In his keynote talk at Stanford in March, Dr Ian Lipkin explained the lengths he goes to as a Microbe Hunter to track down the often complex links between diseases and the bugs that trigger them. This covers everything from using the latest DNA and RNA sequencing methods to taking samples from camels to testing the upper atmosphere for bugs. He went on to say how they are using the same wide-ranging and rigorous approach to CFS, including plans for the crowdfunded microbiome study.
Here are some highlights of the talk …
Once upon a time, proving a link between a pathogen and a disease was more straightforward, and often involved culturing a bug in the lab and creating an animal model. But the bugs responsible from most obviously infectious diseases have already been discovered: TB, smallpox, cholera etcetera. Dr Lipkin and his team now hunt in more difficult territory, where linking disease with a microbial trigger is altogether more complicated.
Most microbes in fact can’t be cultured in the lab, and often there isn’t an animal model — so new ways were needed. Starting in the 1980s with HIV, researchers, including Dr Lipkin, began developing genetic tools to detect tiny quantities of viruses by their genetic material (DNA or RNA). Improved versions of these genetic tools are now the main tool of microbe discovery and have been extended to cover bacteria and fungi.
In the absence of an animal model, or the ability to grow bugs in the lab, Dr Lipkin says the main way of demonstrating a microbe plays a key role in an illness such as ME/CFS is to treat it with drugs and show patients improve or recover – or that vaccinations can prevent it. No doubt this is the kind of proof that patients will warm to.
Finding microbes on the Tree of Life
Dr Lipkin explained how modern pathogen discovery works. Basically you sequence all the DNA/RNA in a sample (eg blood plasma in CFS patients and controls) which generates a whole load of fragment sequences. These are then compared with huge databases of sequences of known microbes – viruses, bacteria and fungi – to identify microbes, or if they are new microbes to see what they are most closely related to.
Tracking proteins to measure ‘shadows of infection’
A new approach that goes further actually looks for antibodies against microbes, rather than microbes themselves — an approach Dr Lipkin has previously called looking for ‘shadows of infection’. Borrowing from an approach used with DNA and RNA, ‘peptide microarrays‘ are used to mimic all the proteins particular microbes might produce, then they look to see if patients have antibodies against these proteins. If they patients do, it indicates a previous or current infection by that microbe. Is this clever, or what?
Following the evidence – wherever it leads
If there’s one theme of Dr Lipkin’s work, it’s never mind the hunch, where does the evidence lead? A classic example is his work in 1999 looking for link between CFS and Borna Disease Virus, a virus that Lipkin himself discovered. A large study ruled out any link, but what he did find was a clear sign of immune activation.
[pullquote align=”full” cite=”” link=”” color=”” class=”” size=””]I have a very open mind in thinking about causes of disease, and I think that ME/CFS is a very complex disease, and we need to be open to thinking about a wide range of interpretations. The only interpretation that I don’t accept is that this is some sort of a psychosomatic illness[/pullquote]
With autism, he was asked to investigate a link with measles virus RNA. Again Dr Lipkin and hsi team showed there was no link, but again they found other very interesting problems: children with autism and gut problems had very low levels of the proteins needed for the uptake of sugars from the gut. The team went on to find microbiome problems in these children, including high levels of a bacterium called Sutterella. The role of sutterella in the illness is now being investigated, but they have already found antibodies against sutterella in some children, suggesting sutterella in the microbiome might be making it’s way into the blood.
Weird and wonderful
Another great example of the kind of elegant detective work done by Dr Lipkin and colleagues, is their work on “PANDAS”, childhood neurological disorders triggered by a simple streptococcus infection such as ‘strep throat’.
Using an animal model and some very clever molecular detective work, they showed that mice exhibited a similar PANDAS-like syndrome if infected by streptococcus, and the problem was caused by antibodies against the bacteria binding to a mouse protein in the brain. This is an example of autoimmunity resulting from ‘molecular mimicry’, where antibodies developed against bacterial proteins by sheer bad luck bind to human proteins too.
The team then showed that children with PANDAS had antibodies against streptococcus that also bind to the equivalent human brain protein that caused the problem in mice (though obviously they didn’t test children’s brains directly). So it seems likely that PANDAS is caused by molecular mimicry too: antibodies against the original streptococcus bacteria by bad luck bind to proteins in patients brains, triggering the neurological symptoms. More about this work
Hunting down microbes with planes
In the extraordinary case of Kawasaki Disease, a rare but sometimes fatal disease of babies and children, outbreaks occurred in clusters — but no-one could work out how the infection was spread or what caused it. Then a smart scientist noticed a connection between outbreaks and changes in wind patterns high in the atmosphere. Military planes were sent up to sample the atmosphere and Dr Lipkin and collaborators found high levels of candida, a fungus, which is now implicated in causing the disease.
Dr Lipkin’s ME/CFS research
Microbe discovery so far
Dr Lipkin, as part of a Chronic Fatigue Initiative project, has looked for microbes in blood. They found no microbes associated with ME/CFS in blood plasma, and have just started looking at white blood cells (some herpes viruses such as EBV and HHV6 hide in white blood cells and won’t necessarily be detected in the plasma). Dr Lipkin presented the initial results, just for HHV6. They found the virus in 13% of ME/CFS cases, and 11% of controls, which is not a significant difference. This work continues.
Cytokines – early findings
The team have looked at a wide panel of cytokines in blood in patients and controls with some interesting early findings. They’ve found higher interferon-gamma in patients who had been ill for under three years.
When they looked at Dr Dan Peterson’s Cerebrospinal Fluid samples they found more cytokine differences, but the pattern was often the reverse of what they saw in blood, with higher levels in controls than patients.
However, this is still unpublished work and more work remains to be done.
Looking at gene expression in white blood cells reveals which genes are active, giving clues as to what might be going wrong in patients’ cells. Dr Lipkin funded a gene expression study out of his own lab’s budget using a new high-tech approach called RNA-Seq, that gives richer information than earlier techniques. The study had been delayed due to a technical hitch but the lab should be getting results around now.
Metawhat? Metabolomics is the “study of the unique chemical fingerprints that specific cellular processes leave behind”. In this case studying small molecules (metabolites) in the blood such as sugars, amino acid and neurotransmitters, that give clues about what’s going on in cells. It’s another way to probe what might be going wrong in ME/CFS. The blood metabalome is affected by the gut microbiome too – as many small molecules from gut microbes make it across the gut wall into the blood – and could well be causing problems in the body.[pullquote align=”full” cite=”” link=”” color=”” class=”” size=””]what we’re trying to do is to find things that are made by bacteria in the gut… that traffic into the bloodstream that we can identify that then can provide clues as to what’s different in the microbiome of individuals who have disease and individuals who don’t – Dr Ian Lipkin[/pullquote]
Proteomics is the protein version of metabolomics, looking at all the proteins that wind up in the blood. Again this is looking for a fingerprint that might indicate what’s going wrong in patients.
Both studies are underway and the lab is probably working on results about now.
Last, and definitely not least, are the plans for a large (100 patients and 100 controls) study of the microbiome, research that this crowdfund appeal can make happen. A smaller and more limited preliminary microbiome study is under way and will inform the larger one.
What emerges from this talk is that Dr Lipkin and his team are extraordinary scientists, searching open-mindedly for whatever the cause might be, wherever that takes them. They are now using the latest technology in large, innovative studies to try to solve ME/CFS. As well as looking for microbes that could trigger the illness, they are hunting for immune markers, other proteins and metabolites – anything that could reveal what’s gone wrong in ME/CFS and what might help fix it.
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