After the Human Genome Project was successfully completed in April 2003 and it was assured that humans are identical in the sequence of their genome by 99.9%, researchers are moving on to find more about the 0.1% left. Although our genome is made out of 3 billion bases (A’s, G’s, C’s and T’s), the 0.1% genetic difference is extremely significant. This is due to the fact that this small percentage holds the key to most of the consequences of such a variation between human beings (e.g susceptibility to diseases and also response to drugs). This introduced the term SNPs (single nucleotide polymorphism) which was found to be highly involved in variation of response to many drugs (like anti-cancers) and the list is increasing every day.
To be more focused on the human variations represented by the 0.1 percent, the NIH led the international HapMap project. Their work was performed on four populations groups: the Yoruba people in Ibadan Nigeria,the Japanese in Tokyo, the Han Chinese from Beijing and Utah residents from western and northern Europe. The work began in October 2002 and successfully ended in October 2005. The work of three successive years helped the researchers invent a shortcut for studying SNPs. Scientists believe that there are about 10 million SNPs distributed among 3 billion base pairs which make up our genome, so scanning the whole genome of millions of people for such SNPs would be extremely expensive. After the HapMap project, researchers demonstrated that variants usually tend to cluster into neighborhoods (called haplotypes) and thus the number could be reduced to 300,000 SNPs only. This means that they could reduce the work load by about 30 folds.
The genome-wide association (GWA) studies aim to pinpoint the genetic differences, which cause a certain disease (or a biological trait) by comparing a group of people (who have the trait under research) to a control group (people who are free from this trait). Utilizing thousands of SNPs markers, we can identify regions (loci) which are statistically different between patient and control groups. Thus, we can identify the genetic difference between sick and control people, even though the difference was subtle. This means that the combination of slightly altered genes plus environmental factors could be well studied. The conventional ways, usually used to study genetic differences, are mainly based on selecting the candidate gene based on knowing or suspecting the mechanism of the disease. GWA helps scanning of the whole genome in a comprehensive unbiased manner. It will let us get the whole picture about other, non expected, contributing genes. In this way, GWA studies will help us study the multi-factorial diseases (like cancer and diabetes) in a more rationalised way.
Another challenge has come up: What about the genetic variations due to geographic ancestry? It is also a significant contributing factor to variation among humans and all the efforts are directed towards making a somewhat universal map of human genome to help develop individualized drugs. A group of scientists led by David Reich, an assistant professor at Harvard Medical School, described a quantitative method that can correct such errors due to geographical ancestry known collectively as “population stratification”. It will help if the disease groups, sharing the same trait, have differences in their geographic ancestry.
Tags: Genome wide association studies
, Genome-wide association studiesX
, Hap-Map project
, human variome project
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Posted by: rose in Immunology
Cancer: An abnormal uncontrolled proliferation of cells. The immune system doesn’t show any response, as the cancer cells are just some normal cells, which have gone crazy. That means that they could deceive our immune system, which recognizes them as normal.
The point of weakness
Cancer cells usually express so-called cancer-specific antigens, which are not otherwise expressed by normal cells.
Using these antigens as a method of differentiation, we have to teach our immune system to wipe out these cells without affecting the innocent normal ones.
Whole cell vaccines: using tumor cells, derived from a patient or many patients or use human tumor cell lines designed in lab. This will elicit the immune response for all the antigens on cancer cells.
Antigen vaccines: using a specific antigen on the cancer cell through identifying a certain gene, then cloning the gene, which encodes for it.
Adjuvants: using chemical substances to enhance T-cell response such as Interleukin-2 “IL2”.
Vector: using viral vectors to deliver the gene of interest to cells, which makes the cancer more visible to the immune system.
One major obstacle facing cancer vaccines is that the response is not readily measurable. For chemotherapeutic drugs development, the end point is usually progression-free survival, which has shorter-term outcomes. Cancer vaccines are characterized by longer-term outcomes and increased survival rate.
For more information, Read here.
, antigen vaccine
, cell lines
, survival rate
, viral vector
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Have you ever heard about the terrorist attacks of anthrax via mail in 2001 in US that finally ended with Bruce Ivins, the chief suspect in those attacks, committing suicide?
What about tracing microbe sources as in the Salmonella US outbreak?
This is microbial forensics.
Microbial forensics uses biological analysis such as genome sequencing, protein and carbohydrate fingerprinting to figure out the source of a biological agent. 2001 was not the year which witnessed the emergence of microbial forensics. Actually, in 1998 a doctor from Louisiana, who intentionally infected his former mistress with HIV, had a trial in court. But the anthrax attacks were really the reason for the amplification of the role of microbial forensics as it was followed by government funding and developing the sequencing techniques into more cheaper ones.
As a result, microbial forensics is extrapolated to further applications beyond biocrimes. In molecular epidemiology, it will help figuring out the source of food or water borne diseases such as in recent US Salmonella outbreak. The field also is expected to provide help in hospital-acquired infections. Many people sue hospitals every year claiming they got MRSA (methicillin-resistent Staphylococcus aureus) infection from a certain hospital. Tracing the source of the infection will prove whether those people really contracted the infection from the hospital or from any other outside source.
, nosocomial infections
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Aetiology and epidemiology
Meninigococcal meninigits and septicemia are devastating diseases caused by Neissieria meningitidis. Although infants and young children are the most susceptible to the disease, adults are also affected but with less incidence. N.meningitidis is a gram negative capsulated bacterium that has been classified into five serogroups (A, B, C, Y, W135) based on their polysaccharide capsule.
Over forty years, developing a vaccine against this dangerous bacterium proceeded with little success. In 1960, the purified polysaccharide antigens were used to develop a vaccine against four groups (A, C, Y, W135) . Unfortunately, this vaccine was highly effective in adults but didn’t give protection to young infants and children who represent the age group most susceptible to the disease. Another challenge is that this vaccine didn’t show success against serogroup B (known as:MenB).
More attempts to overcome the new challenge
Using capsular polysaccharide antigen, as a vaccine against MenB, wasn’t a very good idea. This is due to the fact that MenB capsular polysaccharide is highly similar (nearly identical) to N-acetyl neuraminic acid which is widely distributed in human tissue and that means it is a self antigen. The new vaccine was poorly immunogenic (and thus provide poor protection) and also it might elicit auto-antibodies.
As scientists never give up, they switched into the new trend in vaccinology: Reverse vaccinology. Due to the formerly mentioned, N.meningitidis was expected to be a very promising candidate for reverse vaccinology.
RV provides help
Using in silico technique (computational biology), the genome sequence was fed into a computer, 570 proteins of the bacterium surface were predicted. Going for more refinement, only 350 of these proteins were successfully expressed in E.coli and and used to immunise mice. The sera assays allowed the identification of certain proteins that elicit bactericidal antibodies and surprisingly, were conservative among different strains and this meets the criteria for a good vaccine.
As research is proceeding to deprive our pathogen from enjoying its life inside our bodies, this work suggested further research of other pathogens such as: Streptococcus pneumoniae, Chlamydia pneumoniae, Bascillus anthracis, T.B and group B Streptococcus.
A universal vaccine against serogroup B meningococcus.
, reverse vaccinology
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For many decades, conventional vaccinology has faced many obstacles. One major problem is that among several antigens of the microbe, you have to identify the most immunogenic (and thus protective) antigens (such as virulent factors, toxins, surface-associated proteins, etc.) suitable for vaccine development. This process is very fastidious and costs a lot as it relies mainly on traditional biochemical and microbiological methods. As a summary, it is carried out as following:
- Firstly, you have to cultivate the microbe and harvest proteins.
- Then you have to identify the antigens one by one.
- After that you can pass to vaccine development stage.
Introducing genomics has greatly contributed to providing a new impulse to vaccinology field. The major role it plays is in the antigen discovery stage. As the genome sequence of many microbes has been identified, the integration between the sequence, proteomics and microarray has introduced what is called “reverse vaccinology” . Reverse vaccinology (RV) means to identify and characterise the antigen using bioinformatics. In RV, you start from the genome and not from the pathogen itself i.e. you start from the opposite direction, that’s why it is called “reverse”.
RV will provide solutions to some problems that usually come up during vaccine development as:
- It will provide fast access to almost all antigens including:less common antigens and antigens not expressed in vitro.
- It represents a new approach for non culturable microorganisms.
On the other hand, the major disadvantage of RV is that it cannot be applied to non-proteinaceous antigens such as lipopolysaccharides and glycolipids.
, reverse vaccinology
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Many drugs can’t do it, but GBS can!
Bacterial meningitis is one of the leading causes of death and disability among childrens. Meningits occurs when bacteria cross blood brain barrier (BBB) after interacting with human Brain microvascular endothelial cells (hBMECs) . Although these cells are exhibiting tight junctions and lacking pinocytosis, some bacteria could cross it and this demonstrates an interplay between host cells and some bacterial factors.
Scientists at USCD school of medicine used a process involving generating and screening of many group B streptococcus (GBS) in tissue culture model of human BBB (consisting of immortalised hBMECs) . This culture maintained the normal function of human BBB.
They identified a gene called iagA gene encoding for a glycosyltransferase. A predicted product of the iagA glycosyltransferase is the glycolipid diglycosyldiacylglycerol involved in anchoring lipoteichoic acid (LTA) and consequently, enhances BBB invasion.
Allelic replacement of the iagA gene,so that the resulting mutants are lacking the gene, shed LTA into the media. As a result, mice infected with mutant gene exhibited less mortality rate -up to 90 percent- compared to wild-type infected mice. Mutant-type infected mice developed bacteremia as WT which proves the fact that iagA gene plays the central role in BBB invasion without significantly affecting adhesion or blood survival.
Since bacterial meningitis may cause infected children death or many complications as permanent cognitive deficits, blindness, deafness or seizures, an early treatment may help reduce high rates of death and disability.
This early treatment may be much more easily designed after these findings by blocking LTA anchoring on bacterial cell surface.This will help preventing meningitis even though bacteriemia has taken place.
The journal of clinical investigation has the full story.
Tags: drug targets
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