthritis." In 1946 Edward Kendall''s compound E (cortisone) was first synthesized by Louis Sarett at Merck.
On Sept. 21, 1948, Mrs. Gardner, a 29-year-old who had had severe, erosive rheumatoid arthritis received the first injection of Compound E. Three days later there was an astonishing change – less muscular stiffness and soreness. Over the next seven months, trials were completed on 14 patients with severe or moderately severe rheumatoid arthritis. All showed marked improvement. In 1950, Kendall and Hench received the Nobel Prize in Physiology and Medicine – sharing the prize with Dr. Tadeus Reichstein of Switzerland, who had simultaneously isolated the hormones of the adrenal cortex. Soon, the side-effects of high doses became all too apparent. However, by the time of Hench''s retirement in 1957, cortisone had become a readily available standard treatment for several conditions. Hench''s sensitivity on the subject of his seminal contribution to medicine was somehow unfortunate, in view of his original intention to present his discovery as an investigative tool rather than as a therapeutic breakthrough. Hench''s real achievement however was much greater than demonstrating cortisone''s improvement of rheumatoid arthritis. He opened the way to the understanding that many illnesses share the unifying feature of being caused by uncontrolled or excessive inflammation .
Ulf von Euler – discovery of substance P, prostaglandins and norepinephrine
In 1930, when at the age of 25, Ulf von Euler worked for 6 months in the laboratory of Sir Henry Dale in London, where he had learnt to isolate and identify biologically active substances in tissue extracts. Even during that short stay he had experienced the potency of this approach when, together with Sir John Henry Gaddum they discovered an atropine- resistant factor that lowered blood pressure and contracted isolated intestinal smooth muscle. This factor was later named substance P and recognized as the first identified neuropeptide .
The discovery of substance P fueled von Euler''s interest in hypotensive factors. This commitment culminated in the identification some 3 years later of a lipid-soluble organic acid with hypotensive- and smooth muscle-stimulating activity in accessory genital glands and human semen. He called this factor of unknown biological significance prostaglandin (von Euler, 1935). In 1945 von Euler persuaded Sune Bergström to extend the chemical analysis of lipid extracts of sheep vesicular glands. In 1949, after purifying the crude extract about 500 times, Bergström found that it was composed of unsaturated hydroxyl acids that lacked a nitrogen moiety. In 1957, Bergström and his colleagues isolated the prostaglandins PGE1 and PGF1α. Fortunately, when Bergström''s group moved to the Karolinska Institute in the late 1950s, mass spectrometry became available for analysis. So, by 1962, Bergström and his colleagues were able to identify six prostaglandins in a number of different tissues and then determine their respective chemical structures.
In the 1930s the observations that many effects of sympathetic nerve stimulation could not be mimicked by ''adrenin'' (commercially available preparation of adrenaline), led W Cannon and A Rosenblueth at Harvard to doubt that adrenaline is the sole mediator of sympathetic neurotransmission. In 1933 they proposed the hypothesis that ''……a substance, ''''sympathin'''', is set free when the smooth muscles are subjected to sympathetic nerve stimulation….'', that ''...sympathin differs from adrenin…'', and that in fact ''…two kinds of sympathin are produced – sympathin E, excitatory, produced by structures stimulated, and sympathin I, inhibitory, produced by structures inhibited by sympathetic impulses''. The hypothesis was curiously obscure but stimulated the debate, and thus, it became necessary to differentiate epinephrine (adrenaline) from norepinephrine (noradrenaline) [8,9].
It was not until the mid-1940s that Ulf von Euler used various pharmacological and chemical assays to correctly identify the major catecholamine as norepinephrine (noradrenaline) in extracts of adrenergic nerves isolated from a lymphoid organ, the spleen. In 1945, in a letter to his former teacher, Dale, he reports that ''ordinary alcoholic extracts of cattle spleen contain the somewhat surprising amount of some 10 mg adrenaline pressor equivalents per kg. After purification the active substance was found to differ somewhat from adrenaline, and, it emerged that it resembled definitely more an amino-base like nor-adrenaline than adrenaline or methylated compounds ''…So it may be that the substance found is really sympathin E…'' In 1946 von Euler went one step further and proposed that as, like noradrenaline, his extracts from sympathetic nerve fibres had both excitatory and inhibitory properties, there was no longer any need to retain Cannon''s term, sympathin. His hypothesis and discovery was now that noradrenaline is the main neurotransmitter at postganglionic sympathetic nerve endings . In addition to demonstrating the presence of norepinephrine in almost all sympathetically innervated tissues of mammals, von Euler and his colleagues built upon these findings by later showing that adrenal glands of various mammalian species not only contained varying amounts of epinephrine and norepinephrine but also released them differentially, depending upon the mode and duration of stimulation [8,9].
Until 1946 one did not even know what sympathetic nerve terminals look like, still less of course where neurotransmitters are located. That year Hillarp, in Lund found by classical histological staining (methylene blue, silver impregnation) the answer: they look like ''beads on a string'', forming a long series of swellings (''varicosities'') 1 μm in diameter, separated by 4 μm small calibre intervaricose sections. Fifteen years later he and his colleagues, notably B. Falck, had developed a fluorescence histochemical method in which treatment with dry formaldehyde gas converts noradrenaline into an intensely fluorescent compound .
In 1948, Raymond Ahlquist at the Medical College of Georgia reasoned that if the rank order of potency of a series of catecholamines was the same in all tissues, then the variation in their relative activities must be due to differences in their chemical structure. However, if the rank order of potency varied from tissue to tissue, the observed variations must be due, at least in part, to inherent differences in the receptors. Ahlquist postulated in 1948 that the action of norepinephrine on postsynaptic sites was mediated by two types of adrenergic receptors, which he called α and β. It is of interest to note that the original manuscript submitted by Ahlquist was rejected by the Journal of Pharmacology and Experimental Therapeutics, later published in the American Journal of Physiology – again the scientific community was reluctant to accept this concept because of its novel approach to pharmacology. Ahlquist''s ideas however were later adopted by Sir James Black, and Ahlquist''s concept provided the conceptual framework for the development of β-receptor blockers in the 1950s and 1960s, which was to earn Black the Nobel Prize .
In 1943 Selye described that, in chickens, the Bursa of Fabricius is also extremely sensitive to steroid hormones. In 1946 he also presents data that white blood cell counts rise invariably during stress, regardless of the stressor used. The changes in the adrenal cortex and of thymus involution are also illustrated histologically. The thymus shows a depletion of cortical thymocytes. He points out that lymph nodes, the spleen and other lymphatic organs are almost as markedly affected as the thymus, although they do not involute quite as rapidly, and their involution cannot be completely prevented by adrenalectomy. Selye made all these contributions without knowing the function of the thymus, lymph nodes, or the Bursa of Fabricius. The function of these organs was understood only years later . In 1946, Selye had formulated his general concept of stress and its effects on the organism in his article: "The general adaptation syndrome and the diseases of adaptation" in The Journal of Clinical Endocrinology and Metabolism. According Selye ''…a fully-developed general adaptation syndrome consists of three stages: the alarm reaction, the stage of resistance, and the stage of exhaustion. Most of the physical or mental exertions, infections, and other stressors, which act upon us during a limited period, produce changes corresponding only to the first and second stages. Normally, in the course of our lives, we go through these first two stages, many times. Otherwise we could never become adapted to all the activities and demands which are man''s lot. Even the stage of exhaustion does not always need to be irreversible and complete, as long as it affects only parts of the body''. About the diseases of adaptation he wrote: ''…many maladies are due not so much to what happens to us as to our inability to adapt, and they have therefore been called "diseases of adaptation". The most common of such diseases are peptic ulcers, high blood pressure, heart accidents, and nervous disturbances. Yet this is a relative concept. No malady is just a disease of adaptation….''
In 1948, Astrid Fagraeus, at the National Bacteriological Laboratory, Stockholm demonstrated the antibody production in plasma B cells. She wrote in her thesis that the "formation of antibodies takes place side by side with and during the development of the reticulum cells into plasma cells. In case of an intense antibody formation a differentiation of these cells into plasma cells takes place". In a Journal of Immunology paper, the same year, she concluded that "antibodies are formed by cells of the R.E.S. (reticuloendothelial system), passing through a chain of development, the final link of which is the mature plasma cell".
Rediscovery of cellular immunity, immunological tolerance
In 1942, a definitive proof that cells played a role in immunity came from the classic experiments of Karl Landsteiner and Merrill Chase. They showed that both delayed coetaneous hypersensitivity to chemicals and the tuberculin reaction in guinea pigs could be transferred between animals using peritoneal lymphocytes, but not serum – cells from guinea pigs, which had been immunized with Mycobacterium tuberculosis or hapten, were transferred into naive guinea pigs. Later, when antigen or hapten was injected into these guinea pigs, they elicited an immune recall response that was not present in the naive controls. This did not happen when the serum fraction was transferred. Similar results were obtained using a contact dermatitis model. Thus, the dichotomy of immediate (antibody-mediated) and delayed type (cell-mediated) hypersensitivity had become firmly established by the 1940s, although the identity of the cell that conferred the latter response was unknown. It was not until the pioneering experiments of Gowans in the early 1960s that lymphocytes were recognized as being essential to immunity.
In the 1940s Sir Peter Brian Medawar, a British scientist (born in Brazil of a British mother and a Lebanese father) revived the scientific interest in cellular immunity and marked a major shift in modern immunology. Peter''s most important work was to demonstrate that the rejection of donor grafts was due to an immunological reaction and that tolerance could be built up by injections into embryos. Thus was born the idea of acquired immunological tolerance, an idea that is still spawning new research to this day. The body''s capacity for reacting to foreign proteins can be reduced by repeated exposure to the protein, preferably in small, graded amounts. Although most of his work was immediately accepted and appreciated by his peers, Peter is often quoted for the famous quip: ''The human mind treats a new idea the way the body treats a strange protein – it rejects it''.
Medawar''s earlier research, done at Oxford, was on tissue culture, the regeneration of peripheral nerves. When he showed the first draft of his manuscript describing the research to Howard Florey, the co-discoverer of penicillin, Florey pulled no punches in denouncing the paper, saying that "it sounds more like philosophy than science." While conducting his research, Peter also worked in Florey''s laboratory, where everyone studied wound and burn healing to aid in the World War II effort in Britain. At that time, people with severe burns were kept alive with blood transfusions and sulfa drugs to combat infections. He became intrigued with the fact that skin grafts only worked if the grafted skin came from the same person receiving the graft. One of his first clues was the observation that a second graft of "foreign" skin did not last as long as the first one. This suggested that the body had some kind of memory of the first graft experience. The mechanism allows the body to adapt to or reject foreign intrusion. Moreover, the resistance system can retain a residual memory and respond even more vigorously at the second time of exposure. When he moved to Birmingham in 1947 he continued to work on it, in collaboration with R. Billingham, and together they studied there problems of pigmentation and skin grafting in cattle, and the use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. He realized that each individual develops his own immunological system and that the length of time a graft lasts depends on how closely related the recipient and donor are. He found that grafting was successful not only between identical twins but also between nonidentical, or fraternal, twins. In this work they took into consideration the work of R. D. Owen that the red-cell precursors are exchanged between twin fetuses, and concluded that the phenomenon that they called «actively acquired tolerance» of homografts could be artificially reproduced.
This led to the suggestion by Sir Frank Macfarlane Burnet, an Australian virologist that the immunological system is not developed at conception but is gradually acquired. In 1949, he introduced the concept of "self" and "non-self" to immunology. Burnet regarded that the "self" of the host body was actively defined during its embryogenesis through complex interactions between immune cells and all the other cells and molecules within an embryo. If an antigen were injected into an animal before birth it should develop an immunological tolerance to that antigen, and consequently fail to produce antibodies if ever exposed later in life. But, Burnet discovered, that this did not happen. While a young chick exposed to the antigen as an embryo would fail to develop antibodies, such chicks in adulthood display the usual intolerance and produce antibodies to the appropriate antigen. Burnet had failed to realize that the exposure to the antigen must be continuous for tolerance, not only to develop, but be maintained. The point was later established in 1953 by Peter Medawar, Rupert E. Billingham and Leslie Brent when they showed that splenocytes could be engrafted by intravenous infusion into mice in utero or just after birth and that when these mice matured, they could accept skin and other tissues from the donor but not from any other mouse strain. For their work on immunological tolerance Medawar and Burnet were awarded the 1960 Nobel Prize for physiology and medicine.
In 1951, von Euler raised a crucial question: ''If the neurotransmitter (noradrenaline) was present in the adrenergic axon, as we knew it was, how could it survive there in constant amounts? Obviously such levels of a substance as potent as noradrenaline could not exist in free solution inside nerve terminals; there has to exist ''some special storage mechanisms''. And he was right: in 1953 it was shown conclusively by H. Blaschko and A. Welch in Oxford, and by Hillarp and colleagues in Lund, that catecholamines in cells of the adrenal medulla largely occur in a particulate, sedimentable form, as ''granules'' enclosed in ''chromaffin storage vesicles'' – this was the first evidence of subcellular localization of hormone and transmitter. Might these findings have any bearing on storage of transmitters in nerves? At about this time it was reported that vesicles had been seen electron-microscopically in nerve terminals in the brain, Palade 1954. Soon functional data appeared that the synaptic vesicles might also be involved in transmitter release, and in 1957, J. Del Castillo and B. Katz proposed the ''vesicle hypothesis for quantal release'', according to which the acetylcholine contents of a vesicle may represent the standard-sized multi-molecular packet of transmitter (the ''quantum'') released either spontaneously or by a nerve impulse. The same year, von Euler and Hillarp, in a simple but crucial experiment showed, by differential centrifugation of homogenates of sympathetic nerves, that noradrenaline indeed occurs intra-neuronally in sedimentable form, as vesicle enclosed ''nerve granules''. This was the first conclusive demonstration of what would later turn out to be a universal principle, namely that neurotransmitters (with the exception of nitric oxide) are stored intra-neuronally in specific vesicles .
At approximately the same time, in 1953, James Riley and Geoffrey West discovered that the mast cell granule was the major source of histamine in the body, when a fluorescent histamine-liberator was traced to its site of action in the mast cells of the rat . Subsequent pharmacological investigations confirmed the mast cell as the source of the histamine, and that mast-cell tumors from dogs and the skin lesions of urticaria pigmentosa in man, composed of mast cells, may contain milligrams of histamine per gram of tissue. Thus, histamine on which Sir Henry Dale has spent so much of his working life should eventually be located in a cell discovered by his former teacher, Paul Ehrlich. It was believed that mast-cell histamine is virtually static, waiting for trauma to release it. In 1961, R Schayer, proposed that there is a second source of tissue histamine, formed as a response to stress .
In 1953, Dougherty and Frank described about a 400% increase within 10 min after subcutaneous injection of epinephrine (adrenaline) of what they called "stress-lymphocytes". These cells had the morphology of large granular lymphocytes or natural killer (NK) cells, whose function and characteristics were described in the late 1970s.
In 1955, in his review article in Science, entitled "Stress and disease" Selye proposed that deficient host defense due to abnormalities of neuroendocrine factors may lead to disease  – he discussed the role of ''….an absolute excess or deficiency in the amount of adaptive hormones…..an absolute excess or deficiency in the amount of adaptive hormones retained (or `fixed'') by their peripheral target organs….the production by stress of metabolic derangements, which abnormally alter the target organ''s response to adaptive hormones…''. The prediction by Selye of the pluricausal nature of most diseases is recognition that living organisms have evolved multiple mechanisms to defend themselves against harmful agents, and thus the necessity to interfere at more than one point to control disease. Furthermore, he stated that ''Pasteur, Koch, and their contemporaries introduced the concept of specificity into medicine, a concept that has proved to be of the greatest heuristic value up to the present time. Each individual, well-defined disease, they held, has its own specific cause. It has been claimed by many that Pasteur failed to recognize the importance of the ''terrain'', because he was too preoccupied with the pathogen (microorganism) itself''.
Although Selye''s view that stress responses are nonspecific has been recently challenged, his legacy of empirical stress research remains extremely influential and valid today. In the 1990s the World Health Organization called the stress of everyday life "a worldwide epidemic." Americans spend on stress-management about $18 billion each year, and the American Institute of Stress claims that stress is "America''s number 1 Health Problem" . However, stress probably still remains, as Selye stated ''….a scientific concept, which has suffered the mixed blessing of being too well known and too little understood''. But, as recently stated by Gerald Weismann, the editor of The FASEB journal: ''When we eventually understand the biology of the stress syndromes, we''ll have Hans Selye, the experimental pathologist, to thank'' .
In the 1950s, Thomas Holmes, a charismatic and iconoclastic Seattle physician, studied the association between stress and tuberculosis – although lacking the sophistication of modern biostatistics, several of Holmes'' studies suggested that persons who had experienced stressful situations, such as divorce, death of a spouse, or loss of a job, were more likely to develop tuberculosis and less likely to recover from it. Thomas Holmes and Norman Hawkins reported that patients who contracted tuberculosis had suffered an increase in stressful life events during the 5 years preceding the onset of the disease. In 1957, Hawkins and Holmes compared 20 Firland employees who had become tuberculous between 1949 and 1954 with 20 non-tuberculous sanatorium employees matched for age, sex, race, income, duration of employment, and skin test status at the time they began working at the sanatorium. The "previous occurrence of psychosocial stresses" was measured by the Schedule of Recent Experience, an instrument devised by Hawkins. Examining the same type of variables as before, Hawkins and Holmes found that the tuberculous employees reported increasing numbers of "disturbing occurrences" during the 2 years preceding their illnesses. Moreover, for each stressor, the persons with tuberculosis were more likely than the controls to have experienced such increases. In the final major study conducted by Holmes, titled "Experimental Study of Prognosis," published in 1961, Holmes and colleagues prospectively studied 41 randomly selected patients using the Berle Index, an instrument that identified psychological and social factors characteristic of recovering patients. A high Berle score predicted recovery. When 26 patients who had achieved normal or high Berle scores were located 5 years after testing, none had been classified as a treatment failure. In contrast, 5 of the 15 patients with low Berle scores had become treatment failures .
In 1959, James Gowans at Oxford discovered lymphocyte circulation. In 1953, Howard Florey suggested he should investigate the lymphocyte, a cell whose life history was at that time completely obscure. The main debate at that time, centered on the possibility that lymphocytes migrated from the blood into the bone marrow, where they became precursors of erythrocytes and granulocytes. In 1959, Gowans collected lymphocytes from the thoracic duct of the rat, labeled them, and re-infused them into the blood. According to him, this was probably the most gratifying experiment he ever did: When P32-labelled lymphocytes were re-infused, a large amount of cell-associated radioactivity appeared in the thoracic duct lymph. The simplest interpretation was that lymphocytes did not have a ''destination'', and, thus Gowans discovered that the small lymphocyte continuously re-circulated from the blood to the lymph and back again to the blood. Later, in the 1960s, he also demonstrated that this cell was at the centre of immunological responses.
Clonal selection theory
Prior to the 1950s, it was not known how antibody diversity was generated. Between 1955 and 1959, three scientists, Jerne, Talmage and Burnet, working independently, developed what is widely referred to as the clonal selection theory. In 1955, Jerne published a paper that described a "selective" hypothesis, which held that every animal had a large set of natural globulins that had become diversified in some unknown fashion. According to Jerne, the function of an antigen was to combine with those globulins with which it made a chance fit. The antigen would serve to transport the selected globulins to antibody-producing cells, which would then make many identical copies of the globulin presented to them. In 1957, Talmage wrote: "...it is tempting to consider that one of the multiplying units in the antibody response is the cell itself…" According to this hypothesis, only those cells are selected for multiplication whose synthesized product has affinity for the antigen injected. According to Burnet, the clonal selection theory states: 1) Animals contain numerous cells called lymphocytes. 2) Each lymphocyte is responsive to a particular antigen by virtue of specific surface receptor molecules. 3) Upon contacting its appropriate antigen, the lymphocyte is stimulated to proliferate (Clonal expansion) and differentiate. 4) The expanded clone is responsible for the secondary response (more cells to respond) while the differentiated ("effector") cells secrete antibody. The modern understanding of this concert is that each naïve lymphocyte has a different receptor specificity, each of which can bind a different antigenic determinant. When a pathogen is recognized by the cells, in this case by two different antigenic determinants, then the cells that bind to these determinants are selected to proliferate or undergo clonal expansion, and then differentiate into effector cells that either secrete antibody or mediate various effector mechanisms of cell-mediated immunity . In 1960, along with Peter Medawar, Burnet was awarded the Nobel Prize, "for discovery of acquired immunological tolerance" rather than the clonal selection theory. Jerne would later win the Nobel Prize in 1984 "for theories concerning the specificity in development and control of the immune system".
Discovery of the first cytokine – interferon
While aiming to develop an improved vaccine for smallpox, two Japanese virologists, Yasu-ichi Nagano and Yasuhiko Kojima working at the Institute for Infectious Diseases at the University of Tokyo, noticed that rabbit-skin or testis previously inoculated with UV-inactivated virus exhibited inhibition of viral growth when re-infected at the same site with live virus. They hypothesized that this was due to some inhibitory factor, and began to characterize it by fractionation of the UV-irradiated viral homogenates using an ultracentrifuge. They published these findings in 1954 in the French journal now known as "Journal de la Société de Biologie". While this paper demonstrated that the activity could be separated from the virus particles, it could not reconcile the antiviral activity demonstrated in the rabbit skin experiments.
In 1957 the Scottish virologist Alick Isaacs and the Swiss researcher Jean-Jacques Lindemann, at the National Institute for Medical Research in London, noticed an interference effect caused by heat-inactivated influenza virus on the growth of live influenza virus in chicken egg membranes, in a nutritive solution chorioallantoic membrane. Thus, they discovered that when cells in laboratory cultures are infected by a virus, they secrete a substance that protects other cells from infection. Isaacs and Lindemann traced this effect to a protein and coined the term ''interferon'', and today that specific interfering agent is known as a ''Type I interferon''. Human and animal cells produce it in a rapid "first wave" response to infections. Although the initial hopes for interferons as broad spectrum antiviral agent''s equivalent to antibiotics have faded, interferons were the first cytokines to be discovered and studied in detail.
- Goldstein DS, Robertson D, Esler M, Straus SE, Eisenhofer G. Dysautonomias: clinical disorders of the autonomic nervous system. Ann Intern Med 2002; 137(9): 753-63.
- Gray J, Hoffenberg R. Thyrotoxicosis and stress. Q J Med 1985; 54(214): 153-60.
- Noble D. Claude Bernard, the first systems biologist, and the future of physiology. Exp Physiol 2008; 93(1): 16-26.
- Silverstein AM. Cellular versus humoral immunology: a century-long dispute. Nat Immunol 2003; 4(5): 425-8.
- Kelley KW. From hormones to immunity: the physiology of immunology. Brain Behav Immun 2004; 18(2): 95-113.
- Prull CR. Part of a scientific master plan? Paul Ehrlich and the origins of his receptor concept. Med Hist 2003; 47(3): 332-56.
- Maehle AH, Prull CR, Halliwell RF. The emergence of the drug receptor theory. Nat Rev Drug Discov 2002; 1(8): 637-41.
- Rubin RP. A brief history of great discoveries in pharmacology: in celebration of the centennial anniversary of the founding of the American Society of Pharmacology and Experimental Therapeutics. Pharmacol Rev 2007; 59(4): 289-359.
- Stjarne L. Catecholaminergic neurotransmission: flagship of all neurobiology. Acta Physiol Scand 1999; 166(4): 251-9.
- Silverstein AM. Autoimmunity versus horror autotoxicus: the struggle for recognition. Nat Immunol 2001; 2(4): 279-81.
- Benschop RJ, Rodriguez-Feuerhahn M, Schedlowski M. Catecholamine-induced leukocytosis: early observations, current research, and future directions. Brain Behav Immun 1996; 10(2): 77-91.
- Huber B. [100 years of allergy: Clemens von Pirquet - his idea of allergy and its immanent concept of disease]. Wien Klin Wochenschr 2006; 118(19-20): 573-9.
- Tansey EM. Henry Dale and the discovery of acetylcholine. C R Biol 2006; 329(5-6): 419-25.
- Randall DC. Discovering the role of the adrenal gland in the control of body function. Am J Physiol Regul Integr Comp Physiol 2004; 287(5): R1007-R1008.
- Peterson PK, Chao CC, Molitor T, Murtaugh M, Strgar F, Sharp BM. Stress and pathogenesis of infectious disease. Rev Infect Dis 1991; 13(4): 710-20.
- Biondi M, Zannino LG. Psychological stress, neuroimmunomodulation, and susceptibility to infectious diseases in animals and man: a review. Psychother Psychosom 1997; 66(1): 3-26.
- Chrousos GP, Gold PW. The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis [published erratum appears in JAMA 1992 Jul 8;268(2):200]. JAMA 1992; 267(9): 1244-52.
- Le Fanu J. 1949: Cortisone. In: Le Fanu J, ed, The rise and fall of modern medicine. New York: Carroll & Graff Publishers, Inc. 2000; 16-25.
- Berczi I. Neuroimmune Biology - An Introduction. In: Berczi I, Gorczynski RM, eds, New foundation of biology. Elesevier Science B.V. 2001; 3-45.
- Riley JF. HISTAMINE AND SIR HENRY DALE. Br Med J 1965; 1(5448): 1488-90.
- Weissmann G. The experimental pathology of stress: Hans Selye to Paris Hilton. FASEB J 2007; 21(11): 2635-8.
- Lerner BH. Can stress cause disease? Revisiting the tuberculosis research of Thomas Holmes, 1949-1961. Ann Intern Med 1996; 124(7): 673-80.
- Abbas AK, Janeway CA, Jr. Immunology: improving on nature in the twenty-first century. Cell 2000; 100(1): 129-38.