Dr Beryl Beynon OBE, MBChB and Dr Chris Newton PhD Well-One Clinic, Beverley, East Yorkshire and Centre for Immuno-Metabolism Microbiome and Bio-Energetic Research Introduction: I should firstly thank Demetrios Loukas for organizing this event. It has been no small undertaking by any means. I should also say that this is one of the most difficult talks that I have had to give because of you…… the audience.   Many times one can give a talk where, as an invited speaker, one is some kind of an ‘expert’. This is not the case here. I am no expert when it comes to Lyme disease, it is you……… members of this audience and the many individuals around the world who have Lyme disease, who are the true experts and I mean this not just from the unfortunate familiarity that you have with the symptoms. I mean it from the strong familiarity that you have with the science and medical practice behind the diagnosis (or lack of it) and the treatments for this horrible disease. Unfortunately the reason that you have become such great experts is necessity. It can certainly be argued that our medical system has let you down. But it must also be said that in most instances this is not the fault of individual physicians and/or scientists, rather it is that we have a system in the UK that is far too ridged where ‘so called guidelines’ seem to have to be followed as laws (or the particular consulting physician will get into all sorts of problems). Also as Physicians and Scientists we need to acknowledge that our knowledge when it comes to Lyme disease is often lacking: a very legitimate excuse for this is that Lyme is one of the most complicated diseases known to us at the present time.

Tick bite depositing microbiological ‘cargo’ through epidermis into dermis- the immuno-competent layer of the skin Now I must apologise to members of this audience who have good reason to believe that their infections are not just due to Borrelia burdorferi sensu lato but I refer to Borrelia and not the other potential co-infecting organisms. We are all well aware that there are problems with the detection and therefore diagnosis of Lyme disease. To a great extent our problems with detection are not just technical they are biological. The initial immune reaction is very much determined by the skin’s response to the Borrelia. (SLIDE 2- DESCRIBE A LITTLE). If the skin response is strong there may be no subsequent problems -the battle has been won in the skin- however, if the response is weak and the particular strain of Borrelia etc does not activate the complement system and sufficient numbers of organisms are able to leave the site via the capillary system of the dermis to colonize other areas of the body before a strong enough phagocytic dendritic cell response and antigen presentation in lymph nodes, this is a not so good. This may mean that a sufficient immune response is not mounted and an antibody response in the short-term, will not be detected. A further obvious problem of course is that individuals may not know that they have been bitten and if they do, they are unlikely to be able to get antibiotics administered before testing is done- testing that is less than definitive anyway particularly with respect to time needed for a sufficient immune response. So the outcome most often is that a tick-bite and possible symptoms of malaise (flue like symptoms) go untreated. Also of course, flu-like symptoms may be taken as exactly that and the GP will not provide antibiotics for what might be considered a viral-related condition. Outcome- the bug goes untreated to find a niche within the body where it can plunder resources- in other words, it can ‘dig in’. From then-on the outcome is uncertain and symptoms may range from none (up-to the now) to a whole range of symptoms that can be confused with may other conditions…… as many of you are most aware….. and some of which are presented in the next slide.  

Lyme disease: the great imitator (confuse all concerned in making clinical diagnosis) From: From: Smith et al. (2014) Borrelia burgdorferi: cell biology and clinical manifestations in latent chronic Lyme. Open Journal of Medical Microbiology. 4: 210-223

Hierarchy of influence on general health Ageing of immune system Emotional stress acute/chronic Physical stress Endogenous ‘infection’ gut microbiome Exogenous infection/ environmental factors Dietary components: minerals/vitamins carbohydrate fats proteins/fibre Metabolism mTORC1 Endocrine System (Pit./Adrenal/Thyroid) Autophargy Local inflammation IMMUNE SYSTEM (genetics make-up e.g. HLA markers/ etc) (FUTILE inflammation) Systemic inflammation © Newton CJ 2015

Typical scenario (1) for interaction with NHS physician Worst case scenario: present at GP with a range of symptoms, one may not know that one has been bitten, so how does one know how to point GP in right direction-long haul, much frustration and pain! ‘Partial’ solution- education- have to make people aware (without wishing to alarm) that Lyme disease could be responsible Throughout the 1990s, living in a small village just North of Munich always checked kids for ticks-it was what everyone did) If tick still embedded (but don’t ignore other bites-but what does one do?), what about chance of infection (number of ticks infected) and time tick attached (time needed to transfer infection into skin)?

Prevalence of tick infestations in continental Europe and in UK and transmission time   Cook MJ. (2015) Lyme borreliosis: a review of data on transmission time after tick attachment. International Journal of General Medicine. 8: 1-8 We don’t have a great deal of data yet for UK but Michael has referenced work from Switzerland, where the proportion of ticks carrying Borrelia ranges from 9 to 47% depending on region. For the UK, I would make a guess (but I have no solid data) that the incidence of infected ticks carrying Borrelia is considerably less than the 9% reported for some areas of Switzerland Transmission time for transfer of organisms after tick attachment from studies using animal models <16h

Data from UK: Veterinary and Parsitology and Ecology Group Uni Data from UK: Veterinary and Parsitology and Ecology Group Uni. Bristol -2011 Study Smith et al. (2011) Prevalence, distribution and risk associated with tick infestation of dogs in Great Britain. Medical and Veterinary Entomology 25: 377-384   Examination of 3,534 dogs at random over 9 months. Found 810 dogs carrying at least 1 tick Incidence of tick attachment in June 0.096, so per 10 walks in June, expect 1 tick Per year therefore, around 12 ticks

Same group (Veterinary and Parsitology and Ecology Group Uni Same group (Veterinary and Parsitology and Ecology Group Uni. Bristol) more recent and different study protocol   Jennett AL et al. (2013) Tick infestation risk for dogs in a peri-urban park. Parasites and Vectors 6: 358-367  If a dog was walked once per week in this area of land then should expect 1 tick per year, but a daily walk would reveal on average 7 tick infestations per year. Conclusion from this paper: In peri-urban green spaces, tick-biting risk for dogs may be high and related to exposure frequency. While tick-biting is of direct veterinary importance for dogs, dogs also represent useful sentinels for human tick-exposure.

Calculations on tick-bites per typical summer day in large peri-urban green space In a large woodland/park area with 100 people/dogs (equivalence?) per day in June   At rate of 10 ticks per year per dog/person (based on daily walks) Then…….. 10/365 x 100= 2.7 people get bitten per day This is just ticks, what about other vectors such as mosquito?

Other vectors and means of transmission of Borrelia and associated microorganisms The bottom line is that the overall number of just tick bites throughout the UK’s major parks and indeed back gardens is not insignificant. (We have ticks in our garden in Hull evidenced by our dogs) More research needed on extent of tick infestation and the means of vector transmission i.e. can other insect vectors transmit Borrelia and other microorganisms (web literature -non peer review-suggests they can) Not mentioned so far and despite lack of peer review evidence (as far as I am aware), Borrelia spirochetes may be sexually transmitted and may be passed through placenta. If so, this would amplify the incidence of infection quite considerably

But there are these data…….. Conclusion The culture of viable B. burgdorferi in genital secretions suggests that Lyme disease could be transmitted by intimate contact from person to person. Middelveen MJ Journal of Investigative Medicine • Volume 62, Number 1, January 2014

So, returning to the GP scenario: typical scenario (2)   Better case scenario? So one has convinced GP to do get blood taken and do test, but the result is negative What are the alternatives to NHS testing for second opinion? 1) Live blood microscopy-Dark Field/Phase Contrast Microscopy (Peter Kemp/Alan MacDonald/Michael Cook/ Laane and Mysterud 2) Labs in States and labs in Continental Europe. Western blot testing, Line blot testing, LTT testing, PCR testing/FISH But results of labs outside UK are not generally accepted by NHS, so still no antibiotics

Treatment with antibiotics despite NHS-lab test negative result Is there a rational to provide antibiotics even in a (NHS) lab test negative scenario given that the clinical picture screams Lyme (despite similarity of symptoms for many other conditions). In my opinion and as an informed scientist, I would say Yes, particularly if person has provided evidence of testing (with positive result) from other labs Lets not be too arrogant and except the fact that other European labs are as good as NHS labs! Let us not hide behind national accreditation systems-ours are better than yours! Without a truly definitive testing paradigm, one could argue that many other conditions with similar symptoms to Lyme disease might involve an infective agent and possibly a Lyme-related bacteria, parasite or mycoplasma

Is there any testing that could be truly definitive? The most optimal testing scenario would be to use the very best antibody testing strategy (which in my opinion would be to use a line blot methodology), not to bother with an ELISA for pre-screening and combine this with an LTT test and DNA methodology -both PCR and/or FISH-microscopy Screening could therefore be done with Line blot serology test and well validated very high sensitivity nested PCR as for example Lee et al. 2014

Returning to the GP scenario for last time: typical scenario (3) Best-case scenario so far: GP is persuaded, test is made and is positive- get antibiotics but….. may not be for long enough or may be less effective due to time elapsed from bite to treatment- the greater the interval the greater the chance that the bugs will have escaped from skin to find sites throughout the body-nervous system, muscle, joints, biofilms, etc A biofilm is any group of microorganisms in which cells stick to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS)- Wiki definition New antibiotics-will come back to this

Dr Alan MacDonald’s work on Parkinson’s/ Alzheimer’s and association with Borrelia and microscopic observations on various forms of Borrelia with FISH methodology Results of Research Study on human peripheral blood with Fluorescence In Situ DNA Hybridization(FISH method) and Borrelia burgdorferi family Specific DNA Probes BBO 0147 and BBO 0740 Images and Experimental Methodology and DNA probes design, manufacturer, quality control, and external multicenter Validation of DNA probe unique specificity for binding to Borrelia burgdorferi group (Sensu lato) DNA completed by Alan B. MacDonald MD. FCAP All images Copyright All intellectual content Copyright year 2013 by Alan B. MacDonald, MD

Dr Alan MacDonald’s Fluorescent in-situ hybridization (FISH)

Dr Alan MacDonald’s FISH-work

Dr Alan MacDonald’s Fluorescent in-situ hybridization (FISH) I am really not doing Alan’s work justice but I am sure that he would be happy for those who are interested to get a copy of this document. I can provide his email address for contact. Also I believe that he has sent a video for presentation so that he will explain the method far better than I would be able to.

Post GP/Consultant/Specialist If individuals with suspected Lyme disease can get treatment with appropriate antibiotics (and this leaves a very large number of individuals who cannot get treatment) what do we do if the antibiotics fail? Try others- what about new antibiotics? Pay attention to gut microbiome? Gut dysbiosis is one of the effects of long-term antibiotic treatment and a change in gut bacteria or lack of certain species in particular may play a role in chronic inflammation that accompanies chronic Lyme symptoms and long-term antibiotics

Gut Microbiome   Dysbiosis –microbial imbalance in body. Most common in gut and has been associated with inflammatory bowel syndrome (that is very often confused with gluten intolerance and Celiac disease when not checked for), chronic fatigue syndrome, obesity, cancer etc Long term (non-specific) antibiotics may fail perhaps not (just) from an inability to reduce the bacterial load of the spirochete, but because they provoke ‘leaky gut syndrome’ Antibiotics destroy beneficial bacteria and promote the growth of fungi Leaky gut- gaps between gut epithelial cells open allowing the passage of components of gut contents (including bacteria and parts of) through gut epithelial wall-can induce systemic inflammatory response (will occur where any damage to gut epitelium is present-ulcers / Celiac disease)

Gut Microbiome and other pro-inflammatory factors What about microbiome of individuals who contract Lyme disease? Are some individuals more susceptible to Borrelia and associated microorganisms due to underlying (pre-existing but clinically below threshold for symptoms) inflammatory conditions? How does the stress axis impinge on the disease. Chronic long terms stress would be expected to deregulate the inflammatory arm of the immune system (Th1/Th17)? Stress systems and inflammation reduce the function of the protein to which stress hormones bind (glucocorticoid receptor) What about MTHR (methylenetetrahydrofolate reductase) pathways and methylation cycles?

New antibiotics? Use new antibiotics, but none have begun their development in the last 25 years (statement out of date by several day!)   Longitude Prize? But we need NEW ANTIBIOTICS, and in particular, those that show specificity for Borrelia

New antibiotic identified: Teixobactin January 2015, a collaboration of four institutes in the US and Germany together with two pharmaceutical companies, reported that they had isolated and characterized a new antibiotic, killing "without detectable resistance- Wiki entry

Development of new antibiotics directed to Borrelia burgdorferi sensu lato-possible targets (1) Borrelia synthesize very few macromolecules and have to steal them from us Cannot make purines

Development of new antibiotics directed to Borrelia burgdorferi sensu lato-possible targets (2) There are no genes for the synthesis of fatty acids in Borrelia! The cell membrane of Borrelia is composed of phosphatidylglycerol (PG) phosphatidyly choline (PC) sterol galactoside and monofalatosyl diacylglycerol (MGalDAG) There is higher proportion of polyunsaturated fatty acids (PUSFAs) in Borrelia membrane than in the membranes of many other bacteria, possibly as they scavenge PUSFAs- Most likely the reason for neuroborreliosis symptoms and MS-like symptoms, Motor Neurone Disease? Fatty acid decoys? Borrelia differ from other bacteria (and spirochetes) in that they only contain PG and PC and unlike other bacteria that use a methylation pathway to synthesise PC, Borrelia directly combine choline with (CDP)-diacylglycerol using the enzyme phosphatidylcholine synthase to make PC

No tame pharmaceutical company-what to do when antibiotics fail? Pulsed Magnetic Field Therapy (PMFT) Established work with Dr Beryl Beynon at Well One Clinic using a modified Doug Coil System (often called a Rife machine, but strictly not what Royal Rife was doing) Built system based on: 1) Variable frequency audio oscillator 2) High output audio amplifier (1000W RMS) 3) Large magnetic field coil Audio frequencies used over range 200Hz to 5KHz but typically between 200 Hz and 1000Hz

How might PMFT work? Gene coding for Heat Shock Protein 70 (Hsp70) has an Electromagnetic Radiation Response Element (ERE) Heat shock proteins act a molecular chaperones to protect other proteins and also signal that a stressing agent/modality is present Possibly activate the immune system by acting as PAMPs/DAMPs But response at specific frequencies suggests strong ‘coupling’ and narrow ‘Q’ (frequency range over which effect observed-narrow bandwidth-like tuning of AM/FM radio to specific station)

Does PMFT target “Bugs” or does it target immune system? Jarisch- Herxheimer (Herx) reaction often apparent after PMFT Asymptomatic ‘condition’ seems to correspond to lack of PMFT- effect (no Herx) at frequencies specific for Borrelia My view is that there are specific effects on the ‘Bugs’ as well as effect on immune system via a Hsp-like mechanism (2012 paper below and activation of TLR4). Hsps give immune system a ‘prod’.