Biology 466    Unsolved Problems Fall 2010

Background on Immunology (in one easy lesson)

1) Humans and other vertebrates have the ability to "attack", inactivate and/or kill an UNLIMITED RANGE OF DIFFERENT MATERIALS including not only germs but also otherwise harmless materials like pollen, grafted tissues and (unfortunately) even substances which are normal parts of our own bodies! They even attack artificial materials to which no ancestor of ours was ever exposed!

2) This capacity to "attack" a particular substance is known as immunity and results from the activities of a specific set of differentiated cells, collectively called the immune system.

3) When the material attacked is one which is not harmful in itself, so that the side-effects of the immune attack are more unpleasant than are the direct effects of the material itself, then we refer to the consequences of the immune attack as an "allergy".
(When someone claims that he or she is "immune to poison ivy", what do they really mean?)

4) The immune system can also attack parts of your own body. This is known as "autoimmunity" (meaning self-immunity = self-allergy). There are many different auto-immune diseases, differing with respect to which of the normal parts or molecules of the body are attacked by the victims' immune systems.

    Pernicious Anemia -- the enzyme which absorbs vitamin B12 is attacked
    Multiple Sclerosis -- the myelin sheath around nerve axons is attacked
    Rheumatic Fever -- the extracellular matrix of heart valves is attacked
    Lupus Erythematosis -- collagen, DNA, other cellular contents are attacked
    Myasthenia Gravis -- acetyl choline receptors on muscle cells are attacked
    Male sterility following Mumps -- sperm are attacked; first formed at puberty!

5) A major part of immunity (the best understood part) is the synthesis and secretion into the blood and other fluids of a class of special proteins called "antibodies". These are also called immunoglobins, because they are part of the fraction of blood proteins called globins. Specifically, they make up the subset of these called the gamma-globulins.

6) Each antibody molecule has 2 (or 10, in IgM) binding sites (analogous to the active sites by which enzymes bind to their chemical substrates) by which the antibody molecules bind very specifically and very tightly to whatever other molecules have exactly the right shape to fit into this binding site. The molecule to which an antibody binds is called its "antigen". The apparent etymology of the word antigen implies that the antibody's synthesis is somehow stimulated by the antigen to which its active sites bind; but this is not always true. The newer word "epitope" refers to the molecular site to which a given antibody binds.

7) The specificity of antibody molecules for their corresponding antigen epitope can be very great. Like the specificity of enzymes for their substrates, the specificity of antibody binding results from an exact conformational fit between the two shapes. Experimental biologists often take advantage of this extreme specificity in order to determine the positions of certain molecules within cells. Antibodies "against" the antigen of interest are elicited, borrowed or purchased, and fluorescent chemicals are then covalently bound to these antibodies so that their location can be observed by "immuno-fluorescence microscopy". Modern pregnancy tests also depend on the specificity of antibody binding to certain protein hormones, as do the "radioimmune assays" whose precision has revolutionized endocrinology.

8) Antibodies are synthesized by a particular kind of white blood cell called a B-lymphocyte (often called simply B-cells). When these are secreting, they are also called "plasma cells".

9) (a key fact) Each individual B lymphocyte (and all of its daughter cells, when it divides) will secrete antibody molecules having exactly the same binding specificity. All of the antibody molecules which this cell and its siblings will synthesize and secrete will have exactly the same shaped binding sites and will bind to the same antigens or epitopes. This restriction of B cell clones to just one binding specificity is perhaps the most crucial single fact about the mechanism of our immune system! Make sure you understand this!

10) B lymphocyes (or, rather, their precursor cells) respond to exposure to molecules of "their" antigen (the one to which their antibodies will bind) by increased growth and division, as well as by increased secretion of antibody molecules. This stimulation process can take several days to produce enough antibody to kill pathogens. In fact, this stimulation and response is what is going on during the several days that it takes to start getting well from an infectious disease. Once stimulation by the antigens of certain pathogen has occurred, the quantity of antibody molecules and of lymphocytes which secrete them will remain high enough to prevent reinfection by that pathogen -sometimes this protection will last for the rest of your life. This is why you only get certain diseases once: for example measles, mumps, chicken pox, polio.

11) "vaccination" is based on deliberately stimulating the body to produce more antibodies against pathogenic viruses and bacteria. This stimulation is usually achieved by a deliberate exposure of the body to antigens of these pathogenic organisms, in the form of isolated antigens, killed pathogens, or even living but weakened forms of the pathogens.

12) It was known in ancient times (the historian Thucidides mentions it) that people become insusceptible to certain diseases as a result of having contracted them once. In the 1700s, the idea of deliberately innoculating people with mild cases of smallpox was introduced into England from Turkey by Mary Montague. Later, Edward Jenner championed the folk-practice (then said by nearly all physicians to be a superstition!) of deliberately infecting people with the (mild) disease "cow-pox", in order to induce immunity to the (much more serious) related disease, smallpox. (Notice how the word vaccination comes from the romance language root for "cow", as in Spanish "vaca"; thus, vaccination = "cowification").

13) Among the many achievements of the great French scientist Louis Pasteur (later 1800s) was the development of procedures for isolating and deliberately weakening pathogenic organisms so that they can then be used for vaccination. Otherwise, this approach would be limited to just those diseases where there already happened to be a related, but weaker, form of the disease. Essentially, he was looking for methods to "make your own cowpox" for any given disease.

14) The mechanistic explanation for immunity which Pasteur believed in, and which therefore motivated and guided his research, was completely mistaken! He believed that the body contained various trace elements which were needed for the growth and survival of each particular pathogenic organisms, and that these trace elements were used up by these organisms when you have that particular disease; he believed that you therefore become insusceptible to the disease because your body then lacks enough of the trace elements for the germs to live. (Notice how mistaken theories can sometimes make correct predictions, and lead to medical breakthroughs!)

15) It was later found that injection with killed germs, and even only with proteins isolated from them, can (sometimes) produce immunity. An example of the latter is use of vaccines made from formaldehyde-treated protein toxins from tetanus and diphtheria bacteria ("toxoids"). Notice that the "trace element depletion" type of hypothesis would predict that only living germs would be able to produce immunity. Their development began around 1900.

16) It was also found that isolated serum from an already vaccinated person or animal could produce some degree of immunity when injected (this is called "passive immunity" as opposed to the "active immunity" which results from making your own antibody molecules).

17) Soon afterward (first decades of the 1900s) it was realized that the reason why blood transfusions (which had been tried for many years) often failed so catastrophically was that the body "attacks" the transfused blood as if it were germs. One of the principal scientists involved in these discoveries was Karl Landsteiner (who was Austrian, but came to America after World War I).

18) Because immunity to blood (of types other than one's own) did not seem to require previous exposure (previous transfusions to "vaccinate" you), it was long believed that there were "natural antibodies" which one synthesizes in large quantities even without any prior to exposure to their antigen. The true explanation turned out to be that there are certain ubiquitous species of bacteria that have surface antigens very similar to the human blood group antigens; it is prior exposure to these bacteria which has sensitized you to the blood group antigens.

19) Until well after the turn of the century, it was assumed that the ability to synthesize antibodies against each particular antigen must be an inherited capacity which our ancestors had evolved by natural selection in the usual way, i.e. those who lacked the ability to make antibodies against disease X would tend to die of that disease, while those who had the genes for making those antibodies would tend to survive; thus the genes for making each antibody would increase in frequency in population. This is a form of "selectionist mechanism" for antibody specificity. This quite reasonable assumption is implicitly the basis for H.G. Wells' ending of his novel "The War of the Worlds". Incidentally, Wells himself really wanted to be a research biologist, rather than a novelist, and did earn a PhD. in biology. The same explanation is often given for the greater susceptibility of American Indians to European diseases. Did you once assume it was true?

20) A quite different type of selectionist mechanism was proposed by the great immunologist Paul Ehrlich; this was the side chain theory; it suggested that cells of the immune system might start out with many thousands of special molecules ("side chains") on their surfaces, each with a different shape, and therefore capable of combining with a different antigen; when an antigen came along which bound to one of these side chains, then this binding would somehow stimulate the cell to make lots more of that particular kind of side chain, and to quit making the other kinds; the idea was that the antibody molecules were released copies of those kinds of side chains which the cells had been stimulated to make because of these side chains' abilities to bind to the antigen. This idea foreshadowed Jerne and Burnet's later clonal selection hypothesis, and both the similarities and the differences between the different theories are worth careful thought.

21) However, around the time of the first world war, Landsteiner discovered that the body can also make antibodies against very artificial synthetic chemicals. Because there was no chance that our ancestors could have been exposed to these chemicals (much less that their survival and reproduction could have depended on becoming immune to such chemicals!) these observations therefore seemed to prove (wrongly!) that the antibody molecules must somehow "instruct" our cells as to what shape the antibody binding sites should have (equivalent to making a glove to fit a specific hand, rather than finding one in stock that fits). If you go to the shoe store and pick out some shoes that have just the right conformational fit for your feet, then that is a selective mechanism. But if the people in the store have to measure your feet and then manufacture a shoe to fit it, then that is an instructional mechanism.

22) "Instructional" mechanisms": Several kinds of hypothesis were developed to explain how the same amino acid sequence could be shaped into many different alternative conformations. One such instructional theory was that the antibody molecules, during their synthesis, might somehow be wrapped around a sample antigen molecule, so that they would be molded to its shape. Linus Pauling proposed such a theory, and published experiments supporting it. One vesion involved the relatively large number of disulfide bonds in antibody molecules: the idea was that the approximately 10 cysteine groups might bond in many different alternative permutations, thus producing many different antigen binding sites. (Can you calculate the number of different possible shapes?)

23) Much later, during the 1960s, it was proven that if you denature an antibody molecule (unfold the amino acid chain, and break all the disulfide bonds) and then let the molecule fold back spontaneously into whatever pattern it "wants", then the binding site of the resulting antibody molecule will have exactly the same binding specificity (antigen specificity) as it had before being denatured. This shows that the specificities of antibody molecules are determined by their amino acid sequences; conversely, antibodies with different specificities have different amino acid sequences.

24) Until the later 1950s, all sensible, church-going immunologists still believed confidently in one or another instructional theories of antibody specificity (the antigen must somehow "tell" the body what shape to make the binding sites on the antibody molecule). A foolhardy young man named Niels Jerne proposed a quite different sort of explanatory hypothesis in which selection takes the place of instruction. Jerne called his revolutionary hypothesis the "natural selection theory of immunity", but it was quickly superseded by a modification of this theory invented by Burnet called the "clonal selection hypothesis". (Note, Jerne was in the same Danish lab as Watson!)

25) The basic ideas of the clonal selection hypothesis are the following: A) During embryonic development each individual animal (somehow!!) generates many different clones of lymphocytes each of which can make antibodies against a different antigen.
B) Each lymphocyte would make antibodies against only one particular antigen, and when this lymphocyte divides the daughter cells would continue to have the same specificity, making only antibodies against its one, randomly-chosen antigen.
C) During development, exposure of lymphocytes to "their" antigen (the one that their antibodies will bind to) would cause the death or inactivation of these clones of lymphocytes ("forbidden clones"). This is how the mechanism explains self-tolerance.
D) Later during life, lymphocytes respond to "their" antigens in the opposite way, by being stimulated to grow, divide and secrete antibodies, thus producing immunity.
E) The majority of lymphocyte clones would never be exposed to "their" antigens (to which their antibodies would bind) so that these clones survive in an inactive form.

Incidentally, Jerne's original version of the theory was still influenced so much by instructionist thinking that it postulated the generation and selection of clones of chemical templates (rather than clones of antibody-synthesizing cells) around which the antibody molecules were supposedly molded into shape! This is why we would be unjust to think of Burnet as having "stolen" Jerne's idea (but note that the Swedes gave the Nobel prize for this to Burnet, not Jerne, although he did get it much later for other theories, including his "network theory"). As Gore Vidal has said "Never underestimate the Scandanavian sense of humor".

26) The clonal selection hypothesis was tested experimentally by injecting antigens into embryos and showing that the animal then became tolerant to those antigens. It was for this that Peter Medawar shared the Nobel Prize with Burnet.

27) The mechanism which generates all this million-fold diversity on the genes for the variable sequence regions has (somewhat irreverently) been called the "Generator Of Diversity" (i.e. G.O.D. !). It was confidently predicted that the discoverer of its mechanism would win the Nobel Prize, and one of the discoverers did win it in 1987 (Tonegawa). The hypothetical mechanisms proposed were divisible into two main categories "germ line" and "somatic line" theories, but the true answer turned out to be a strange mixture of both.

28) It was very difficult to determine the amino acid sequences of antibody molecules because so many different antibody molecules make up gamma globulins of an ordinary individual. It was like trying to determine the amino acid sequence of a whole collection of different enzymes, all mixed together (since different antibodies have different amino acid sequences.)
This problem was circumvented by using "Bence-Jones proteins" instead of normal antibodies. Bence-Jones proteins are found in high concentrations in the blood and urine of patients suffering from a certain kind of cancer called "multiple myeloma"; this is a cancer of the B lymphocytes and the Bence-Jones proteins turn out to be fragments of antibody molecules. The advantage is that for any one patient with this disease all of the Bence-Jones proteins have exactly the same amino acid sequence. This is because all of the billions of cancerous B cells in any given patient are all descended from just one original B cell that became cancerously transformed, and all of these cancerous B-cells secrete antibody molecules with the same amino acid sequence as this original transformed cell (albeit often a defective form of this original sequence). In a sense, Bence-Jones proteins are the original monoclonal antibodies. These Bence-Jones proteins were discovered about 1850, but it was not understood what they were until much later, in the 1960s. It also turns out to be possible to induce this form of cancer in certain strains of mice ("Balb-C") by injecting mineral oil into their coelomic cavities. (!*&?)

29) These and other determinations of amino acid sequences showed that antibody molecules contain some regions in which the sequence is quite different between one antibody molecule and another; these are called variable sequence regions. But most of the molecule consists of constant sequence regions in which the amino acid sequence is the same from one antibody to another (or from one Bence-Jones protein to another). These are called constant sequence regions. As we will see later, the variable sequence regions make up the binding site, while the constant sequence region forms the rest of the molecule.

30) It also turns out that there are several different kinds of antibody molecules (called immunoglobin G, immunoglobin M etc.; abbreviated as IgG, IgM, IgA, IgD and IgE). Each of these have somewhat different constant sequence regions. In fact, each of them consists of a combination of different protein molecules: for example, each IgG molecule is made up of two "heavy chains" (each about 50,000 molecular weight) and two "light chains" (each about 25,000 molecular weight). Each heavy chain has a constant sequence region of about 300 amino acids and a variable sequence region of about 100 amino acids. Each light chain has a constant sequence region of about 100 amino acids and a variable sequence region of about 100 amino acids. The 4 chains are bound together by disulfide bonds.
As you may have guessed, the binding site of the IgG molecule is formed by the combination of the variable sequence regions of one heavy chain and one light chain. Each IgG molecule has two such binding sites and they both have the same specificity (bind to the same epitope).

31) The IgM type of antibody molecule is even more complex, having 10 light chains and 10 heavy chains, with a total molecular weight of around 900,000. The structure is analogous to 5 IgG molecules combined together and they have 10 antigen binding sites instead of 2. When first exposed to an antigen, you at first synthesize mostly IgM, and then later you synthesize mostly IgG.
The other classes of immunoglobins (IgA, IgD and IgE) have 2 binding sites each.

32) Potential antigens will (usually!) not stimulate the immune system to synthesize antibodies unless the original antigens are parts of relatively large molecules (molecular weights of around 20,000 or more). Once the antibody molecules are made, however, they will bind perfectly well to antigens of low molecular weights (molecular weights as low as 100). An interesting example is the allergy which people sometimes develop to the antibiotic penicillin, even though it is a relatively small molecule; the original sensitization to this compound depends on the fact that penicillin tends to bond covalently to proteins; when attached to a protein, its effective molecular weight is then large enough to produce the needed stimulation of the immune system, although the antibody molecules which eventually result from this stimulation will also bind to individual penicillin molecules.
Thus, to immunize an animal against a small molecule, you usually need to combine the small molecule to some sufficiently big molecule. In addition, it is found that there are certain oily materials (called "adjuvants") which have the effect of increasing the sensitivity of the immune system to injected antigens, when they are injected mixed with the adjuvant.

33) When the immune system fails (or refuses!) to make antibodies against a certain antigen, this is called "tolerance" (or "immune tolerance"). Our day-to-day survival depends upon this refusal of our immune systems to attack the many thousands of potential antigens which are normal parts of our bodies. Failure of this self-tolerance in the case of even just one or a few of our normal constituent molecules results in autoimmune diseases, some of which were listed above: many are fatal.
One consequence of self-tolerance is that it can be difficult or impossible to stimulate animals to make antibodies against proteins which happen to be evolutionarily conservative, such as actin. The mechanism of self-tolerance remains one of the least understood aspects of immunity. A tendency to take tolerance for granted has long characterized the study of immunity.

    Question for class discussion: How could you try to explain self-tolerance in terms of either the original evolutionary selection type of explanation for immunity (#19 above), or in terms of the later instructional type of explanation (#22 above).

34) Monoclonal antibodies are made by growing clones of antibody-synthesizing lymphocytes in culture. Since the antibodies made by each lymphocyte and its mitotic progeny all have exactly the same amino acid sequence in their variable sequence regions, they all have exactly the same specificity. So you can get lots and lots of antibody molecules all of which have exactly the same specificity (exactly the same shaped binding sites). The essential trick was to fuse cancerous lymphocytes (immortal, fast-growing) with normal lymphocytes (slow-growing, but antibody synthesizing); one then selects progeny cells which both grow rapidly and secrete antibody molecules. Quite recently, people have isolated antibody genes and transformed them into bacteria.

35) The differences between B lymphocytes and T lymphocytes: It has been discovered that the effector cells of the immune system fall into two distinct categories. The cells that actually secrete the antibodies are B lymphocytes (or "B cells"), responsible for "humoral immunity". The "T cells", on the other hand, do not secrete antibodies; but some of them produce equivalently specific binding proteins on their surface membranes ("called T cell receptors") that serve the analogous purpose in selectively killing cells that have antigens on their surfaces to which these reecptors will bind. This killing process has a degree of specificity approximately equivalent to that of antibody binding and is the central part of "cellular immunity".
In addition, there are other sub-classes of T cells whose functions are to control the activities of the B cells -sometimes by assisting in their sensitization to antigens, sometimes by stimulating them and sometimes by inhibiting them. T cells can function without B cells, but the B cell part of the immune system becomes non-functional if deprived of T cells. It is for this reason that people born without the B cell part of the immune system can survive much better than those born without the T cell part; and it is also why the disease AIDS, which selectively kills a sub-class of T cell needed for the activation of killer T cells as well as B cells, results in the loss of both humoral and cellular immunity (and was the first evidence of helper T cells).
In general, one can say that resistance to bacterial infection is primarily the result of B cell (humoral) immunity, while resistance to viruses, fungi, protozoa and cancer are primarily the job of the T cell (cellular) immunity. The T cell system is supposed to have evolved first.

36) Graft rejection is believed to be largely due to the activities of T cells attacking the graft tissue, as opposed to being a result of the effects of antibodies. Likewise, the unpleasant effects of poison ivy are (supposedly) due to T cells: somehow the plant's toxin ("urushiol") changes exposed skin cells in such a way that T cells regard them as alien and therefore "reject" the skin cells as they would a graft. More needs to be known about this interesting phenomenon, and such knowledge may prove very useful not only for the treatment of poison ivy poisoning itself but also potentially for the deliberate use of this and comparable toxins to induce the rejection of cancerous cells.

37) There is a theory of "Immune Surveillance" according to which cancers actually arise much more frequently than we realize but that nearly all of them are killed off by the immune system before they can do any harm. This theory was originally proposed by none other than the well-known medical essayist Lewis Thomas and was very popular for several years before becoming somewhat passe. The most relevant supporting evidence is the much higher frequency of cancers which is observed in people whose immune systems are somehow suppressed (for example, by anti-rejection drugs following organ transplants, as well as in AIDS victims). Although this is exactly what the theory predicts, an alternative explanation is that the immune system is protecting us from cancer causing viruses and (perhaps) chemicals, rather than from already-transformed cells.

    Second set of questions for class discussion: How to use the immune system to reject cancer cells, even though they are part of the body itself. Actually, it is not unusual for a person's immune system to attack cancer cells. That may be the cause of many thousands of "spontaneous" cures of what had seemed incurable cancers? Much research has and is being focused on the possibility of stimulating immune attacks specifically on cancer cells, without attacking normal cells.
    a) But if the cancer cells are part of your body, why shouldn't your body be tolerant to them?
    b) And if cancer cells are often attacked by T-cells and antibodies, what does this imply about cancer cell antigens?
    c) How might the immune system be made more likely to attack "self" cancer cells?
    d) How might you try to increase the sensitivity of cancer cells to immune attack?
    e) Suppose that there were forms of uncontrolled cell growth in which the immune system always did successfully attack and destroy the abnormal cells: would such a disease be classified as a form of cancer?
38) The differentiation of T cells has been found to depend upon the cells' ancestors having lived for a while in the thymus gland during embryonic development and early childhood. (That is why they are called T cells: T is for thymus.) This organ is in the throat region, and as recently as two decades ago was thought to be vestigial; it develops from 2 of the pairs of embryonic "gill" clefts, and because it is comparatively large in the embryo and child, but then shrinks in adulthood, the thymus was said to be recapitulating its phylogenetic loss and to have no function. Later it was thought that the lymphocytes arise by the dispersal of the epithelial cells which make up the thymus, but then it was proven that the lymphocyte precursors actually originate somewhere else (apparently the bone marrow), subsequently migrate to the thymus and stay there for periods of months or years, and then disperse to become T lymphocytes. But without this residence in the thymus, they won't differentiate.
These facts were discovered in part because of a type of birth defect in humans ("DiGeorge's syndrome") and in mice, rats ("nude mice", "nude rats") etc. in which the thymus gland either fails to form or is very underdeveloped. The resulting organisms have little ability to resist infections or to reject grafts. It was a puzzle why victims of DiGeorge's syndrome also happen to have a defective calcium and phosphate metabolism, but this was explained by the parallel underdevelopment of the parathyroid gland, which regulates calcium metabolism, and which happens also to be formed embryonically by the same 2 pairs of gill cleft evaginations! However, it still remains a puzzle why the gene which causes thymus underdevelopment in mice should also cause an underdevelopment of hair follicles (causing the mice to be "nude"); a mutation for thymus underdevelopment has also been isolated in rats, and it likewise produces hairlessness! No one understands the connection.

39) It is believed that differentiation of B cells depends upon transient residence in some glandular organ (which thus serves the inductive maturation function equivalent to that served by the thymus for the T cells). In the case of birds, it was proven experimentally (although inadvertently) that this function is served by a diverticulum from their hindguts that is called the "Bursa of Fabricius". If this organ is prevented from developing then the resulting bird is unable to make antibodies because it has no B cells. In fact, the term "B" cells is derived from b in "bursa". Curiously, however, this organ is peculiar to birds and is not found in mammals; so presumably some other organ serves the same function in mammals, possibly it might be the appendix, or the "Peyer's Patches", or the bone marrow itself, but no one knows. There is a widespread hope that the name of the organ responsible will happen to start with the letter B!
Animals lacking B cells cannot make antibodies, of course. This weakens, but does not eliminate their immune resistance to disease. Their T cells can still fight off many pathogens, as well as reject grafts, even without the help of B cells. In contrast, animals that lack the T cell part of the immune system have little or no immune capacity, even to make antibodies. This was once a puzzle; but the explanation seems to be that the stimulation of the B cells to make antibodies depends on the B cells' interactions with certain kinds of ("helper") T cells. Of course, this is also what is believed to to be that basis of immune deficiency in AIDS.

40) The mechanism of the "Generator of Diversity": (G. O. D. as it was called)

The following is a list of some of the alternative hypothetical mechanisms that were seriously considered as possible explanations for how you get the genes coding for a million-plus different variable sequence regions (do you remember which one(s) turned out to be correct?):

A) There could simply be a million different antibody genes (i.e. in the eggs and sperm), each individual gene identical in the part coding for the constant sequence region, but each different (and unique) in the part coding for the variable sequence region.

B) The genome could contain a million different genes for just the variable sequence regions, with one of these being chosen at random (in each lymphocyte clone) and somehow spliced onto the gene for one of the different classes of constant sequence regions.

C) There could be a very, very high rate of somatic mutation in the part of the antibody gene that codes for the variable sequence region..

D) There might not actually be gene for the variable sequence region, at least not in the germ line cells or in cell types other than lymphocytes. Instead, as part of the differentiation, there might be some mechanism for random addition of nucleotides. The net result would be equivalent to the high rate of somatic mutation.

E) There might be only two different genes for the variable sequence region; during the differentiation of lymphocytes, a process of genetic crossing-over (equivalent to that known to occur in meiosis) might occur between these two original variable sequence regions of DNA, even though these regions differed greatly in base sequence. Such crossing-over between genes of very different base sequences would thus generate many, many new sequences (although with certain predictable patterns of either/or regularity; you might want to think about what sort of regularity this ought to have been!)

F) Different clones of lympocytes might have different populations of special t-RNAs with specificities different from those of the usual genetic code.

G) There might be some kinds of special base-altering enzymes that would act on the variable sequence regions of the m-RNAs coding for antibody molecules (with each lympocyte clone having a special set of such enzymes).

H) The variable sequence region of each the different antibody gene might be spliced together out of 2 or 3 randomly selected fragments from series of 4 - 100 (or so) alternative fragment sequences (perhaps something along the lines of "one from column A, one from column B,would you like a potato or rice...and which kind of dressing on your salad: French, thousand island..?).

Do you see why some types of theories (such as A and B) were sometimes called "germ line theories", to contrast them from other types of theories (such as C and D) that were called "somatic line theories"?

    Third set of questions for class discussion: Pretend that you don't already know which one of the 9 theories above turned out to be true (or maybe you don't have to pretend?), and discuss how each of the following sets of facts could be used to argue for or against these 9 alternatives.

(alpha) Mutations had been found that corresponded to amino acid substitutions in the constant sequence regions of the antibody molecules, and these mapped genetically in a simple Mendelian fashion, as if located at a single site on a particular chromosome

(beta) The total amount of DNA in a mammal genome would be barely adequate to code for several million different proteins of the molecular weight of the immunoglobins.

(gamma) The binding site of antibodies is formed along the zone of contact between the variable sequence of the heavy chain and the variable sequence of the light chain.

(delta) As a given animal responds to a particular antigen, it first makes mostly IgM antibodies against it, but later shifts to making mostly IgG antibodies with the same specificity. This occurs at the single cell level, in the sense of individual B-cells shifting from secreting IgM antibodies secreting IgG molecules (i.e. as if keeping the same variable sequence region, but shifting to a different constant sequence region).

(epsilon) Variable sequence regions from different myeloma clones were found to differ much more in some parts ("hypervariable regions") than in the rest.

(zeta) Except in the hypervariable regions, the amino acid sequence of variable regions from antibodies from different myeloma clones tended to fall into patterns in which each site was occupied by either of only 2 (or sometimes of only 3, or 4) different alternative amino acids. For example, at site #22, you might have valines in the antibodies from 8 of 14 myeloma clones, and leucines in the antibodies from the other 6 clones. Likewise, sequence of amino acids tend to be steroetyped, alternating between one particular sequence and another.

(eta) X-ray diffraction crystallography of antibody molecules shows that the variable sequences of both heavy and light chains consistently fold into beta pleated sheet patterns that are very similar to one another (even for antibodies against different antigens) except at the binding site itself (which is small relative to the whole variable sequence regions).

41) Histocompatibility antigens: A graft of tissue from one person to another (unless the two are identical twins) will almost always result in the "rejection" of the grafted cells by the immune system of the host, which attacks them much as if they were pathogens. Much research has been devoted to identifying which particular antigens are most responsible for this immune rejection of grafts (analogous to the A and B antigens, rhesus factors, etc. that are responsible for the analogous rejection of blood transfusions). A whole technology if "tissue typing" has been developed, analogous to the typing of blood. Unfortunately, it has turned out that there are a great many more different variable forms of genes which govern the graft rejection; this means that the probability of finding a "match" between two individuals is enormously lower than is the case with blood. Imagine that instead of just having antigens A and B, our blood had A, B, C, ...X, Y, and Z antigens, a difference in any one of which would result in an immune reaction.
Research on graft rejection has been concentrated in mice and humans. Ironically, this work began with grafts of cancers from one mouse to another; as Medawar once wrote, "people started out thinking that they were using immunology to study cancer, but it turned out that they were really using cancer to study immunology"! Although there are many different molecules at the cell surface, and differences in any of these can potentially stimulate and be the targets of some degree of immune attack, it turned out that there are a few special classes of cell surface molecules which stimulate the immune system much more strongly than any others. These are called histocompatibility antigens, although the term has come to be confined specifically to those coded for by genes located in a cluster called the major histocompatibility locus. They have been most intensivily studied in mice, where they are called the H-2 antigens; the equivalents in humans are called the HLA antigens.
In most species (in mice and humans, for example; but not in Syrian hampsters) there are many alternative forms of these genes, thus reducing the chances of any 2 people being compatible; and this situation is made still worse by the presence of multiple genetic loci in each set of chromosomes, so that each person has several different forms of the antigen, a situation which almost seems fiendishly designed to make tissue and organ grafting nearly impossible. Presumably there is no evolutionary selection pressure against the acceptance of tissue grafts (although some would answer that such selection pressures might have existed in some ancestral invertebrates, such as sea squirts or sponges). We therefore need to ask what functions these antigens have, of which graft rejection is an unfortunate by-product.
The answer was suggested by the structures of the major histocompatibility antigens themselves, which are very similar to antibody molecules and even have antigen binding sites that hold 10-20 amino acid peptides! It is now thought that their normal function is as a sort of molecular "holder" for the purpose of "presenting" partially digested fragments of potential antigen molecules to other cells of the immune system. This is part of the cell-cell signalling system by which the specificity of antibody-antibody binding is ascertained. Thus, when these holder molecules are themselves alien, the immune system responds with special diligence and ferocity.

42) Type I and type II Histocompatibility antigens It also turns out that there are two main classes of major histocompatibility antigens. The type I antigens are the ones originally discovered by tissue grafting; these are found on the outside of the plasma membranes of nearly all the cells of the body, with red blood cells, sperm, and the outer (trophoblast) surface of the early mammal embryo being the only exceptions. In contrast, the type II histocompatibility antigens are found only on the surfaces of a few cell types of the immune system itself (macrophages, thymus epithlial cells, dendritic cells and certain kinds of B cells). The existence of the type II antigens resulted from studies of "immune response genes", some mutations of which are expressed as inabilities to make antibodies or other immune responses to certain specific antigens. These genes code for proteins with molecular structure very similar to the type I antigens, and are very closely linked to them (i.e their structural genes are close together, on chromosome 6 in humans).
There are still many puzzling aspects to histocompatibility antigens:

    A) Why do most species have so many variant genetic forms in the population? B) Why is the possession of certain ones of these variant forms correlated with greatly increased probabilities of certain autoimmune diseases? C) Why are the genes for different forms of these antigens so closely linked to one another and to other genes which regulate the activity of the immune system, such as those for several lymphokines?

43) The activities of the various kinds of T and B cells are controlled by feedback cycles, many of which involve sending signals from one cell to another. The signaling mechanisms can be divided into two categories, those that involve direct contacts between cell surface molecules, and those that are accomplished by hormone-like proteins that are secreted by one cell and reach others by diffusion. These proteins are called "lymphokines", and around a dozen have been identified. Interferon is one of the best known (one should rather say, "the interferons" since there are 3 main kinds). The artificial synthesis of interferons and other lymphokines has been accomplished by cloning the genes for them into bacteria; this is a very active area of pharmaceutical research because of the prospect of being able to control and manipulate the activities of the immune system. Interferons and several other lymphokines are now being used as experimental treatments for cancer, following more conventional chemotherapy. Unfortunately, the interferons produce the same side effects as having the flu fever, headache, aching joints, etc.! When you have the flu, your body is stimulated to produce lots of interferon, which is a large part of what makes you feel so bad.

44) A radically selectionist hypothesis about learning and the brain: You might be interested to know that Gerald Edelman, who shared the Nobel Prize for work on the molecular structures of antibodies, and later discovered new kinds of cell-cell adhesion proteins, has now moved on to neurophysiology. He has proposed a new and exciting hypothesis to explain how people learn new skills. He calls this new theory "Neural Darwinism" and has written a book with that title. His proposal is reminiscent of Jerne's and Burnet's, the basic idea being that the brain initially generates a large number of different neural wiring circuits (sort of like computer chips, or maybe pocket calculators) made with many different randomly-chosen wiring patterns, so that each circuit differs in capabilities. Edelman's idea is that these circuits play approximately the same role as the lymphocyte clones. Learning is then supposed to be selective, in the sense that each different circuit gets "tried out" to see which ones produce desirable consequences. The circuits that produce bad results are discarded or destroyed, while those that yield good results are kept and reduplicated. This may sound impractical or crazy, but so did clonal selection, at first. Do you think neurophysiology could now undergo a revolutionary paradigm switch from instructionism to selectionism?
There are some interesting parallels to Socrates' theory that education was a process of recalling of forgotten information, probably from previous reincarnations!! This notion was the original motivation for his famous "Socratic method" of teaching, although few people realize this.
Past thinking about learning has nearly all what one might call "instructionist", going back to John Locke's metaphor of the mind as "Tabula Rasa", Latin for "blank slate", on which experience writes things into your memory. Piaget's ideas also seem very "instructionist" to me. Learning is assumed to be a matter of putting something into the brain, or of creating something new there, as opposed to picking and choosing between different things that are already there (or differentially strengthening and weakening pre-existing patterns). On the other hand, Noam Chomsky, (MIT professor) revolutionized linguistics by providing strong evidence that babies and young children could not possibly learn correct grammar as rapidly as they do (i.e. having heard as few spoken sentences as they have) unless some kind of very abstract grammatical rules were already genetically programmed into the brain at birth (sort of like the ROM chip in a Macintosh computer!). Note that there would have to be one set of abstract rules for all languages. Several people have tried unsuccessfully to deduce what these rules might be. The great British zoologist J. Z. Young had previously proposed theories comparable to Edelman's "neural darwinism" based on many years of experiments on behavior and learning in captive octopuses!! Science moves along strange paths!

    Fourth set of questions for class discussion: What do you think goes on in your brain when you learn a new concept? Do you suppose that it's more like a multiple choice test, a true-false test, or a fill-in-the-blank? Are some people really smarter than others? Is it that smarter people have learned more; that they can learn more; or perhaps that they are faster or otherwise more skillful at learning? If Edelman were right, what would I.Q. be, at cellular and molecular levels?

1) Do you see why it is essential that each clone of B lymphocytes should make antibodies against only one single antigen (i.e. that all the antibodies made by a given clone should have exactly the same amino-acid sequences in their variable sequence regions)? Suppose that each lymphocyte clone made two different antibodies, with binding specificities for two different antigens. What would be some of the undesireable consequences of having some lymphocyte clones make more than one antibody? Would there be any advantages?

2) Mass epidemics of smallpox and other diseases decimated the native populations of the Americas soon after Columbus' voyages (and a comparable epidemic of syphilis occurred in Europe ). The usual explanation is that the natives had not "evolved immunity" to the germs. Is this consistent with current theories of immunity, or does it seem to reflect older assumptions? Discuss what the true explanation might be, whether this might require some further "revolution" in our theories, and what sorts of experiments or observations might be relevant to the question.

3) To what extent have the various theories of immunity conformed to Karl Popper's ideas about the best hypotheses being those that are most susceptible to disproof? Can you suggest some examples in which immunological theories now believed to be correct would once have actually seemed to have been conclusively disproven?

4) To what extent does the intellectual history of immunology conform to Thomas Kuhn's ideas? What were the different "revolutions" that occurred in this field, and what were the alternative "paradigms" that replaced one other? In particular, can you trace a "swing of the pendulum" back and forth between instructional and selectionist types of theories.

5) It has been said that "Any complicated phenomenonon has to be invented before anyone can discover it. Unless you have already considered the possible existence of a phenomenon, then how will you be able to recognize it even if you see it.?" Do you think that this is generally true in science? To what extent do you think that it may be true in daily life, in general? Can you give some examples, either from the history of immunololgy or from some other field, where this assertion seems to you to be true, or where it seems to be untrue? Do you think that maybe it tends to be more true of complex phenomena? If so, then how complicated does a phenomenon have to be before this statement becomes true?

6) Can you give some examples, either from immunology or from some other field, in which erroneous theories nevertheless made correct predictions.

7) As new theories develop, are modified, or replace one another, they often are shown to cover a wider and wider range of different phenomena. Conversely, progress sometimes takes the form of realizations that certain phenomena, although previously thought to share the same cause, are really separate. Can you describe some examples of this from the history of immunology? Consider the following list of phenomena: which ones are (and which are not?) explicable by today's immunology?

    (a) immunity to diseases from which one has recovered.
    (b) allergy.
    (c) "immunity" to such things as poison ivy.
    (d) "immunity" to poisons which one has been exposed to repeatedly
    (as in the ancient story of King Mithridates, and in one of the Peter Wimsey murder mysteries).
    (e) reactions to blood transfusions.
    (g) graft rejection.
    (h) rejection of grafts of cancers from one animal to another.
    (i) occasional spontaneous recoveries from cancer.
    (j) accumulation of proteins in the urine of some lymphoma patients.
    (k) autoimmune diseases.
    (l) correlation of immunodeficiency syndromes with calcium imbalances.
    (m) failure of hair development in immunodeficient mice.
The greatest remaining unsolved problem in immunology (in my opinion) is self-tolerance, the reasons why self-tolerance fails in autoimmune diseases, and finding methods for re-estabishing normal self-tolerance in order to cure Lupus, Multiple Sclerosis etc. Any method for restoring tolerance in victims of these diseases would lift an enormous weight of suffering off the backs of millions of human beings.

Another unsolved problem is how to use the immune system to kill cancer cells specifically. Because cancer cells are merely behaviorally-abnormal versions of your own cells, you should expect every animal to be just as self-tolerant to its own cancer cells as it is to all of its other cells. In fact, however, there is often evidence of immune attacks on tumors; many cases of "spontaneous" remissions of cancer in humans seem to result from some activity of the immune system; in addition, much medical research has been devoted to stimulating the immune system to attack cancer cells.
Injection of cytokines, more specifically interferons, following cancer chemotherapy, has been tried experimentally for about the past ten years, but with mostly disappointing (i.e. no) results.

In the specific cases of B-cell and T-cell lymphomas (almost? all Non-Hodgkin's lymphomas fall into one or the other of these categories), it is to be expected that all of the cancerous cells in a given patient will be members of a single original clone, so that their antibodies (in the case of B-cells) or their T-cell receptors (in the case of T-cells) will have binding sites of a given amino acid sequence and shape. Perhaps you can imagine cures based on inducting other cells of the immune system could to attack specifically any cells having surface molecules shaped like these binding sites (i.e. the binding sites specific to that particular person's lymphoma cells). Reading between the lines of some of the newspaper stories about former Chancellor Hooker's attempted "experimental treatment at Johns Hopkins" may have been based on this general approach. Newspapers strive to exclude factual information that some of its readers would not understand; better nobody should know the facts than have some of the readers receive information they are not educated enough to understand?

A special class of T-lymphocytes is needed to stimulate the function of B-cells and other T cell. These "helper T-cells" are selectively killed by the AIDS virus, thereby weakening the whole immune system to the degree that the person dies of bacteria, fungi or other pathogens to which everyone is normally immune. Preventing this, or re-establishing helper T-cells, or perhaps substituting for their functions (in some other way stimulating B & T-cell function) are all possible ways of curing AIDS.

The specific enzyme(s) that serves to recombine the V(D)J sequences is called RAG. It is an important question when and where the genes for the RAG enzyme are expressed.

There have been reports that the RAG genes are expressed (=the messenger RNAs transcribed & the proteins made) only during the early development of B-cell and T-cells.

There have also been reports that RAG may be expressed in certain cells of the developing brain!

It has been controversial to what extent RAG genes may be expressed (& V(D)J recombination occur?) later in life in B cells as part of their response to exposure to their antigen! There is a report in this week's Nature that GFP (Green Fluorescent Protein) - RAG fusion genes are expressed in B-cells after antigen presentation. Copies of that paper will be handed out in the next class.


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