Amsterdam 16Point

The Role of Biofilms in Chronic Infections

◼ The National Institutes of Health (NIH) estimates
that 60% of all human infections and 80% of
refractory inf’s are attributable to biofilm colonies

◼ Biofilms are made up of cells & extracellular
polymeric substance (EPS), creating a matrix

◼ They provide a physical barrier for antibodies,
antibiotics, & shield persisters from the immune
system. Bacteria can genetically exchange material

◼ Ex’s biofilm inf’s: C Diff, Salmonella, borrelia, C.
albicans (yeast), staph, Klebsiella, Porphrymona g.

Stationary Phase Persister/Biofilm

Microcolonies Cause ↑ Disease

B. burgdorferi in the tick could develop variant forms that may represent different forms of persisters (Cabello)

Article Published in the Author Account of

Jie Feng

Stationary Phase Persister/Biofilm Microcolony of Borrelia
burgdorferi Causes More Severe Disease in a Mouse Model
of Lyme Arthritis: Implications for Understanding
Persistence, Post-Treatment Lyme Disease Syndrome
(PTLDS), and Treatment Failure

Published on March 28, 2019

Author: Jie Feng
Specialty: Microbiology, Infectious Diseases, Immunology
Institution: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns
Hopkins University
Address: Baltimore, Maryland, 21205, United States
Author: Tingting Li
Specialty: Microbiology, Infectious Diseases, Immunology
Institution: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns
Hopkins University
Address: Baltimore, Maryland, 21205, United States
Author: Rebecca Yee
Specialty: Microbiology, Infectious Diseases, Immunology

Treatment failures due to Persistence of Lyme
Borreliosis: Cystic Forms (round bodies, S-
forms, L-forms, CWD forms)

Preac-Mursic, V et al, Formation and Cultivation of Borrelia burgdorferi Spheroplast-L-form
Variants, Infection 24 (1996);No 3:218-26
Brorson,O et al, Transformation of cystic forms of Borrelia burgdorferi to normal, mobile
spirochetes, Infection 25 (1997); No 4:240-45.

Treatment failures due to Persistence of Lyme

Borreliosis: Round Body Forms/Cystic Forms

◼ Brorson,O et al, Transformation of cystic forms of Borrelia
burgdorferi to normal, mobile spirochetes, Infection 25 (1997); No
4:240-45.

◼ Brorson, O et al, A rapid method for generating cystic forms of
Borrelia burgdorferi, and their reversal to mobile spirochetes, APMIS,
106 (1998):1131-41

◼ Preac-Mursic, V et al, Formation and Cultivation of Borrelia
burgdorferi Spheroplast-L-form Variants, Infection 24 (1996);No
3:218-26

◼ Alban PS et al, Serum-starvation induced changes in protein synthesis
and morphology of Borrelia burgdorferi, Microbiology (2000),
146:119-27

◼ MacDonald, A. Concurrent Neocortical Borreliosis and Alzheimer’s
Disease: Demonstration of a Spirochetal Cyst Form. Ann NY Acad Sci.
1988. 468-470

◼ MacDonald A. Spirochetal cyst forms in neurodegenerative
disorders,…hiding in plain sight. Med Hypotheses (2006)

Comprehensive Treatment for
Persistent Lyme May Need To Address:

◼ Log Phase/Actively dividing forms: Penicillin's:
(Amoxicillin, Augmentin, IM Bicillin), Cephalosporins:
Ceftin [cefuroxime axetil], Omnicef [cefdinir], IV Rocephin
[ceftriaxone], IV Teflaro [5th generation], IV Vancomycin..

◼ Round body/cystic forms (L-forms, S-forms, CWD forms):
Plaquenil (hydroxychloquine), GSE (grapefruit seed
extract), Flagyl (metronidazole), Tindamax (tinidazole)

◼ Intracellular forms: tetracyclines (doxy, mino), macrolides
(azithromycin, clarithromycin), rifampin, rifabutin,
quinolones (cipro, Levaquin, moxifloxacin)

◼ Stationary Phase Persisters/biofilm forms: DDS (dapsone),
PZA, ? Daptomycin, ? Disulfuram + Stevia, Oreg. oil, Biocidin

Address Co-infections Simultaneously:

Polymicrobial Infection in ticks!

◼ Ticks in NYS: 71% harbored 1 organism, 30% had a
polymicrobial inf (2), and 5% had 3 or more
microbes: Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma

phagoctyophilum, Babesia microti, & Powassan virus

◼ Vector-Borne and Zoonotic Diseases. Tokarz et al, Sept 2009

◼ Ticks in Europe: 34% of Danish ticks were positive

for tick-borne microorganisms: 2 types Borrelia, 2 types
Babesia, Borrelia afzelii (39%), Rickettsia spp. (16%, 1° R. helvetica),

Borrelia burgdorferi sensu lato (15%), Babesia spp. 8% (Babesia
microti, 82%; Babesia venatorum (‘EU1’), 18%), Candidatus
Neoehrlichia mikurensis (1%), and Bartonella spp (0.6%)

◼ Babesia spp. and other pathogens in ticks recovered from domestic dogs in Denmark. Stensvold, C. et al.
Parasites & Vectors (2015) 8:262

◼ Ticks in France: Co-infections are the rule: 45% w/5 org’s

◼ Moutailler S, et al. (2016) Coinfection of Ticks: The Rule Rather Than the Exception. PLoS Negl Trop Dis
10(3): e0004539.

Number of Coinfections

Horowitz, R., Freeman P. International Journal of General Medicine 2019:12 101–119

◼ Participants tested positive for exposure to
between 0 and 16 coinfections (M=5.87, SD=2.29)

◼ 0.5% had no evidence of coinfections
◼ 26% had between 2-4 coinfections
◼ 64% had between 5-8 coinfections
◼ 8% had between 9-12 coinfections
◼ 1.5% had more than 12 coinfections
◼ Most Frequent: Babesia, Bartonella, Chlamydia

pneumonia, EBV, HHV6, and Mycoplasma

Coinfections: Indirect/Direct Testing

◼ Bacteria: Anaplasma (13.5%), Bartonella (B. henselae and

B. quintana [N=93, + titer, PCR, FISH, VEGF 46.5%), Brucella
(10%), Chlamydia Pneumonia (51%), Ehrlichia (14.5%), H.
pylori (7.5%), Mycoplasma (M. pneumonia, M. fermentans,
M. penetrans (82%), Q-Fever (Coxiella burnetti [8.5%),
Rocky Mountain Spotted Fever (10%), tularemia (16.5%),
typhus (10,5%)

◼ Parasites: Babesia (B. microti and B. duncani [N=104,

52%]), Toxoplasmosis (N=23, 11.5%)

◼ Viruses: HSV1 (23%), HSV2 (11.5%), HHV6 (81%), CMV

(37%), Coxsackie, (7.5%) EBV (80%), Parvovirus (11.5%),
WNV (6.5%)

◼ Horowitz, R., Freeman P. International Journal of General Medicine 2019:12 101–119

Co-infection Status N=200 Dapsone

64% of patients had between 5-8 coinfections

Horowitz, Freeman: International Journal of General Medicine 2019:12 101–119

Comprehensive Treatment of Chronic

Disease: Use The 16 Point MSIDS Map

◼ Pasteur’s postulate 1800’s: 1 cause for 1 disease: old
paradigm (“Chronic Lyme”: Lyme-MSIDS)

◼ Advantage of the MSIDS model: Takes into account
simultaneous overlapping etiologies causing the same
symptoms. Ex: fatigue, ? Infections (bacterial, viral,
parasitic, fungal), immune dysfunction, inflammation with
cytokines, environmental toxins (heavy metals, mold),
detoxification problems with lack of adequate GSH,
hormonal problems, vitamin deficiencies (B12, MMA),
mineral deficiencies (zinc), sleep disorders, mitochondrial
dysfunction… All of these can contribute to persistent
symptoms of fatigue

Defining, Diagnosing & Improving The
Treatment of Chronic Lyme Disease

◼ Two year study: A patient symptom survey &
retrospective chart review of 200 patients with “Chronic
Lyme disease”/PTLDS. Three aims :

◼ Better define “Chronic Lyme disease”

◼ Evaluate the efficacy of dapsone combination therapy
(DDS CT) in those with Chronic Lyme disease/PTLDS
failing traditional therapy

◼ Diagnose abnormalities on the 16-point MSIDS map
potentially affecting health & pinpoint overlapping
sources of inflammation and their downstream effects
to improve clinical outcome

◼ Horowitz, R., Freeman P. International Journal of General Medicine 2019:12 101–119



16 Point MSIDS Map: Evaluate all of
the Sources of Inflammation

◼ Primary Sources: ◼ Downstream effects:

◼ 1) Chronic infections ◼ 7) Endocrine disorders:
low T, low adrenal (f)
◼ 2) G.I.: Dysbiosis of
intestinal bacteria ◼ 8, 9) Neurological,
Psychological dysfunction
◼ 3) G.I. : Leaky gut w/ Food
allergies and sensitivities ◼ 10) POTS/dysautonomia

◼ 4) Sleep disorders:↑ IL-6 ◼ 11) Mitochondrial Dys(f)

◼ 5) Environmental toxins ◼ 11) Pain Syndromes
(heavy metals, mold…) ◼ 12) Liver Dysfunction

◼ 6) Nutritional Deficiencies ◼ 13) Autoimmune phen.

Several Biochemical Pathways Are
Involved in Inflammation

◼ NF Kappa B: mediator of inflammation: Cytokines

IL-1, IL-6, TNF alpha, interferon gamma, IL-17..

◼ Chemokines (CXCL-9, CXCL-10, CCL 19)

◼ Nrf2 and ARE genes

◼ NO pathway: peroxynitrite (stimulates NF Kappa
B & macrophage induced cytotoxicity)

◼ Bradykines, Prostaglandins, leucotrienes

◼ Mast cell mediators: histamine, heparin…

◼ https://www.merckvetmanual.com/pharmacology/anti-inflammatory-agents/chemical-
mediators-of-inflammation

NF-kB:
A Mediator of Inflammation

Glutathione
Lipoic Acid
Antioxidants

Figure from Evans
JL, Goldfine ID et al
(2002) Endocrine
Reviews 23: 599-622.

Lower Inflammation By Blocking NFK-B,

Activating Nrf2 and ARE genes

◼ Nrf2 acts as sensor in the cytoplasm that regulates
redox balance & stress response: it is activated by
oxidative stress. ROS→ Nrf2 detaches from Keep-1

◼ Nrf2 then goes into the nucleus and binds to
Antioxidant Response Element (ARE) genes.

◼ These are DNA binding sites that primarily activate phase II enzymes
(minor effect on phase I) plus numerous other cytoprotective
enzymes

◼ ARE gene activation enhances detoxification,
decreases inflammation & inhibits cancer growth

◼ This mechanism may explain many observed
beneficial effects of detoxifying phytochemicals. A
variety of substances activate Nrf2: Sulforaphane,
Resveratrol, Green Tea (EGCG) & Curcurmin

Use NrF2 Activators to Address
Inflammation/Detoxification

◼ Curcurmin: translocates Nrf2 to the nucleus, activates

ARE genes, ↓ inflamm by ↓COX 1 & COX 2, leukotrienes,
prostaglandins, nitric oxide, & TNF-alpha..(2-8 grams/d)

◼ Sulforaphane (glucoraphanins/broccoli seed extract):

↑ phase II liver pathways, antioxidant, anti-
inflammatory, tumor inhibition of p53 gene (100-300 mg)

◼ Resveratrol: affects sirtuin genes, stabilize Aβ (.5-2 gm)

◼ Green Tea (EGCG): translocates Nrf2 (250-500 mg)

◼ Yadav, et al. Immunomodulatory effects of curcurmin. Immunopharmacol Immunotox. 2005;27
(3):485-97; Valenzano DR, Terzibasi E, Genade T, Cattaneo A, Domenici L, Cellerino A. Resveratrol
prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr Biol.
2006 Feb 7;16(3):296-300; 7th Clinical Trials Confer. on Alzheimer's Disease (CTAD): Abstract OC2.
November 20, 2014

The NO/ONOO Cycle in

Inflammation/Chronic Illness

◼ NO can be stimulated by infections (bacterial, viral),
toxins, trauma →↑ peroxynitrite →↑ oxidative stress →
stimulates NF-κB. NF-κB → increases inflammatory
cytokines, such as ↑ IL-1, IL-6, IL-8, TNF-α, IFNγ which
contribute to symptoms and signs of these varied illnesses

◼ Classical therapy: immune modulators (Plaquenil,
DMARD’s), drugs with an anti-inflammatory effect
(macrolides, tetracyclines), IVIG for ↓ immunoglobulins

◼ CAM Therapy: focus on down-regulation of NO/ONOO
cycle biochemistry: Antioxidants, CoQ10, B vitamins, α-
lipoic acid, Mag++, Zn++, omega 3 FA’s, GSH precursors,…

◼ Glassford, J. The Neuroinflammatory Etiopathology of Myalgic Encephalomyelitis/Chronic Fatigue
Syndrome (ME/CFS). Front. Physiol., 17 February 2017; Pall M.L., Common etiology of posttraumatic
stress disorder, fibromyalgia, chronic fatigue syndrome and multiple chemical sensitivity via elevated
nitric oxide/peroxynitrite. Med Hypotheses, 2001; 57,139-145

Oxidative Stress and Inflammation
Underlie Chronic Illness

Positive Correlation with Chronic Disease and Aging

Figure from Evans, JL et al (2000) Diabetes Technol Therap 2:401-413.

How Can We Lower Inflammation?

◼ 1. Block NFKappa-B and Activate Nrf2 with
antioxidants (curcurmin, green tea, resveratrol) and

phytochemicals (sulforaphane/broccoli seed extract..)

◼ 2. Block Activation of Glial cells in the brain: LDN:

Blocks NFKappa-B and TLR4 signaling (decreasing glial cell
activation) and shifts immune responses from TH2 to TH1

◼ 3. Do an Anti-inflammatory diet: ↑ omega 3, ↓

omega 6 FA (Medit diet)→ ↓ arachidonic acid, avoid
allergic/sensitive foods, reduce simple sugars, red meat,
eggs, dairy, gluten, ? Histamine

◼ 4. Replace minerals (zinc, copper, magnesium)

◼ 5. Get proper sleep and exercise (insomnia:↑ IL-6)

How Can We Lower Inflammation and
Heal the Body from Lyme?

◼ 6. Treat the Infections causing inflammation, + immune

dysfunction (The 3 I’s): antibiotics and natural therapies

◼ 7. Detoxification: Remove environmental toxins &

inflammatory cytokines causing inflammation

◼ 8. Balance the hormones, cytokines, & the microbiome

◼ 9. Heal the Damage to the Body: Repair the

mitochondrial damage from free radicals and oxidative
stress (may improve energy; neuro/card)

◼ 10. Heal the Damage to the Mind/Emotions: role of

meditation, love and compassion

Laboratory Testing of MSIDS Variables

Horowitz, R., Freeman P. International Journal of General Medicine 2019:12 101–119

◼ Several national reference laboratories (Quest

Diagnostics, LabCorp, BioReference, PacTox)

◼ Local state laboratories (i.e., Sunrise, NorDx,

Affiliated Laboratory Inc., AccuReference)

◼ Specialty laboratories for tick-borne diseases

(Imugen, IgeneX, MDL Laboratory, Stonybrook Lyme
Disease Laboratory, Milford Molecular Diagnostics,
Galaxy diagnostics, Immunosciences

◼ Functional medicine laboratories (Aeron Lifecycles,

Labrix, Genova Diagnostics, Great Plains, Diagnos-Tech,
Doctor’s Data, RealTime Laboratory)

Data Mining of MSIDS Variables in 200
Patients on DDS CT: Inf’s/Immune Dys(f)

◼ 1) Infections: 100%, high % Babesia (52%), significant

% with IC infections, including: Bartonella (46.5%),
Mycoplasma (82%), tularemia (16.5%), Brucella (10%)

◼ 2) Immune Dysfunction: (positive ANA, RF, HLADR2,

HLADR4): 145 (72.5%) of participants had immune
dysfunction, 13.5% had elevated IgM antibodies, and
up to 85% had some form of immune deficiency:

◼ o 20.6 % had total IgG deficiency

◼ o 19.3% had IgM deficiency;

◼ o 15.9% had IgA deficiency

◼ 85.5% had combined IgG subclass deficiencies 1-4

II: Immune Dysregulation

◼ Many patients with Lyme disease present with
autoimmune phenomenon & immune dysfunction

◼ Positive ANA’s, RF’s, Anti-thyroid antibodies, anti-
cardiolipin antibodies, anti-ganglioside antibodies, anti-

myelin antibodies, autoimmune neuropathy..

◼ 1) Role of Bacteria: (i.e., Lyme) & other bacteria (LPS)

◼ 2) Role of Viruses: CMV, EBV, Herpes Viruses

◼ 3) Role of Toxins: BPA, Fine particle pollution

◼ 4) Role of the Gut and Microbiome: Prevotella, Clostridium

◼ 5) Role of Galectin-3: initiates pro-inflammatory signaling
cascades, orchestrates immune suppression, forms
adhesive lattices for biofilms, drives metabolic dysfunction

II: Immune Dysregulation & Bacteria

◼ Animal and human data suggest that bacterial
organisms play some role in the initiation and
propagation of RA as well as other forms of
arthritis, such as osteoarthritis

◼ Bacterial cell wall components, called
lipopolysaccharides (LPS) appears to upregulate
inflammation in cartilage (chondrocytes)

◼ Bacterial Lipopolysaccharides Form Procollagen-Endotoxin Complexes That Trigger Cartilage
Inflammation and Degeneration: Implications for the Development of Rheumatoid Arthritis. Lorenz, W.
et al. Arthritis Res Ther. 2013;15:R111

◼ LPS appears to trigger the NF-kappaB pathway of
inflammation through interactions with toll-like R4

◼ FSTL-1 is a protein/immune modulator in Lyme with
an established role in autoimmune arthritis

◼ Follistatin-like protein 1 is a critical mediator of experimental Lyme arthritis and the humoral response
to Borrelia burgdorferi infection, Campfielda, B. et al. http://dx.doi.org/10.1016/j.micpath.2014.04.005

II: Immune Dysregulation and Toxins

◼ BPA is an endocrine disruptor, affects mitochondrial
(f) by disrupting the cytochrome P450 system, &
may provoke AI reactions, causing molecular
mimicry

◼ Kharrazian, D. The Potential Roles of Bisphenol A (BPA) Pathogenesis in Autoimmunity. Hindawi
Publishing Corporation, Autoimmune Diseases, Volume 2014, Article ID 743616, 12 pages

◼ Beyond Asbestos: Autoimmunity, Pollution, and
Particles. Aug 13, 2014, Nancy Walsh, MedPage Today:
Nanoparticles may provoke AI reactions. Ex:
cigarette smoke (epigenetic effect), fine particle
pollution→ strong environmental risk factor for RA,
JIA, Lupus, scleroderma

◼ Pfau J, et al "Autoimmunity and asbestos exposure" Autoimmune Dis 2014; DOI: 10.1155/2014/782045

Autoimmune Disease & MSIDS

◼ 10 Factors on the MSIDS model associated w/ AI dx:

1. Bacterial infections: Lyme, other borrelia species, Mycoplasma/Chlamydia sp.,
Candidatus Neoehrlichia mikurensis, Bartonella, periodontal infections
(Aggregatibacter) + their biofilm formation + GAL-3

2. Viral infections: CMV, EBV, H. Zoster, Chikungunya v.
3. Parasitic infections: ↑ TH9 cells & autoimmunity
4. Environmental toxins: small particle pollution, Hg..
5. Imbalances in the gut: leaky gut, Prevotella sp.
6. Sleep disturbances: ↑ in RA
7. Vitamin D deficiency: association with SLE, MS (? Marker of IC infections)
8. Hormonal Dysregulation: prolactin, estrogen
9. Autonomic Nervous System Dysfunction
10. Genetics: lack of circular RNA’s, genetic predispositions (dsDNA, CCP…)

◼ Konig, M., et al. Aggregatibacter actinomycetemcomitans–induced hypercitrullination links periodontal infection to
autoimmunity in rheumatoid arthritis. Science Translational Medicine 14 Dec 2016: Vol. 8, Issue 369, pp. 369ra176

◼ C.-X. Liu et al. Structure and degradation of circular RNAs regulate PKR activation in innate immunity. Cell. Vol. 177,
Published online April 25, 2019.

Mechanisms Behind Autoimmune

Manifestations with Borreliosis

◼ Borrelia Outer Surface Proteins (Osp’s) are pro-
inflammatory, leading to increases in cytokine formation
(especially 31kdA)

◼ Blebs (pieces of DNA secreted from the organisms surface),
cause immune dysregulation, ↑ pro-inflammatory
cytokines

◼ Molecular Mimicry: w/ Osp A & myelin
◼ FSTL-1 (Follastatin-like protein 1): immune modulators are

produced ↑ arthritis

◼ Fraser at all.,1997; Casjens et al.; 2000; Szczepanski and Benach, 1991; Weis et al, 1994
◼ Whitmire WM; Garon CF. Specific and nonspecific responses of murine B cells to membrane blebs of

Borrelia burgdorferi. Infection & Immunity, 1993 61:1460-1467
◼ Alaedini A, Latov N. Antibodies against OspA epitopes of Borrelia burgdorferi cross- react with neural

tissue. J Neuroimmunol. 2005;(159):192–195.
◼ Follistatin-like protein 1 is a critical mediator of experimental Lyme arthritis and the humoral response

to Borrelia burgdorferi infection. Brian T. Campfielda, et al. J.Micropath.2014

Autoimmune Antibodies Associated with
Lyme disease & Other Conditions

◼ Lupus/SLE: + ANA, antiphospholipid, anticardiolipin
antibodies (dsDNA, Smith Ab= specific lupus)

◼ Rheumatoid Arthritis: + RF (CCP=specific RA)
◼ Multiple Sclerosis: + Antimyelin antibodies
◼ Hashimoto’s Thyroiditis: + Antithyroid Ab’s (anti-TPO, TG)
◼ Autoimmune Neuropathy: Peripheral neuropathy,

POTS/dysautonomia: + Antiganglioside Ab’s (GM1, Mag,
ASI)
◼ Autoimmune Encephalopathy: + Anti-dopamine, Anti-
Tubulin, Anti-lysoganglioside, CaM Kinase II (Cunningham)

II: Immune Dysregulation and Lyme

◼ Positive ANA, RF & other autoimmune markers
(Plaquenil). True Lupus=dsDNA, True RA=+CCP

◼ Increased severity with genetic HLA markers
(HLA DR2, 4) Molecular mimicry can occur…

◼ Elevated pro-inflammatory cells:-IL-6, TNF-α, IFN gamma

Propensity to excessive proinflammatory response in Lyme borreliosis. Kisand et al, APMIS. 2007
Feb; 115(2):134-41
Interleukin-6 is expressed at high levels in the CNS in Lyme neuroborreliosis. Pachner et al.
Neurology 1997 Jul;49(1)c147-52
INF-gamma alters the response of Bb activated endothelium to favor chronic inflammation. J.
Immunol. 2007 Jan 15;178(2):1172-9

◼ Decreased anti-inflammatory cells: IL-10 & abnormal
helper/suppressor cell ratio (CD4/CD8)

◼ IgG, IgA and IgM deficiency as well as IgG subclass
deficiencies have also been associated with Lyme disease

◼ Horowitz, Freeman: International Journal of General Medicine 2019:12 101–119

◼ Frequencies and Percentages of Low,
Normal, High Immunoglobulin Levels:

A Significant % Have Low IgA, Low IgM and Low IgG levels & subclasses

Horowitz, Freeman: International Journal of General Medicine 2019:12 101–119

Table 1: Frequencies and Percentages of Low, Normal and High Immunoglobulin Levels

Low Normal High
N* Frequency Percent Frequency Percent Frequency Percent

IgA 170 27 15.88 139 81.76 4 2.35

IgM 171 33 19.30 115 67.25 23 13.45

IgG 175 36 20.57 131 74.86 8 4.57

SubClass1 163 45 27.61 115 70.55 3 1.84

SubClass2 164 30 18.29 126 76.83 8 4.88

SubClass3 164 51 31.10 112 68.29 1 0.61

SubClass4 164 14 8.54 142 86.59 8 4.88

Immune Dysfunction in Lyme-MSIDS

◼ More than 72% of the 200 pts had immune dysfunction

◼ Total IgG deficiency was found in 20.6%; 19.3% had IgM
deficiency (13.5 % had elevated IgM antibodies); 15.9%
had IgA deficiency (? ↑ food allergies) & > 85% had
combined IgG subclass deficiencies 1-4.

◼ 85% had low IgG subclasses 1 and 3→can affect
phagocytosis & antibody-dependent
cellular/complement-dependent cytotoxicity

◼ Some patients: ↓ antibody response to a pneumovax

◼ Possible etiologies of immune dysfunction: Lyme, co-
infections (Anaplasma), heavy metals, gliotoxins..

◼ Olano JP and DH Walker. Human Ehrlichiosis. Medical Clinics of NA. 2002. 86(2):375392.

Similar Immune Dysfunction in Lyme
Was Seen in Infected Mice with Bb

◼ Nicole Baumgarth: infection with Bb in mice affects
lymph nodes & production of IgG antibodies, by affecting
germinal centers, ↓functional & long-lived AB responses

◼ Borrelia subverted a B cell response in that study &
caused T cell independence → IgM skewed profile

◼ We found 20.6% with IgG deficiency & 7% w/CVID

◼ 13.5% had ↑ IgM AB’s and 19.3% had an IgM deficiency:
? 2° immune complexes with active disease

◼ Tunev, et al. Lymphoadenopathy during Lyme Borreliosis Is Caused by Spirochete Migration-Induced
Specific B Cell Activation. PLOS Pathog. 2011, 7, e1002066;

◼ Schutzer, S. E., et al. Sequestration of antibody to Borrelia burgdorferi in immune complexes in
seronegative Lyme disease. Lancet Lond. Engl. 1990, 335, 312–315;

Subclass Deficiencies in Lyme-MSIDS

◼ 85% of our patients had IgG subclass 1+3 deficiency

◼ Prior examination of the IgG subclass distribution in
Lyme borreliosis showed the predominating subclasses in
both serum and CSF were IgG1 and IgG3

◼ The T helper type 1 (Th1) IFN-gamma-predominated
immune response seen in Lyme borreliosis, results in the
production of IgG1 and IgG3 subclasses that are
complement activating and opsonizing. It has been
hypothesized that increased levels of these two
subclasses early in disease might contribute to recovery
and counteract the development of chronicity

◼ Widhe, M., et al. IgG subclasses in Lyme borreliosis: a study of specific IgG subclass distribution in an
interferon-gamma-predominated disease. Scand. J. Immunol. 1998, 47, 575–581.

Immune Dysfunction Can Affect
Treatment Outcomes

◼ Immune dysfunction/Immune deficiency (CVID):

◼ Occasionally required SQ or IVIG, as immunoglobulins are
necessary to fight infections (CVID), modulate immunity,
heal (small fiber) neuropathy, POTS, and address AI
encephalopathy. We found 7% CVID, 20% w/ low IgG

◼ A 2018 study showed that robust B cell responses predict
rapid resolution of Lyme disease

◼ Kisand, K. E., et al. Propensity to excessive proinflammatory response in chronic Lyme borreliosis.
APMIS 115, 134–141, doi:10.1111/j.1600-0463.2007.apm_538.x.

◼ Younger, D. S.; Orsher, S. Lyme Neuroborreliosis: Preliminary Results from an Urban Referral Center
Employing Strict CDC Criteria for Case Selection Available online:
https://www.hindawi.com/journals/nri/2010/525206/ (accessed on May 28, 2018).

◼ Blum, L. K., et al. Robust B Cell Responses Predict Rapid Resolution of Lyme Disease. Front. Immunol.
2018, 9, doi:10.3389/fimmu.2018.01634

Intravenous & Subcutaneous
Immunglobulins (IVIG)

◼ Getting approval: 4 Different indications:

◼ 1. CVID (low immunoglobulins & or multiple subclasses);

◼ 2. Poor AB response to a pneumococcal vaccine;

◼ 3. Positive small fiber biopsy with/without CDIP or
Multifocal Motor Neuropathy (MMN)

◼ 4. Autoimmune encephalopathy (+ Cunningham

panel for brain autoantibodies)

◼ Examples: IV Gammaguard, IV Gammaplex, SQ

Hizentra, SQ Cuvitru..CI: severe IgA deficiency, allergies

◼ Katirji B, Koontz D. Disorders of peripheral nerves. In: Daroff RB, Fenichel GM, eds. Bradley's
Neurology in Clinical Practice. 6th ed. Philadelphia, PA: Elsevier Saunders; 2012:1915-2015.

III: Inflammation: Lyme disease ↑
Inflammation in the PNS and CNS

◼ Signs and symptoms depend on genospecies: Bb sensu stricto

(US), Borrelia afzelii (Europe, ACA), Borrelia garinii (neuroborreliosis)

◼ Bb spirochetes express lipoproteins on the outer surface
membrane of the Borrelia cell wall that are known to be
pro-inflammatory (LPS)

◼ These lipoproteins attract neutrophils & are actively
increase production of inflammatory cytokines. Molecular
mimicry may occur in the PNS, with a cross reaction
between Bb specific antibodies against the tail of the
spirochete and our nerves causing neuropathy

◼ Szczepanski and Benach, 1991, Weis et al., 1994, Alaedini and Latov, 2005

III: Inflammation in Lyme-MSIDS

◼ 3) Inflammation: Elevated ESR, CRP, TGFB1, C3a,
C4a, TNF, VEGF)

◼ These markers of inflammation are not specific for
Lyme. CRP is an indirect marker of ↑ IL-6 (an
inflammatory cytokine seen in Lyme disease)

◼ C4a can be seen in Lyme and mold toxicity

◼ 139 (69.5%) participants had markers of
inflammation

◼ Horowitz, R.I.; Freeman, P.R. Precision Medicine: The Role of the MSIDS Model in Defining,
Diagnosing, and Treating Chronic Lyme Disease/Post Treatment Lyme Disease Syndrome and
Other Chronic Illness: Part 2. Healthcare 2018, 6, 129.

◼ Edmondson, D. A. et al. Immune Response among Patients Exposed to Molds. Int. J. Mol. Sci. 2009,
10, 5471–5484, doi:10.3390/ijms10125471.

◼ Pachner, A. R.; Steiner, I. Lyme neuroborreliosis: infection, immunity, and inflammation. Lancet
Neurol. 2007, 6, 544–552, doi:10.1016/S1474-4422(07)70128-X.

Decrease Inflammation & Immune
Dysregulation: Nrf2, NFK-B, LDN..

◼ 1) LDN: 1st clinical study: Crohn’s Disease: 2/3
patients went into remission, 89% had a + response
to LDN Low-Dose Naltrexone Therapy Improves Active Crohn’s Disease. Smith. Amer Jnl

Gastroenterology Apr 2007;102:1-9.

◼ 2) LDN: Fibromyalgia: Single-blind, small clinical trial at

Stanford Medical Center. Fibromyalgia Symptoms are Reduced by Low Dose

Naltrexone: A Pilot Study. Younger et al. Pain Med. 2009 May-Jun;10(4):663-72.

◼ 3) LDN: Multiple Sclerosis: LDN was well tolerated, with a

1.6 point improvement on the Pain Effects Scale (P=.04) Pilot trial of low

dose naltrexone and quality of life in MS. Cree et al. Online Annals of Neurology 19 Feb,
2010

◼ 4) LDN: Lyme Disease: Horowitz open labeled study at HVHAC

(2009-2016): over 1000 patients: 75% improvement sx

Mechanism of Action of LDN

◼ Mechanisms of Action of LDN: raises endorphin and

encephalin levels & promotes healing, inhibits cell growth
(cancer cells), & reduces inflammation

◼ Blocks TLR4 signaling (decreasing glial cell activation) Since
glial cells release pro inflammatory cytokines and
chemokines, LDN decreases cytokines (? C.I. Hashimotos)

◼ Modulates T and B lymphocyte production

◼ Shifts immune responses from TH2 to TH1

◼ Blocks IL 6, IL 12, TNF alpha and NFKappa-B

◼ 2013 & 2014 LDN Research Trust Conference Summaries:

http://www.ldnresearchtrust.org/node/338
◼ http://www.ldnresearchtrust.org/node/245

IV: Toxicity and Lyme-MSIDS

◼ Environmental toxins: Chemical Sensitivity/

Environmental Illness (E.I.) is due to exposure to a
multitude of chemicals in the environment (PCB’s, dioxins,
plastics, pesticides, heavy metals, TCE, VOS’s..) We will
focus on 3 major categories:

◼ 1) Heavy Metals (Hg, Pb, As, Cd, AL)

◼ 2) Mold (aflatoxins, ochratoxins, gliotoxins, trichothecene)

◼ 3) Biotoxins 2º to Lyme and associated co-infections/

External toxin exposure→ Proven to play a role in A.I. dx,
Autism, ADHD, MS…Also endotoxemia (G.I., dental: LPS)

◼ http://www.natureworldnews.com/articles/9137/20140921/206-million-pounds-chemicals-hit-
waterways-one-year.htm

◼ http://www.mindbodygreen.com/0-13432/extreme-levels-of-herbicide-roundup-found-in-food.html:
GMO soy has 9 mgs of Roundup/kg

IV. Toxicity: Urine Toxic Metals

Appx 85% Tested + w/ Lyme-MSIDS

Horowitz, R.I.; Freeman, P.R. Precision Medicine: The Role of the MSIDS Model in Defining, Diagnosing,
and Treating Chronic Lyme Disease/Post Treatment Lyme Disease Syndrome and Other Chronic Illness:

Part 2. Healthcare 2018, 6, 129.

Data Mining of MSIDS Variables:
4. Environmental Toxins

◼ Heavy Metals: 84.5% had one or more heavy
metals using a 6-hour urine DMSA challenge

◼ 159 (79.5%) had ↑ lead levels (73 were ↑, 59 were very high)

◼ 136 (68%) had ↑ mercury levels (77 were ↑, 59 were very high)

◼ 30 (15%) had ↑ cadmium levels (26 were ↑, and 4 were very ↑)

◼ 25 (12.5%) had ↑ aluminum levels (N=25 were elevated)

◼ 5 (2.5%) had ↑ arsenic levels (3 were elevated, and 2 were very
elevated)

◼ Clarkson, T. W.; Magos, L. The toxicology of mercury and its chemical compounds. Crit. Rev. Toxicol.
2006, 36, 609–662

◼ Horowitz, R.I.; Freeman, P.R. Precision Medicine: The Role of the MSIDS Model in Defining,
Diagnosing, and Treating Chronic Lyme Disease/Post Treatment Lyme Disease Syndrome and Other
Chronic Illness: Part 2. Healthcare 2018, 6, 129.

IV: Role of Heavy Metals & Toxins

◼ Ubiquitous, accumulates slowly over years

◼ They can cause the same symptoms seen in Lyme-
MSIDS patients: fatigue, pain, neuropathy, mood
disorders, memory/concentration problems

◼ Escapes recognition since regular testing is not
generally performed by most PCP’s

◼ Produces oxidative stress and ROS driving
inflammatory pathways in pain, & have been linked
to neurodegenerative disorders

▪ Jomova et al. Metals, oxidative stress and neurodegenerative disorders. Mol Cell Biochem 2010
Dec;345(1-2): 91-104

▪ Metals, Toxicity and Oxidative Stress. Current Medicinal Chemistry Vol 12, 10
▪ Studies on Heavy Metal Burdens among Lyme pts (Horowitz, 16th Intl Sci Conf on Lyme disease, 2003)

Importance of Detoxifying Heavy Metals
and Pollutants: Pregnancy & Autism

◼ Harvard study 2014: Number of Utah 8-year-olds w/

autism has almost tripled since 2002 (1/47 Utah vs 1/210 in
Alabama). This study concluded that "exposure in the
womb to mercury, lead, manganese, methylene chloride &
especially diesel exhaust was “signif associated with ASD”

◼ UC Calif Davis 2014 (EHP): ↑ risk pesticides & ASD

◼ Pesticides and ASD/Neurodevelopmental Disorders, June 2014. Environ Health Perspect;
DOI:10.1289/ehp.1307044

◼ Lancet Neurology 2014: Chemicals ↑ risk of ASD,

ADHD, dyslexia, cognitive impairments

◼ Neurobehavioural effects of developmental toxicity. Grandjean, P., et al. Lancet Neurol 2014; 13: 330–
38.

Data Mining of MSIDS Variables:
IV. Environmental Toxins

◼ Mold: 30/42 (71.4%) had 1 or more ↑ mold levels

◼ 13/25 (52%) had elevated aflatoxins: RealTime Labs, urine mycotoxin

◼ 18/26 (69%) had elevated ochratoxins: RealTime Labs urine test

◼ 20/26 (76.9%) had elevated trichothecenes: RealTime Labs urine test

◼ 17/17 (100%) had elevated gliotoxins: RealTime Labs urine test

◼ 7/18 (38.9%) had “other” elevated mold (Stachybotrys exposure)

◼ Pesticides: 5 (2.5%) tested positive for pesticides*Not all

patients were tested for mold or pesticides: only those with a history with significant
chemical sensitivity and/or Parkinson’s symptoms

◼ Edmondson, D. A.; Barrios, C. S.; Brasel, T. L.; Straus, D. C.; Kurup, V. P.; Fink, J. N. Immune Response
among Patients Exposed to Molds. Int. J. Mol. Sci. 2009, 10, 5471–5484, doi:10.3390/ijms10125471.

◼ Horowitz, R.I.; Freeman, P.R. Precision Medicine: The Role of the MSIDS Model in Defining, Diagnosing,
and Treating Chronic Lyme Disease/Post Treatment Lyme Disease Syndrome and Other Chronic Illness:
Part 2. Healthcare 2018, 6, 129.

Toxicity Associated Symptoms & Conditions
w/ Chemical Sensitivity/Environmental Illness

◼ Headaches ◼ Panic attacks
◼ Mineral imbalances
◼ Kidney dysfunction ◼ Memory loss
◼ Fertility problems w/
◼ Abnormal pregnancies ◼ Parkinson’s disease
◼ Immune system depression
◼ Chemical sensitivities ◼ Broad mood swings
◼ Fibromyalgia
◼ Recurrent yeast infections ◼ Fatigue
◼ Tinnitus
◼ Contact dermatitis ◼ Chronic fatigue syndrome
◼ Learning disorders
◼ Cancer ◼ Muscle weakness

◼ Unusual response to meds or
supplements

◼ Increasing sensitivity to
exogenous exposures: odors,
medications, etc.

◼ Worsening of symptoms after
anesthesia or pregnancy



Why Detoxify?

◼ CDC 2003: 6.5 million $ study w/ 2500 patients:
found 116 different pollutants (13 heavy metals,
14 combustion byproducts, 10 pesticides) One of
those toxins TCE, caused a leukemia outbreak in
Woburn, MA, & frequently causes learning
disabilities, paresthesia's...

◼ Testing: Genova Labs, Doctors Data, Real Time Labs

Mycotoxin Panel (Ochratoxins, Aflatoxins,
trichothecenes, Gliotoxins), PacTox, Local labs (LabCorp..)

◼ Among the population, 12.8% report medically diagnosed
MCS and 25.9% report chemical sensitivity

◼ CDC Report on Environmental Toxins. Joan Stephenson, PhD. JAMA. 2003;289(10):1230-1233.
◼ National Prevalence and Effects of Multiple Chemical Sensitivities. Anne Steinemann. Journal of

Occupational and Environmental Medicine. Publish Ahead of Print():, JAN 2018

Why Detoxify?

◼ Miscarriage, fetal death, decreased birth weight,
and other fetal developmental effects ↑

◼ Jianying Hu, PhD. Environ Sci Technol. 2015;49:10651-10657;

◼ Children: Environmental toxins ↓ IQ, ↑ ASD risk

◼ Attina TM, et al. Exposure to endocrine-disrupting chemicals in the USA: a population-based disease
burden and cost analysis. Lancet Diabetes Endocrinol. 2016;4(12):996-1003

◼ Adults: Parkinson’s Dx, Dementia Risk ↑

◼ Chen H, et al. Living near major roads and the incidence of dementia, Parkinson's disease, and multiple
sclerosis: a population-based cohort study. Lancet. 2017 Jan 04; Lee PC, et al.

◼ Coronary Artery Risk ↑ (severe CAD, risk MI)

◼ Kaufman, D. et al. Association between air pollution and coronary artery calcification within six
metropolitan areas in the USA: a longitudinal cohort study. Lancet, Volume 388, No. 10045, p696–704,
13 August 2016