Thursday, 31 March 2011

Working from the Ground Up - Genome Research

Back in the 1980's DNA sequencing was carried out 250 base pairs at a time, often a 3-4 day process. The DNA in even a simple species (i.e. its genome) such as Escherichia coli contains around 5 million base pairs, so you can imagine how long it would take to read the entire genome. The genome size of much larger organisms such as ourselves are a thousand times larger!

Such is the importance of being able to read the genomes of many species (e.g. we need to know what makes a dangerous bacterium 'tick' so we need its sequence), that DNA sequencing technology has been massively developed and speeded up. We now routinely use robots to generate DNA sequences at a furious rate, sequencing up to 5-9 million base pairs per day.

 So successful has this effort been that the international repositories for DNA sequences (EMBL, NCBI &  DDBJ currently hold over 2000 billion base pairs from thousands of different species.

Clearly the problem we have in being able to understand how cells work is no longer one of being able to read DNA sequences! But what about the next step? DNA codes for RNA & protein and it is those products that are used to construct all parts of all living things. We now have the DNA sequences and can use these to predict which proteins can be made, giving us a big clue as to what makes different organisms diferent, including what makes them dangerous to us.

However it isn't as simple as all that. Deciding which proteins are expressed is a difficult business at best and with many millions of base pairs to sort through (estimating the average size of a proteing to take up 1000 base pairs) it is a long process. Naturally computers can help us and there are many examples of 'automated annotation' of genomes available, all of which have been carried out by computer software (see detailed explanation of the technical procedures here).

Unfortunately computers can only work from known information, so to get a full accurate appreciation of a genome and to discover new information from that sequence humans must also annotate manually - as you might imagine that is a slow process akin to sequencing 250 base pairs at a time back in the 1980's.

There seem to be 3 complete Aspergillus genomes available at NCBI, and there are two specialist Aspergillus resources that host another 11 genomes: CADRE hosts 9 with mainly automated annotation and the Aspergillus Genome Database hosts 2 which are fairly extensively manually annotated.

A recent project funded by NCBI has attempted to take automated annotation to a higher level of detail (Gnomon). The genome information known about 8 Aspergillus genomes has been pooled and compared with other similar organisms about which a lot more is known (e.g. S. cerevisiae, S pombe - yeast). Gradually more information is gleaned when similarities between areas of each genome are found and compared - effectively a shortcut to maximising the information known about all fungal species by using the information already established from one or two species.

This researc will ultimately be able to tell us about all of the genes in our bodies, when they are expressed and how they are expressed. There is a long way to go but back in the 1980's it was barely believeable that we would be able to read the DNA sequence of one complex organism in our lifetime. That problem was solved very impressively so perhaps in 30 years time we will look back and wonder why we ever doubted it could all be done - possibly while running computer models of entire cells!

Wednesday, 23 March 2011

Presence of Mould Species Correlate with Presence of Asthma

Last week we featured a paper that appeared to demonstrate that the presence of some moulds at a very early age correlated with the lowering of the susceptibility of children to asthma in later life. The authors postulated a 'protective effect' caused by the triggering and presumably modulation of infection response pathways involved in atopy and asthma.

In that paper the 'mouldiness' of the houses examined was determined by the collection of dust samples from those houses.

A new paper takes a look at this method of sampling, its accuracy and reliability and compares it to the results of a newly developed test based on Polymerase Chain Reaction technology (PCR). This new test looks at the DNA 'signatures' left by more than 100 specific fungi and can accurately detect and reliably identify each species and how much of each species is present (mould specific quantitative PCR (MSQPCR)).

Older, traditional technology is fraught with potential difficulties and sources of inaccuracy and in some ways the new test is inherently more accurate, faster and gives us more information. Both still rely on dust samples which may or may not be representative of current mould levels in any house at a particular time but they can usually be a reasonable guide to the overall state of mouldiness of a house.

MSQPCR was used to compare mould levels in damp houses with those in dry houses and was able to develop an index of which mould species tend to be associated with damp houses - the Environmental Relative Mouldiness Index (ERMI).

Using this index the researchers found that houses suffering from moulds that are present in damp houses tend to house children with asthma - that is to say particular moulds correlate with asthma in children.

You might now be saying "This is the opposite conclusion to that made by the paper we covered last week isn't it??". In fact the paper published last week was making its conclusions based on information that was not as detailed as this new paper. In that paper they could not differentiate individual species and it is the precise finding of this new paper that only specific species are correlating with damp & asthma. The findings of the first paper where that a particular group of moulds (possibly containing dozens of species) correlates with lack of asthma, but we don't know which species are involved, only that a broader range of species are involved compared with homes that are associated with atopy & asthma.

This second paper effectively follows on from the first rather than disagrees. Hopefully now that we have been able to start to identify 'bad' moulds (as mentioned last week) we will be able to make more progress in preventing childhood asthma.

Friday, 18 March 2011

Childhood Exposure to Aspergillus Protects Against Asthma

A recent paper published in the highly prestigious journal 'The New England Journal of Medicine' looked at the breadth of exposure to microbes of children who grow up in rural homes and their frequency of developing asthma.

The rational goes as follows: the rural & farming environment exposes children to a far wider range of microbes compared with children in a city environment so we should be able to detect differences between the two population with regard to their health - in this case asthma and atopy. If these microbes are bad then those children should suffer from more asthma. The study showed the opposite to be true - children exposed to a wider range of microbes - including moulds such as Aspergillus - suffered from less asthma.

There is similarity to the report published 2 years ago that children who played in dirt more often were prone to less allergy (BBC report). In that study researchers found that Staphylococci microbes actually protected children from over-reacting to common allergens.

In this more recent study the same has been found for Aspergillus and bacteria such as Listeria. Exposure at a young age to these microbes seems to protect children against developing asthma.

Perhaps that camping holiday in the countryside does more good for your children than simply providing fresh air and exercise, perhaps it exposes them (especially at a young pre school age) to beneficial microbes which help prevent asthma.

Does this mean that living in a damp house is beneficial to your child? Absolutely not. It has been demonstrated several times that damp housing is bad for the health of your child. There is no mention of the housing standards of the children involved in this study and perhaps children from farms had less damp houses? Regardless there is something about damp housing that is bad for our health.
Perhaps when this research proceeds further we will be able to identify particular microbes that are good, and some that are bad (as the preceding study on allergy did). Perhaps it will show that bad microbes tend to overgrow in damp housing.

At the moment all we have are conclusions based on identification  of very broad groups of microbes potentially containing many hundreds of different microorganisms. We have a general observation that exposure to a broader range of microbes is good for atopy & asthma. We now need more specific work to be carried out. Nonetheless we have moved one step forward in the battle against asthma.

Friday, 11 March 2011

New Treatment for Chronic Granulomatous Disorder (CGD)

Chronic Granulomatous Disorder (CGD) is a genetically inherited severe immune deficiency that effects one in 200 000 people per year worldwide. Its sufferers have a reduced ability to fight off infection and because of the inherited nature of the disorder and the severity of the consequences many die at a young age. This is predominantly an illness that effects children and young people.

Children with CGD often suffer life threatening infections and one of the worst is infection with Aspergillus. Aspergillus is able to infection people with normal immune systems who have damaged lungs but those infections are usually limited to the site of infection and can sometimes be removed surgically with good results. CGD patients cannot limit the spread of Aspergillus as efficiently as non-CGD people as their immune system is weaker, so once Aspergillus infects a CGD patients it is much more difficult to prevent it spreading. CGD patients have to be treated more often with antifungal medication.

New research has found a new method to treat CGD patients. One of the ways that the immune system of a CGD patients does not work properly is in the production of reactive chemicals by specific immune cells (neutrophils) that are used to kill invading microbes. It has also been found that neutrophils also respond to infection by forming a structure called a Neutrophil Extracellular Trap (NET). These literally form a network of fibres rather like a spiders web that trap microbes and then exude reactive chemicals to kill microbes once trapped. One of the chemicals produced is calprotectin which has antifungal properties and this research has pinpointed that calprotectin is important in the battle against Aspergillus infection. Neutrophils from CGD patients cannot form NET's or produce calprotectin.

How do we treat this absence of calprotectin in CGD patients? Bianchi used gene therapy techniques to restore the ability of CGD neutrophils to produce calprotectin and found that they were also able to form NETs and successfully fight off a pre-existing Aspergillus infection in human patients. This patient already had a life threatening infection prior to treating with gene therapy. Gene therapy effectively 'cured' some of the immune deficiency and the restored immune system was successful in defeating the aspergillosis.

This extraordinary result implies that gene therapy now has the potential to be a routine treatment for aspergillosis in CGD patients - it can be used when there is an ongoing infection. In practice this treatment is only being recommended for use in infections where antifungal therapy is ineffective as the current method involves the use of a DNA construct based on a disabled virus. This virus is presumably infected into neutrophils (details of exactly how this has been done had eluded me) and there are risks associated with that strategy. Until more is known about those risks gene therapy will be used with caution.

This research has been supported by Chronic Granulomatous Disorder Research Trust, United

Friday, 4 March 2011

Secondary Prophylaxis with Antifungals for Leukemia Patients - recommended or not?

A recent study looked at how well patients suffering from acute leukemia recovered after they had contracted an invasive fungal infection - something that affects 5-15% of all acute myeloid leukemia patients. Those that get a fungal infection often have to have their chemotherapy delayed while the infection is treated as chemotherapy can reduce the abilty of the patient to fight the fungus, leaving him/her vulnerable to a serious invasive infection. Unfortunately delaying chemotherapy can cause a worsening of the leukemia - this is truly a 'catch 22' situation of the most demanding kind with severe consequences. Shall the doctor attempt to limit the infection or the cancer first? - if the doctor neglects either it is potentially going to cause problems for the patient but going forward with chemotherapy before treating the fungal infection adequately will also cause problems later on.

Not too surprisingly the paper published finds that patients who get a fungal infection tend to do less well than those who do not. They recommend protecting patients due for chemotherapy by giving a course of antifungal drugs prior to the chemotherapy whether or not invasive fungal infection is suspected (primary prophylaxis).

Diagnosis of a fungal infection in these patents is very problematic as by definition they have a disease of their immune system, therefore using the same immune system to check for infection isn't going to work well in some cases and some diagnostic techniques rely on the patient having a good immune system. Primary prophylaxis thus compensates for any undetected infections by treating anyway!

The second recommendation is to provide secondary prophylaxis - this is treatment to protect against any infections that happen during or after subsequent rounds of chemotherapy.

In a letter to the editor (Cordonnier)  it is argued that secondary prophylaxis is warranted if the fungal infection is not active (i.e. 'quiescent') after transplant. I assume that means an initial infection has been treated and may well not have completely gone but is not actively growing. The antifungal prophylaxis may then keep it in check - this is open to experimentation to provide evidence.

There is another risk that these patients face and that is from new fungal infections after the transplant. These are likely to be highly active  (i.e. not 'quiescent') and Cordonnier seems to argue that secondary prophylaxis is useful to prevent these infections as well. Girmenia argues that in this context the usefulness of secondary antifungal prophylaxis is far less clear and that this should be the subject of more research before coming to firm conclusions.

It is easy to see that the best procedures for the treatment of patients undergoing treatment for leukemia are still a subject for much discussion & research. It is clear that much progress has been made and in many cases where a patient with leukemia and an invasive fungal infection (usually aspergillosis) would have been given little hope some time ago, there is now a much better chance of survival. This is still a serious combination of illnesses with relatively high levels of mortality but slowly and surely they are being brought under our control.

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