Elsewhere in this blog, I have referred to how DNA damage can be repaired through a combination of homologous recombination and base excision repair. The well known mediators of homologous recombination are BRCA1 & BRCA2. Women who carry defective BRACA 1 or 2 allele are at increased risk of breast, and in the case of BRCA2 loss, breast and ovarian cancer. Men who have lost a wild type BRCA2 allele are also at increased risk of breast cancer.
It is thought that breast or ovarian cancer develops when the remaining healthy allele becomes mutated in the adult- a process called somatic mutation, to differentiate it from germline mutations, which are inherited.
Those with defects in both alleles of BRCA1 are thought unlikely to survive beyond a very early stage of their life- perhaps succumbing in utero or in early childhood. But what happens to those with two defective BRCA2 alleles at birth?
We didn't know, until now.
An illuminating article in the New England Journal of Medicine reveals that BRCA2 is in fact identical to FANCD1, one of the twenty proteins for whom defective genes have been found in Fanconi's anaemia, an autosomal recessive childhood onset severe anaemia with reduction of other cell lines as well, resulting in an aplastic picture.
It is thought that patients with Fanconi's anaemia are very susceptible to DNA damage through cross linking, such as by exposure to radiation. These patients are also susceptible to neoplastic processes such as acute leukaemias, which are curiously much more sensitive to agents such as cisplatin than ordinary leukaemias. It is thought that this is because of their cross linked DNA.
The twenty defective genes in Fanconi's anaemia are thought be responsible for DNA repair through homologous recombination. The process is started off by coming together of two proteins called FANCD2 and FANCI, which then associate with other downstream proteins, one of which is FANCD1....or BRCA2.
It is now realised that those with loss of both BRCA2 alleles through germline mutations develop Fanconi's anaemia, while those with loss of one allele are at increased risk of breast and ovarian cancer in life. Both processes are due to an inherent defect in DNA repair through homologous recombination.
Not surprisingly, defects in some of the twenty genes responsible for Fanconi's anaemia have also been described in other cancers. An innovative way of increasing the susceptibility of such cancers to cisplatin like chemotherapeutic agents is to expose such cells to DNA cross linking agents, as was demonstrated in Fanconi's anaemia.
Another disorder that epitomises the consequences of defective DNA repair is ataxia-telangiectasia, in which the fault lies with a gene described as ataxia-tengiectasia-mutated. Such patients present with immunodeficiency in addition to the obvious symptoms of ataxia & telangiectasia.
It's quite clear that some cancer genes give rise to more than one phenotype...and not just cancers either.
Sunday 30 May 2010
Sunday 23 May 2010
The Hardy Weinberg Distribution
The Hardy Weinberg distribution allows you to find out the prevalence of homozygotes and heterozygotes for given alleles if you know the overall prevalence of the disease.
Thus, say a trait has two phenotypes, a and b. "a" has an allele called p and "b" has an allele called q.
The prevalence of homozygotes for p or q would be p^2 and q^2 respectively. The prevalence of heterozygotes would be 2pq.
This is easy to understand. Say if you dip into a bag containing two types of marbles- a & b, whose proportions are p & q respectively. The chances that you pick out "a" first time is of course, p. The chance of picking "a" twice in a row is p*p, and similarly the chances of picking "b" back to back is q*q.
If you picked "a" first, the chance that you'd pick "b" next is of course p*q. If you pick "b" first the chance that you'd pick "a" next q*p. Thus the chance that you'd pick a combination of a & b in two attempts is 2pq.
Let's look at two applications of this.
An African boy has sickle cell anaemia, an autosomal recessive disease with a prevalence of 1 in 500 among Blacks. His sister is getting married to a man of same ethnic origin, and wants to know the odds that her partner, who is unrelated to her, is a carrier.
For an autosomal recessive disorder, cases are obligate homozygotes, and therefore will have their distribution given by p^2. Thus, p^2= 1/500, or p= sq root 1/500= 1/22 (approx).
Thus, the likelihood that her husband would be a heterozygote would be 2pq or 2*1/22*21/22= 1/11 (approx).
Let's take another example. Haemophilia has a population prevalence of 1 in 5000. What's the likelihood that a random female would be a carrier?
Since haemophilia is an X linked trait, the prevalence of the disease is the same as that of the defective allele (since affected boys carry the defective allele on their only X chromosome). Thus, p= 1/5000.
Therefore the chance that an unselected female would be a carrier is 2pq= 2*1/5000*4999/5000= 1/2500.
The Hardy Weinberg distribution assumes that there is no inbreeding among relatives. In practice, many rare disorders express themselves more frequently in communities who inbreed. I'll deal with ways to get around this in another blog entry.
Thus, say a trait has two phenotypes, a and b. "a" has an allele called p and "b" has an allele called q.
The prevalence of homozygotes for p or q would be p^2 and q^2 respectively. The prevalence of heterozygotes would be 2pq.
This is easy to understand. Say if you dip into a bag containing two types of marbles- a & b, whose proportions are p & q respectively. The chances that you pick out "a" first time is of course, p. The chance of picking "a" twice in a row is p*p, and similarly the chances of picking "b" back to back is q*q.
If you picked "a" first, the chance that you'd pick "b" next is of course p*q. If you pick "b" first the chance that you'd pick "a" next q*p. Thus the chance that you'd pick a combination of a & b in two attempts is 2pq.
Let's look at two applications of this.
An African boy has sickle cell anaemia, an autosomal recessive disease with a prevalence of 1 in 500 among Blacks. His sister is getting married to a man of same ethnic origin, and wants to know the odds that her partner, who is unrelated to her, is a carrier.
For an autosomal recessive disorder, cases are obligate homozygotes, and therefore will have their distribution given by p^2. Thus, p^2= 1/500, or p= sq root 1/500= 1/22 (approx).
Thus, the likelihood that her husband would be a heterozygote would be 2pq or 2*1/22*21/22= 1/11 (approx).
Let's take another example. Haemophilia has a population prevalence of 1 in 5000. What's the likelihood that a random female would be a carrier?
Since haemophilia is an X linked trait, the prevalence of the disease is the same as that of the defective allele (since affected boys carry the defective allele on their only X chromosome). Thus, p= 1/5000.
Therefore the chance that an unselected female would be a carrier is 2pq= 2*1/5000*4999/5000= 1/2500.
The Hardy Weinberg distribution assumes that there is no inbreeding among relatives. In practice, many rare disorders express themselves more frequently in communities who inbreed. I'll deal with ways to get around this in another blog entry.
Saturday 15 May 2010
Forensic Genetics- Righting A 30 Year Old Wrong
Recently, Raymond Towler was freed from a US jail on DNA evidence after being cleared of a rape conviction handed down 29 years ago.
Forensic genetics is still work in progress. However, it's not new. Thousands of 9/11 victims were identified through their DNA after the WTC catastrophe from their remains.
The very first use of forensic genetics was in 1986 in the UK. Two women, raped and murdered in Leicester in similar fashion 3 years apart, triggered an arrest. The suspect (bizarrely, as it turned out) confessed to the first murder, but not the second. The police approached Sir Alec Jeffreys at Leicester University, the then Professor of Genetics, for help. Based on DNA analysis from the murder scenes, the suspect was acquitted. A second man, Colin Pitchfork, was subsequently found guilty of both murders. He had escaped justice initially by volunteering his friend for a DNA sample he purported to be his own.
So how does DNA fingerprinting work? There are several techniques, but the most useful one makes use of short tandem repeats (STRs) present in the DNA sequence, usually comprising 4 bases such as GATT. The length of a STR is unique to each person. Each STR of course occupies a certain locus on a given chromosome, and as each chromosome is paired, each person possesses two STRs for a given locus, usually of varying length. Part of a sequence is denoted with a point (for example GATT GATT GATT GATT GA would be denoted as 4.2). If there are 4 repeats of GATT on one cheomosome, and 7 repeats on its allele, that is denoted as 4/7.
The USA has a national DNA database called CODIS, which contains around 5 million DNA fingerprints, a number similar to that held in the UK. CODIS is based on 13 different STRs, giving a likelihood that a given sequence will be matched of 1 in 1 trillion. The UK uses 10 STRs, giving a likelihood of match of 1 in a billion.
The frequency of a given allele in the population is given by the Hardy Weinberg equation. For a homozygous allele p, the frequency is given as p^2, while for heterozygous alleles p & q, the frequency is 2pq. To find the probability that all the STRs present in a given person could be replicated in another person, you multiply the probability of finding each STR in a given ethnic group. Therefore, the more STRs you use, the greater the certainty that no two individuals would have the same profile.
Sometimes DNA from victims can be badly degraded because of time elapsed, making STR analysis difficult or incomplete (say 8 STRs possible instead of 13). Under such circumstances, geneticists exploit single nucleotide polymorphisms (SNPs). These are single base differences that occur every 100 to 300 bases within the human genome. Two out of every 3 SNPs involve replacing a C with a T. By using enough SNPs, usually around 70 where C and T are likely to occur equally often, you can make it virtually certain that a profile cannot statistically belong to another human being on the planet. Thus, if all 70 SNPs were to occur independently, the likelihood of a match would be 2^70, or around 10^21. In practice, though, many of these do not assort independently, a phenomenon called linkage dysequilibrium, which still leaves us with a very high likelihood.
A third technique is to use mitochondrial DNA, which is inherited from the mother. This is highly preserved from one generation to the next, and it is thought that 5% of all Caucasians have the same mitochondrial DNA. It therefore lacks the discriminatory power of STRs. It doesn't degrade easily however, and has been used to solve historical mysteries, such as the one used to disprove Anna Anderson's claim to have descended from the Romanov family. The same investigation also showed that Prince Philip, the Duke of Edinburgh, was a descendant of the Russian Romanovs.
Forensic genetics is still work in progress. However, it's not new. Thousands of 9/11 victims were identified through their DNA after the WTC catastrophe from their remains.
The very first use of forensic genetics was in 1986 in the UK. Two women, raped and murdered in Leicester in similar fashion 3 years apart, triggered an arrest. The suspect (bizarrely, as it turned out) confessed to the first murder, but not the second. The police approached Sir Alec Jeffreys at Leicester University, the then Professor of Genetics, for help. Based on DNA analysis from the murder scenes, the suspect was acquitted. A second man, Colin Pitchfork, was subsequently found guilty of both murders. He had escaped justice initially by volunteering his friend for a DNA sample he purported to be his own.
So how does DNA fingerprinting work? There are several techniques, but the most useful one makes use of short tandem repeats (STRs) present in the DNA sequence, usually comprising 4 bases such as GATT. The length of a STR is unique to each person. Each STR of course occupies a certain locus on a given chromosome, and as each chromosome is paired, each person possesses two STRs for a given locus, usually of varying length. Part of a sequence is denoted with a point (for example GATT GATT GATT GATT GA would be denoted as 4.2). If there are 4 repeats of GATT on one cheomosome, and 7 repeats on its allele, that is denoted as 4/7.
The USA has a national DNA database called CODIS, which contains around 5 million DNA fingerprints, a number similar to that held in the UK. CODIS is based on 13 different STRs, giving a likelihood that a given sequence will be matched of 1 in 1 trillion. The UK uses 10 STRs, giving a likelihood of match of 1 in a billion.
The frequency of a given allele in the population is given by the Hardy Weinberg equation. For a homozygous allele p, the frequency is given as p^2, while for heterozygous alleles p & q, the frequency is 2pq. To find the probability that all the STRs present in a given person could be replicated in another person, you multiply the probability of finding each STR in a given ethnic group. Therefore, the more STRs you use, the greater the certainty that no two individuals would have the same profile.
Sometimes DNA from victims can be badly degraded because of time elapsed, making STR analysis difficult or incomplete (say 8 STRs possible instead of 13). Under such circumstances, geneticists exploit single nucleotide polymorphisms (SNPs). These are single base differences that occur every 100 to 300 bases within the human genome. Two out of every 3 SNPs involve replacing a C with a T. By using enough SNPs, usually around 70 where C and T are likely to occur equally often, you can make it virtually certain that a profile cannot statistically belong to another human being on the planet. Thus, if all 70 SNPs were to occur independently, the likelihood of a match would be 2^70, or around 10^21. In practice, though, many of these do not assort independently, a phenomenon called linkage dysequilibrium, which still leaves us with a very high likelihood.
A third technique is to use mitochondrial DNA, which is inherited from the mother. This is highly preserved from one generation to the next, and it is thought that 5% of all Caucasians have the same mitochondrial DNA. It therefore lacks the discriminatory power of STRs. It doesn't degrade easily however, and has been used to solve historical mysteries, such as the one used to disprove Anna Anderson's claim to have descended from the Romanov family. The same investigation also showed that Prince Philip, the Duke of Edinburgh, was a descendant of the Russian Romanovs.
Sunday 9 May 2010
Synthetic Lethality- Strategies To Target Cancer
One way of targeting cancer cells is to aim for an antigen that's only present in cancer tissue and not in healthy tissues, for example with monoclonal antibodies to the epidermal growth factor called Her/neu present in certain breast cancers, through a now well known agent called Herceptin (Trastuzumab). However, this approach only works with a few cancers. Another ingenious approach is to exploit a phenomenon called Synthetic Lethality.
The DNA we all are made of undergoes thousands of breakages in every cell cycle. If this is not repaired, sooner or later, we'll all develop cancer. To ensure that this doesn't happen, nature has introduced more than one mechanism, a sort of backup that is termed redundancy in medicine. If one mechanism fails, there will be another that will repair the damaged DNA. Redundancy has to be there, because otherwise we'll simply cease to exist.
Here's where the opportunity for fighting cancer comes in. Nature's two most important methods of repairing DNA are through an enzyme called PARP1, which removes the defective DNA base pairs, allowing the defective bit to be repaired, and through a process called homologous recombination, where new DNA is generated from existing DNA. Remember, DNA has two strands, and all the info required to generate a fully functional DNA ribbon is present in any one strand.
This is no big deal and is not unique to human beings. For thousands of years, bacteria have repaired faults through homologous recombination. In 2007, two American & one British scientist were awarded the Nobel Prize in Medicine for describing this phenomenon and its applications in more detail.
Now we come to the nity-gritty. Most people will know by now that one of the biggest risk factors for breast and ovarian cancers is the presence of mutations in two vital genes called BRCA1 and BRCA2. These tend to run in families and increase the risk of these cancers severalfold.
How do you think the BRCA gene works? It codes for homologous recombination. "Defective" families carry one copy of the faulty gene and one healthy copy (called the "wild type allele"- in genetics, "wild" refers to the natural, healthy copy.) They only develop cancer when the healthy gene somehow undergoes a new mutation. The lifetime risk of such cancer is around 40% for carriers of the BRACA1 gene and around 20% for the BRCA2 gene. In desperation, many such poor women undergo "preventative" mastectomy or oophorectomy at a relatively young age for peace of mind.
Yet, now a study suggests that there is a way out of such terrible solutions. Remember the PARP1 enzyme I mentioned, the other part of the double act of DNA repair? When cancer develops in the BRCA mutation carriers because of an acquired defect in the remaining wild type gene, the cancerous cells become totally dependent on the PARP1 enzyme for DNA repair. Yet, the other, non-cancerous cells in the cancer sufferer are not dependent on PARP1 alone because they still have one functioning, healthy copy of the BRCA gene. Therefore, it follows that if you can target the PARP-1 in these women through a pharmacological agent, the cancer cells, having no means to repair their DNA, will die, while the healthy cells will be unaffected.
That's what synthetic lethality means. Two genes are said to be in a synthetic lethal relationship if defect in one can be compensated by the other. However, if both are defective, the cell will die.
Hats off to the cancer researchers. In a few years, it seems, we'll be able to beat familial breast & ovarian cancer, a task that once seemed insurmountable.
The DNA we all are made of undergoes thousands of breakages in every cell cycle. If this is not repaired, sooner or later, we'll all develop cancer. To ensure that this doesn't happen, nature has introduced more than one mechanism, a sort of backup that is termed redundancy in medicine. If one mechanism fails, there will be another that will repair the damaged DNA. Redundancy has to be there, because otherwise we'll simply cease to exist.
Here's where the opportunity for fighting cancer comes in. Nature's two most important methods of repairing DNA are through an enzyme called PARP1, which removes the defective DNA base pairs, allowing the defective bit to be repaired, and through a process called homologous recombination, where new DNA is generated from existing DNA. Remember, DNA has two strands, and all the info required to generate a fully functional DNA ribbon is present in any one strand.
This is no big deal and is not unique to human beings. For thousands of years, bacteria have repaired faults through homologous recombination. In 2007, two American & one British scientist were awarded the Nobel Prize in Medicine for describing this phenomenon and its applications in more detail.
Now we come to the nity-gritty. Most people will know by now that one of the biggest risk factors for breast and ovarian cancers is the presence of mutations in two vital genes called BRCA1 and BRCA2. These tend to run in families and increase the risk of these cancers severalfold.
How do you think the BRCA gene works? It codes for homologous recombination. "Defective" families carry one copy of the faulty gene and one healthy copy (called the "wild type allele"- in genetics, "wild" refers to the natural, healthy copy.) They only develop cancer when the healthy gene somehow undergoes a new mutation. The lifetime risk of such cancer is around 40% for carriers of the BRACA1 gene and around 20% for the BRCA2 gene. In desperation, many such poor women undergo "preventative" mastectomy or oophorectomy at a relatively young age for peace of mind.
Yet, now a study suggests that there is a way out of such terrible solutions. Remember the PARP1 enzyme I mentioned, the other part of the double act of DNA repair? When cancer develops in the BRCA mutation carriers because of an acquired defect in the remaining wild type gene, the cancerous cells become totally dependent on the PARP1 enzyme for DNA repair. Yet, the other, non-cancerous cells in the cancer sufferer are not dependent on PARP1 alone because they still have one functioning, healthy copy of the BRCA gene. Therefore, it follows that if you can target the PARP-1 in these women through a pharmacological agent, the cancer cells, having no means to repair their DNA, will die, while the healthy cells will be unaffected.
That's what synthetic lethality means. Two genes are said to be in a synthetic lethal relationship if defect in one can be compensated by the other. However, if both are defective, the cell will die.
Hats off to the cancer researchers. In a few years, it seems, we'll be able to beat familial breast & ovarian cancer, a task that once seemed insurmountable.
Saturday 24 April 2010
Conjugate Vaccines- Taming Meningococcus C
There are 6 serotypes of Neisseria meningitidis- A, B, C, X, Y and W-135, based on the capsular polysaccharide that imparts the organism its main defense against the host immune system- resistance against phagocytosis.
W-135 and X serogroups are more common in Africa. Group C accounted for 40% of meningitis cases in children in the UK pre-1999, when the conjugate vaccine against this organism was introduced. More about the vaccine in a moment.
The organism is one of the few that is vulnerable to direct lytic attack by the complement membrane attack system, comprising factors Vb-IX. The antibodies that set off the classical pathway for complement activation are unusual in this case, in that they are not produced by B cells that have been primed by helper T cells. Rather, the B-cells are involved through a thymus independent process and are capable of directly responding to the invading meningococci without help from the T cells. Thus, menigococcal antigens are an example of thymus-independent or TI antigens.
There are 2 types of TI antigens- TI-1 and TI-2. A prototype of TI-1 antigens are the lipopolysaccharides released from gram negative organisms, which are capable of activating both mature and immature B cells. In high doses, TI-1 antigens can activate all B cells, irrespective of their antigen specificity. They are thus called B cell mitogens. This represents an useful line of defense for the body against these usually virulent organisms before T-cell mediated adaptive immunity has had time to kick in.
The capsular polysaccharide present in N.menigitidis is an example of TI-2 antigen. Such antigens typically have repetitive epitopes and inactivate immature B cells. They can however activate mature B cells by cross linking the B cell receptors through their repetitive epitopes, but if the cross linking is extensive, even mature B cells can become anergic, as happens with very high levels of antigen.
It is thought that children under the age of two years have mainly immature B cells, which would explain why they have a poor response to vaccines containing the meningococcal polysaccharide. Thus, young children do not have the capacity to respond to TI-2 antigens, unlike adults. Unfortunately, they are also the most vulnerable to this potentially deadly infection.
The problem was solved through a stroke of brilliance by observing that by conjugating TI-2 antigens to an immunogenic protein such as tetanus toxoid, you could convert TI antigens into thymus dependent (TD) antigens. Such "conjugate" antigens are dealt with by T cells, which recognise the peptide fragments presented on MHC Class II molecules by B cells, and stimulate the B cells to produce antibodies against the peptide-polysaccharide complex. Such a strategy works in children.
The United Kingdom was the first country to introduce the conjugate Meningococcus C vaccine (Men C) in November 1999. Within a few years, the proportion of the C serogroup contributing to childhood meningeal infections fell from 40% to 10%. Other countries adopted the vaccine throughout Europe with gratifying results.
Unfortunately, no such vaccine is available against Meningococcus B, which now accounts for 80% of childhood meningitis in the UK. The biggest roadblock is the fact that the polysaccharide antigen in this serogroup resembles an antigen present in neurons in the human brain, thus raising the possibility that a vaccine might cause the body to produce autoantibodies against the brain. Various strategies are being explored to overcome this diificulty.
Conjugate vaccines have also been successfully used against another erstwhile common cause of childhood meningitis- Haemophilus influenzae, group B.
Curiously, the B-cells that are most active against TI-2 antigens are not your usual B-cells. They are two evolutionally primitive cells of B cell lineage called B-1 cells (so called because they were discovered ahead of the much more common "conventional" B cells or B-2 cells) and marginal zone B cells, so named because of their location in the spleen on the periphery of T cell areas. As stated before, these B cells do not depend on the helper T cells for activation, unlike conventional B cells. It is thought however, that they do receive co-stimulatory signals from dendritic cells in the form of BAFF (B-cell activating factor- also called BLyS or B-cell stimulator), which acts on a B cell receptor called TACI. Defects in the latter can lead to a condition called Common Variable Immunodeficiency, which has been discussed elsewhere on this blog.
The importance of TI-2 response is illustrated by sufferers of another X-linked condition called Wiskott Aldrich syndrome, who are incapable or mounting a response to TI-2 antigens, and are thus susceptible to infections by bacteria with polysaccharide capsules such as Meningococcus, Pneumococcus and H.influenzae.
W-135 and X serogroups are more common in Africa. Group C accounted for 40% of meningitis cases in children in the UK pre-1999, when the conjugate vaccine against this organism was introduced. More about the vaccine in a moment.
The organism is one of the few that is vulnerable to direct lytic attack by the complement membrane attack system, comprising factors Vb-IX. The antibodies that set off the classical pathway for complement activation are unusual in this case, in that they are not produced by B cells that have been primed by helper T cells. Rather, the B-cells are involved through a thymus independent process and are capable of directly responding to the invading meningococci without help from the T cells. Thus, menigococcal antigens are an example of thymus-independent or TI antigens.
There are 2 types of TI antigens- TI-1 and TI-2. A prototype of TI-1 antigens are the lipopolysaccharides released from gram negative organisms, which are capable of activating both mature and immature B cells. In high doses, TI-1 antigens can activate all B cells, irrespective of their antigen specificity. They are thus called B cell mitogens. This represents an useful line of defense for the body against these usually virulent organisms before T-cell mediated adaptive immunity has had time to kick in.
The capsular polysaccharide present in N.menigitidis is an example of TI-2 antigen. Such antigens typically have repetitive epitopes and inactivate immature B cells. They can however activate mature B cells by cross linking the B cell receptors through their repetitive epitopes, but if the cross linking is extensive, even mature B cells can become anergic, as happens with very high levels of antigen.
It is thought that children under the age of two years have mainly immature B cells, which would explain why they have a poor response to vaccines containing the meningococcal polysaccharide. Thus, young children do not have the capacity to respond to TI-2 antigens, unlike adults. Unfortunately, they are also the most vulnerable to this potentially deadly infection.
The problem was solved through a stroke of brilliance by observing that by conjugating TI-2 antigens to an immunogenic protein such as tetanus toxoid, you could convert TI antigens into thymus dependent (TD) antigens. Such "conjugate" antigens are dealt with by T cells, which recognise the peptide fragments presented on MHC Class II molecules by B cells, and stimulate the B cells to produce antibodies against the peptide-polysaccharide complex. Such a strategy works in children.
The United Kingdom was the first country to introduce the conjugate Meningococcus C vaccine (Men C) in November 1999. Within a few years, the proportion of the C serogroup contributing to childhood meningeal infections fell from 40% to 10%. Other countries adopted the vaccine throughout Europe with gratifying results.
Unfortunately, no such vaccine is available against Meningococcus B, which now accounts for 80% of childhood meningitis in the UK. The biggest roadblock is the fact that the polysaccharide antigen in this serogroup resembles an antigen present in neurons in the human brain, thus raising the possibility that a vaccine might cause the body to produce autoantibodies against the brain. Various strategies are being explored to overcome this diificulty.
Conjugate vaccines have also been successfully used against another erstwhile common cause of childhood meningitis- Haemophilus influenzae, group B.
Curiously, the B-cells that are most active against TI-2 antigens are not your usual B-cells. They are two evolutionally primitive cells of B cell lineage called B-1 cells (so called because they were discovered ahead of the much more common "conventional" B cells or B-2 cells) and marginal zone B cells, so named because of their location in the spleen on the periphery of T cell areas. As stated before, these B cells do not depend on the helper T cells for activation, unlike conventional B cells. It is thought however, that they do receive co-stimulatory signals from dendritic cells in the form of BAFF (B-cell activating factor- also called BLyS or B-cell stimulator), which acts on a B cell receptor called TACI. Defects in the latter can lead to a condition called Common Variable Immunodeficiency, which has been discussed elsewhere on this blog.
The importance of TI-2 response is illustrated by sufferers of another X-linked condition called Wiskott Aldrich syndrome, who are incapable or mounting a response to TI-2 antigens, and are thus susceptible to infections by bacteria with polysaccharide capsules such as Meningococcus, Pneumococcus and H.influenzae.
Saturday 17 April 2010
The Hygiene Hypothesis
As somebody who grew up in India, it was instructive to see how common allergic disorders are in the west. Peanut allergy, ragweed pollen allergy, dust mite allergy... resulting in conditions such as anaphylactic reactions, hay fever and asthma are all too common. These disorders are relatively uncommon in countries like India.
An interesting hypothesis has been put forward to explain this phenomenon, called the hygiene hypothesis. It goes like this.
There are four types of CD4 or Helper T cells- Th1, Th2, Th17 and Treg. The direction that an inflammatory process, say due to an infection, takes is largely determined by whether the helper cells differentiate into Th1 or Th2 cells. Treg cells are the controller cells- they tend to suppress the other subtypes.
Th1 differentiation occurs in response to IL-12 and interferon-gamma, while Th2 lineage develops mainly in response to IL-4.
In allergy, the T cell that predominates is of the Th2 subtype. Several cytokines produced by these cells contribute to particular cell types or changes vital to allergy. Thus IL-5 leads to accumulation and survival of eosinophils, IL-4 and IL-13 cause isotype switching from IgM to IgE, rather than to IgG or IgA, while IL-3 and IL-9 augment mucus secretion. IL-4 also suppresses the development of the Th1 phenotype.
It is thought that in developing countries, the numerous childhood infections that kids are subjected to, steers the development of helper T cells towards the Th1 subtype. It is thought that cytokines that promote the Th1 phenotype, such as IL-12, also suppress the development of Th2 cells, and thus protect against allergic diatheses. This is the hygiene hypothesis.
There is a major spanner in the works with this hypothesis though. Children in developing countries also carry a much higher load of various parasites, which have been clearly associated with predominance of the Th2 subtype of T helper cells. Why do then such childen not have a higher prevalence of allergic disorders, which is also subserved by Th2 cells? In fact, parasitic infestation is associated with a lower prevalence of allergies. How does one explain this?
To account for this inconsistency, another hypothesis called the counter-regulatory hypothesis has been put forward. This states that any infections, whether associated with the Th1 phenoptype, such as common bacterial chest infections in childhood, or with the Th2 phenotype, such as is found with parasitic infestations, protect against the future development of allergy. It is thought that the body responds to such infections in the long run by increasing the 4th subtype of helper T-cells, called the T regulatory or Treg cells (which carry CD4 and CD25 surface molecules and have been mentioned elsewhere on this blog). Treg cells act by damping down both Th1 and Th2 phenotypes, mainly by secreting cytokines IL-10 and TGF-beta, and it is the suppression of the Th2 phenotype that protects against allergy.
An interesting hypothesis has been put forward to explain this phenomenon, called the hygiene hypothesis. It goes like this.
There are four types of CD4 or Helper T cells- Th1, Th2, Th17 and Treg. The direction that an inflammatory process, say due to an infection, takes is largely determined by whether the helper cells differentiate into Th1 or Th2 cells. Treg cells are the controller cells- they tend to suppress the other subtypes.
Th1 differentiation occurs in response to IL-12 and interferon-gamma, while Th2 lineage develops mainly in response to IL-4.
In allergy, the T cell that predominates is of the Th2 subtype. Several cytokines produced by these cells contribute to particular cell types or changes vital to allergy. Thus IL-5 leads to accumulation and survival of eosinophils, IL-4 and IL-13 cause isotype switching from IgM to IgE, rather than to IgG or IgA, while IL-3 and IL-9 augment mucus secretion. IL-4 also suppresses the development of the Th1 phenotype.
It is thought that in developing countries, the numerous childhood infections that kids are subjected to, steers the development of helper T cells towards the Th1 subtype. It is thought that cytokines that promote the Th1 phenotype, such as IL-12, also suppress the development of Th2 cells, and thus protect against allergic diatheses. This is the hygiene hypothesis.
There is a major spanner in the works with this hypothesis though. Children in developing countries also carry a much higher load of various parasites, which have been clearly associated with predominance of the Th2 subtype of T helper cells. Why do then such childen not have a higher prevalence of allergic disorders, which is also subserved by Th2 cells? In fact, parasitic infestation is associated with a lower prevalence of allergies. How does one explain this?
To account for this inconsistency, another hypothesis called the counter-regulatory hypothesis has been put forward. This states that any infections, whether associated with the Th1 phenoptype, such as common bacterial chest infections in childhood, or with the Th2 phenotype, such as is found with parasitic infestations, protect against the future development of allergy. It is thought that the body responds to such infections in the long run by increasing the 4th subtype of helper T-cells, called the T regulatory or Treg cells (which carry CD4 and CD25 surface molecules and have been mentioned elsewhere on this blog). Treg cells act by damping down both Th1 and Th2 phenotypes, mainly by secreting cytokines IL-10 and TGF-beta, and it is the suppression of the Th2 phenotype that protects against allergy.
Monday 12 April 2010
The All Important X-Chromosome
Although the MHC genes reside on chromosome 6, a host of serious immunodeficiency disorders have been linked with mutations or deletions of genes on the X-chromosome. The X-chromosome is probably one of the most important from an immunological point of view. Naturally, all such disorders express themselves phenotypically in boys. As there is no normal allele to balance out these defects in males, the resulting immunodeficiency is severe, and manifests early in life.
Bruton described a X-linked syndrome with complete absence of immunoglobulins called X-linked agammaglobulinaemia (XLA). The disease is also named after him. The condition is because of a defect in a cellular tyrosine kinase, called btk, that halts the development of B-cells. Female carriers can be discerned due to the phenomenon of X-inactivation, described elsewhere on this blog. Thus, all circulating B cells will have been selected out by virtue of having inactivated the defective X-chromosome. OTOH, cells that do not depend on the defective gene for their function, such as T-cells, will have random inactivation of either X-chromosome, with a 1:1 ratio of such cells in circulation.
The earliest antibody isotype to be made by B-cells is IgM. Later, as the B-cell matures, en route to its ultimate role as the plasma cell, there is switching of the isotype to production of other antibodies, namely IgG, IgA or IgE. Along with this process of isotype switching, random mutations are introduced in the variable region of the immunoglobulin molecule, a process that is called somatic hypermutation. This process increases the affinity with which the B-cell receptor and its secreted immunoglobulin bind their putative antigen, a phenomenon called affinity maturation. The latter occurs only when the B-cell is exposed to its specific antigen in peripheral lymphoid tissues. This whole process cannot occur without the aid of helper T-cells, which are also constantly traversing peripheral lymphoid tissues, having entered through small blood vessels called high endothelial venules. The fact that an antigen specific B cell meets an antigen specific T cell is something of a small miracle- the odds of this happening has been calculated as being of the order of 10^-10. But meet they do, in the Tcell/Bcell border zones of lymph nodes and the spleen. T cells have on their surface a molecule called CD40 ligand (also called CD154). This ligates CD40 on B cells, providing a co-stimulatory signal that allows isotype switching and somatic hypermutaion to occur. It follows therefore, that in the absence of CD40-CD40L interaction, isotype switching will not occur, and the B-cells will continue to express one antibody isotype- IgM. This is exactly what happens in X-linked hyper-IgM syndrome, where there is a defect in the CD40 ligand on T cells. The interaction is bidirectional, and also activates T cells to express other co-stimulatory molecules. Thus, there is also a T-cell defect in this condition.
Other causes of hyper IgM syndrome have been identified. Predictably, defects in CD40 will give rise to a similar condition. A condition called NEMO syndrome leads to the same phenotype, as does deficiency of an enzyme called activation induced cytidine deaminase or AID, which is essential for the process of both isotype switching and somatic hypermutation. This latter only affects B-cells and thus causes a milder immunodeficiency than the first three aetiologies.
A further, much more severe immunodeficiency disorder has been characterised as "Severe Combined Immunodeficiency" or SCID. SCID has either X linked or autosomal recessive forms of inhertance. The X-linked form, which accounts for around 60% of cases, is due to deficiency of the gamma chain of the receptor of Interleukin-2 (IL-2). IL-2 is the most important survival and growth factor for T cells and is induced by co-stimulation of T cells through CD28 by B7 present on APCs, a process that's been described elsewhere on this blog. Upon activation, 3 transcription fators- nFkappaB, AP-1 and NFAT, are induced in the T cell, which leads to a hundred-fold surge in the production of IL-2. IL-2 is an autocrine cytokine- it acts on the T cell itself, through the IL-2 receptor. This receptor is trimeric when fully functional, and has alpha, beta and gamma chains. The beta and gamma chains exist as a dimer in the naive T cell. When the T cell is activated by its antigen and through co-stimulation, the beta-gamma heterodimer associates with the alpha chain, thus making a fully functional receptor, with a very high affinity for IL-2. This cannot happen in the absence of the gamma chain, which is defective in most cases of X-SCID. Other causes of X-SCID have been described including the absence of Janus Kinase-3 (JAK-3) and a defect in the gene that is responsible for the IL-7 receptor. In addition, autosomal forms of this condition exist due to deficiency of RAG-1 and RAG-2 recombinase enzymes which catalyse the somatic rearrangement of DNA in T and B cells that determine receptor specificity, a rare syndrome called ARTEMIS and deficiency of an enzyme called adenosine deaminase.
In addition to all these X-linked immunodeficiency disorders, an autoimmune X-linked condition has also been described. This is called IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy, and X-linkage). This condition is caused by a mutation in the gene for FoxP3, an essential transcription factor for natural Treg(CD4 CD25)cells, and results in severe allergic inflammation, autoimmune polyendocrinopathy, diarrhoea, haemolytic anaemia and thrombocytopaenia.
Since all the above conditions are recessive, female carriers of defective genes are phenotypically normal and do not manifest immunodeficiency.
Bruton described a X-linked syndrome with complete absence of immunoglobulins called X-linked agammaglobulinaemia (XLA). The disease is also named after him. The condition is because of a defect in a cellular tyrosine kinase, called btk, that halts the development of B-cells. Female carriers can be discerned due to the phenomenon of X-inactivation, described elsewhere on this blog. Thus, all circulating B cells will have been selected out by virtue of having inactivated the defective X-chromosome. OTOH, cells that do not depend on the defective gene for their function, such as T-cells, will have random inactivation of either X-chromosome, with a 1:1 ratio of such cells in circulation.
The earliest antibody isotype to be made by B-cells is IgM. Later, as the B-cell matures, en route to its ultimate role as the plasma cell, there is switching of the isotype to production of other antibodies, namely IgG, IgA or IgE. Along with this process of isotype switching, random mutations are introduced in the variable region of the immunoglobulin molecule, a process that is called somatic hypermutation. This process increases the affinity with which the B-cell receptor and its secreted immunoglobulin bind their putative antigen, a phenomenon called affinity maturation. The latter occurs only when the B-cell is exposed to its specific antigen in peripheral lymphoid tissues. This whole process cannot occur without the aid of helper T-cells, which are also constantly traversing peripheral lymphoid tissues, having entered through small blood vessels called high endothelial venules. The fact that an antigen specific B cell meets an antigen specific T cell is something of a small miracle- the odds of this happening has been calculated as being of the order of 10^-10. But meet they do, in the Tcell/Bcell border zones of lymph nodes and the spleen. T cells have on their surface a molecule called CD40 ligand (also called CD154). This ligates CD40 on B cells, providing a co-stimulatory signal that allows isotype switching and somatic hypermutaion to occur. It follows therefore, that in the absence of CD40-CD40L interaction, isotype switching will not occur, and the B-cells will continue to express one antibody isotype- IgM. This is exactly what happens in X-linked hyper-IgM syndrome, where there is a defect in the CD40 ligand on T cells. The interaction is bidirectional, and also activates T cells to express other co-stimulatory molecules. Thus, there is also a T-cell defect in this condition.
Other causes of hyper IgM syndrome have been identified. Predictably, defects in CD40 will give rise to a similar condition. A condition called NEMO syndrome leads to the same phenotype, as does deficiency of an enzyme called activation induced cytidine deaminase or AID, which is essential for the process of both isotype switching and somatic hypermutation. This latter only affects B-cells and thus causes a milder immunodeficiency than the first three aetiologies.
A further, much more severe immunodeficiency disorder has been characterised as "Severe Combined Immunodeficiency" or SCID. SCID has either X linked or autosomal recessive forms of inhertance. The X-linked form, which accounts for around 60% of cases, is due to deficiency of the gamma chain of the receptor of Interleukin-2 (IL-2). IL-2 is the most important survival and growth factor for T cells and is induced by co-stimulation of T cells through CD28 by B7 present on APCs, a process that's been described elsewhere on this blog. Upon activation, 3 transcription fators- nFkappaB, AP-1 and NFAT, are induced in the T cell, which leads to a hundred-fold surge in the production of IL-2. IL-2 is an autocrine cytokine- it acts on the T cell itself, through the IL-2 receptor. This receptor is trimeric when fully functional, and has alpha, beta and gamma chains. The beta and gamma chains exist as a dimer in the naive T cell. When the T cell is activated by its antigen and through co-stimulation, the beta-gamma heterodimer associates with the alpha chain, thus making a fully functional receptor, with a very high affinity for IL-2. This cannot happen in the absence of the gamma chain, which is defective in most cases of X-SCID. Other causes of X-SCID have been described including the absence of Janus Kinase-3 (JAK-3) and a defect in the gene that is responsible for the IL-7 receptor. In addition, autosomal forms of this condition exist due to deficiency of RAG-1 and RAG-2 recombinase enzymes which catalyse the somatic rearrangement of DNA in T and B cells that determine receptor specificity, a rare syndrome called ARTEMIS and deficiency of an enzyme called adenosine deaminase.
In addition to all these X-linked immunodeficiency disorders, an autoimmune X-linked condition has also been described. This is called IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy, and X-linkage). This condition is caused by a mutation in the gene for FoxP3, an essential transcription factor for natural Treg(CD4 CD25)cells, and results in severe allergic inflammation, autoimmune polyendocrinopathy, diarrhoea, haemolytic anaemia and thrombocytopaenia.
Since all the above conditions are recessive, female carriers of defective genes are phenotypically normal and do not manifest immunodeficiency.
Sunday 11 April 2010
The Mpemba Effect
Have you heard of the Mpemba effect? No, didn't think you would have.
Erasto Mpemba was a Tanzanian schoolboy in 1963 when he informed his science teacher that he could make ice-cream faster by putting hot milk in the freezer than when he used cold milk. He was laughed at for his troubles. The findings were confirmed by school inspector Denis Osborne, and the two published an article on this phenomenon in a peer reviewed journal in 1969.
Apparently, the effect is due to super-cooling of water. Cold water doesn't strictly freeze at 0 degree Celsius- it often takes much lower temparatures before it finally turns into ice. OTOH, hot water is not subject to supercooling. It needs to get only to 0 degrees to freeze. Thus, the rapidly cooling hot milk freezes faster than the cold milk, which lingers around the freezing point of water for a while.
The Mpemba effect is not always reproducible. Apparently, it only works under certain conditions.
Erasto Mpemba was a Tanzanian schoolboy in 1963 when he informed his science teacher that he could make ice-cream faster by putting hot milk in the freezer than when he used cold milk. He was laughed at for his troubles. The findings were confirmed by school inspector Denis Osborne, and the two published an article on this phenomenon in a peer reviewed journal in 1969.
Apparently, the effect is due to super-cooling of water. Cold water doesn't strictly freeze at 0 degree Celsius- it often takes much lower temparatures before it finally turns into ice. OTOH, hot water is not subject to supercooling. It needs to get only to 0 degrees to freeze. Thus, the rapidly cooling hot milk freezes faster than the cold milk, which lingers around the freezing point of water for a while.
The Mpemba effect is not always reproducible. Apparently, it only works under certain conditions.
Saturday 10 April 2010
Why Don't Women Mount A Immune Attack On The Foetus?
This is one of the oldest puzzles that have confounded Immunologists. The father, and thus the foetus, often contain major histocompatibility antigens that are distinct from those carried by the pregnant woman. Yet, the foetus is almost never immunologically attacked by the maternal immune system. There are several reasons for this.
The placenta is of foetal origin. The outermost layer of the placenta, called the trophoblast, is poor in both MHC Class I & Class II antigens, thus reducing the risk of attack by maternal T cells. However, not expressing MHC Class I antigens does open up the trophoblast to attacks from Natural Killer (NK cells). It does express a weakly antigenic MHC Class I molecule called HLA-G. This interacts directly with the two main inhibitory receptors on NK cells- KIR-1&2, and thus keeps the NK cells at bay.
Secondly, the placental tissue produces an enzyme called IDO- indoleamine 2,3 dioxygenase. This degrades the tryptophan contained in T cell proteins, and makes these T cells less likely to become activated.
Thirdly, it is thought the specific T cells that the mother does have against major and minor paternal antigens become temporarily anergic to these antigens. This tolerance disappears after the baby is delivered.
Fourthly, regulatory T cells such as CD4 CD25 T-reg cells may play a part by suppressing Th1 responses in particular. Thus, cytokines such as IL-4, IL-10 & TGF-beta predominate in the placenta, which steer the T-cell response away from the more stridently inflammatory Th1 phenotype to Th2 and T-reg phenotypes.
The foetus is not the only "tissue" that's exempt from immune attacks. There are three other sites in the body- namely the brain, the anterior chamber of the eye, and the testes, which are normally sequestered from the immune system. They are thus "immunologically previleged". The brain and the anterior chamber of the eye lack lymphatics, and the brain is also separated physically by the blood brain barrier. This sequestration does occasionally break down, resulting in autoimmune conditions such as multiple sclerosis, but in general, this phenomenon of immune previlege explains why HLA-matching is not required for transplants in these regions such as corneal grafts, and also why such grafts are quite long-lived.
No such tolerance exists outwith pregnancy. When organs such as kidney, etc are transplanted into the (MHC matched) female of the species from the male, the female's T-lymphocytes recognise the foreign proteins translated from genes on the Y chromosome of the male, and attacks them, thus increasing risk of graft failure. This does not happen with female--> male transplants, as both have common proteins translated from the mRNA transcripts of the X-chromosome. This phenomenon is known as H-Y, and explains why a solid organ or bone marrow transplant from female to male is more likely to succeed than a male to female transplant, although modern immuno-suppressive regimes have narrowed the gap.
The placenta is of foetal origin. The outermost layer of the placenta, called the trophoblast, is poor in both MHC Class I & Class II antigens, thus reducing the risk of attack by maternal T cells. However, not expressing MHC Class I antigens does open up the trophoblast to attacks from Natural Killer (NK cells). It does express a weakly antigenic MHC Class I molecule called HLA-G. This interacts directly with the two main inhibitory receptors on NK cells- KIR-1&2, and thus keeps the NK cells at bay.
Secondly, the placental tissue produces an enzyme called IDO- indoleamine 2,3 dioxygenase. This degrades the tryptophan contained in T cell proteins, and makes these T cells less likely to become activated.
Thirdly, it is thought the specific T cells that the mother does have against major and minor paternal antigens become temporarily anergic to these antigens. This tolerance disappears after the baby is delivered.
Fourthly, regulatory T cells such as CD4 CD25 T-reg cells may play a part by suppressing Th1 responses in particular. Thus, cytokines such as IL-4, IL-10 & TGF-beta predominate in the placenta, which steer the T-cell response away from the more stridently inflammatory Th1 phenotype to Th2 and T-reg phenotypes.
The foetus is not the only "tissue" that's exempt from immune attacks. There are three other sites in the body- namely the brain, the anterior chamber of the eye, and the testes, which are normally sequestered from the immune system. They are thus "immunologically previleged". The brain and the anterior chamber of the eye lack lymphatics, and the brain is also separated physically by the blood brain barrier. This sequestration does occasionally break down, resulting in autoimmune conditions such as multiple sclerosis, but in general, this phenomenon of immune previlege explains why HLA-matching is not required for transplants in these regions such as corneal grafts, and also why such grafts are quite long-lived.
No such tolerance exists outwith pregnancy. When organs such as kidney, etc are transplanted into the (MHC matched) female of the species from the male, the female's T-lymphocytes recognise the foreign proteins translated from genes on the Y chromosome of the male, and attacks them, thus increasing risk of graft failure. This does not happen with female--> male transplants, as both have common proteins translated from the mRNA transcripts of the X-chromosome. This phenomenon is known as H-Y, and explains why a solid organ or bone marrow transplant from female to male is more likely to succeed than a male to female transplant, although modern immuno-suppressive regimes have narrowed the gap.
Tuesday 6 April 2010
The Amazing Mr HIV
HIV infects the CD4 T cells. But how does it enter those cells? It has two envelope glycoproteins called gp 120 and gp 41. These are translated from a single unspliced mRNA, which itself is transcribed from the env gene that forms part of the 9-gene HIV genome. The virus relies on a host protease to split the two envelope proteins. However, it needs its own protease to split apart another monolithic protein (called polyprotein), that comprises the viral core protein and another that includes vital enzymes like reverse transciptase, integrase and the protease itself (mRNA transcribed from two genes called gag and pol)- this viral protease is a target for therapeutic protease inhibitors that form a cornerstone of HAART (highly active anti-retroviral therapy).
Gp 120 binds to the CD4 molecule itself, but also to a chemokine receptor on the surface of CD4 T cells, called CCR5. CCR5 is normally a ligand for chemokines called CCL3, CCL4 and CCL5 and serves a vital function in normal T-cell physiology. After gp 120 has anchored the virus to its target CD4 T-cell, the second envelope protein- gp 41- causes fusion of the viral coat with the cell membrane of the T-cell, thus allowing the virus to enter its victim.
As you can expect, CCR5 is vital for virus replication. This has been starkly brought out by a small number of subjects of Caucasian origin, who are completely resistant to HIV infection, despite repeated exposure. As it happens, these individuals are homozygous for a mutated CCR5 exon, which has a 30 base pair deletion resulting in a frameshift mutation and a truncated CCR5 peptide chain, termed delta 30. The defective protein blocks entry for HIV into the CD4 T cell. Currently, there is intense research into developing a pharmacological CCR5 binding molecule that would block entry of HIV into CD4 T-cells.
Curiously, there is another portal for entry for HIV into the CD4 T-cell. This is a receptor called CXCR4 (The 2 Cs stand for cysteine residues, joined to each other by another amino acid, designated X). It is thought that CCR5 acts as the entry point for the virus early in infection, while CXCR4 becomes important much later in the course of infection, around the time that the infected individual develops clinical AIDS. HIV virus that uses the CCR5 co-receptor for entry is also called R5 virus, while virus subgroups that use CXCR4 are called X4 viruses.
Gp 120 binds to the CD4 molecule itself, but also to a chemokine receptor on the surface of CD4 T cells, called CCR5. CCR5 is normally a ligand for chemokines called CCL3, CCL4 and CCL5 and serves a vital function in normal T-cell physiology. After gp 120 has anchored the virus to its target CD4 T-cell, the second envelope protein- gp 41- causes fusion of the viral coat with the cell membrane of the T-cell, thus allowing the virus to enter its victim.
As you can expect, CCR5 is vital for virus replication. This has been starkly brought out by a small number of subjects of Caucasian origin, who are completely resistant to HIV infection, despite repeated exposure. As it happens, these individuals are homozygous for a mutated CCR5 exon, which has a 30 base pair deletion resulting in a frameshift mutation and a truncated CCR5 peptide chain, termed delta 30. The defective protein blocks entry for HIV into the CD4 T cell. Currently, there is intense research into developing a pharmacological CCR5 binding molecule that would block entry of HIV into CD4 T-cells.
Curiously, there is another portal for entry for HIV into the CD4 T-cell. This is a receptor called CXCR4 (The 2 Cs stand for cysteine residues, joined to each other by another amino acid, designated X). It is thought that CCR5 acts as the entry point for the virus early in infection, while CXCR4 becomes important much later in the course of infection, around the time that the infected individual develops clinical AIDS. HIV virus that uses the CCR5 co-receptor for entry is also called R5 virus, while virus subgroups that use CXCR4 are called X4 viruses.
Friday 2 April 2010
Innate & Adaptive Immunity
Mammals have two distinct, but interrelated immune tiers in place- the primitive innate immune system, which they share with other vertebrates, and the much more evolved adaptive immune system. When the body is invaded by foreign pathogens such as bacteria or viruses, it is the innate immunity that kicks in first- this is a generic system, geared to kill pathogens in general, and lacks specificity. Meanwhile, the much more specialised adaptive immune system recognises the specific antigen and somewhat later, mounts a deadly directed assault which, more often than not, wipes out the pathogens.
An example of the workings of both innate and adaptive immunity can be found with the human immune response to viruses. Viruses lack the capacity to make their own protein, such as the one that constitutes their coat. They can only do so by invading host cells, incorporating their own RNA or DNA into the host genome and getting the host to make the viral proteins.
When viruses first attack mammalian cells, they alter certain glyco-proteins present on the cell surface called MHC Class I molecules. These are present on all cells. Viruses downgrade or alter the production of MHC class I molecules with a rather clever motive-these molecules are essential for the next step of the host defence- the adaptive immunity. However, a member of the innate immune system, called Natural Killer cells, or NK cells, has been put in place for just such an eventuality. The NK cells recognise the cells that have altered or diminished MHC class I molecules on their surface and directly kill them by releasing enzymes called granzyme and perforin, which cause the virus infected cell to die in an orderly fashion, called apooptosis. This process is highly directed, so that neighbouring cells don't suffer. Neighbouring healthy cells are also protected by increasing their expression of MHC class I molecules due to the influence of a group of soluble agents called Class I interferons- comprising alpha and beta-interferons. Although many cells produce these two interferons, the principal source are a group of cells called plasmacytoid dendritic cells, which produce 1000 times more class I interferons than any other cells. These interferons upregulate the production of MHC class I proteins by normal cells, thus protecting them from attack by NK cells. They also produce a distinct type of interferon themselves, called interferon-gamma, which facilitates later responses by the adaptive immune system, specifically by activating a class of T-cells called Th1 cells.
For those viruses which manage to evade the NK cells and survive in host cells, backup is required. Enter the cytotoxic T lymphocytes. T cells, or thymus derived lymphocytes, are broadly of 2 types- CD4 and CD8, and form pillars of the adaptive immune system. CD8 lymphocytes, also called cytotoxic lymphocytes, recognise specific foreign antigens, mainly viral, presented on MHC class I molecules by infected cells. (thus they work in an exactly opposite way to NK cells, which recognise cells with low levels of MHC class I molecules). Cytotoxic, or CD8 lymphocytes, kill in exactly the same way as NK cells, by releasing the same destructive enzymes onto the surface of the virus infected cell in a directed way, but being part of the adaptive immune system, they are much more specific. Instead of generically killing any virus infected cell, they recognise a specific virus as it sits within a host cell. They are thus much more specific and powerful killers than NK cells. Mice lacking MHC class I molecules tend to be overwhelmed by viruses, as CD8 cells require MHC class I molecules to recognise their specific viral targets.
We'll talk more about the interplay between the innate and adaptive immune systems in later posts.
An example of the workings of both innate and adaptive immunity can be found with the human immune response to viruses. Viruses lack the capacity to make their own protein, such as the one that constitutes their coat. They can only do so by invading host cells, incorporating their own RNA or DNA into the host genome and getting the host to make the viral proteins.
When viruses first attack mammalian cells, they alter certain glyco-proteins present on the cell surface called MHC Class I molecules. These are present on all cells. Viruses downgrade or alter the production of MHC class I molecules with a rather clever motive-these molecules are essential for the next step of the host defence- the adaptive immunity. However, a member of the innate immune system, called Natural Killer cells, or NK cells, has been put in place for just such an eventuality. The NK cells recognise the cells that have altered or diminished MHC class I molecules on their surface and directly kill them by releasing enzymes called granzyme and perforin, which cause the virus infected cell to die in an orderly fashion, called apooptosis. This process is highly directed, so that neighbouring cells don't suffer. Neighbouring healthy cells are also protected by increasing their expression of MHC class I molecules due to the influence of a group of soluble agents called Class I interferons- comprising alpha and beta-interferons. Although many cells produce these two interferons, the principal source are a group of cells called plasmacytoid dendritic cells, which produce 1000 times more class I interferons than any other cells. These interferons upregulate the production of MHC class I proteins by normal cells, thus protecting them from attack by NK cells. They also produce a distinct type of interferon themselves, called interferon-gamma, which facilitates later responses by the adaptive immune system, specifically by activating a class of T-cells called Th1 cells.
For those viruses which manage to evade the NK cells and survive in host cells, backup is required. Enter the cytotoxic T lymphocytes. T cells, or thymus derived lymphocytes, are broadly of 2 types- CD4 and CD8, and form pillars of the adaptive immune system. CD8 lymphocytes, also called cytotoxic lymphocytes, recognise specific foreign antigens, mainly viral, presented on MHC class I molecules by infected cells. (thus they work in an exactly opposite way to NK cells, which recognise cells with low levels of MHC class I molecules). Cytotoxic, or CD8 lymphocytes, kill in exactly the same way as NK cells, by releasing the same destructive enzymes onto the surface of the virus infected cell in a directed way, but being part of the adaptive immune system, they are much more specific. Instead of generically killing any virus infected cell, they recognise a specific virus as it sits within a host cell. They are thus much more specific and powerful killers than NK cells. Mice lacking MHC class I molecules tend to be overwhelmed by viruses, as CD8 cells require MHC class I molecules to recognise their specific viral targets.
We'll talk more about the interplay between the innate and adaptive immune systems in later posts.
Saturday 13 March 2010
Co-stimulation- Nature's Safety Catch
Why don't the immune cells in our body run amok and attack our own tissues? Well, sometimes they do, resulting in autoimmune diseases like type I diabetes mellitus, pernicious anaemia, vitiligo, etc, but mostly they are kept in check.
The way this happens is through an elaborate system of checks and balances. The first of these occur in the thymus- the site of development and maturation of T cells, where they migrate soon after they are formed in the bone marrow.
In the thymus, young T cells come in contact with the body's own antigens- called "self antigens"- on the natural residents of the thymus- the epithelial & stromal cells. These antigens are recognised when they are "presented" to the maturing T cells, gift-wrapped in MHC molecules- also known as HLA, the major determinants of transplant success in allogenic transplants. T cells that recognise these MHC-self antigen complexes are selected to survive- a process called positive selection.
However, the thymus does not contain many antigens, which are sequestered in far-flung tissues of the body- the pancreatic antigens, for example. When the mature T cells are released into the circulation, there is a real danger that these would then attack the body's own tissues, mistakenly interpreting the hitherto hidden self antigens as foreign.
Why doesn't this happen?
This is because of need for co-stimulation. The mature T cell with its receptor bound to hitherto unfamiliar antigens carried on self MHC proteins is like a loaded gun....but with its safety-catch on. Co-stimulation is that safety catch.
I had alluded earlier to the fact that for T cells to recognise antigens, these must be carried in the groove of self MHC molecules. These molecules are in turn part of specialised cells called antigen presenting cells (APC).
When APCs, carrying the self MHC molecule, holding the antigen in its embrace, meets and binds to a T cell receptor, the T cell asks- "Is that all you have got for me? Where's your co-stimulation molecule?"
Every mature T cell constitutively expresses a marker called CD28. The ligand (something that it ligates) for this is something called B-7, present on APCs. There are two types of B7- B7-1 (also called CD80), and B7-2 (also called CD86). Only when a CD28 on T-cell binds to a B7 on an APC does the T cell become activated. Since this is required in addition to the T cell receptor recognising the putative (foreign) antigen bound to a self MHC molecule in the APC, the process is called co-stimulation.
The nice bit is that co-stimulation molecules on APC- B7- does not become expressed until the APC meets an invading pathogen. These pathogens have certain patterns- lipopolysaccharides on gram negative bacteria, for example- that are called pathogen associated molecular patterns or PAMPs. The PAMPs bind to certain probes on APCs that recognise them- called pattern recognition receptors or PRRs. The most well known PRRs are a group of primitive receptors called Toll-like receptors (TLR), characterised only recently from fruit flies.
Thus when the TLR or a similar PRR on the APC recognises a PAMP on an invading pathogen, B7 becomes expressed on the APC. The APC is now "licensed" to activate a mature T cell. The safety catch is off.
This principle has been exploited in medicine through a drug called Abatacept, used successfully in the treatment of Rheumatoid arthritis. Further attempts at exploiting the phenomenon backfired when volunteers at a drug trial in London became critically unwell after administration of an experimental drug. More on that soon.
The way this happens is through an elaborate system of checks and balances. The first of these occur in the thymus- the site of development and maturation of T cells, where they migrate soon after they are formed in the bone marrow.
In the thymus, young T cells come in contact with the body's own antigens- called "self antigens"- on the natural residents of the thymus- the epithelial & stromal cells. These antigens are recognised when they are "presented" to the maturing T cells, gift-wrapped in MHC molecules- also known as HLA, the major determinants of transplant success in allogenic transplants. T cells that recognise these MHC-self antigen complexes are selected to survive- a process called positive selection.
However, the thymus does not contain many antigens, which are sequestered in far-flung tissues of the body- the pancreatic antigens, for example. When the mature T cells are released into the circulation, there is a real danger that these would then attack the body's own tissues, mistakenly interpreting the hitherto hidden self antigens as foreign.
Why doesn't this happen?
This is because of need for co-stimulation. The mature T cell with its receptor bound to hitherto unfamiliar antigens carried on self MHC proteins is like a loaded gun....but with its safety-catch on. Co-stimulation is that safety catch.
I had alluded earlier to the fact that for T cells to recognise antigens, these must be carried in the groove of self MHC molecules. These molecules are in turn part of specialised cells called antigen presenting cells (APC).
When APCs, carrying the self MHC molecule, holding the antigen in its embrace, meets and binds to a T cell receptor, the T cell asks- "Is that all you have got for me? Where's your co-stimulation molecule?"
Every mature T cell constitutively expresses a marker called CD28. The ligand (something that it ligates) for this is something called B-7, present on APCs. There are two types of B7- B7-1 (also called CD80), and B7-2 (also called CD86). Only when a CD28 on T-cell binds to a B7 on an APC does the T cell become activated. Since this is required in addition to the T cell receptor recognising the putative (foreign) antigen bound to a self MHC molecule in the APC, the process is called co-stimulation.
The nice bit is that co-stimulation molecules on APC- B7- does not become expressed until the APC meets an invading pathogen. These pathogens have certain patterns- lipopolysaccharides on gram negative bacteria, for example- that are called pathogen associated molecular patterns or PAMPs. The PAMPs bind to certain probes on APCs that recognise them- called pattern recognition receptors or PRRs. The most well known PRRs are a group of primitive receptors called Toll-like receptors (TLR), characterised only recently from fruit flies.
Thus when the TLR or a similar PRR on the APC recognises a PAMP on an invading pathogen, B7 becomes expressed on the APC. The APC is now "licensed" to activate a mature T cell. The safety catch is off.
This principle has been exploited in medicine through a drug called Abatacept, used successfully in the treatment of Rheumatoid arthritis. Further attempts at exploiting the phenomenon backfired when volunteers at a drug trial in London became critically unwell after administration of an experimental drug. More on that soon.
Monday 8 March 2010
Why Aren't Women Men?
Why indeed? A woman has XX, men have XY. The Y chromosome is much smaller than the X chromosome, and virtually useless. It carries only around 100 genes. OTOH, The X chromosome contains several important genes, such as the hemophilia gene, the gene that controls the excretion of phosphate by kidneys, and the dystrophin gene, abnormalities in which accounts for Duchenne's Muscular Dystrophy.
If the X chromosome is so important, how then do men manage with only X chromosome, while women need two? The surprising answer is that soon after birth, a woman randomly inactivates one X chromosome in every cell of hers. It might be the paternal X chromosome or the maternal X chromosome that's inactivated, but not the same X chromosome in every cell, as otherwise women would be as prone to X linked disorders as men.
This is done by an extra-genetic mechanism called epigenetics- usually by methylation of cytosine bases in DNA, where they occur next to guanine bases- a pairing known as CpG- p stands for the phospate in the deoxyribose sugar that joins up nucleotide bases like cytosine and gunanine in the DNA. This has the effect of shutting off genes which are downstream of the methylated CpG unit.
Epigenetics is an amazing phenomenon and may explain some of the most amazing events seen in the inheritance of acquired traits. More on that later.
If the X chromosome is so important, how then do men manage with only X chromosome, while women need two? The surprising answer is that soon after birth, a woman randomly inactivates one X chromosome in every cell of hers. It might be the paternal X chromosome or the maternal X chromosome that's inactivated, but not the same X chromosome in every cell, as otherwise women would be as prone to X linked disorders as men.
This is done by an extra-genetic mechanism called epigenetics- usually by methylation of cytosine bases in DNA, where they occur next to guanine bases- a pairing known as CpG- p stands for the phospate in the deoxyribose sugar that joins up nucleotide bases like cytosine and gunanine in the DNA. This has the effect of shutting off genes which are downstream of the methylated CpG unit.
Epigenetics is an amazing phenomenon and may explain some of the most amazing events seen in the inheritance of acquired traits. More on that later.
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