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Lipkin finds biomarkers not bugs

The CDC PCOCA telephone broadcast on 10 September 2013, featured a lengthy presentation from Dr Ian Lipkin who revealed some stunning initial results from the study that is primarily hunting for pathogens in ME/CFS. Simon McGrath and Russell Fleming (Firestormm) review this exciting and possibly game-changing news…

Lipkin - CFI

Dr Ian Lipkin
Lead Researcher
Chronic Fatigue Initiative (CFI)
Pathogen Discovery and Pathogenesis Study

Read the full Lipkin Transcript: Here.

Dr Ian Lipkin has been a human whirlwind in ME/CFS research since he became involved a few years back, and he’s just surprised us all by announcing the first results from the world’s largest ever biomedical ME/CFS study in a public broadcast!

He started off by lowering expectations: their results thus far do not implicate a single infectious agent and they have not discovered the cause of CFS. But he then wowed the hundreds listening to the conference call by revealing they had found strong evidence for immune overstimulation, both in blood plasma and in cerebro spinal fluid; and that they were now hard at work trying to identify what could be causing these abnormalities.

The big pathogen hunt draws a blank thus far

This study is looking exhaustively for any pathogen: viral, bacterial, fungal or parasitic, to see if a chronic infection could explain ME/CFS. Many of the results are in, but so far there is no clear sign of viruses at least. It is every bit a collaborative effort, with patients provided by long-established physicians including Dr Dan Peterson and Professor Jose Montoya.

The Key Players

Lipkin began by thanking the clinicians who had provided the all-important samples. Effectively it was a roll call of America’s top ME/CFS physicians:

Professors Jose Montoya, Anthony Komaroff and Nancy Klimas; and Drs Dan Peterson, Lucinda Bateman, Sue Levine and Donna Felsenstein.

The study itself was run by Dr Mady Hornig (CFI Principal Investigator) at the Centre for Infection and Immunity at Mailman School of Public Health; where Lipkin is also based.

He went on to acknowledged the very necessary support of the Chronic Fatigue Initiative, the Evans Foundation and an anonymous donor supporting Montoya’s work.

First the researchers looked at blood plasma for 285 CFS patients and 201 controls from Jose Montoya at Stanford (these are serious numbers). They searched for genetic evidence of infection using a panel that was able to detect 20 specific bacteria, viruses or parasites; including Epstein-Barr virus, Human Herpes Virus 6, enteroviruses, Influenza A and Borrelia bacteria.

Next they used high throughput sequencing, a method that was pioneered by Lipkin and colleagues that was used to discover over 500 viruses – so they felt fairly confident that if a virus was present, then they would have found it.

But they effectively drew a blank using both methods – only 1.4% of CFS patients were found to have HHV6 infection, but so did 1% of controls.

Other findings…

Lipkin said they also found Anelloviruses in 75% of those studied in plasma samples, but it was not specific for Chronic Fatigue Syndrome (we don’t currently have separate figures for patients and controls). Anellovirus is believed to cause widespread chronic infection in the human population, but has not yet been associated to any specific disease. “I really don’t know at this point what this finding means” said Lipkin.

They also found retroviruses in 85% of the sample pools. However, Lipkin expressed caution:

“It is very difficult at this point to know whether or not this is clinically significant. And given the previous experience with retroviruses in Chronic Fatigue, I am going to be very clear in telling you – although I am reporting this at present – in Professor Montoya’s samples neither he nor we have concluded that there is a relationship to disease …if I were to place bets and speculate, I would say that this is not going to pan out.”

Window on the brain: Cerebrospinal fluid samples

Both Lipkin and Hornig were clearly excited to have had access to the 60 cerebrospinal fluid samples from Peterson, and kindly donated by his ME/CFS patients. Cerebrospinal fluid bathes our brain and spinal cord so it gives access to what goes on in the brain, a prime site of interest to researchers, but hard to access; so these spinal tap samples are precious.

However, using the same molecular techniques to track down pathogens as they had applied to Jose Montoya’s samples, Lipkin’s team again drew a blank.

At the Invest in ME conference in May, Hornig said they had  tentative findings that there might just be a novel pathogen or pathogen candidate in these samples – but it was too early to say for sure. In reply to a question yesterday, Lipkin said there is no more to report:

we do not yet have a novel pathogen nor do we have a pathogen candidate in which I would have any confidence. So at this point we have no plans to publish anything.

Nonetheless, he also said they hadn’t finished all the DNA sequencing work on these samples, so this is not yet a dead end.

Immune stimulation and biomarkers

Up to this point we’d heard a lot of very impressive negative findings, based on large, well-defined samples of patients and controls, and using powerful research techniques. But what we most wanted to hear about was positive leads – and Ian Lipkin duly obliged:

plasma and blood

Plasma is the clear fluid left when blood is separated

They took plasma from the CFI cohort of 200 very well-defined ME/CFS cases and 200 controls collected by clinicians (See ‘Key Players’ above). Then they searched for cytokines and chemokines, which are regulators and controllers of the immune system.

The big finding was a decrease in levels of Interleukin’s IL-17, IL-2, IL-8 and in TNF-alpha – all of which have a role in pro-inflammatory responses. Lipkin believes these significant differences are worth follow-up in larger cohorts to better understand what they mean.

Professor Jonathan Edwards (who is an adviser to the planned UK Rituximab trial) commented on this forum yesterday, that it was a surprise to see TNF-alpha levels decrease, and suggested it could mean that ‘inflammation’ is too crude a concept for the process.

Lipkin and colleagues also found upregulation in Leptin, a hormone that plays a key role in regulating energy uptake and use, and Serpin, a protein family with multiple roles. More obscure cytokines, and and their cousins chemokines, were also affected but Lipkin didn’t give any further detail.

Two new patient types revealed

Ian Lipkin said they found something very surprising in their data: there appear to be substantial differences in profiles between those people who have had the disease for 3 years or less and those who have had the disease for more than 3 years. And that this is important because, “it could have implications for therapy as well as for diagnosis”.

In the ‘early group’, who have been ill for less than 3 years, there seem to be a number of markers suggestive of some sort of allergy aspect. For example there are often increased numbers of eosinophils in blood, with more differences in cytokines. But while this is tentative, he was more confident in the finding the IL-17 was elevated in these ‘early’ patients, compared with reduced levels in patients ill for over three years.

Lipkin said that he thought this 3-year division could be very important and that it hadn’t been something they were looking for; but would be looked at in any future work they did.

Cerebrospinal fluid: different differences

Lipkin’s team also found differences in cerebrospinal fluid biomarkers between patients and controls. I’m not sure from what he said if there were significant differences between the ‘early’ group and patients who had been ill for more than three years. However, they did find that patients had elevated levels of the TH-2 type cytokines IL-10 and IL-13 and elevated levels of four TH-1 cytokine: IL-1 beta, TNF-alpha, IL-5, and IL-17. Lipkin said this is compatible with a profile of some particular types of response that may provide insights into immunological dysregulation in Chronic Fatigue Syndrome.

So, overall they found clear differences between patients and controls in both plasma and spinal fluid. Many immune differences have been detected before, but findings are not very consistent and none had used such a large and robustly-defined patient cohort (meeting Fukuda and Canadian criteria), or with such carefully matched controls.

HEALTH WARNING

Dr Ian Lipkin again was very clear that he did NOT recommend anyone act on these provisional findings: he doesn’t want anyone to get a spinal tap, or measure the cytokine levels and hope to modulate them with drugs based on this research: it’s too early for that.

The lost years…

Back in 1999 Ian Lipkin published his first paper on CFS, which ruled out a connection between the illness and Borna Disease Virus (one of the many viruses he’s discovered). But crucially they found evidence of polyclonal B-cell reactivity. He commented that back then:

“there was a very strong sentiment in some portions of the scientific and clinical communities – not always and not everywhere – but in some portions of the community, that this was a psychological illness. What I said was that based on our findings we had very strong evidence that people with Chronic Fatigue Syndrome were truly ill with a physical illness and they deserved a “Deep Dive” to find out why they were ill.”

He noted that was a long time ago and dryly added “I am pleased to see that people are now paying much more attention to this disorder and what we can do about it”.

What next?

“I still believe the primary cause is likely to be an infectious agent.”

Having found clear signs of cytokine abnormalities, Lipkin wants to find out what is causing these changes; he currently thinks that an ongoing stimulus is causing immune system activation which might be resulting in the symptoms we associate with the disease. And interestingly, he still believes that  infection lies at the root of it all – despite not yet finding any infections.

The team – working closely with the clinicians he’s already mentioned – plan even more extensive work on a cohort of 200 CFI patients and controls. They will further test for viruses and crucially they will also look for bacteria and fungi by sequencing ribosomal RNA (which should detect unknown bacteria and fungi, as well as known ones).

Previous infection detection

The group has looked for current pathogens in the blood and spinal fluid samples they have – and has so far drawn a blank, though there is more work underway. However, what if the trigger for ME/CFS is an earlier infection that has since been cleared: the ‘hit and run’ scenario Hornig has described? Lipkin says they are looking for “shadows” of previous infections by searching for antibodies against specific infections, rather than the infections themselves (the body will continue to produce low levels of specific antibodies years after an infection has been cleared (the basis of much immunity)).

They will use a technique called LIPS to look for antibodies against particular known infection. In addition, as in their viral sequencing work, they will use an ‘unbiased’ system (using peptide arrays) that should detect all potential viruses – or rather antibodies against them. They don’t currently have an ‘unbiased’ system that works on bacteria or fungi.

Fecal future?

What excites Ian Lipkin most is the gut microbiome, the community of micro-organisms that live in our gut – which might seem odd given his focus so far on pathogens and the immune system. However, it turns out that the microbiome can have a profound influence on the immune system and may be important in many diseases. Hornig and Lipkin have already developed techniques to study the microbiome, and have shown it could be a factor in autism and even in the severity of colon cancer.

It turns out the best way to find out what lives in the microbiome is to measure what comes out of the gut: fecal matter, or ‘poop’ to you and me. Their ME/CFS microbiome project has started, but Lipkin felt there wasn’t enough material in the first attempt at gathering samples, so things have been delayed as they switch to a new technique (the ‘special’ collection cups).

The Big Ask: “we can’t do this without you”

Microbiome research is very expensive (analysis, not sample collection), and they currently only have the funds to do 10% of what’s needed. In fact, throughout the call, Lipkin was stressing the need for more funds, because good research costs and ME/CFS is woefully underfunded: there just isn’t enough money to do the work needed. The current science budget cuts (Sequestration) in the US makes the climate even harder.

“It is probably inappropriate for me to be passing the hat, but that’s precisely what I am doing.”

He urged patients to contact their representatives in Congress, demanding more funding for ME/CFS research – pointing out that in the early days of HIV, there was little funding until patients demanded it.  He also said that if patients were able to afford it, they should invest in research themselves.

Conclusion

ME/CFS research is coming of age. Ian Lipkin has reported these early – and soon-to-be published results – from a huge study, using 285 of Jose Montoya’s patients, the 200 patients from the Chronic Fatigue Initiative, plus the 60 ‘gold-dust’ spinal fluid samples from Dan Peterson’s patients. Using state-of-the-art techniques, they have largely ruled out a role for a specific pathogen – though that work is incomplete. And they have strong evidence of ongoing immune stimulation in ME/CFS patients, with abnormalities found in both the blood and spinal fluid. The next stage involves deeper searching for bacteria and fungi, looking for ‘shadows’ of previous infections and investigating the gut microbiome. These are exciting times!

Thanks to the CDC for organizing the presentation and to Firestormm for providing a transcript of this talk, and above all to Dr Ian Lipkin for taking time out to talk to patients.

If you would like to donate to Ian Lipkin’s research, click here. In the comments box, put “for M.E/C.F.S Study” to make sure it goes to the right place.

 

Simon McGrath tweets on ME/CFS research:

Sign the petition to get ME/CFS a research budget of $250 million a year!
There's a US petition and a global solidarity petition here with a long-term target of 50,000 signatures. Time we were on an equal per-patient funding basis with similar diseases!

{ 165 comments… add one }

  • lansbergen September 21, 2013, 10:48 am
    Forbin

    Ideally, with the new knowledge of this shift, I’d think that you would now want to follow a group of “under three year” patients so you could compare their pre- and post-shift profiles, get data from closer to when that shift happens, and also get the profiles of any of them that “recover.”

    I agree

  • aimossy October 3, 2013, 4:19 am

    I may have missed a post.
    but would anyone take a wild guess at how long it will be before lipkin publishes?
    miss impatient I know!
    lol

  • Firestormm October 3, 2013, 7:49 am
    aimossy

    I may have missed a post.
    but would anyone take a wild guess at how long it will be before lipkin publishes?
    miss impatient I know!
    lol

    Before the end of the year? Only a guess, I mean, hell, we are still waiting for the Norwegians to publish what they announced back in May, and which has been embargoed ever since then 'pending publication'. Drives you round the bend, doesn't it? 😡 😀

  • aimossy October 3, 2013, 11:54 am

    haha ABSOLUTELY. thanks for the guess!

  • Firestormm October 28, 2013, 9:14 am

    Morning :)

    Mark was contacted by someone who noted others wanting to donate to Lipkin's work specifically and who were wondering how to do it. This was the email Mark received explaining how it can be done:

    Hi there,

    After reading your article – Lipkin finds Biomarkers not bugs, September 12, 2013 and noticing all the comments about where to donate, I contacted Lipkin's team to find out.

    Here's the link:

    https://giving.columbia.edu/giveonline/?schoolstyle=5881&alloc=12636

    Perhaps this could be added to the article and to the forum thread that carried a poll and discussion on this topic:

    http://forums.phoenixrising.me/inde…r-prof-lipkins-study.25213/page-4#post-399155

    Thanks

    Have just donated and the link goes straight through to Centre for Infection and Immunity, Lipkin Laboratory. I just put into the notes section 'for M.E/C.F.S Study'

    The link has also been added to the article.

  • Firestormm November 6, 2013, 4:41 pm

    Science Article: Nov 4, 2013

    Gut Bacteria May Cause Rheumatoid Arthritis

  • Bob November 6, 2013, 5:12 pm

    This is not directly ME related, but there was another story re gut bacteria in the news yesterday.

    A strain of Lactobacillus brevis (KB290) has been found to give an immune boost to mice against a flu virus.
    "The researchers noticed that the bacteria enhanced the production of not just flu-related antibodies but also the immune system molecules IFN-α."
    Interesting that it boosts IFN-α (Interferon alpha.)
    I haven't read the research paper yet, so I don't know how effective it's supposed to be, or what sort of immune boost it gives.
    They haven't tested it in humans yet.

    Scientists Prove That Special Japanese Pickle Prevents Flu Infection
    http://www.scienceworldreport.com/a…anese-pickle-prevents-flu-infection-study.htm

    Preventing flu: Scientists find bacteria in Japanese pickled turnips which could boost immune system
    http://www.independent.co.uk/life-s…-which-could-boost-immune-system-8924768.html

    Japanese Turnip Pickle has Flu-Preventing Bacteria
    http://www.natureworldnews.com/arti…panese-turnip-pickle-bacteria-prevent-flu.htm

  • Bob November 6, 2013, 5:18 pm

    I've created a new thread for the previous two posts, so that they are seen more widely on the forum:
    http://forums.phoenixrising.me/inde…ritis-related-and-flu-immunity-related.26297/

  • Simon November 7, 2013, 3:36 pm

    RE @Firestormm: Science Article: Nov 4, 2013 Gut Bacteria May Cause Rheumatoid Arthritis

    The mysterious microbial world within us is beginning to reveal its secrets

    New research linking gut bacteria to Rheumatoid Arthritis throws more light on the complex relationship between illness and the gut, which may yet prove key to solving ME/CFS. As this thread's blog said:

    Fecal future?
    What excites Ian Lipkin most is the gut microbiome, the community of micro-organisms that live in our gut – which might seem odd given his focus so far on pathogens and the immune system. However, it turns out that the microbiome can have a profound influence on the immune system and may be important in many diseases. Hornig and Lipkin have already developed techniques to study the microbiome, and have shown it could be a factor in autism and even in the severity of colon cancer.

    Our gut bacteria, aka the 'microbiome', 'microbiota' or 'microflora' are becoming ever more important in our understanding of comlex long-term conditons:

    eLife commentary

    It has been known for decades that proper development of the immune system requires the gut microbiota, and that alterations in the repertoire of microbes are often associated with immunological disorders, in particular autoimmune diseases

    The new study reported in the prestigous new open access journal eLife shows a strong link between just one bacteria and Rheumatoid Arthritis (RA): 75% of new cases of RA had Prevotella Copri bateria compared with just 21% of healthy controls. New cases are important as they are unmedicated, and drugs themselves could affect the bacterial composition of the gut.

    And while the sample size was small (44 newly-diagnosed RA vs 28 healthy controls), the 21% rate of P. Copri seen in healthy controls is similar to that seen in larger population studies of bacteria, making it less likely that this is a statistical freak. Nonetheless, replication of these findings is critical.

    Assuming the finding is robust, the question is what is its significance? there are 3 possibilities:

    1. Prevotella Copri plays a causal role in RA, perhaps as a trigger
    2. RA leads to changes in the gut, with the effect that Prevotella Copri increases
    3. There is a third, unknown factor that leads to both RA and higher rates of Prevotella Copri, a relationship known as co-association.

    So P.Copri could be a cause of RA, or just a marker.

    Clearly it can't be the cause of RA because only 75% of RA cases had P. Copri, and 21% of healthy controls had the bacterium too. However, RA is known to be a complex illness with many predisposing features so it could still play a causal role for some people, and that would be hugely significant.

    Presumably this is exactly the sort of thing that Ian Lipkin is looking for in his ME/CFS microbiome study.

    Background: why it's hard and expensive to study gut bacteria
    Getting hold of gut bacteria is easy: 60% of the dry weight of faeces is bacteria so there is no shortage of raw material.

    The much harder part is identifying all the bacteria (there are typially around 500 different species in each person's gut). The standard method for identifying bacteria has been to first culture (grow) them in the lab – but unfortunately most gut bacteria won't grow under any lab conditions.

    The best current method for identifying bacteria is through their genetic material, specifically their ribosomal 16S RNA. Ribosomes are the protein-building factories of all cells, for us and bacteria included. Except bacterial ribosomes have slightly different RNA: human and all other eucaryotes have a 18S RNA while bacteria have 16S RNA, making it fairly easy to separate bacterial from human ribosomal RNA. Better still, the precise sequence of 16S RNA is different for every bacterial species, so by sequencing the 16S RNA, each species of bacteria can be identified. A similar approach can be used to work out how much of each species exists in the gut.

    More information
    The Human Microbiome Project was set up in 2008 to kick off systematic study of the human microbiome (note that actually the microbiome covers fungi and protozoa too, not just bacteria).

  • Bob November 7, 2013, 4:27 pm

    Prof Edwards has also expressed some thoughts about the arthritis study:
    http://forums.phoenixrising.me/inde…ximab-trial-30-july.24499/page-39#post-401745

  • Firestormm November 7, 2013, 10:19 pm
    Simon

    RE @Firestormm: Science Article: Nov 4, 2013 Gut Bacteria May Cause Rheumatoid Arthritis

    The mysterious microbial world within us is beginning to reveal its secrets

    New research linking gut bacteria to Rheumatoid Arthritis throws more light on the complex relationship between illness and the gut, which may yet prove key to solving ME/CFS. As this thread's blog said:

    Our gut bacteria, aka the 'microbiome', 'microbiota' or 'microflora' are becoming ever more important in our understanding of comlex long-term conditons:

    The new study reported in the prestigous new open access journal eLife shows a strong link between just one bacteria and Rheumatoid Arthritis (RA): 75% of new cases of RA had Prevotella Copri bateria compared with just 21% of healthy controls. New cases are important as they are unmedicated, and drugs themselves could affect the bacterial composition of the gut.

    And while the sample size was small (about 44 RA vs 28 healthy controls), the 21% rate of P. Copri seen in healthy controls is similar to that seen in larger population studies of bacteria, making it less likely that this is a statistical freak. Nonetheless, replication of these findings is critical.

    Assuming the finding is robust, the key question is what is its significance? there are 3 options:

    1. Prevotella Copri plays a causal role in RA, perhaps as a trigger
    2. RA leads to changes in the gut, with the effect that Prevotella Copri increases
    3. There is a third, unknown factor that leads to both RA and higher rates of Prevotella Copri, a relationship known as co-association.

    So P.Copri could be a cause of RA, or just a marker.

    Clearly it can't be the cause of RA because only 75% of RA cases had P. Copri, and 21% of healthy controls had the bacterium too. However, RA is known to be a complex illness with many predisposing features so it could still play a causal role for some people, and that would be hugely significant.

    Presumably this is exactly the sort of thing that Ian Lipkin is looking for in his ME/CFS microbiome study.

    More information
    The Human Microbiome Project was set up in 2008 to kick off systematic study of the human microbiome (this actually covers fungi and protozoa too, not just bacteria)
    BBC piece on early Human Microbiome Project finds
    Carl Zimmer piece in the New York Times
    Learning to love our microbiomes – radio piece

    The piece I cited talked about T17 cells from what I remember and without checking – because I am knackered.

    Here's another piece from science that mentions T17 cells in the intestine in association with lack of sleep being a trigger for immune response and possibly leading to disease:

    7 November 2013

    Why Late Nights Are Bad for Your Immune System

    These cells are a type of immune cell known as a T cell. They get their name from a signal they produce, called interleukin 17, which tells other T cells to increase the immune response. In normal numbers, TH17 cells, which live in the intestines, help the body fight bacterial and fungal infections. But when there are too many, the immune defense begins to cause illness rather than prevent it. Boosting NFIL3 levels in T cells growing in lab cultures resulted in fewer of them turning into TH17 cells, the researchers found, suggesting that the protein's job is to prevent T cells from going into that area of specialization. The absence of the protein, the team concluded, leads to runaway TH17 activity.

    At this point, the researchers had no reason to suspect a connection to our body’s internal timekeeping system—also known as our circadian clock—which responds to daily cycles of light and dark. But as they continued to explore the connection between NFIL3 and TH17 cells, they found that some of the proteins produced by the body’s "clock genes” attach to the NFIL3 genes. What's more, cultured cells and mice whose clock genes were experimentally tampered with produced fewer TH17 cells. The researchers surmise that a key protein in the clock network binds to the NFIL3 gene to keep the production of TH17 cells synchronized with periods of light and darkness. And the team found that normal mice produce less NFIL3, and thus more TH17 cells, during the day than at night.

    In a final experiment, the researchers gave the mice jet lag. "We didn't fly them anywhere," Hooper jokes. Instead, the team shifted the rodents' light/dark cycles by 6 hours every 4 days. "It would be like flying from the U.S. to Europe, India, and Japan and spending 4 days in each country," she explains. Mice with altered light cycles had nearly twice as many TH17 cells in their spleens and intestines, compared with mice having a normal day, the team reports online today in Science. The jet-lagged mice also mounted a stronger inflammatory response to irritation by an experimental chemical—a test used to gauge immune-system sensitivity that hints the animals may be more prone to inflammatory disease.

    The finding adds to a growing body of research showing that a healthy pattern of light and dark, sleeping and waking, is essential to keep the immune system in balance, Hooper says. She notes that inflammation is the basis of many chronic disorders, such as heart disease, asthma, chronic pain, and many things ending in "-itis," like bursitis and dermatitis. Inflammatory conditions are more prevalent in developed countries, where people's circadian rhythms are chronically disrupted. Even people who don't work shifts or cross time zones still wake and sleep out of sync with light and darkness, Hooper says. "We all have screwed up light cycles. We stay up late, keep the lights on, look at our lit-up iPhones at 2 a.m."

    About time Science and science started producing some interesting articles. It's been rather dull lately :)

  • knackers323 October 8, 2014, 5:24 am

    Why didn't they look for enterovirus in the gut lining etc. like Dr Chia?

  • alex3619 October 8, 2014, 6:08 am
    knackers323

    Why didn't they look for enterovirus in the gut lining etc. like Dr Chia?

    Because they used a sensitive assay, and it should show up in the faeces assays as the gut lining sheds lots of cells? This was publicly replied to by Lipkin, but I forget the details. Lipkin is running many different kinds of assays, and not all are finalized yet.

  • Kati October 8, 2014, 7:24 am
    alex3619

    Because they used a sensitive assay, and it should show up in the faeces assays as the gut lining sheds lots of cells? This was publicly replied to by Lipkin, but I forget the details. Lipkin is running many different kinds of assays, and not all are finalized yet.

    They also cited the difficulty in obtaining these samples (stomach biopsies), it would be very difficult to get this through ethics committee since there are anesthesia and procedure related risks to a stomach biopsy when the risks for blood and fecal matters are next to nothing.

  • knackers323 October 8, 2014, 7:29 am

    Yes that's true but maybe this is what needs to be done to find some answers