A, B, O and We

I had an interesting discussion with Ganaraj, that ended up in some unanswered questions on Blood Group Antigens and Antibodies. He was so interested in the Immunophysiology of the Blood Group antigens that he really dragged me to the basics. By the way, I love it when something goes to its basic. Before I throw up those questions, let me give you some background story under which he came up with those questions.

We were discussing on the issues of importance of blood groups in determining the possible consanguinity in a marriage. I told him, it's hard to estimate the genetic resemblance of the partners by blood group alone. Blood Group antigens obey a certain inheritance pattern and represent one or a few specific genes but there are million others to be considered! He then raised his question of the child getting affected by the mother's immune system if the child belongs to a blood group other than that of the mother. He was concerned about the Hemolytic Disease of the New Born. To explain that, I had go back to the basics. By the way, Ganaraj is a techie and he is no way "academically" concerned with the life sciences. Here's what I explained him.

What is an Antigen? Before jumping on the properties of an Antigen and all that stuff, Antigen is basically a Protein. And so, it has all the basic characteristics of a protein, plus some other special qualities. Further dissecting, Proteins are complex molecules, with amino acids as their building blocks. There are 20 different amino acids which all the living beings on Earth use to synthesise a protein. The "number" of amino acids and the "sequence" of the amino acids in a protein is determined by the Master Code, "DNA." So, that makes it clear - One "set of DNA (or a Gene)" codes for one 'set of amino acids' (i.e., a Protein)! So far so good? "Yes," he said, "Continue."

Alright, Antigen is a protein, mostly a protein. There are some Polysaccharide Antigens and many others, but to keep it simple, I am not considering them. Once again, antigen is a protein, made up of amino acids, the number and sequence of which are determined by a Gene. That is common to any protein. Apart from that, Antigen is special in that it is found on the surface of a cell. In other words, Antigens are found in the membranes of the cell and that makes Antigen a cell-membrane-protein! At the molecular level, these antigens are the "identity marks" of a cell which are very important in "communicating" with other cells. It is like an "Username" and "Domain" for you email address - using the same alphanumeric code (A-Z, 0-9) multiple email addresses can be created which are "unique" to an user. Similarly, the amino acids can be arranged (as dictated by their DNA sequence) and used as "username" by the cells while they communicate. "I am with you," Ganaraj allowed me to continue.

Let's go back to the time when we slept comfortably in our mothers' wombs. Ahh.. How comfortable it was! Anyways, while we slept carefree, there were million things happening at the molecular level because of which we are able to now breathe comfortably. We began our journey as a single cell with its DNA containing every single information needed to build this heavy functional machinery. That cell underwent divisions and multiplications to form a big bunch of cells. All of them had the same DNA but somehow, one group of cells decided to become Liver, some went on to become Heart, some became Brain and so on. How did this happen? Though they contained information about everything, they chose to express only what they wanted. They sort of "specialised" in their expression of the Genetic code. So, each of those cells chose an "username" for themselves. The Immune System was also developing at this time; to keep it simple, Immune system means White Blood Cells (WBCs), and it was quietly building its "Contacts List." As blood circulated to almost all the cells, these White Blood Cells went on communicating with them. Every time it got a "friend request," it identified the "Antigens" on the cell membranes and added those "Usernames" to its "contacts list." By 5th to 6th month of our life in the womb, its "Address Book" becomes complete and it would no longer accept any friend requests. The Spam Guard is now turned on, so to say!

So, from now onwards, any emails from persons NOT in the address book will be reported spam and dealt seriously; I mean, after this maturation of Immune system, if any protein other than the "Self Protein" enters the blood stream, it will be recognised as "Foreign" and an antibody response will be triggered. I have explained in my previous blog "Molecular Battlefield," how an antibody generation is triggered. Antibodies are produced by "specialised" White Blood Cells when the body is exposed to a "Foreign" Antigen. Antibodies are complimentary in structure to that of the Antigen and they fit exactly into their respective antigens like a "lock and its key." Oh, by the way, even antibodies are proteins and these are "dissolved" in the "fluid" of the blood, unlike antigens which are found on the membranes of the cells.

"Wait a minute," Ganaraj interrupted me at this time. "What about all those proteins we eat daily? Do they generate an antibody response?" Great question, you see. Perfectly logical. Yes, they would trigger an antibody response, IF ONLY they were to directly enter our blood stream. But our digestive tract is very efficient in digesting the proteins. The Hydrochloric Acid in the stomach and other digestive juices and enzymes in the intestines break down the proteins to their basic building blocks. No matter what protein you ingest, ultimately, it gets broken down into those 20 amino acids which will be absorbed by the intestines and sent into the blood stream. Amino acids are too "light" in terms of Molecular weight and hence, they do not trigger an Antibody response.

Now let's consider the Antibodies to these Antigens. According to Landsteiner's Law (after Karl Landsteiner, an Austrian Pathologist), if an Antigen is present on the membranes of the RBCs, it's corresponding Antibody will be absent in the blood; if an Antigen is 'absent' on the membranes of the RBCs, then it's corresponding Antibody 'must be present in the blood.' Computing this law for the ABO system of the blood groups, we can tabulate certain conclusions as depicted in the following diagram:

"Sounds logical," said Ganaraj and allowed me to continue. I then, described the other medically important system, Rh. The "Rh Positive" individuals have an "Rh" antigen on their RBCs and hence, do NOT contain Anti-Rh Antibodies in their blood. The "Rh Negative" individuals do NOT contain "Rh" Antigen on their RBCs... "I got it," Ganaraj interrupted. "And therefore, they contain Anti-Rh Antibodies in their blood!"

"No. That's wrong," I continued. "An Rh-Negative individual does NOT contain Anti-Rh Antibodies in his blood! This is an exception to Landsteiner's Law." "Oh! OK," he allowed me to continue, but he was not satisfied with the explanation. I continued, "But when an "Rh Negative" individual gets exposed to "Rh" Antigen, he would then start producing Anti-Rh Antibodies." This one was logical, and he accepted this.

So, let's consider a woman who is "Rh Negative." If she gets pregnant with an "Rh Positive" baby, what would happen? "Nothing. Why should something happen?" Ganaraj was clear but I wanted him to know something more. Yes, nothing would happen until the delivery but during the delivery, some amount of baby's blood would enter the mother's blood due to rupture of Placenta. Now, the baby's RBCs carrying "Rh" Antigen would be treated as "intruders" by the mother's immune system and she would start producing Anti-Rh Antibodies in about 72 hrs. "By this time," Ganaraj added correctly, "the baby would be out and safe!" Yes. He was right. By the time she produces Anti-Rh Antibodies (3 days after delivery), the baby is no longer dependant on the mother's blood for nutrition! But the problem is, the next time she gets pregnant with an "Rh Positive" baby, she would have already had Anti-Rh Antibodies circulating in her blood and that would cross the placenta and attack the second baby's RBCs. That is what is medically termed "Hemolytic Disease of the New Born."

"Can we NOT prevent this blood-mixing at the time of delivery?" he questioned again. It's practically impossible, as the mixing occurs at microscopic levels. But there's hope. After the delivery, we have 3 days at our disposal to prevent the mother's immune system from producing Anti-Rh Antibodies. What we can do is, inject some "Neutralising Antibodies" which would mask the baby's RBCs carrying "Rh" Antigen from being recognised by the mother's immune system and that's exactly what is done routinely.

"But, I have another question," he continued. His earlier suppressed doubt now surfaced up. "Well, going back to the basics, Antigen is something that is present since birth. Antibodies are produced when the immune system is exposed to "Foreign" antigens. How can a O+ve individual produce Anti-A and Anti-B antibodies without being exposed to the "A" and "B" Antigens? I mean, what's the basis of Landsteiner's Law?" His question was perfectly logical. Even I had this question in my mind when I was in my med school. With all due "respects" to my Alma Mater, my physiology professors were so lousy that I dared not to ask them questions, that made sense! Fortunately, now I am associated with someone who is at the other end of the spectrum and he doesn't mind even if I ask him the silliest of the questions. So, I paused the discussion with Ganaraj until I discussed it with my professor.

Meanwhile, I opened this discussion on Facebook and some of you contributed to that. Anirudh came up with an idea of exposure to "A" and "B" antigens through gut (digestive tract). I was apprehensive about two issues at that time - how would an "A Positive" blood enter the digestive tract of a O+ve individual, unless that O+ve individual "drinks" it (as a contaminant)? OK, even if it enters, it should be broken down into amino acids by the digestive juices. Even if it "somehow" enters the blood stream triggering Antibody response, why doesn't the same phenomenon occur to Rh system? Then, Anirudh came up with another interesting approach. Exposure of "A" and "B" Antigens through the placenta during our "womb-days." I had two more issues at this time - what if the mother is also O+ve? How can she expose her baby to the "A" and "B" Antigens? And also, if it is exposed during the "womb-days," the immune system should recognise those antigens as "Self-Antigens" and should NOT mount an Antibody response!

With all these doubts, I went to my professor. Before he answered, he first took time to appreciate the questions! That made me comfortable and curious as well. He explained, "There are certain strains of E. coli (an harmless bacteria in our digestive tract) and some of their antigens resemble the Human "A" and "B" Antigens in their molecular structure. This is a sort of 'Antigenic Mimicry'!! Blood group "AB" individuals recognise these antigens as "Self Antigens" and they do NOT mount any response; the "A" individuals mount response only to "B-resembling" Antigen; the "B" individuals mount response only to "A-resembling" Antigen; and the "O" group individuals mount response to both "A-resembling" and "B-resembling" Antigens.

"Wow," I exclaimed, "that's interesting!" "That's not all," he continued. "Mounting these antibodies is believed to help the individual in fighting off the E. coli, if it 'misbehaved' and so, blood group "O" individuals are able to better tolerate the infection than "AB" individuals. That's one of the explanations as to why there are more "O" group individuals in the world than are the "AB" group individuals!"

"So, this can be explained by Darwin's theory of Natural Selection?" I became even more curious. He explained me that I was right in guessing. "If you look at the DNA sequence of the Gene coding for "A" Antigen, it very closely resembles the DNA sequence of the related Gene in "O" group individuals. So, it means, the "A" Gene must have mutated and got silent, without expressing any Antigen (that's what is "O" group) and this mutation must have given a "survival benefit" to the individuals in fighting off the bacterial infection and hence must have been Naturally Selected and propagated!"

"Ahhh..." I took a nice long audible breath. That was absolutely a brilliant explanation. But my professor, had got something more to add to the believed theory. "This observation of 'there are more O group individuals than A' holds good only in the tropical countries. If you see the Scandinavian countries, there are equal or more "A" group individuals than "O." My experience on the study of Malaria parasite tells me that there are certain "Malarial Antigens" which resemble "A" and "B." So, I believe, this "O" group must have added benefit against Malarial infection also!"

"That gives us a whole new dimension to look at Malaria and the blood groups!" I was thrilled. "But what about the Rh system?" I asked him again. "Oh, Chethan, that's another interesting story," he continued. "Karl Landsteiner and Alexander Wiener were working on the blood groups when they were unable to explain an adverse reaction. Some "A" group individuals developed adverse reactions even if they were given another Group "A" blood. This made them think, there must be another system of blood group and they went in search of that. Wiener observed that humans and monkeys (a Rhesus monkey) shared a common antigen. He took the blood of the monkey, injected into Guinea pigs to get trigger antibodies against that antigen and used those antibodies to classify the human blood. Blood of some individuals "reacted" with those antibodies while blood of other individuals did NOT "react" to them. Those that reacted, were believed to be having a "Rhesus" Antigen and they were termed "Rh Positive" (Rh stands for Rhesus) and others were "Rh Negative." Including this system in the blood grouping reduced the no. of 'adverse reactions' and this came to be known as the "Rh System."

"But there was some confusion about the claim. An obstetrician, during those times, had observed the babies getting affected by mother's immune system. He used the serum (fluid part of the blood) of those mothers to classify the human blood. Some blood samples reacted positive and some reacted negative. This classification very closely resembled Wiener's Rh System. And hence, there was dissatisfaction regarding the claim of "Rh" Antigen. Careful studies have now shown that, what Landsteiner and Wiener had discovered is NOT "Rh" Antigen but something else. It is now named "LW" Antigen, LW standing for Landsteiner-Wiener. What the obstetrician had discovered was the actual "Rh" Antigen. Therefore, although Rh stands for Rhesus, Rh antigen is not found in monkeys. It's unique to human blood. "LW" antigen is what humans share with monkeys!"

His explanation was not at all esoteric. It was an easy listening to me, as if I was listening to a piece of instrumental music - a soothingly joyful experience. In simple words, he had explained me a whole chapter of physiology. It was as if teaching me, "If you want to convey something effectively, keep it simple!"

MOLECULAR BATTLEFIELD

I would like to take you into a different world. I am no J.K. Rowling to create a fictitious world. The one we are going is real. Discovered a few centuries ago, this is an elusive world with impeccably arranged moieties which pose perpetual challenges to the understanding of their functions. There are times and places where these moieties exist in reconciliation; but many a times they are engaged in the battles fiercer than the battle of Kurukshetra or the World Wars I and II. There exist peace-keeping bodies like UNO but these battles are inevitable in the sense that they aren’t fought for greed or jealousy. These battles are fought for survival where there is absolutely no mercy for the timid.

I would not have taken you to this world if it were not for Ganaraj who insisted me to take him there. Though it wasn’t the first time I was visiting, with him I found it more vivid than before. The more I explained him, the more I learnt myself. I remember, my physics teacher (Mr. K. S. Murugesh Sir) used to tell us, “You should write your answers in such a way that even the last fool in the universe should understand physics by reading them.” Well, that’s the way I learnt physics. But now, I was asked to answer a biology question in that way…

Ganaraj asked me, “Why does one get fever?” It was 13^th of February. “Well, it isn’t uncommon for a guy like you to get fever on the eve of Valentine’s!” I chuckled, not knowing that he was running a mild fever since some days. He is a techie who analyses the problems to depth before trying to fix them. And so, I had to explain him Immunology in a way I had never done before.

Just as a kingdom’s territory that has some natural barriers like mountains or river or sea at its boundary, the human body too has some natural barriers – viz. Skin (the largest organ in the body), Hydrochloric acid in the stomach that kills microbes in the contaminated foods, the hair and mucous in our nose that trap microbes in the contaminated air etc..

If the bugs somehow manage to cross these barriers and try to gain access into our body, the war begins. Most tissues have got cells called ‘Macrophages’ (literally meaning, ‘Big Eaters’) which function as scavengers. They keep ‘eating’ dead materials (from turnover of worn-out cells) and when they feel there’s some foreign material, they get alert and ‘blow the conch’ for the battle. The white blood cells (WBCs) are the soldiers that are constantly circulating in the blood stream. So, when the macrophages need help, they release ‘cytokines’ or ‘chemical messengers,’ that signal the circulating ‘soldiers’ (WBCs) to move into the place of battle; just as you dial 911 (or 100 in India) when you are in danger. One such cytokine is Interleukin-1 which alters the “temperature regulator” in the brain. It is like setting the ‘thermostat’ at a higher temperature and the body temperature goes up. That’s how Ganaraj was having fever! (So did we all!)

There are different types of WBCs, each of which is a ‘specialist’ in the use of different ‘weapons.’ First ‘Neutrophil’, a kind of WBC, visits the battlefield, makes a general survey and releases some more ‘chemical messengers’ to wake up the ‘sleeping soldiers’ and attack the enemy.

As I said earlier, this battle is going to be fierce and in battles it’s not just the invaders that die; thousands of soldiers and civilians too. To minimise as much damage as possible, the army works on sharpening the target. Once macrophages ‘gulp’ the invading microbe, they process it and the ‘key features’ (antigens) are documented and conveyed to the ‘soldiers’ so that they can distinguish the enemies from the civilians – the process is termed “Antigen Processing.” This is something like the FBI’s way of taking the photographs of the criminals and circulating them with a “Wanted” stamp. Once the enemy is identified (or the antigen is processed) it is taken to the Helper T-cells or the “Commander-in-chief” of the army. Helper T-cells or CD4+ T-cells are also a kind of WBCs. This is the prime target of the notorious virus HIV; HIV kills the commander-in-chief of the army, soldiers go helter-skelter and the army disintegrates. And so, the open door tempts even a saint!!

The commander-in-chief (Helper T-cell) coordinates between the other specialists of the army. It does so by means of ‘cytokines’ or chemical messengers. There are varieties of cytokines, each one carrying a specific message. It is like a Morse code. One specialist of the army is Cytotoxic T-cell, a kind of WBC, comparable to a ‘bombing expert.’ Once the Helper T-cell sensitises the Cytotoxic T-cell with the ‘photograph’ (antigen) of the enemy, it goes in search of the microbe and kills it. As I said, it does so with ‘bombs’ which claim the lives civilians as well!

The other specialist is a B-Lymphocyte or B-Cell, again a kind of WBC, comparable to a ‘missile expert.’ Again the commander-in-chief, Helper T-cell awakens the B-cell by showing the enemy’s photographs (antigens). B-cell generates antibodies (missiles) against the antigens. The antigen’s structure is assessed by the B-cell and the antibody it generates is complementary in structure to that of the antigen, so that the two fit exactly into each other; something like a lock-and-its-key or a box-and-its-lid or simply a USB-port-and-USB-connector! These missiles or the antibodies are released into the blood stream and wherever they encounter the microbe they fasten themselves to their respective antigens. With antibodies sticking on to them, the microbes become docile and thereupon they can easily be destroyed. Again scavengers (macrophages) come to site of destruction and ‘mop up’ the debris. Later on new cells deftly replace the lacunae.

That’s not the end of it all. Some of the ‘specialist’ cells remain quiescent. Not because they are sloths but because they are used as ‘Immunologic Memory.’ It is like adding a copy of the file to the database for future references. In due course of time, should the same microbe invade the body again, the ‘soldiers’ need not waste time in analysing the antigens. The memory cells form copies instantly and attack the bug more vigorously and efficiently. This amazing property of our immune system is the basis for Immunisation. Immunologists artificially process the bugs and present the antigens in the form of vaccine. The immune system gets acquainted with the bug’s antigens and forms ‘memory’ and whenever the infection with that bug occurs; it will be a cake walk for the ‘soldiers.’ The ‘boosters’ we receive every-once-in-five-years or so are meant to trigger our fading Immunologic ‘memory.’

What an immaculate mechanism! I had wondered. It may have taken millions of years of evolution but every day is worth it, I had felt, during the first year of my medical school. But if it were to be as perfect as I had imagined, I would not have to study the subjects in the later years of my medical school! Oh, yes, I mean it! As I moved on, I understood that despite such a composite organisation, things go wrong. I saw a few cases where the ‘Morse code’ was ‘misread’ and resulted in catastrophes beyond imagination. These are the Disorders of Immune System – autoimmune disorders, hypersensitivity (commonly referred to as ‘Allergies’) and Immunodeficiency disorders etc. I also saw the specimens in the pathology museum; sufferings in almost all the wards of the hospital because of bugs that somehow managed to delude the immune system.

No amount of complexity is sufficient. The bugs take it as a challenge and evolve into ‘stronger’ bugs. Antibiotics are no reason to open champagne. Bugs are amazingly brilliant in developing alternate biochemical pathways to elude the harmful chemicals. If we don’t struggle we lose the battle; and complacency is our greatest enemy. So, the quest for perfection continues… Who’s next?