Wednesday, 29 September 2010

New Research to Help Identify Targets for New Antifungals

One of the first requirements to begin development of a new antifungal drug is to identify what target the drug will be aimed at. The target must be specific and not exist in human cells so that the drug does not affect the patient, and it must be effective in that it must attack part of the fungus that is vital for its growth.
Currently we have a series of antifungal drugs that fall into three broad categories,
  • those that target the cell wall (something that does not exist in mammals) that the fungus relies on for physical cell integrity e.g. echinocandins: caspofungin, micafungin, and anidulafungin. These tend to be very safe and have fewer side effects.
  • those that target the cell membrane e.g. amphotericin B, azoles (Itraconazole, voriconazole, posaconazole etc). Mammalian (including human)  cells have a cell membrane so this is a less specific target. These drugs target chemicals (ergosterols) that are far more plentiful in fungal cell membranes than they are in mammalian cell membranes so fungi are hit a lot harder than mammalian cells by these drugs. Consequently these drugs are effective at tackling fungal infections but do become toxic to humans if the dose is too high - a lot of care is taken with the use of these drugs to ensure dose is optimised and side effects are minimised. 
  • those that target DNA/RNA synthesis e.g. flucytosine. Mammalian cells make extensive use of DNA & RNA so this drug lacks specificity. Difficult to use.
Clearly the best existing target in terms of specificity is the cell wall. In this news report it states that this research group have just announced the $1.5 million funding of new research into drugs that will act on a component of the construction of cell walls (UDP-galactopyranose mutase) in fungi with the aim of disrupting the process and thus making it very difficult for the fungus to make its own cell wall and grow normally. This particular target has not been utilised for antifungal drugs before now and we already know that if the gene for UDP-galactopyranose mutase is knocked out by genetic manupulation the ability of Aspergillus fumigatus to infect mammals is significantly impaired (reduced virulence) so it is likely that inhibiting its activity using a new drug will inhibit infection.

Galactofuranose (the chemical made by UDP-galactopyranose mutase) is extensively used by A. fumigatus and is already being  investigated as a molecule we could use to detect infection by this fungal pathogen, so further research into this molecule has the potential to improve diagnosis, detection and treatment of A. fumigatus infections.

The prospects for this antifungal target look good though of course there are many hurdles to overcome yet, but this is certainly one of the possibilities for improving the treatment of fungal infections, possibly serious fungal infections caused by Aspergillus fumigatus.

References
Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus
Targeted Gene Deletion of
Leishmania major UDP-galactopyranose Mutase Leads to Attenuated Virulence

Thursday, 23 September 2010

Aspergillus Genomics and Metabolic Pathways - AsperCyc

Genomics is the study of the  effect of genes and gene expression on the entire genome. Originating in the1970's the huge advances in DNA sequencing techniques, in particular sequencing carried out by robots in the last 10 years or so at huge dedicated centres has led to large numbers of organisms having their entire genomes sequenced, including humans.
There are thousands of bacteria and virus genomes sequenced as they are small and simple in terms of genome size. It is a far larger task to sequence the genome of fungi, plants and animals - collectively referred to as eukaryotes - so fewer of these have been sequenced but even there the process is accelerating.

We have sequence information for the whole genomes of at least 9 species of Aspergillus (CADRE and aspGD) with many more planned. This means that we have access to the entire book of life for each species, each consisting of 7 or 8 volumes and a total of 30 million 'letters' (base pairs) or 10 million 'words' (codons). To give you an idea of how big that is, the combined plays of Shakespeare number 39 in all with a total of less than 1 million words. The Bible totals less than 600 000 words. We are storing enough information to fill 150 Bibles 0r 100 collections of the plays of Shakespeare!

Just as books looked at word by word are less informative that when we read whole pages, codons are not particularly informative unless we can link them together into functional genes. Aspergillus codons form nearly 30 000 genes of which 10 000 are known to be in use - already we have a big piece of information that we could only guess at before genome sequencing. 20 years ago scientists would have looked at an expressing gene, cut it out of the genome, sequenced it and characterised its expression. All of that could have taken several years to complete. Even then we would have only gathered information about that particular gene and often only the parts of the gene that are actually expressed - little was known about how neighbouring DNA sequences influence the expression of that gene.

Now we already have all of the gene information we need for every gene in an organism and it is stored in the context that it is stored in in our cells so we can look at all of the sequences either side of the gene - and this is all at the tips of our fingers in freely available computer databases. We can now look for sequences either side of a gene that are known to control the expression of similar genes, we can locate unique sequences and extract whole genes for examination very quickly, and we can even construct genes completely artificially if we need to - this is technology beyond the wildest dreams of 30 years ago  which along with other similarly revolutionary techniques is massively speeding up the accumulation of knowledge.

Massively important though all this is, we have still only begun to look at single pages in the genome 'book', much more information is available. Single genes are only capable of one function each. Most of the substances that our cells make that enable us to stay alive are complex and are built up from other materials. Substrates have to be broken down and then rebuilt into a useful form and no gene is capable of doing that on its own. Several, sometimes dozens of genes must be expressed and their products utilised sequentially, each one providing the means for another step in pathways that can be many steps long. These are known as Metabolic Pathways.

Genome information and pathway information are currently in the process of being put together for each sequenced organism (MetaCyc, BioCyc). This is the equivalent af starting to put the pages of our genome 'book' together into chapters. We are helping this process for Aspergillus by the introduction of AsperCyc. So far we are largely relying on computers identifying potential pathways as there is such a huge amount of data to be processed but there is a slow process of manual curation ongoing where a human will cross check what the computer has decided with what is known through published papers.

This is the next step in the ultimate dream of being able to give a computer a genome sequence (or even some DNA) and then stand back as it detects the genes, assesses their potential for expression, assigns them to pathways, and then runs a full simulation of how all of the genes interact with each other to form a living cell. We could then introduce changes to gene expression and watch the consequences, discover new drug targetsand ultimately test new drugs. To continue the analogy we would be reading the whole genome 'book' and analysing the full meaning of the contents. This could have innumerable benefits, for example if someone has a genetic disorder we would be able to work out how to counter the effects of the mutation as we would have a full picture of all of the effects of that disorder presented to us by the computer.

There is some way to go yet but progress is being made and computer power is getting cheaper all the time - adopt an optimistic attitude it is not too difficult to see a positive conclusion!

Tuesday, 21 September 2010

Be Aware of the Risks of Inhaling Cannabis

Recent reports show that the number of cannabis farms in the UK may well be on the rise with twice as many detected and closed down in 2009/10 compared with 2007/8 according to a report from the Association of Chief Police Officers in the UK.
Increase in production is not something restricted to the UK as rises in production are also reported in Mexico with detection rates also on the rise in the US.

In the face of these increases in supply and presumably use it is worth making the point again that plant material such as marijuana is an excellent food source for fungi such as Aspergillus. Once cut the material must be dried rapidly and consistently to a very low moisture level to avoid it becoming mouldy. Once dried it must be stored in completely dry conditions to prevent it becoming damp and once again quickly becoming mouldy.
Storing marijuana or any other plant material in small sealed containers or wrapped in plastic will only help if the material is completely dry in the first place, otherwise you are effectively locking the mould in with its own supply of food and water whereupon it will flourish. Mould does not need light or much heat to grow.

Once it has run out of food it will sporulate, emitting billions of tiny spores that are small enough to float on the slightest draught of air. If this material were to be wrapped up in a paper tube or placed in a pipe, one end lit and inhaled then billions of spores would very easily penetrate the lungs of the smoker to its deepest depths as they are small enough to spread to the smallest air pockets in the lungs.

 Even if the smoker has a completely normal immune system it would only be able to clear so many spores in a given time. If they have any pre-existing debris or scar tissue in their lungs the spores may well be able to evade the immune system and start to grow in a similar way that the bacteria that cause tuberculosis (Tb) grow and cause cavities in the lung. Tuberculosis can be treated successfully using strong antibiotics. Deep infections by Aspergillus are far harder to eradicate and tend to become chronic infections, even with all the latest antifungal medication attacking it - the marijuana smoker is more likely to have lifelong infections that become highly debilitating.

Marijuana is a special case as it is produced and stored under a wide variety of conditions. The material you purchase is of unknown origin so its mould status is unknown. Checking its dryness on delivery is no guide to how quickly it has been dried and thus no guide to how many spores it may carry. Needless to say there is no official monitoring system controlling the quality of the supply.

Aspergillus can be black, green, brown, white, yellow or blue in colour - it is all bad and you will not necessarily be able to see it at all. Picking a few bits of mould off the material makes no difference. If you inhale in the form of a cigarette you are inhaling smoke from the burning tip through an entire tube of mouldy material - it will not be sterilised.

Breathe this material in at your own risk in full knowledge of the potential hazards.

Tuesday, 14 September 2010

Mouldy Films Can Project More Than Movies

An examination of old film stock by members of the Dept Biology, Chemistry and Health Science at Manchester Metropolitan University has revealed something more sinister than damaged movie footage. If the film is subjected to a 'mock projection' it can release enough spores into the air than is safe for the person working with the film to breathe in.

The team at Manchester Metropolitan University (MMU), headed by Professor Joanna Verran recently reported their finding to the Society for General Microbiology autumn meeting (September 2010). Research student Gavin Bingley presented his work describing how they collected a range of contaminated film from archives at the North West Film Archive and the British Film Institute National Archives and subjected the film to mock inspections, much as an archivist would carry out with a view to assessing the film for further preservation work. On the worst affected films the levels of spores released during this process where considered to be dangerous to health - Aspergillus was a common fungus found and is a known allergen & pathogen..

Traditionally photographic film is largely made out of cellulose & gelatin and as such is vulnerable to microbial attack if not stored correctly. Any damp getting into the film stock can quickly lead to fungal growth as the gelatin makes an excellent food source. Older films are thus actively collected and assessed for damage. Many (most) have already been lost and many techniques have been developed to halt this loss with a lot of resources now going into preserving what is left, hopefully forever.

The MMU group are working to assist the safe continuation of this valuable work by developing a sensor that will indicate if viable mould is present in a can of film and thus signal to the person handling the film whether or not it is safe to open the can without taking further precautions e.g. using an airflow hood to suck spores away from the person concerned and face masks designed to prevent inhalation of mould spores.

Of course even if there is no viable fungal material present then there could still be dead fungal matter that could be released into the air and could still cause allergy problems, so an assessment of the past state of the film is also important from the point of view of the health of the people working on the film. The MMU team are also working on standard recommendations for the safety of people handling mouldy film, viable or not.

Friday, 10 September 2010

Why are some people vulnerable to Aspergillosis?

Acute invasive aspergillosis is mainly a threat to patients who have a severely compromised immune system. By definition they are poor at fighting off infection and thus run a high risk of infection by all micro-organisms, including Aspergillus.

Once of the reasons why people become severely immunocompromised is when they are undergoing a stem cell transplant (HSCT) for the purpose of replacing their entire immune system. This is most often carried out as a treatment for leukaemia or myeloma, cancers of the immune system, and to treat other blood disorders. HSCT requires a donor to contribute the healthy immune cells and requires the complete removal of the recipient's immune cells. This leads to a short period of a few days when the recipient has few immune cells while they wait for the donors cells to start working fully and it is at this time that the infection can occur. Once infected the fungus is difficult to eradicate, even for the rapidly returning immune cells. Some people seem to be more vulnerable to this infection that others and we don't yet know why.

Dectin is known to be important in our immune response to Aspergillus infection, switching on the inflammatory response when exposed to Aspergillus fumigatus spores. Dectin, in common with other genes, has small variations in its DNA code (Single Nuclear Polymorphism - SNP) from person to person. Most SNP's are rare and have small if any detectable effect but some do and there is one in Dectin (named the DECTIN1 Y238X polymorphism) that has a severe effect on the gene.  Theoretically it might be that those people who carry this SNP are more susceptible to fungal infection. If we knew the answer to that question we would be able to screen HSCT recipients and donors for the SNP and reject the donor or take extra steps to reduce the chances of fungal infection if the recipient is effected.

This recent paper has carried out experiments to try to provide answers.
  • It turns out that recipients who carry the Y238X polymorphism have twice the chance of developing invasive aspergillosis up to 36 months post transplant compared with those who do not. 
  • If the donated cells carry the Y238X polymorphism then the recipient has twice the chance of developing invasive aspergillosis
  • If both the recipient and the donor carry the Y238X polymorphism the chances of developing invasive aspergillosis is even higher at 2.5 to 3x those who do not carry the faulty gene.
This result is fairly clear - HSCT patients need to know their Y238X SNP status and that of their donors.

There is an interesting extra experiment in this paper. The authors set up a mouse model system for HSCT using a mouse strain that lacked Dectin. Those mice that expressed Dectin showed a mild inflammatory response  when Aspergillus fumigatus conidia where used to infect their lungs whereas mice that had no Dectin became highly inflamed.

The conclusions are that Dectin controls resistance to A. fumigatus infection and also controls inflammation in response to A. fumigatus conidia. The latter may well be linked to the former as inflammation is a two ended sword for our ability to fight off infections, including aspergillosis. Inflammation that follows shortly after an infection is part of the normal process of eradicating that infection, but inflammation can also prevent eradication of an infection if it becomes uncontrolled. Examples included Chronic Granulomatous Disorder where there is chronic inflammation but persistent infection at the same time (link).

This paper illustrates the progress being made to identify people vulnerable to infection by Aspergillus and provides hope that in this case at least we now know a little more about preventing aspergillosis.

Friday, 3 September 2010

Aspergillus is Associated with Worsening Asthma

Recent studies have led us to start to think that the presence of fungus colonising the lungs of asthma sufferers contributes to the severity of their symptoms & disease. This paper takes a look at the current relationship between:
  • Sensitisation to Aspergillus (i.e. the patient has been shown to be reacting to Aspergillus by looking for the present of specific antibodies to Aspergillus in their blood)
  • Asthma status (GINA scale)
  • Presence of A.fumigatus in sputum (i.e. an indication that Aspergillus is living in their lungs)
79 patients were involved in the trial, divided up into 3 different groups according to their level of sensitivity to Aspergillus fumigatus.

The numbers of patients who had Aspergillus fumigatus recovered from their sputum correlated with their level of sensitivity to Aspergillus, suggesting that sensitisation is a consequence of their exposure to the organism living in their lungs - an observation that is not as obvious as it might seem as we all breathe in Aspergillus in the air without necessarily becoming colonised.

Patients sensitised to Aspergillus also had lower lung function, more bronchiectasis, and more neutrophils.

This study concludes that there is an association between Aspergillus in sputum and sensitisation, numbers of neutrophils and reduced lung function.

It is easy to construct a model system using these results whereby an asthmatic person becomes colonised by Aspergillus and as the colonisation proceeds the asthma deteriorates, the latter being a consequence of the former. The earlier paper mentioned seems to show that if that group of people are treated with an antifungal their asthma improves, and that would support this model.

Does Aspergillus therefore cause at least some severe asthma? Perhaps but there is some way to go yet before we can come to firm conclusions. Some centres are already treating fungally-sensitive severe asthmatics (SAFS) with antifungals with good results so the future for this hypothesis does look promising.

NB Colonisation could be defined as an organism living in the lungs without causing noticeable symptoms. This is distinct from inflammation which is where the organism is causing a response from the immune system of the host i.e. inflammation.

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