CONTENTS

In order of relevance

(ALS) Amyotrophic Lateral Sclerosis or (MND) Motor Neurone Disease are referred to as ALS/MND. PALS is short for People (or a person) with ALS.

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My*co*plas*ma (noun): any of a genus (Mycoplasma) of minute pleomorphic gram-negative chiefly nonmotile micro-organisms without cell walls that are intermediate in some respects between viruses and bacteria and are mostly parasitic usually in mammals called also pleuropneumonia-like organism.

intermediate  (noun)  : a usually short-lived chemical species formed in a reaction as an intermediate step between the starting material and the final product.

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Experimental Clinical Study on ALS Infections
High Frequency of Systemic Mycoplasmal Infections in
Gulf War Veterans and Civilians with Amyotrophic Lateral Sclerosis (ALS)
Journal of Clinical Neuroscience 2002

Garth L. Nicolson PhD1,2*, Marwan Y. Nasralla PhD1,3, Joerg Haier MD, PhD
1,4 and John Pomfret PhD2 - 1 The Institute for Molecular Medicine, Huntington Beach, California, USA; 2 Gulf War ALS Research Project, Parsons Walk, Wigan, England, UK; 3 International Molecular Diagnostics, Inc., Huntington Beach, California, USA; 4 Department of Surgery, Wilhelm University, Muenster, Germany
Correspondence: Prof. Garth L. Nicolson, The Institute for Molecular Medicine, 15162 Triton Lane, Huntington Beach, California 92649 USA; Tel: +1-714-903-2900; Fax: +1-714-379-2082; Website:http://www.immed.org

ABSTRACT The presence of systemic mycoplasmal infections in the blood of Gulf War veterans (n=8) and civilians (n=28) with Amyotrophic Lateral Sclerosis (ALS) and age-matched controls (n=70) was investigated by detecting mycoplasma gene sequences with forensic Polymerase Chain Reaction (PCR) and back-hybridization with a radiolabeled internal oligonucleotide probe.

Almost all ALS patients (30/36 or ~ 83%) showed evidence of Mycoplasma species in blood samples, whereas <9% of controls had blood mycoplasmal infections (P<0.001). Using PCR ALS patients with a positive test for any mycoplasmal infection were investigated for the presence of M. fermentans, M. pneumoniae, M. hominis and M. penetrans in their blood.

All Gulf War veterans with ALS were positive for M. fermentans, except one that was positive for M. genitalium. In contrast, the 22/28 civilians with detectable mycoplasmal infections had M. fermentans (13/22, 59%) as well as other Mycoplasama species in their blood, and two of the civilian ALS patients had multiple mycoplasma species (M. fermentans plus M. hominis).

Of the few control patients that were positive, only two patients (2/70, 2.8%) were positive for M. fermentans (P<0.001). The results support the suggestion that infectious agents may play a role in the pathogenesis and/or progression of ALS, or alternatively ALS patients are extremely susceptible to systemic mycoplasmal infections.

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Before reading further please note that the following is information regarding the pilot study only and, although the above evidence would appear to support the approach described, nothing has been scientifically proven to be effective in treating ALS/MND.  The following is extremely encouraging and coincides with many of my own findings and theories.  I include it here for your information only. Steven Shackel

Experimental Clinical Study on ALS Infections: IMM Clinical Trial (June 2000)
Prof. Garth L. Nicolson

This experimental study will be conducted in Huntington Beach, CA.  Below is a summary of the trial that should answer your questions, except for cost. This will vary, depending on whether you are a Gulf War veteran (zero cost for the trial) or civilian and on whether we receive funding from an ALS foundation that is interested in helping with costs.  This message may be sent in parts to be able to communicate it to all those who requested information. Prof. Garth Nicolson

PILOT COMBINATION CLINICAL TRIAL FOR ALS PATIENTS?
CHRONIC BACTERIAL AND VIRAL INFECTIONS
Principal Investigators:  Dr. Garth Nicolson and Dr. Darryl See Institutions: Immune Institute of Southern California (DS) and The Institute for Molecular Medicine (GLN)
Addresses: The Immune Institute of Southern California, 18800 Delaware Street Suite 900, Huntington Beach, CA 92648; Tel: 714-842-1777; Fax: 714-375-4670. The Institute for Molecular Medicine, 15162 Triton Lane, Huntington Beach, CA 92649; Telephone: 714-903-2901; Fax: 714-379-2082.
 
Abstract

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, idiopathic, progressive generative disease. Although the cause of ALS remains unknown, there have been several hypotheses.  Of these, the role of chronic infections has attracted recent attention with the finding of enterovirus (EV) sequences in 15/17 spinal cord samples. 

The possibility that two or more infectious agents could interact to produce ALS remains a distinct possibility.  Our recent findings indicate that (28/31) patients have mycoplasmal infections, including 6/6 Gulf War veterans with ALS.  Thus we propose to investigate the biological relevance of multiple chronic viral and bacterial infections in ALS by initiating a pilot clinical trial based on these findings.

These are our specific aims: (1) Examine the frequency of EV and mycoplasmal coinfections in ALS, and (2) Follow 20 patients enrolled in a pilot treatment program based on treating these infections.  The pilot trial will be an unblinded, open trial that utilizes antibiotics and antivirals to treat chronic infections in ALS patients. 

Patients will be assessed by medical and laboratory tests before, during and after trial.  We hope to gather evidence for the possible role of viral and bacterial infections in ALS morbidity.

Introduction
Patients with ALS show gradual progressive weakness and paralysis of muscles due to destruction of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord, ultimately resulting in death, usually by respiratory failure [1-3].  The overall clinical picture of ALS can vary, depending on the location and progression of pathological changes found in nervous tissue [3]. 

ALS has no known cure, and in general it is thought to be a terminal disease. Although the cause(s) of ALS remains unknown, there are five main hypotheses on its pathogenesis: (1) accumulation of glutamate causing excitotoxicity; (2) autoimmune reactions against motor neurons; (3) deficiency of nerve growth factor; (4) dysfunction of superoxide dismutase; and (5) chronic infection(s) [3-11]. 

Of these hypotheses, the role of chronic infections has attracted the most recent attention with the finding of chronic EV sequences in 15 of 17 spinal cord samples from ALS patients by Polymerase Chain Reaction (PCR) [11].  Although another group failed to detect EV sequences in spinal cord samples from patients with or without ALS, possibly because of the techniques that they used [12], a distinct possibility remains that two or more infectious agents could interact to produce ALS or promote its progression [13].

We have been working on the presence of chronic infections in ALS patients, and in particular, systemic mycoplasmal infections in ALS patients.  The reason for this is our discovery of a high rate of incidence of mycoplasmal infections in various chronic diseases.  We first discovered the presence of systemic mycoplasmal infections in Gulf War Illness (GWI) patients [14-15], including GWI patients with ALS. 

Although this finding flew in the face of official pronouncements that GWI was caused mainly by stress related phenomenon [16], recent results from a large clinical trial conducted by the Dept. of Veterans? Affairs and Dept. of Defense at 32 VA and DoD hospitals indicate that we were right all along.  We previously published that ~45% of GWI patients had systemic mycoplasmal infections, primarily M. fermentans, in their blood leukocytes by Nucleoprotein Gene Tracking [14,15]. 

The recent results presented by the PI of this VA/DoD study at a recent CFS Co-ordinating Board meeting at NIH indicate that out of over 1,500 GWI patients examined, approximately 40% had mycoplasmal infections, and over 80% of these were infections with M. fermentans.  This result is very close to what we previously published [14, 15].

Using PCR procedures developed to detect micro-organisms buried deep inside cells with complexed DNA, we developed Forensic PCR which overcomes the problem of false negative results due to protein-DNA complexing and the presence of PCR inhibitors in blood, a commonly found situation in clinical specimens.  This new approach overcame the initial negative findings from the DoD on mycoplasmal infections in GWI patients, and indicated that the use of appropriate methods is extremely important. 

Importantly, patients with identified mycoplasmal infections have been successfully treated with antibiotics that suppress mycoplasmal infections, but not with antibiotics that do not suppress these infections. 

When the recovered patients were retested for the presence of mycoplasmal infections, they were found to have reverted to a mycoplasma-negative pheontype.  These results and the fact the species of mycoplasmas found in GWI patients were of the pathogenic variety, suggested that mycoplasmal infections play a role in this chronic illness.

Interestingly, patients that recovered from GWI on the antibiotic protocol were also less environmentally sensitive, suggesting that these infections may also sensitize patients to environmental stressors, such as chemicals, allergens, etc.

Using PCR procedures we began to examine the incidence of mycoplasmal infections in other chronic illnesses, such as CFS/ME, FMS and Rheumatoid Arthritis.  Similar to GWI, we found high rates of systemic mycoplasmal infections in CFS/ME (~60%), FMS (~65%), RA (~45%) and other chronic conditions [16-18]. What was of particular interest was that in contrast to GWI where almost all patients have only M. fermentans infections, most of these civilian chronic illness patients had multiple mycoplasmal infections involving M. fermentans, M. pneumoniae, M. hominis, M. penetrans, among others [17, 18].

We found that the patients that had been ill for longer periods of time tended to have more different species of infections than patients sick for shorter periods of time.  Also, the severity of signs and symptoms was related to the number of mycoplasmal infections in these patients [17]. 

Similar to GWI, we determined that successful treatment of mycoplasmal infections in chronic illness patients with long term antibiotics and immune boosting supplements and vitamins resulted in recovery from signs and symptoms, suggesting that these infections are causing much of the clinical signs and symptoms. 

Also similar to GWI analysis of patients that had recovered indicated that the infection(s) could no longer be detected in their blood.  Since only certain antibiotics worked in these treatment studies, and they were antibiotics that could suppress mycoplasmal infections but not those that were not active against mycoplasmas, we concluded that the mycoplasmal infections caused patient morbidity. Thus we were able to fulfil most of the criteria, including animal model studies of the illnesses by others (see below), that are necessary to be able to conclude that patient morbidity was due, at least in part, to chronic infections. 

Our findings did not necessarily mean that the infections that we found were the initial causes for the above illnesses, but it did indicate that much of the patient morbidity could be due to chronic infections.  Mycoplasmal infections are associated with a wide variety of chronic and acute illnesses in man [19-21]. Before systemic mycoplasmal infections can be considered important in causing disease, certain criteria must be fulfilled [22,23]:

[a] The incidence rate among diseased patients must be higher than in those without disease.  This has been found for M. fermentans.  Although this mycoplasma has been found in asymptomatic adults, the incidence is low, usually only a few percent (0-9%) compared to 40-65% of CFS/ME, FMS, GWI and RA patients [review: 25]. 

In addition, most of these patients have more than one species of mycoplasma, especially if they have more severe signs and symptoms or have been ill for several years [17].  In contrast, if the few controls that are found to have mycoplasma in their blood, they have only have one species [17].

[b] More of the mycoplasma must be recoverable from diseased patients than from those without disease.  This has been found [see review: 24].

[c] An antibody response should be found at higher frequency in diseased patients than in those without disease.  This has been seen, but usually not until the disease has progressed.  According to Lo et al. [21,27] M. fermentans hides inside cells and does not elicit a strong immune response until near death.

[d] A clinical response should be accompanied by elimination of the mycoplasma. This is exactly what has been found [14-16].

[e] Clinical responses should be differential, depending on the type of antibiotic.  Only antibiotics that are effective against the pathogenic mycoplasmas result in recovery, and some antibiotics, such as penicillins, can worsen the condition [15,16].

[f] The mycoplasma must cause a similar disease in susceptible animals.  The best description comes from Lo et al. [27] where injection of M. fermentans into monkeys resulted in development of a fulminant disease that leads to death.  These animals display many of the chronic signs and symptoms found in human with similar infections, but in monkeys this infection is fatal.

[g] The mycoplasma must cause a similar disease when administered to volunteers.  This has not been done, because of ethical considerations.

[h]  A specific anti-mycoplasma antibody reagent or immunization protects against disease.  This has not yet been done to our knowledge.  Therefore, six out of eight of the above criteria have been fulfilled, at least in the case of M. fermentans, suggesting strongly that these micro-organisms are involved in illness [24,25,26].

We have proposed that chronic infections are an appropriate explanation for much of the morbidity seen in a rather large subset of CFS/ME, FMS, GWI and many RA patients, but certainly not every patient will have this as a diagnostic explanation or have the same types of chronic infections [24,25].

Some patients may have viral, chemical or other exposures or other environmental problems as the underlying reason for their chronic signs and symptoms.  In these patients, chronic infections may be opportunistic.  In others, somatoform disorders or illnesses caused by psychological or psychiatric problems may indeed be important.  However, in these latter patients antimicrobial treatments should have no effect whatsoever, and they should not recover on antibiotic therapies. 

The identification of specific infectious agents in the blood of chronically ill patients may allow many patients with CFS/ME, FMS, GWI or RA to obtain more specific diagnoses and effective treatments for their illnesses [24,25].

It is important to state again that these infections need not be the cause illness to be important in the pathogenesis.  They could act as cofactors or progression factors and be important sources of patient morbidity [20].

Morbidity may be caused by a number of biological properties of pathogenic mycoplasmas.  They can stimulate lymphocyte activation, increase production of cytokines, produce hydrogen peroxide, compete for nutrients and biosynthetic precursors, and most importantly, they can stimulate autoimmune responses [reviews: 19,20].  

Mycoplasmas have also been documented to be able to penetrate into the CNS and cause neurological signs [21,26,27]. In addition to bacterial infections, chronic illness patients also have viral infections. 

In ALS patients enteroviruses (EV) have been found [10-12]. EV are small, encapsulated RNA viruses that have been associated with acute syndromes such as upper respiratory infections, exanthmas, myocarditis, pleuritis, pancreatitis and aseptic meningitis. 

In addition, chronic infections have been identified in human idiopathic dilated cardiomyopathy [28], hepatitis [29], diabetes mellitus [30] and chronic aseptic meningitis.  In addition, EV are neurotropic [31] and can persist in the CNS [32].  Several scenarios can be envisaged by which a CNS virus infection could cause, at least in part, the pathology observed in ALS.

Recent studies indicate that cell death following virus infection is often apoptotic and that neurons, unlike most other cells, are relatively resistant to apoptosis following virus infection [33].  This can result in virus persistence, which may or may not be productive and has been shown to be capable of causing neuronal dysfunction and eventually death [34].

The concept that persistent neuronal infections could lead to nerve dysfunction by interfering with cellular gene expression and stealing nutrients and cellular materials while producing toxic substances opens the possibility that persistent infection of neurons could result in nerve cell death similar to what has been found in other cell types [review: 20].

For example, infections can promote the production of free radicals or interfere with protective mechanisms, leading to oxidative stress and eventually cell death.  Cytokine dysregulation in infected cells, dysregulation of neurotransmitter receptors, or changes in neurofilament or ion channel gene expression are other possibilities.

Patterns of viral and bacterial dissemination may explain the different patterns of disease progression in each patient, and such has been shown for paralytic poliomyelitis [35]. An early study for EV sequences in spinal cord samples from patients from ALS by RT PCR and in situ PCR showed that a high percentage were positive for EV-specific nucleic acid sequences [8].  However, a study the following year failed to detect EV RNA [10]. 

Most recently, a study from France showed that EV RNA was present from spinal cord samples from 15/17 ALS patients [11].  Our data supports the finding of EV sequences in ALS patient's blood along with mycoplasma sequences.

Preliminary Results with ALS Patients
Clinical Analysis of ALS Patients. Patients with ALS show gradual progressive weakness and paralysis of muscles due to destruction of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord, ultimately resulting in death, usually by respiratory failure [1-3]. 

The overall clinical picture of ALS can vary, depending on the location and progression of pathological changes found in nervous tissue [3].  All of the ALS patients in our studies were diagnosed by neurologists on the basis of standard clinical criteria [1-3].

Analysis of ALS patients used in obtaining our preliminary data indicates that none had familial ALS, and they often had unexpected signs and symptoms. In addition to muscle weakness and wasting, speech and swallowing problems, fasciculation, cramping, among other signs, several ALS patients had rheumatoid arthritis signs/symptoms (7/27), history of asthma, bronchitis or pneumonia (9/27), allergies (7/27), rashes (14/27), night sweats (11/27), diarrhea (8/27), digestive problems (10/27), sleep problems (12/27), nausea (11/27), overall fatigue (22/27), dental problems (11/27), and infections (25/27) [Note: 4 patients are being evaluated]. 

The signs/symptoms unrelated to ALS diagnosis are similar to those usually found in FMS and CFS patients [24,25].  Gulf War veterans with ALS also had signs and symptoms of Gulf War Illness (see refs. 14-16 for a list) which include: disabling fatigue, intermittent fevers, night sweats, arthralgia, myalgia, impairments in short-term memory, headaches, skin rashes, intermittent diarrhea, abdominal bloating, chronic bronchitis, photophobia, confusion, transient visual scotomata, irritability and depression and other signs and symptoms that until recently have defied appropriate diagnoses [14-16].

These symptoms are not localized to any one organ, and the signs and symptoms and routine laboratory test results were not consistent with a single, specific disease.

Preliminary Study of Chronic Mycoplasmal Infections in ALS Patients We have studied the presence of systemic microbial infections in a preliminary number of ALS patients.  For example, we found that 6/6 Gulf War veterans diagnosed with ALS all have systemic M. fermentans infections. These Gulf War veterans include 4 patients from the USA, one from England and one from Australia. 

Also, in 22/25 civilians from USA and England we have found mycoplasmal infections, most were positive for M. fermentans; however, we did find two with M. hominis, two with M. pneumoniae and one with M. genitalium infections.  Using an internal probe these infections were all confirmed with Southern back-hybridization of the PCR product [see refs. 16,17].  This technique is extremely sensitive and can specifically detect mycoplasma DNA down to 1-10 pg in a clinical sample [16,17,36].

Validation of PCR Procedures using Blinded Samples from NIH In a study funded by the DoD we determined whether we could correctly identify coded samples of M. fermentans and other species of mycoplasmas down to the level of a few pg DNA or a few organisms in a blood sample. 

The coded samples were from cultures of mycoplasmas and clinical samples confirmed with M. fermentans infections.   We and two other laboratories (AFIP, Univ. of Texas, San Antonio) that we trained under the contract all correctly identified the coded samples (provided by the Mycoplasma Laboratory at NIH) containing M. fermentans and other mycoplasmas, indicating that the PCR techniques that we developed are extremely sensitive, specific and reliable [17]. 

In addition, routine PCR can prove problematic in detecting chronic intracellular infections due to protein-DNA complexing and the presence of inhibitors in clinical specimens.  We have developed methods to overcome these problems, such as the use of Chelex to remove inhibitors and chaotrophic agents to remove proteins from DNA complexes that can block the polymerase chain reaction.  This is why we were able to show why the DoD did not find these infections in their initial studies.  Using our procedures the DoD is obtaining data almost exactly the same as what we previously published [14-16].

Preliminary Study of Chronic Enterovirus (EV) Infections in ALS Patients Previously 15/17 ALS patients were found to be positive for EV infections [11]. This virus was found in the motor neurons that degenerate in ALS. One of us (D.S.) has studied the type of EV found in ALS, and it appears from sequence data to be similar to an Eco-7 EV.  We have designed specific PCR probes to find this virus in blood and tissue samples from ALS patients.  In preliminary experiments we have found that 10/11 ALS patients were positive for this EV in their blood.

The possible reason that not all researchers have found EV in ALS patients [10] is that very sensitive and specific methods must be used, and inhibitors of the PCR reaction must be removed. We have developed procedures that can overcome some of the problems associated with PCR (discussed above).

Possible Heavy Metal Contamination in Some ALS Patients Many patients with neurological or degenerative diseases have some evidence of heavy metal exposure. Some metals, such as mercury, may result in high levels of oxidative stress or reactive oxygen species (ROS) [37], which have been implicated in neurological diseases [38].  Mercury can also form conjugates with thiol compounds, such as glutathione and cysteine and cause depletion of glutathione, a necessary metabolite to counter or mitigate reactive damage. 

Eggleston [39] found that mercury amalgam fillings or nickel dental materials caused suppression of T-lymphocytes and impaired T4/T8 ratios.  In one very preliminary study 12/13 ALS patients tested showed positive lymphocytes to heavy metals in vitro [40]. 

There are case reports of patients with severe signs and symptoms similar or the same as ALS improving after treatment for mercury poisoning [41].  Although these reports are not compelling or conclusive, they do point to one possible complication in MND such as ALS.  At a minimum such possible complications need to be explored in each ALS patient.

Pilot Combination Clinical Trial for ALS Patients
Patients and Specimens Clinical evaluation of ALS patients: Each patient (total=20) shall have examinations every month while on the study as well as a pre-visit and post-visit.  A board-certified Neurologist shall conduct each examination.

Blood will be obtained and sent for appropriate laboratory studies.  A complete physical examination will be conducted at the week -2 pre-visit and at visits at month 3, month 6, month 12 and the post-trial examination at day +30.  At each additional monthly visit, targeted neurological examinations will be performed.

Any non-neurologic symptoms will be evaluated between or on regularly scheduled visits.  Muscle testing will be evaluated on each group on a standard 1 to 5 scale.  At week -2, month 6, and month 12, a full 4-limb Nerve Conduction Velocity  (NCV) test will be performed along with an electromyelogram (EMG) of all major muscle groups. Speech shall be evaluated on a scale of 1 to 10 (1 being the worst impediment). 

A full mental status examination will be performed on each visit and scored for short and long-term memory; judgement; insight; depression; orientation; and alertness on a scale of 1 to 10 by a neurologist. Voluntary healthy controls (n=70) will be selected from comparable geographical areas without the clinical signs and symptoms described for ALS patients. They will be chosen after a routine clinical examination.  Age and gender of control subjects will be comparable to ALS patient's group.

Blood samples will be taken freshly under the same conditions as ALS patient's blood as described below. Control samples will be run together with patient specimens at the same time. Mycoplasma and EV and all other tests will be performed on all specimens in a blinded matter.  Only the physicians collecting the blood will know the patient diagnosis until the code is broken.

Blood From Patients for PCR. Blood will be collected in EDTA-containing tubes and immediately brought to ice bath temperature as described previously [16,17,28]. Samples will be fresh or shipped refrigerated or on wet ice by over night courier for analysis. Whole blood (50 ml) will be used for preparation of DNA using Chelex (Biorad, Hercules, USA) as follows. Blood cells are lysed with nano-pure water (1.3 ml) at room temperature for 30 min.

After centrifugation at 13000 x g for 2 min, the supernatants were discarded. Chelex solution (200 ml) will be added, and the samples were incubated at 56C and at 100C for 15 min each. Aliquots from the centrifuged samples will be used immediately for PCR or stored at -70C until use. Multiple micro-organism tests will be performed on all patients.

Amplification of Gene Sequences. Amplification of the target gene sequences (for example, Table 1 for mycoplasma PCR) will be performed in a total volume of 50 ml PCR buffer (10 mM Tris-HCl, 50 mM KCl, pH 9) containing 0.1% Triton X-100, 200 mm each of dATP, dTTP, dGTP, dCTP, 100 pmol of each primer, and 0.5-1 mg of chromosomal DNA. Purified mycoplasmal DNA (0.5-1 ng of DNA) will be used as a positive control for amplification.

The amplification will be carried out for 30-40 cycles with denaturing at 94C and annealing at various temperatures.  For example, we will use 60C for genus-specific primers and M. penetrans or 55C for M. pneumoniae, M. hominis, M. fermentans. Extension temperature will be 72C in all cases. Finally, product extension will be performed at 72C for 10 min. Negative and positive controls will be present in each experimental run. The efficiency of the PCR process will be monitored by amplification of b-actin mRNA. (See Appendix for details).

Southern Blot Confirmation. The amplified samples will be run on a 1% agarose gel containing 5 ml/100 ml of ethidium bromide in TAE buffer (0.04 M Tris-Acetate, 0.001 M EDTA, pH 8.0). After denaturing and neutralization, Southern blotting was performed as follows. The PCR product will be transferred to a Nytran membrane. After transfer, UV cross-linking was performed.

Membranes will be prehybridized with hybridization buffer consisting of 1x Denhardt's solution and 1 mg/ml salmon sperm as blocking reagent. Membranes will then be hybridized with 32P-labeled internal probe (107 cpm per bag).  After hybridization and washing to remove unbounded probe, the membranes will be exposed to autoradiography film for 7 days at -70C.  In some cases, the film will be scanned and densitometery used to determine relative probe amount compared to known controls.

Detection of EV RNA from PBMC of ALS Patients. PBMC will be separated from other blood elements by Ficol-Hypaque density-gradient centrifug-ation and RNA extracted by standard methods.  RT PCR will be carried out on 2 ul of extracted RNA by using the EV-specific primers: 5?-CCTCCGGCCCCTGAATGCGGCTAAT-3? (upstream) and 5?-ACCGACGAATACCACTGTTA-3? (downstream).  The DNA will be amplified for 30 cycles using standard cycle parameters, and the product evaluated for a 154-bp product by agarose-gel electrophoresis. 

The efficiency of the PCR process will be monitored by amplification of b-actin mRNA. The presence of amplifications inhibitors will be evaluated by spiking all EV-negative RNA samples with 2 ul of RNA isolated from Coxsackievirus B4 stock.  EV-specific oligonucleotides in the PCR product will be identified by Southern Blot and dot-blot hybridization using a 21-mer internal probe:(5-ATGAAACCCACAGGCACAAAG-3?) 3?-end-labelled with digoxigenin ?UTP.

Severity Score of Signs and Symptoms and SF-36. Illness survey forms (See Appendix) will be given to each patient that analyzed the most common signs and symptoms of chronic illnesses at the time the blood sample was drawn, before and after onset of illness.

Patients mark the intensity of ~120 signs/symptoms prior to and after onset of illness on a ten-point self-rating rank scale (0: none; 10: extreme). The questions were then grouped into 28 categories containing 3-9 questions each. An average score for each category will be calculated as the average change of the intensity of all questions in the category (score = sum of differences between self-rating values prior to and after onset of illness / number of questions in the category).

The data from prior to onset of illness and after onset as well as within the last week before the blood was drawn will be compared. A significant difference is obtained if the score after onset/within the last week was three or more points higher than prior to the illness [17].

Additionally, the average score change with illness and the duration of illness can be correlated with the results of different infections. Surveys of ~70 patients with negative mycoplasma test results will be used to compare score values. In addition, each patient will be given the SF-36 form to assess quality of life. All survey and quality of life forms will be completed before entering the trial and after the trial (week  12). Survey and clinical data will be statistically analyzed using Spearman Rank correlation and Mann-Whitney tests (StatMost32, Dataxiom, USA).

Pilot Combination Treatment Trial Twenty (20) mycoplasma-positive, EV-positive patients will be enrolled in open-label pilot combination treatment trial that will last for 8 weeks (not including follow-up monitoring).  Study subjects will have baseline, week 2, week 8, week 12 and week 14 studies.  All patients will receive the same therapy.  For the first 3 weeks of the study, the subject will be required to come into the Immune Institute for approximately 4 hours per day Monday through Friday. 

Intravenous Ciprofloxacin (Cipro, an antibiotic) will be administered at a dosage of 400 mg/day.  Also, intravenous 5,6 semi-benzylated ascorbic acid (SBA, an antiviral) will be administered a dose of 50 grams per day. After the first three months, participants will be allowed to continue treatments at home but must return to the Immune Institute at months 6, 12 and 13 for full testing and evaluation. 

At home participants will receive oral Doxycycline, 100 mg twice daily for the first three months, followed by 500 mg of Clarithromycin twice daily for three months, then oral Ciprfloxacin at a dose of 1500 mg per day for three months and finish with oral Azithromycin, 500 mg twice daily.  The reason for the rotation of the antibiotics is to prevent any drug resistance and to account for multiple bacterial infections. For the duration of the study at home, participants will receive oral SBA at a dose of 2 grams per day.

Inclusion Criteria for the Study: · Age > 18 years · Signed informed consent · Diagnosis of ALS disease based upon full evaluation by a neurologist · Positive test for Enterovirus and Mycoplasma species

Exclusion Criteria for the Study: · Pregnant or nursing women · Metabolic dysfunctions such as hypoglycemia, hyperparathyroidism, hyperthyroidism and vitamin deficiencies. · Negative NCV · Antibiotics, antivirals, mercury chelation, hyperbaric oxygen, or growth factors within the past 30 days · Involvement of sensory neurons

Endpoints: · Nerve Conduction Velocity · PCR for Enteroviruses and Mycoplasma species · Quality of life (SF 36 Form) · Natural killer cell function · EMG · Muscle strength

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Mycoplasma infections have been implicated as causal, triggering or complicating factors in many chronic illnesses including neurodegenerative disorders such as ALS/MND.

A mycoplasma is a pathogen that infects plants, animals and humans. It is not a bacteria or virus but a member of the mollicute family, having no cell wall, and is a virus-like infectious agent, somewhere between a virus and bacteria in complexity. They can spread on an airborne or casual contact basis or by direct contact between individuals.

Mycoplasmas can comprise part of the normal mucosal flora of healthy persons but a normal blood specimen should be sterile, as should cerebrospinal, pleural, abdominal and joint fluids and bone.

Mycoplasmas can hide intra-cellularly, unlike common bacteria which typically exist inter-cellularly. They cause no inflammatory response so the sed-rate/ESR diagnostic assay will show negative. Mycoplasmas can evade detection by conventional laboratory diagnostic assays that attempt to culture these pathogens and they escape the immune system by antigenic surface variation and molecular mimicry.

As mycoplasmas do not trigger an elevated white blood cell count, systemic mycoplasma infection will not show on a routine complete blood count (CBC) used to determine elevated white blood cells typically associated with a bacterial infection.

Uninformed physicians may diagnose virus as there is no elevated white blood cell count and  antibody tests can result negative in early stages of infection. Viral infection can't be helped by antimicrobials but if the true problem is mycoplasmal infection this can be treated with long-term antibiotic therapy.

Mycoplasmas require specialized environments to be cultured. They grow well at high-altitude/low-pressure conditions and will otherwise culture false-negative.  Only polymerase chain reaction (PCR) testing, otherwise known as dna-amplification or dna-probe is sensitive enough to ascertain mycoplasma infections reliably. A positive test confirms the presence of live mycoplasma genome and active infection.

PCR assays and subtyping are not available at the majority of commercial clinical laboratories but the correct approach to mycoplasma PCR analysis is to obtain a mycoplasma species test and, if positive, proceed to subtype the pathogen. As well as testing for Mycoplasma infection it is advisable to also test for Rickettsiae, Chlamydia, IL2 and IL6.
[See below for instructions for shipping blood].

The other laboratory analysis include, but are not limited to, natural killer cell cytotoxic activity/function, absolute lymphocyte sub population, the myelin basic protein antibody, the 2-5A Synthetase/RNase-L antiviral pathway used to confirm the presence of a chronic infection and provide a benchmark for tracking therapeutic clinical results, lymphocyte immune function test also known as T-cell proliferation to mitogens, antigen and cytokine tests, and quantitative comparative PCR which measures and tracks mycoplasma-load over an antibiotic treatment protocol.

There are hundreds of different mycoplasma subtypes and numerous isolates (strains) within any given subtype.  A partial list of mycoplasmas may be obtained from the American Type Culture Collection under the category of bacteria.

Mycoplasmas can cause numerous and various pathogenic mechanisms including extragenital systemic infection, production of super antigens, abnormal stimulation of cytokines such as interleukin-2, generation of toxic oxygen radicals contributing to oxidative stress observed in infected individuals (antioxidants can help), development of lesions in the heart, liver, kidneys, and other organs, induction of apoptosis (programmed cell death), aphthous ulcerations, thrombocytopenia, central nervous system disease, problems with cell-mediated immunity, and numerous other destructive actions.

Mycoplasmas can target the host white blood cells (lymphocytes/WBC) for intracellular infection, and these cells have the unique ability to cross the blood-brain barrier over into the spinal fluid and thereby carry infection into the host central nervous system (CNS). Once inside the CNS, mycoplasmas have been reported to activate the CNS hypothalamus/pituitary/adrenal axis. The hypothalamus and pituitary glands are part of the endocrine system producing hormones to regulate bodily functions.

Mycoplasmas interact with the lymphoreticular system as they are immunomodulating pathogens that can compromise cellular immunity (T-lymphocytes). T-Suppressors (T8) will move towards a high index when infected with mycoplasma hominis and T-Killers (T3) will decrease due to mycoplasmosis. Natural killer cell function deteriorates as a result of extended systemic mycoplasma infection.

When cellular immunity becomes compromised, ordinarily latent pathogens, such as the herpes viral family (epstein-barr, CMV, HHV-6 and others), can proliferate to detectable levels. Epstein-barr or CMV can be diagnosed although these are only symptoms (markers for immunity) of an underlying mycoplasma infection.

A systemic mycoplasma infection can trigger immunological problems and indirectly lead to chronic illness through the unchecked proliferation of herpes and other pathogens. Certain mycoplasmas will cause multistage oncogenic processes leading to chromosomal alteration.

The antibody response, may not be measurable during systemic mycoplasma infection until a patient is nearing death. This is because mycoplasmas can evade detection by the immune system (and thereby cause a characteristically chronic illness) through various mechanisms including antigenic surface variation and molecular mimicry. Also, the absolute sub population of B-lymphocytes will be observed to increase as a result of a systemic mycoplasma infection.

Since mycoplasmas carry ribosomal genes and are a prokaryote and not a virus, they have been classed as a virus-like infectious agent. Certain mycoplasmas can inhibit normal DNA to messenger-RNA transcription and cause messenger-RNA translation to abnormal protein antigens. This can cause abnormal intracellular proteins (and proteins are the building blocks of the cell), immune dysregulation and abnormal cytokine production.

This dna-recombinent issue may (unless confined to the WBC fraction) implicate serious limitations from a therapeutic cure standpoint, including sophisticated gene therapy techniques which are not widely available.

Some mycoplasmas are being detected with unusual DNA sequences, such as the HIV-1 envelope gene which codes for a surface glycoprotein, gp120, involved in pathogen attachment and entry into cells.

The gp120 recognizes receptors on the lymphocytes and other cell surfaces which can result in opportunistic cell attachment and penetration . Since the receptor recognized by gp120 is present on many cell types, these modified mycoplasmas could be capable of invading most body tissues with unprecedented associated morbidity.

The many different mycoplasma subtypes/isolates can cause a variety of symptoms depending upon the patient and degree of infection. Some of the more pathogenic mycoplasmas, which can cause fulminate illness, include subtypes hominis, fermentans (incognitus), pirum, genetalium, pneumoniae and penetrans.

Acute/primary systemic mycoplasma infection can present with parkinsons-like twitching, motor disorder, shaking, weakness, night sweats, neuropathy, rashes, pharyngitis, sleep disorder, heart palpitations, sensation of terror and/or irritability (hypothalamus controls emotions such as rage, fear and pleasure), muscle and joint pain, sensory and reflex hypersensitivity (e.g. sound intolerance), adenopathy, confusion and anxiety, coagulated ejaculate, spleenomegaly, bleeding gums, rapid weightless, nausea, racing metabolism (thyroid/endocrine), sepsis (overwhelming infection), diarrhoea and bowel disorder, temperature fluctuations, chills, drooling, blurred vision, metallic taste in mouth, numbness in extremities and back of head during attempted sleep, extreme spine, neck and back pain, difficulty turning neck, skin irritated by fabric coverings, and elevated herpes antibody titers.

The onset of mycoplasma infection can be insidious and/or localized depending on the particular mycoplasma subtype and isolate involved.

* The chronic clinical picture can include central nervous system disease, learning disability, cognitive disorder, memory loss, fatigue, myoclonus, abdominal pain, painful granulomas under armpits, rib malformation and prominence above spleen in young children, dark circles under the eyes, chostochonderitis (inflamed sternum/cartilage), headaches, nose-bleeds, hair loss, bone pain from metastasize, stammering, stunted growth, bruises, cancers (glioma, blastoma, etc.), and organ failure.

* A typical symptom of systemic mycoplasma infection, resulting from abnormal stimulation of cytokines, involves a chronic red discoloration of the anterior pharyngeal pillars. Stand in front of a mirror with your mouth open wide. Point a flash-light into the mirror so that the beam reflects back into your pharynx. On either side of your throat, behind the molars and in front of the tonsils, the crescents may be an intense crimson color along the margins of both  pharyngeal pillars. In patients without tonsils, the crimson crescents would appear further back.

* Overcoming systemic mycoplasma infection will depend on the characteristics of the specific mycoplasma involved, and many cases will respond to combined antibiotic/steroid treatment, naturopathic treatment and avoidance of stress and certain foods.

* Where a mycoplasma pathogen targets host white blood cells for infection, the disease can initiate a vicious cycle of immunological dysregulation where the lymphocytes actually start replicating and attacking themselves for being infected with the immunomodulating mycoplasmas. In order to break the vicious cycle combined antibiotic/steroid treatment protocols can be used.

* Mycoplasmas are susceptible to long-term antibiotic therapy. With an appropriate course and brand of antimicrobal therapy mycoplasma patients can revert back to permanent asymptomatic status. Detecting the infection early, such as during acute infection, would shorten treatment duration.

* Chloramphenicol  is a preferred antibiotics for treatment of systemic mycoplasma infections. Other antibiotics that have demonstrated efficacy against extragenital mycoplasma infections include doxycycline, minocycline, zithromax, rifampin, gentamicin, the lincosamides, trovafloxacin (for M. hominis) and sparfloxacin. Resistance needs to be ascertained to achieve any clinical benefit and, where culture techniques are ineffective, anti-microbial sensitivity determinants can be ascertained on a dna-amplification basis.

* Mycoplasma shares similar capabilities to viruses in that it can recode DNA and even alter its own characteristics, which is why it is so compatible with viral proteins.

* Penicillins  are contraindicated and will actually exacerbate the clinical picture since mycoplasmas have no cell-wall.

* Certain mycoplasma infections can be addressed with a sophisticated derived blood product, monoclonal antibodies.

* In addition to their role between mycoplasma infection and the endocrine system, some steroids suppress the production of white blood cells and thereby starve some mycoplasmas.

* Mycoplasmas generate toxic oxygen radicals that contribute to oxidative stress in infected individuals. Antioxidants will not cure the underlying mycoplasma disease but may help in managing the mycoplasma infection.

* Cholesterol and the amino acid arginine (commonly found in chocolate) stimulate growth of mycoplasmas and should be avoided, although Mycoplasma hominis characteristically produces arginine so avoidance of arginine rich foods may not help in this case.

* Stress should be avoided since it contributes to progression of mycoplasma disease.

* Mycoplasma grows well in low-pressure conditions so avoid unnecessary airline travel while infected with this disease.

* Among other promising immunomodulators, bacterial polysaccharides from clavibacter michiganense (potato ring rot) has been reported to correct the T8 and T3 (cell-mediated) irregularities otherwise associated with a natural proceeding of mycoplasmosis.

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Date   : Thu, 06 May 1999 09:32:42 +0000
From  : "Prof. Garth Nicolson" Subject: Re: ALS in Gulf War vets

John Pomfret (U.K.) and I have initiated a joint study of ALS Gulf War veterans, and we would like to obtain blood samples from as many as possible. Thus far, working with about 20 U.K., U.S. and Australian veterans we have found that 100% have progressive M. fermentans infections that are likely to play an important role in their disease progression. These are complicated cases, and although we do not feel that mycoplasmal infections are a causative agent, they could be very important in the progression of the disease and patient morbidity.

We would be very interested in obtaining additional ALS patients in order to examine their blood for infectious agents. The Institute for Molecular Medicine and I will take care of all cost for the blood analyses, except for the cost of drawing and shipping the blood to our facility. Each patient (and their physician) will be appraised of their results and of the study results when completed. We are particularly interested in seeing whether we can arrest or slow the progression of the disease using
therapies developed at the Institute for Molecular Medicine.

Please contact me for further details and information. Please circulate this information to veterans groups and interested individuals.  This will not be a part of the ongoing VA clinical trial (Co-operative Clinical Study Program #475) on the diagnosis and treatment of mycoplasmal infections in GWI patients that just started at 30+ VA Medical Centers and 2 DoD hospitals.

Prof. Garth Nicolson
Chief Scientific Officer
The Institute for Molecular Medicine 15162 Triton Lane, Huntington Beach, CA 92649
Tel: 714-903-2900    Fax: 714-379-2082

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Excerpts from Dr Nicolson's Findings
Dr. Garth L. Nicolson and Dr. Nancy L. Nicolson Institute for Molecular Medicine, June 26, 1997 to the Subcommittee on Human Resources and Intergovernmental Relations, House Committee on Government Reform and Oversight.

After examining (Gulf War Illness or "GWI") patients' blood for the presence of chronic biological agents, the most common infection found was an unusual micro-organism, Mycoplasma fermentans (incognitus strain), a slow growing mycoplasma located deep inside blood leukocytes (white blood cells) of slightly under one half of patients studied.

This micro-organism is similar to a bacterium without a cell wall, and although mycoplasmas are often found at superficial sites in humans, such as in the oral cavity, they are rarely  found in the blood. When they are in the blood, similar to other bacteria, they can cause a dangerous system-wide or systemic infection. In addition, cell penetrating mycoplasmas, such as Mycoplasma fermentans, may produce unusual autoimmune-like responses to host cell antigens carried on the mycoplasma surface.

Our detection of mycoplasmal infections in the blood leukocytes of~45% of the patients examined (76 out of 170 patients) ...indicate that systemic infections may be a major contributor to GWI. ...The Uniformed Services University of the Health Sciences, the U.S. military's medical school, has been teaching its medical students for years that this type of infection, although rare in the U.S. population, is very dangerous and can progress to system-wide organ failure and death.

...we have found that this type of infection can be successfully treated with multiple courses of specific antibiotics, such as doxyxycline (200 mg/day for 6 weeks per cycle), ciprofloxacin (or Cipro, 1500 mg/day for 6 weeks per cycle), azithromycin (or Zithromax, 500 mg/day for 6 weeks per cycle), chromycin (or Biaxin, 500-1000 mg/day per 6 week cycle) orminocycline (200 mg/day for 6 weeks per cycle), along with other nutritional recommendations. Multiple treatment cycles  are required, and patients relapse often after the  first few cycles, but subsequent relapses are milder and patients eventually recover.

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...We found that 73 patients on antibiotic therapy relapsed within weeks after the first 6-week cycle of therapy, but 58 of the 73 recovered after up to six cycles of therapy. Patients who recovered from their illness after several (3-7) 6-week cycles of antibiotic therapy were re tested for mycoplasmal infection and were found to have reverted to a mycoplasma-negative phenotype.

We hypothesize that the therapy takes a long time because of the microorganism(s) involved (a mycoplasma) is slow growing and is localized deep inside cells in tissues, which are more difficult locations to achieve proper antibiotic therapeutic concentrations. Multiple cycles of therapy result in eventual recovery in a high percentage of  mycoplasma-positive patients.

Although anti-inflammatory drugs can alleviate some of the signs and symptoms of GWI, the signs and symptoms appear to quickly return after discontinuing drug use. If the effect was due to an anti-inflammatory action of the antibiotics, then the antibiotics would have to be continuously applied and they would be expected to eliminate only some of the signs and symptoms of GWI.

In addition, not all antibiotics, even those that have anti-inflammatory effects, appear to work. Only the types of antibiotics that are known to be effective against mycoplasmas are effective; most have no effect at all on the signs and symptoms of GWI/CFS/FM, and some antibiotics make the condition worse. Thus the antibiotic therapy does not appear to be a placebo effect, because only a few types of antibiotics are effective and some, like penicillin, make the condition worse.

We also believe that this type of infection is immune-suppressing and can lead to other opportunistic infections by viruses and other micro-organisms or increases in endogenous virus titers.

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Dr Mercola has information on Mycoplasmal infections

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