Friday, 29 July 2011

Growth of Fungi: Time Lapse Video

Though not all Aspergillus fungi, this excellent video shows clearly the many growth phases of filamentous fungi on laboratory media going from initial growth to sporulation (0:53). Then the culture becomes infected by insects for a while (3:23) and ultimately the fungus dies.
Shot by someone we only know as 'Nick' it is also worth listening to the accompanying music!

We can get some idea of the level of specialised equipment needed for this work by looking at his setup below
We can see the round  white Petri dish containing the growing fungus at the centre of the picture with what looks like a device to allow the camera to rotate around the dish to which the camera is clamped, or perhaps the dish rotates in front of a stationary camera.

One point worth making, and if you are reading this Nick please take note, if that is Aspergillus fumigatus on that plate (amongst other fungi) you should be thinking about being very careful breathing the air around that apparatus especially if the plate is usually uncovered. Growing such a concentrated collection of fungi leaves you at a high risk of breathing in large numbers of spores which are very easily dislodged when touching the plate, moving the plate or with any disturbance of the air. Needless to say disposing of the plate should be done asceptically. Aspergillus fumigatus is a very dangerous pathogen if you or anyone else in the area are in any way susceptible to infection, we don't yet understand how to tell if someone is susceptible when they are immunocompetent so you will not know if you are in danger.

A good idea would be to enclose the plate in a cabinet which would take the air away from your face (that doesn't look practical here) or to wear a good HEPA grade facemask, prefereably one that includes cover for your eyes too but at the very least one of those described here!

Tuesday, 26 July 2011

Research to Prevent Mucus Overproduction in Airways in Aspergillosis - a new clue

Excessive mucin production is a major debilitating feature of allergic bronchopulmonary mycoses such as ABPA. The mucin characteristically causes major blockages of bronchial airways and is very difficult to remove and is tends to be very sticky and clinging in nature. Mucin production is an important part of the mechanisms that protect our airways as it can bind to harmful microorganisms and prevent them growing, but in some circumstances there appears to be too much mucin produced and it can start to cause problems - not the least of which is suppression of the fine hairs lining our airways (cilia) that brush debris and micro-organisms out of our lungs.
In conditions such as ABPA mucin overproduction also make it difficult to detect and sample the strain(s) of Aspergillus that are infecting, as it can make it very difficult to isolate samples of the fungus and grow it on in laboratory culture plates. It may well also inhibit antifungal drugs from getting at the fungus - for fungal infections mucin is not at all helpful!
If we could control mucin production we should be able to prevent it being overproduced and thus be able to overcome many of the problems faced by both the patients and the team trying to treat them.
This recent paper shows that at least in the laboratory mucin production by cells that line the walls of our airways can be selectively stimulated by the presence of Aspergillus fumigatus extracts via the gene MUC5AC.
The suggestion is that one of the ways A. fumigatus can establish itself in our lungs is by overstimulating mucin production, hindering our natural defence mechanisms and allowing the fungus to persist. Importantly the authors show that the induction of mucin can be diminished by administering substances that block the activities of TGF-alpha or epidermal growth factor (EGF), both well characterised gene regulating factors.
It looks like TGF and EGF induction by (as yet unknown) factors in Aspergillus fumigatus might be causing a cascade of induction of mucin causing overproduction via other known intermediaries, the result of which we apparently see happening in human patients. We may now have several new potential targets to use to improve treatment of ABPA and similar diseases
NOTE: we mentioned genome sequencing in earlier blogs, but an equally important relatively new technology that has followed from the ability to sequence an entire genome is that of microarrays where we can quickly determine the expression of all of the genes in an organism under specific circumstances. This paper being discussed in this blog is an example of a discovery made using this technology.

Friday, 22 July 2011

NewsBite: Aspergillus sojae Genome Sequenced

The complete genome of Aspergillus sojae has been published. Sequence was compared to that of Aspergillus oryzae (another species widely used for fermentation to prepare food - Soy sauce in this case) and genetic similarities noted alongside functional similarities in the hope of understanding differences between these strains and other sequenced Aspergillus species.

Wednesday, 20 July 2011

Lung Regeneration to Cure Chronic Lung Disease - Getting Closer to Science Fact not Fiction

Striking new research published over the last couple of months gives hope to everyone with chronic lung disease. The world's media recently covered the story of a synthetic windpipe made using stem cells from the recipient seeded into an artificial framework which then grew to form a complete replacement organ.
The key to growing new organs is the isolation of stem cells. These are elusive and difficult to purify but it appears that it is possible in human lung tissue and the resultant cells are capable of regenerating lung structures in animal models.
In vitro the isolated cell lines (isolated using the expression of c-kit as a marker for stem cells) proved to be able to self renew, to lack any detected features of differentiation (unlike earlier cell lines), were clonal in origin and had the ability to differentiate into a range of cell types. In this case these stem cells were shown to be able to differentiate into lung cells but not cardiac cells (multipotent).
Chapman puts this paper into the context of our current understanding of how lung tissue is formed during development of the embryo. Formerly it was thought that the mature lung was made up of at least two cell lines from distinct cell lineages. This means it would be impossible to make complete lung tissue from a single cell - at least two different cell lines (mesoderm and epithelial tissue) are needed. This paper suggests otherwise as the injected human stem cells - all derived from a single cell - were able to form all of the types of structures seen in the distal lung and in only 14 days
The fact that these stem cells could be isolated from mature (fully differentiated) distal lung tissue is just as striking. Are these cells actively repairing lung tissue in situ all the time? Can they be 'persuaded' to multiply and repair our lungs given the right treatment? As yet we do not know the answer.
In the meantime we should note that experiments have already been successfully carried out which involve stripping all the cells from a set of lungs leaving only a supporting structure in place. That structure has then been repopulated using lung cells, replaced into an animal model and shown to work, albeit for only up to 2 hours. Perhaps here is a hint that if we can do something similar with human lungs, stem cells will be able to do a much better job of regrowing complete lungs. 
Time will tell, but something that was science fiction only 5 years ago is starting to look like it has a chance of becoming reality. People who have severely damaged lungs may be able to benefit by regrowing part for new lungs which will be made from their own cells so they will not have to take drugs to combat rejection. Regrowth could conceivably take the form of regrowth alongside their current organs, regenerating them gradually (either by injecting isolated stem cells into the area or perhaps by stimulating existing stem cells), or it could even mean growing tissue outside of the body and transplanting it into the patient. There are interesting times ahead!

Friday, 15 July 2011

NewsBites: Confocal Laser Scanning used to Quantify Host/Fungus Interaction

Scientists in Switzerland have devised a method to automatically count the numbers of fungal conidia being engulfed by our immune system (macrophages). Aspergillus fumigatus mutants were analysed for ability to evade capture and thus continue to infect the host. Mutant pksP was particularly adept at avoiding capture and this technique also revealed other changes in how its conidia behaved when confronted by macrophages i.e. a greater tendency to clump together - not previously noted. This systematic technique shows promise for the further investigation of conidia/macrophage interaction and its effect on pathogenicity

Tuesday, 12 July 2011

A vaccine for aspergillosis?

Successful vaccines have been created to protect against pathogenic bacteria and viruses. Why aren't there any for combating fungal infections? A recent article in The Scientist by Brad Spellberg discusses the possibilities.

"Invasive fungal diseases often take hold when a person’s natural defenses are weakened. These infections frequently occur in hospital settings, after a patient’s normal bacterial flora is wiped out by antibiotics, or the skin and gut mucosa are breached by surgery or central venous catheters including for intravenous nutrition. In fact, candidiasis, an infection caused by one of several species of the yeast Candida, is now the fourth most common bloodstream infection in hospitalized patients both in the United States and in many European countries. And the death rate from such Candida infections remains about 30 to 40 percent, even after treatment with antifungal therapy. Given their increasing frequency and unacceptably high morbidity and mortality rates, prevention of invasive fungal infections has become of paramount importance. Vaccination is a promising strategy for prevention, since it has the potential to permanently protect individuals from fungal infection."

The premise that all patients who develop life-threatening fungal infections have profound defects in their immune system -implying that their immune systems would be too weak to respond to vaccination - has led to incorrect assumptions that would limit the usefulness of an anti-fungal vaccine.
However if you consider patients who develop serious candida infections of the bloodstream- only 10-20% are seriously immunocompromised- the remainder have developed infection because their susceptibility has increased whilst in hospital -due to broad spectrum antibiotic usage, surgery, IV catheters etc. Such patients have relatively intact immune systems - besides there is significant evidence in the literature that even patients with very weakened immunity - can generate adequate immune responses to vaccination - for example - to the influenza or pneumonia vaccines - which are often given to leukaemic patients, HIV and those on corticosteroids.
A candida vaccine is currently entering phase I trials - based on the agglutinin-like group of proteins expressed on the cell surface of candida albicans. When injected into mice it prevented widespread lethal candidiasis. Moreover this vaccine targeted TH1 and Th17 CD4+ T helper cells which recruit and activate phagocytic cells that can engulf and destroy the fungus in tissue.

The author points out that vaccination against fungal pathogens very likely requires enhancement of phagocytic host defences whether via antibody mediated or non antibody methods. Also vaccine responsive T cells can provide phagocytic enhancement in the absence of protective antibodies, and contrary to widely held assumption - it is not necessary to develop antibodies that neutralize virulence factors- ie the toxins or disease causing proteins expressed by the pathogen- in order to achieve protection with a vaccine.

So where does that leave us with respect to Aspergillus infection - the second most common cause of hospital acquired invasive fungal infection ? It is more of a challenge to develop a vaccine for Aspergillosis since virtually all patients with invasive illness are highly immunocompromised. The risk factors for aspergillosis include white cell depletion from chemotherapy, leukaemia or bone marrow transplant and long term corticosteroid or immunosuppressant treatment. Since the infections tend to set in after multiple weeks of an at - risk situation - the suggestion that clinicians could vaccinate before infection sets in - is promising.
Early studies using crude extracts of Aspergillus fumigatus as vaccines in mice have shown protection against subsequent infection- however these have now been refined to identify a fungal surface antigen Asp f3 which appears to be the active antigen at least in mice. If this antigen can be produced according to good manufacturing practice it is a potential candidate for a development of a specific Aspergillus vaccine.

Another approach would be to develop a pan fungal vaccine which targets most serious fungal infections. Evidence recently published (Stevens et al) suggests that a combination of glycan (cell wall component) with an immunogenic protein - based on studies from heat killed Saccharomyces (yeast), may lead to developing such a pan fungal vaccine.

Thursday, 7 July 2011

NewsBites: Accurate Identification of Aspergillus Species using Rapid Laser Technology

Traditional identification of Aspergillus species relies upon skilled eyes scanning microscope slides and is prone to error. The authors of this paper utilise MALDI-TOF mass spectrometry to develope a reference spectra which achieves 100% accuracy distinguishing 28 clinically relevant species.

Tuesday, 5 July 2011

15 minute Genome to Help Prevent Aspergillosis

The human genome was sequenced several years ago (2001) with a final 'fine detail' version finished a few years later (2004) after a monumental international effort taking 10-15 years in all.
Analysis tells us many of the 'physical' features of the genome, not least telling us how many  genes there are (expressed and non-expressed), where they are, whether they are likely to be expressed and their DNA sequence.
However the human genome is not a fixed entity. There are differences that occur regularly between the DNA of individuals and groups of people and some of those differences caused differences in how the genes coded by the DNA work - there can be many different variants for a single gene in a population of people. For example eye colour is controlled by many variants of several genes

Our immune systems are also controlled in part by our genes. Variations in those genes can make us resistant or vulnerable to infection, depending on the gene and on the mutations. It is easy to conclude that people who are genetically predisposed to getting infections will be relatively easy to identify once we know which mutations in which genes are important to resist for example an aspergillosis infection. Research is currently focussing on this question (Read this review for more details).

This sounds great  as in theory we will be able to identify mutations in individuals at risk from aspergillosis or other infection and to treat them before they get infected. Unfortunately it isn't as easy as that as to do that we would need to sequence their genome, or at least the parts we know we need to look at. To get a complete picture we may need to sequence the entire genome and as we know that takes 10-15 years to achieve - or does it??

This article in the Economist newspaper shows us the latest new technology for sequencing DNA. Nanopore sequencing offers a massive speed upgrade compared with traditional sequencing methods which date back to the 1970's. Feeding DNA strands through nanopore molecules may allow us to sequence an entire genome in a few minutes. Marry that with powerful computers to read the DNA sequences and theoretically it may become possible to sequence everyone's DNA - The Economist suggests 'while you wait' but that might be a little optimistic.
Is this the technology of the far distant future and only of potential use to our grandchildren? Apparently not! The Economist suggests such technology may be available within 2 years.

Friday, 1 July 2011

NewsBites: Aspergillus tubingensis Helps Cleans Up Fly Ash Pollution

Babu et. al. find that treating toxic ash from combustion of coal with a combination of Aspergillus tubingensis and arbuscular mycorrhizal fungi reduces the toxicity of the ash and enhances its ability to support plant growth, enhancing nutrient uptake and reducing metal translocation.

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