Impaired Neuronal Communication Syndrome (INCS) as Novel Neurological Side Effect to Botulinum Toxin Type A Therapy with 16 Case Reports

  

    
 
    
Past Medical History 
    
Family History 
    
General and Neurological Examination 
  
  

    
Case #9 
    
Hypothyroidism: thyroid hormones before the toxin therapy: thyroid    stimulating hormone: normal, T3 1.87 ng/dl (2-4.90), T4: 0.71 ng/dl    (0.76-1.6), vagal syncope since teenage, 24     000 premature ventricular contractions on 24 hours heart holter not    responding to ablation but treated successfully with beta blockers, mild    essential tremor
    
Father with cancer
    
Word registration 2/3 on first attempt, and 3/3 on second, 29 animals    in the first 30 seconds, no animals generated despite significant mental    strain in the next 20 seconds and a few more animals named in the last 10    seconds, 5-/5 tiring weakness of neck flexion and in the proximal legs, mild    right>left and postural>action hands tremor, draws spirals with mild    bilateral tremor (due to presence of old and mild essential tremor)
  
  

    
Case #10 
    
one successful in vitro fertilization and natural  birth before toxin therapy, one miscarriage    after toxin therapy when anti-cardiolipin antibodies were found    positive.  Initial 90% recovery from    the toxin syndrome but then relapsed after the second in vitro fertilization,    mild asthma at age 8
    
Father: cancer
    
2/3 words recall at 5 minutes, literal paraphasias, visual-spatial    abnormalities on clock drawing, able to draw it right only on 5th attempt, names less frequent objects with significant delay, reflexes 2+ in    the upper and 1+ in the lower limbs.
  
  

    
Case #11 
    
Controlled hypothyroidism, left elbow reconstruction after trauma with    normal muscle strength, right breast lumpectomy
    
Cancer in the family
    
Left eye afferent like papillary reaction with normal vision, hearing    decreased on the right to fingers rub, minimal facial weakness: cannot hold    air in mouth with cheek pressure, 4/5 neck and hip flexion weakness, 3+    reflexes, decreased speed of foot tapping bilaterally
  
  

    
Case #12 
    
Cancer of uterine cervix 8 years before the toxin therapy: surgically    healed with conization, at age 5 left leg osteoid osteoma: cured surgically
    
Non-relevant
    
Normal
  
  

    
Case #13 
    
3 months post-partum depression, generalized anxiety and compulsive    cleaning, one week before the toxin therapy had a swollen lymph node in the    neck which cleared spontaneously
    
Father: cancer
    
Pulse 90, normal neurological examination
  
  

    
Case #14 
    
None
    
Father and sister: major depression,
      
3 family members with cancer
    
Slight fatigability of neck flexion, minimal dysmetria on    finger-to-nose test, slight reduction of vibration sense in legs
  
  

    
Case #15 
    
None
    
Father: depression
      
Grand mother: cancer
    
Normal
  
  

    
Case #16 
    
At age 12 and 18 months    episodes of neck and head fixation with 2 days duration, hands fixation in    claw position for seconds or minutes (suggesting elements of generalized    dystonia), social anxiety: treated, Helicobacter pilory ulcer: treated and    cured, Hepatitis B (active carrier state as per gastro-enterology consult:    positive e and surface antigene, vial load 116 UI/ml.  Patient asymptomatic before the toxin    therapy.  The hepatitis virus    discovered after the toxin injection. Cervical dystonia after one dose of    metoclopramide.  Cholecystectomy after    the toxin therapy to relieve toxin related symptoms with only minor relief    from the procedure. Smoker. Magnetic resonance imaging head before toxin    therapy:  non-specific T2 lesion in    right external capsule most likely due to peri-natal trauma, which remained    unchanged after the toxin therapy (on second imaging).
    
Essential tremor and epilepsy in family, Father and sister with    hepatitis
    
Severe rash: red pimples blanching to some extent, on the back and    upper chest (persistent for months) frequent throat clearing and coughing    during the examination, some visual-spatial distortion on clock drawing,    named a pretzel a snake, 3+ reflexes and tandem difficulties.
  

Table 3b:  

We composed and analyzed 9 groups of clinical symptoms as phenomenology expression of INCS:

Central Nervous System (CNS)

Patients often use the following expressions: “I am in a fog… …I am in a bubble…I feel detached…My body is not listening to my brain…I am disconnected…Somebody else is operating my body”. The most common symptoms are impaired concentration, word finding difficulties, impairment of memory, significant head pressure, phono- and photophobia and inability to multitask. Processing speed is decreased. Events happening at a faster speed are not perceived well, such as: retrieving all information from a person speaking quickly or from a group of people speaking at the same time reading and looking at fast moving objects. Most of all, there is a mental fatigue which often leaves the patients debilitated and with the ability to only do a few tasks before exhaustion sets in. Other reported symptoms in order of frequency are: internal shaking, electric feeling in the head or along the spine, headaches, episodes of speech arrest with duration of 10-15 minutes, loss of smell, myoclonus, abnormal movements, distortion of the visual fields, gait ataxia and tremors.

Peripheral nervous system

The most common symptoms are pain and/or paresthesia in limbs, face and/or torso and a “buzzing” feeling in the nerves. Many times the pain is severe and is described as, “crushed glass under my skin” or “acid dripping”, leaving the patient suffering 24/7. Feelings of pins and needles, itching and numbness are less frequent symptoms, but can be, as well, pronounced. Cranial nerves, such as the acoustic nerve, can be affected with tinnitus, and the optic nerve with tunnel vision. Maximum pain at the area of the injection is one sign which can be used to establish connection to the toxin. Sensation disturbances may occur in the vaginal area, without presence of infection.

Autonomic nervous system

Most common and disabling are the following symptoms: shortness of breath, arrhythmia, heart racing, palpitations, chest pain, dizziness, dry mouth, dry eyes and stuffy ears, nose and head. Sometimes the intensity is severe, especially of the shortness of breath. The patents describe it as “not being able to take a deep breath”, “involuntary arrest of breathing for several seconds” and “need to constantly remind the self to breathe”. When present and intense, the later symptom can linger for years deeply affecting the quality of life of the patient. Other reported symptoms are: urine incontinence, frequent urination, straining on initiation of urination, nausea, vomiting, constipation, diarrhea, blurred vision, eye strain, icy cold limbs, night sweats, abdominal pain, runny nose, hoarseness and chest pressure. Constipation can be very severe.

Psychiatric

Psychiatric symptoms are very common and debilitating such as severe anxiety, panic attacks, feeling desperate, hopeless, negative and fixated on the symptoms. There is a deep feeling that something is wrong and the patients feel terrified or fearful for a long time. The patient’s whole world is absorbed by the disease. It is common for the patient to wake up at night with agonizing fear that death is imminent. Usually, depression sets in as the patient is unable to receive help or acknowledgment of his/her symptoms from others or, at times, from the medical providers. Psychosis is less common but no less crippling. One patient had constant visions of killing others, seeing aliens and other people in her room.

Hypothalamic syndrome

Weight loss, loss of appetite, insomnia, reduction of libido, change in menses or sperm, change in tolerance of cold and warm, hot flashes and increased thirst are frequent symptoms. Increased hunger is rare. Weight loss (6-35 pounds), though most of the time transient, could be extreme at times. Insomnia is often disabling and prolonged. Amenorrhea can last for months. The one male patient reported in this series said that his sperm looked like water for one year after the occurrence of the side effects. Patient #10 had initial normal thyroid tests and then pattern suggesting “central” hypothyroidism.

Fatigue, lack of endurance, muscle twitches and muscle stiffness

Fatigue is usually profound and present in all patients. Sometimes even the slightest effort tires the patients, such as holding a book for several minutes when reading or rigorously stirring tea. Muscle stiffness and tightness are very common and disturbing to the patients. Myoclonic jerks and other twitches occur, but are rare.

Muscle weakness

It is very important to note, that although the patients experience and report lack of muscle power and complain of weakness, the muscle power on manual testing is almost always normal suggesting more central than peripheral nervous system origin of the symptom. If present, the weakness is usually mild, transient and easily can be missed if more gross or quick neurological examination is carried out, especially because most of the times the patients look “generally well”. The most common features are fatigability of the neck and proximal hip flexor muscles, ptosis and swallowing deficit. The swallowing deficit may persist for months though rarely were swallowing studies performed.

Atrophic syndrome

Patients complain of skin atrophy, subcutaneous tissue loss, lumping of the subcutaneous tissue, hair loss and nail impairment.

Loss of muscle mass, at times significant, usually occurs in the first year of the syndrome, though it can occur later too.

Autoimmune syndromes

Seventy-five percent of the patients have some autoimmune marker or clinical symptom suggestive of immune activation at some time during the course of their disease. However, those markers and symptoms are usually non-specific and many times only transiently present. It is important to note that, at times, the markers can reappear shortly during subsequent bouts of the disease. In these case-series, the only persistent and visible damage over the nervous system had been the presence of small fiber polyneuropathy with skin biopsy performed when the patient had significant peripheral nervous system symptoms. Unfortunately, this was done only in 13.3% of the patients who had clinical symptoms of peripheral nervous system involvement. Flu-like symptoms are rare.

Other prominent features

Increased frequency of infections: One-third of the patients have increased rate of infections. The most common are upper respiratory tract infections, pneumonia, urinary tract infections and sinusitis. Activation of dormant diseases such as herpes may occur.

Sensitivity to food and medications: This peculiar symptom is present in 62.5% of the patients. The sensitivity is especially pronounced to serotonin uptake inhibitors, coffee, sugar and alcohol which almost uniformly worsen the symptoms of the patients. Steroids either worsen the symptoms or are ineffective. Patients have adverse reactions to most of the CNS modulating drugs with the exception of benzodiazepines (alprazolam, lorazepam, clonazepam) in a low dose, which helped some of the psychiatric symptoms in some patients. Here is the list of the drugs which the reported patients were not able to tolerate: paroxetine, fluoxetine, sertraline, pregabaline, escitalopram, duloxetine, desvenlafaxine, trihexyphenidyl, lamotrigine and quetiapine. Usually, with time and attempted treatments, the patients become afraid of trying any new drugs or therapies.

Crises and waxing and waning of the symptoms: This is a uniform feature throughout the population. The patients have bad and good days with fluctuation of their symptoms. Different symptoms can be more pronounced at different times. In addition, there are sudden crises where some or all symptoms worsen. We identified common triggers such as medications, food, pushing through fatigue and stress. Most patients become very fragile and afraid of activity, food, and medications since the reaction is often unpredictable and can be severe. Crises continue to occur many years after the injection, with new symptoms developing over time, though the majority of the new symptoms develop during the first year.

Symptoms onset: The symptoms onset ranges from minutes to weeks with most common frame of time onset 24-72 hours.

Years of toxin treatment and reaction to prior injections: Our data demonstrate that a generalized spread of the toxin can occur even after years of uneventful toxin therapy. Patient #7 had 30 successful treatments over a decade before the adverse events occurred. Prior uneventful injections cannot be a predictor for toxin safety, because in our experience, generalized spread can occur at any time and with any injection. Forty-four percent of the patients develop a reaction after their first time injection. From those who had more than one injection, 50% had an adverse event to prior injections which remained unrecognized and lead to devastating effects with next injections. Our data suggests that with each subsequent toxin reaction the time of symptoms onset may shorten, as the patient’s immune system sensitizes further to the toxin. Some of the injections were done together with Lidocaine which increases the injected volume and, with this, the possibility of generalized spread. Some patients were injected more frequently than the recommended 3-month inter-injection interval, which may have created a cumulative effect and an unnecessary stimulation of the patient’s immune system. Some patients were injected as “models” during BoNT/A conferences with no prior establishment of doctor to patient report and without documentation for the event. Others were given BoNT/A because it was offered for free to the staff at injector’s clinics during drug representatives visits.

Functionality, disability, course of disease and prognosis: Before the BoNT/A therapy, all patients were 100% functional and highly active individuals, apart from patient #4 who had mild pre-morbid impairment due to postural orthostatic tachycardia syndrome. In all patients, disability occurred after BoNT/A therapy with duration from months to years (at the present moment of observation it is 8 years or more). The disability is often significant: patient #7 was wheelchair bound for 1.5 years. The maximum disability duration is unknown at present. Many patients loose their jobs and relationships. Apart from the 3 patients who recovered completely (19%), all others continued to have symptoms for years after the injection. Though, in our experience, mild and slow yearly improvement takes place as the toxin is cleared from the system, the majority of the patients do not reach his/her pre-morbid state of health at 5-6 years time mark after the last injection. The only BoNT/A condition that seems to persist over the years, with no or very little improvement, is the small fiber polyneuropathy, since it is an auto-immune disease. In our experience, if the patients have no yearly (even slow) improvement of their symptoms in the first 3-5 years, the prognosis for recovery is less favorable. Since the syndrome is not yet well recognized, it is not surprising that when a physician encounters normal neurological examination, normal or minimally positive laboratory results and normal imaging studies in the face of multiple patient’s complains, the symptoms are assumed to be due to anxiety or somatization. This, often, leads to referral to psychiatry or to the start of psychiatric drugs which then usually leads to worsening of the symptoms. The patients often go from doctor to doctor seeking help. Thousands of dollars worth of investigations are conducted, with little or no help or relief from their symptoms and/or establishment of the true diagnosis. The majority of the cases reported here were admitted to a hospital or ER at least once during the course of their disease. Many times the affected patients end up alone, depressed, broken and terrified, prisoners to their homes with restricted social contacts and very few people who believe them. The impact of INCS on the patients’ quality of life is enormous. From the three patients who recovered, patient #3 and #13 came to see me within 1-2 months from the onset of the adverse event. I advised them not to inject the toxin again, not to vaccinate, I put them on activated charcoal, echinacea and diet therapy and supported them emotionally. Patient number #7 did daily enemas with activated charcoal and coffee, in slow dose increments reaching a maximum of two table spoons of charcoal in 3 cups of coffee. The treatment persisted for 1.5 years and the patient did not stop the therapy when temporary worsening of her symptoms occurred after each enema. However, there were patients who did not respond to charcoal/echinacea therapy suggesting that there is likely a subpopulation in this patients’ group. For example, charcoal may worsen patient’s symptoms if severe constipation is present, since no elimination can occur and toxin re-absorption from the gastrointestinal tract and secondary hematogenic spread may occur.

Therapy: All therapies are in a research stage of development. Here is a list of modalities which helped some of the patients reported here: Diet: gluten free, organic, vegan; chlorella; celery juicing; abstinence from sugar, alcohol and caffeine; avoidance of CNS modulating drugs, with the exception of benzodiazepines, which can be used in small doses for anxiety and panic attacks therapy; bananas (likely because of the high potassium content); benadryl (the symptoms in many patients suggest high histamine state); aspirin (for the small fiber polyneuropathy); valerian root (for the internal shaking); magnesium gluconate (for the muscle tightness and spasms); hyperbaric oxygenation (each treatment may induce several hours of healing crisis with temporary worsening before improvement occurs); activated charcoal; and echinacea.

Discussion, Hypotheses and Algorithm

Discussion and hypotheses of the phenomenology of INCS

Central nervous system: Research has established that botulinum can block many mediators in CNS [6- 9], though its preference is upon the cholinergic pathways. On central level, the peripheral acetylcholine (Ach) specificity is lost suggesting that the target of botulinum is common to many, and may be all central neurons of mammals as Bigalke et al. concluded [10]. Ach plays a role in neural plasticity, word finding, concentration, attention, learning, abstract thinking, creativity, brain speed processing, noise/ signal ratio improvement and intra-cortical transfer of information. It is not a surprise, that the patients presented here frequently display cognitive deficiency. Luvisetto et al. [11] injected the brain ventricles of live mice with botulinum and demonstrated that the animals produced dementia-like features. Some of the important principles of CNS functions are mediators’ balance and neuronal nets interactions. When one mediator pathway is impaired the brain adapts by altering the remaining mediators balance. Most of the patients presented here exhibit combined features of depletion of some mediators and increment of others, such as: Ach depletion with impaired concentration, memory, focus, learning, thinking clearly and coherently, recall and ability to process information; dopamine depletion with affection of memory, attention and concentration and problem-solving, slowness of movement and processing, low libido, nausea, vomiting, lack of appetite and depression; hyper-adrenergic Central nervous system: Research has established that botulinum can block many mediators in CNS [6- 9], though its preference is upon the cholinergic pathways. On central level, the peripheral acetylcholine (Ach) specificity is lost suggesting that the target of botulinum is common to many, and may be all central neurons of mammals as Bigalke et al. concluded [10]. Ach plays a role in neural plasticity, word finding, concentration, attention, learning, abstract thinking, creativity, brain speed processing, noise/ signal ratio improvement and intra-cortical transfer of information. It is not a surprise, that the patients presented here frequently display cognitive deficiency. Luvisetto et al. [11] injected the brain ventricles of live mice with botulinum and demonstrated that the animals produced dementia-like features. Some of the important principles of CNS functions are mediators’ balance and neuronal nets interactions. When one mediator pathway is impaired the brain adapts by altering the remaining mediators balance. Most of the patients presented here exhibit combined features of depletion of some mediators and increment of others, such as: Ach depletion with impaired concentration, memory, focus, learning, thinking clearly and coherently, recall and ability to process information; dopamine depletion with affection of memory, attention and concentration and problem-solving, slowness of movement and processing, low libido, nausea, vomiting, lack of appetite and depression; hyper-adrenergic

Peripheral nervous system: The symptoms of peripheral nervous system involvement suggest autoimmune process affecting the small fibers confirmed with skin biopsy in two patients. Increased pain perception due to prolonged hypocholinergic state [12] may be in play as well.

Autonomic nervous system: It is well established that botulinum affects the motor and the autonomic fibers to equal extent. The toxin can spread via blood and retrograde axonal transport through both motor and sensory nerves [13-15]. Vagus nerve is the major autonomic nerve in the body, therefore blocking it may create hypersympathetic tone in the affected organs with symptoms of tachycardia, nausea, diarrhea, chest pain, weight loss, stomach pain and spasms, poor digestion, and difficult urine control which were frequently experienced by the presented cases. In addition, the toxin acts on the potassium channels [10] on which the heart is rich. Cardiac deaths after botulinum therapy have been reported in the post-marketing period (Botox® insert). Blocking the vagus nerve can also, affect the related central pathways such as brainstem, hypothalamus and limbic system. The normal breathing studies, performed during time of significant breathing problems in some of the presented patients, suggested a central breathing pacemaker impairment and central hypoventilation problem similar but not equal to Ondine’s curse syndrome. Only one patient had a polysomnography demonstrating a central apnea episode without desaturation. The vital capacity, the forced vital capacity and the negative inspiratory pressure are rarely measured, while they may be a helpful tool when significant shortness of breath is present. Stone et al. demonstrated that locally applied toxin leads to long lasting arteriolar dilatation which may explain the common complaint of nose, head, ears or sinuses congestion, without presence of infection. It is well established that Ach plays a role in the autoregulation of the cerebral blood flow.

Psychiatric: Deficiency in acetylcholinergic pathways has been related to anxiety, bipolar behavior and delirium. We speculate that retrograde toxin spread to the limbic system or shift in the global brain mediator balance are possible causes of psychiatric symptoms experienced in these patients’ series. Hyper-histaminic state has been considered in schizophrenia patients and there is a report of a patient who’s mental illness responded well to treatment with the histamine receptor antagonist famotidine [18].

Hypothalamic syndrome: Theoretically, the toxin can reach hypothalamus via retrograde vagus or cranial nerves’ transport, or via blood spread. Though the blood brain barrier protects the brain, it is thinned at the hypothalamus in order for monitoring of body homeostasis to occur. Boroff et al. [16] demonstrated that radiolabeled botulinum crosses the blood brain barrier. Thirunavukkarasu et al. [17] showed that, once injected, the toxin alters the expression of 227 genes including those responsible for transmigration of monocytes and T-cells across the blood brain barrier. In respect to the alteration of the mediator balance, the highest concentration of histamine in the brain is in the hypothalamus. Histamine is involved in the regulation of cognition, circadian rhythms (including sleep) and neuroendocrine balance [18]. Increased concentration of histamine is reported in serum and spinal fluid of Alzheimer dementia patients [18].

Fatigue, lack of endurance, muscle twitches and muscle stiffness: The pathophysiology of fatigue has always been a challenging and complex question. The symptom has been reported to persist from 6 to 13.5 years after botulinum food poisoning [19,20]. It is likely mediated by cholinoreceptors in the ventromedial hypothalamus [21]. Alteration of brain circulation may be involved too [22]. Studies on chronic fatigue syndrome suggest involvement of hyper-serotoninergic mechanisms, hypoactivity of the hypothalamus-pituitary-adrenal axes [23] and the presence of cytokines inflammatory mediators [24]. Profound fatigue has been reported in chronic and advanced cases of multiple sclerosis, human immunodeficiency syndrome and neurolyme, suggesting central etiology of the syndrome. Mazzoni et al. [25] studied Parkinson’s disease patients and demonstrated that patients with Parkinson’s move slower than controls when moving naturally but, if encouraged, they can execute movements at a normal speed. We contemplate if the INCS may have a “central motor drive deficiency”, as well. Muscle spasms and tightness are frequently reported in our patient population and in post-foodborne-botulism [20]. This is most likely due to affection of the Renshaw neurons in the spinal cord, which have been shown to be targeted by the toxin [26].

Muscle weakness: Is related to the direct actions of the toxin on the neuromuscular junction. Since the junctions effects persist for 3-6 months, a true muscle weakness has a relatively short duration in these patients with the exception of diaphragmatic and swallowing deficits when the duration is unknown. It is unclear, at present, if swallowing and breathing problems are due to true weakness (some patients had abnormalities in pulmonary function test) or to discoordination of the peripheral and central neuronal pathways, or to both.

Atrophic syndrome: Weight loss associated with BoNT/A has been demonstrated in animal studies [27]. When trophic syndromes are present we need to consider the enormous effect of nonneuronal Ach on the body systems. Research has demonstrated that extraneuronal Ach is crucial for the survival and the function of the cells: blockage of Ach receptors on non-innervated cells results in cell dysfunction and death; adding Ach antagonists to cell cultures induces almost immediate cell shape change with shrinkage and cell separation; Ach regulates skin regeneration with keratocytes containing 100 times higher Ach levels than airway cells; Ach is crucial for almost any cell to cell contact [28]. Ach nicotinic receptors are involved in appetite and food seeking behavior.

Autoimmune syndromes: The presence of BoNT/A blocking antibodies in one of the cases presented here did not prohibit the presence of good wrinkle abolishing effect suggesting that, autoimmune mechanisms other then just the production of toxin blocking antibodies are at play. Oshima et al. [29,30] have shown that human lymphocytes are activated by the toxin irrespective if the patients’ blood had or did not have blocking antibodies. This suggests that assays of toxin blocking antibodies may not be an efficient marker for the presence or the absence of autoimmune reaction to the toxin. Singh et al. [31] showed that the toxin-associated proteins, injected in the body together with BoNT/A and consisting of 2/3 of the complex, are 5 times more immunogenic than the toxin itself. Reports of rash or eosinophils elevation, as in the presented cases, suggest toxin connection. The published reports of botulinum induced autoimmune encephalitis [32] and Guillain-Barre syndrome [33-35] indicate that BoNT/A is capable of inducing an auto-immune reaction in humans.

Discussion of other features of INCS and hypotheses

Increased frequency of infections: Since the acetylcholinergic pathways are involved in regulation of immunity, it is not a surprise that this symptom is common in the patients’ population presented here. Pneumonia was documented in animals sacrificed to the toxin [5].

Sensitivity to food and medications: Serotonin given to animals after BoNT/A injection increased the toxin’s activity up to 1000 times [36]. This may explain why, almost uniformly, the cases presented here had worsening with serotonin-reuptake inhibitors therapy. Large amounts of sugar intake has been related to suppression of Ach release. Only one patient was vaccinated (flu shot) after the toxin exposure and had worsening of her symptoms, likely due to stimulation of an already upset immune system. Note that while steroid therapy is likely not indicated for treatment of INCS, steroids may be useful in botulinum induced autoimmune encephalitis [32].

Crises and waxing and waning of the symptoms: The latest research of Bomba-Warczac et al. [37] demonstrated unequivocally that the toxin travels via retrograde axonal transport and transfers between neurons while remaining enzymatically active. This suggests that the occurrence of new symptoms or worsening of old symptoms may co-inside with the time of BoNT/A transfer and engagement of new neuronal nets. Polley et al. [38] demonstrated in animals, that 5 minutes after intravenous injection of BoNT/A there was cessation or significant decrement of EEG recorded cortical activity associated with loss of consciousness. The animals recovered in 30-60 minutes to have another 3-4 similar episodes of EEG depression in the next 24 hours. It is difficult to explain the repetitive appearance of the cortical depression, after initial recovery, unless we accept the theory that the time of appearance of cortical depression is a marker of the time of intoxication of new neuronal nets by botulinum. Herrero et al. [5] gave lethal dose BoNT/A to monkey, an though most of the monkeys died shortly after the toxin exposure, some recovered and died 40 days later, again suggesting an existence of transneuronal passage of the toxin, or later absorption of the toxin from peripheral peri-neural depots. Pharmacokinetic studies in animals suggest that the clearance of the toxin is a significant issue. Each biological system has parameters of detection and operation below or above which the data may be regarded as noise. Due to the minimal substrate cleavage and nano-dose concentrations, the toxin seems to stay under the radar of the elimination mechanisms of the body such as liver and other metabolic cells [39]. It seems that BoNT/A just squats on the inner layer of the pre-synaptic membrane of the neuron, locked into its substrate: the synaptosomal associated protein – 25 (SNAP-25) [40], and jamming the transmission until the neuronal membrane is recycled, which occurs at a very slow rate. Once injected in the muscle, only a small amount of the total toxin load is absorbed in the neuronal terminals [39] making the rest of perineuronal-pool toxin available for further absorption over time. Re-absorption from the micro-compartments could be responsible for the patients’ crises and can explain why some patients report worsening after having massage or after rigorous detoxification procedures. Research has shown that once in the neurons, BoNT/A can persist more than one year [39], suggesting that the elimination of the toxin may take years, as it is our experience with the cases presented here. Keller et al. [41] showed that in spinal neuronal cultures the enzymatic activity of BoNT/A decreased only with 50% in 78 days. This is significant because the toxin needs to preserve only 6% of its enzymatic activity in order to block a synaptic transmission [40]. Since blood does not alter the toxin at all, it has been regarded by some researchers as a toxin holding compartment [39]. Unless methods of toxin detection become very sensitive, readily available and frequently used, the true duration of toxin presence in humans cannot be established. A mouse assay was reported positive in the blood of a patient, 2 years after onset of her symptoms (author’s correspondence). None of the cases cited here had mouse bioassays or other direct assays for detection of the toxin.

Symptoms onset: It is possible that the symptoms occurring within the first minutes to a few hours after the injection are due to hematogenic spread and autonomic nerve endings blockage, while those occurring later are due to retrograde axonal transport, which is slower. It is unclear, at present, of how many hours are needed for the toxin to generate axonal spread related symptoms. Moreno- Lopez et al. showed that reduction of field potential of cranial nerve IV occurred 3 hours after toxin injection in the lateral rectus muscle of cats and that the effects were dose dependent [42].

Dose and toxin brand: The general belief in the medical community has been that generalized BoNT/A spread may occur only above certain toxin doses. However, we need to keep in mind that we do not have any studies on safety of hematogenic or retrograde axonal transport BoNT/toxicity in humans. Our data shows that spread can occur with a dose as low as 10 units (0.08 units/kg) signifying that likely no dose is safe when a generalized spread occurs. Matak et al. [43] demonstrated that 3.5U/kg of BoNT/A, injected in the whisker pad of rats, reached CNS in 72 hours and remained enzymatically active. It is important to note that BoNT/A organ toxicity dose, which is a dose that affects the function of the organs, is much lower then the lethal dose (LD50) (the one that kills 50% of the injected animals). Therefore LD50 cannot be used as a safety measure for organ toxicity and disease occurrence but can be used only to assess risk of death. Young at al. [44] reported generalized spread symptoms in patient case series after a toxin dose as low as 6.8 units. Switching a patient from one brand drug to another, as it occurred in patient #16, does not increase safety since most drug brands contain BoNT/A. As well, botulinum toxin types A and B carry cross reactivity, due to their similar molecular structure.

Examination and past medical history: Almost all patients, presented here, had a weakened immune background with presence of past personal or family history of cancer, immune, autonomic or allergic diseases. It is possible, that such patients’ population may be more prone to toxin reaction, but this is to be established in the future in larger population. A significant finding is that only two patients had pupillary reaction impairment (minimal one). This fact together with the usually normal muscle power examination indicates that the INCS differs in symptomatology from that of foodborne botulism. Less frequent pupillary involvement has been demonstrated in animals injected with the toxin [5].

Diagnosis and clinical markers: At present, the diagnosis is a real challenge since there is not a high alertness in the medical society about this significant problem, there are no guidelines for diagnosing iatrogenic botulinum side effects and there are no easily available methods to prove the toxin-symptoms connection. In foodborne botulism, the diagnosis is usually established by testing for clostridium bacteria and toxin in food and/or stool samples. The gold standard for testing is lethal mouse bioassay which detects the biologically active toxin in 1-4 days. Its sensitivity of detection is 20- 30 pg/ml [45]. ELISA (Enzyme Linked Immunosorbent Assay) uses polyclonal antibodies. The method is faster (8 hours) and its sensitivity is 0.2 ng/ml [45]. However, it usually uses amplification methods, which require culturing live bacteria (not present in iatrogenic cases) to increase and detect toxin production. The same applies to the live time PCR method which detects 5 pg/ml and uses monoclonal antibodies [45,46]. Since it is very likely that the serum toxin levels in INCS are much lower than that in foodborne botulism, where bacteria are producing toxin continuously, it is possible, that we may need more sensitive methods such as mass spectroscopy where not only the toxin, but as well parts of the toxin can be detected. It seems that detection of the entire toxin or the entire light chain in serum is preferable. There is a laboratory in Poland (Dr. Ella Kukier: ph: +48 818 89 31 93, fax: +48 81 889 33 54) which is conducting research on mass spectroscopy methods for detecting the toxin and its particles in human serum. The same laboratory offers lethal mouse bioassay to the public. In the USA, the mouse assay is not available to the public and requires approval of the state or the federal health authorities when the diagnosis of botulism is suspected. As the Center for Disease Control reported, in the USA, due to the nature of the toxin, even foodborne botulism has been substantially under-diagnosed or misdiagnosed with whole disease clusters and deaths related to the toxin not being detected [47].

Possible, future therapies

Activated charcoal: Charcoal has been studied to have no toxicity, even in high doses. In research, charcoal, when given trough the mouth, enhances the elimination of multiple intravenously administered drugs [48]. When BoNT/A was injected intraperitoneally in increasing concentrations in two groups of mice, none of the mice in the treated with activated charcoal group developed botulism or died, while all the mice in the untreated group developed botulism and died in progressive numbers with each increment of the toxin potency. The highest potency group, which was not exposed to charcoal, had 100% mortality [49]. Our observation with charcoal therapy is that almost all patients who took it had initial good effects and some alleviation of their symptoms. After that the group divided in two considering the effectiveness of the therapy: the first group kept getting better and improving, though at times mild crises may have occurred; the second group had more pronounced crises of temporary worsening and the patients stopped the therapy. However, there were two patients who pushed through the therapy despite the temporary worsening and had beneficial results at the end. One of them, as we mentioned above, achieved full recovery, despite that she had the worst clinical picture from all the cases, which is an encouraging finding. This unexpected split response prompted us to look deeper into the mechanism of the effect of activated charcoal upon the elimination of BoNT/A from the body. It looked like the crashes occurred more often in patients who took a higher dose of charcoal on a daily basis or if they had constipation. The pharmacokinetics in animals clearly suggest that the liver is about the only organ via which the toxin can be eliminated, since blood leaves the toxin unchanged and the BoNT/A molecule is too big to pass the 50 kDa size of molecule elimination-kidney threshold [39]. This has been supported by autopsies of animals who died from botulism where the highest accumulation of botulinum has been found in the liver and then in the spleen [50]. We speculated that if charcoal pools the toxin out of the tissue micro-compartment and blood into the bowel lumen, via liver elimination while bowel motility is slower than normal, a partial re-absorption of the toxin may occur. This can induce secondary BoNT/A re-introduction in the blood stream, which may present as worsening of the clinical picture. As well, it is possible that there is a liver threshold of accumulation of toxin before elimination. If the threshold is surpassed by pooling more toxin from the depots to the blood than the liver can handle at a given moment of time, the unfiltered amount of toxin may relodge into the nerve terminals. We found that 1 capsule of 280mg charcoal through the mouth taken every other day or twice a week was generally safe (no crises, or very mild ones) unless the patient had severe constipation. In our opinion, charcoal should not be used until the constipation is resolved. The fact that the two cases whose symptoms resolved 100% within the first 4 months of the initial insult after taking charcoal early into the disease course is encouraging too.

Research has shown that Echinacea plant has some similarity to botulinum toxin, and may compete with it [51]. Some of the reported here patients improved while taking 400mg of Echinacea daily.

Yuliang at al. [52] suggested that the Chinese herb toosendanin can inhibit the translocation of the light chain of the toxin. Zhou et al. [53] stated that toosendanin has been an effective cure of experimental botulism. The substance made synaptosomes of a rat brain completely resistant to BoNT/A. Even after toxin entrance into the cells, toosendanin was still able to partially antagonize BoNT/A substrate cleavage. The researchers concluded that the effect is likely due to blocking the toxin approach to the substrate. Mice, which developed botulism, cured completely in seven days after being treated with toosendanin. Since toosendanin has a low toxicity profile, further research and clinical trials are highly desirable.

3-4 diamino pyridine (3-4 DAP): Is a reversible inhibitor of voltage gated potassium channels and cholinergic agonist. Zakhari et al. [54] demonstrated that the 3-4 DAP facilitates recovery of post-botulinum intoxication by blocking the potassium channels but there were some toxic effects. A less toxic cousin of 3-4 DAP, 4-aminopyridine (dalfampridine) has been available on the market for treatment of multiple sclerosis but has never been used or studied in BoNT/A affected patients.

Young et al. [44] reported improvement of botulinum induced constipation with pyridostigmine.

Benadryl, which is an anti-histamine agent, has been beneficial to some of the cases presented here, likely due to effect of tuning down the discussed above hyper histaminic state.

Boroff at al. [36] showed in animals that BoNT/A activity can be blocked by reserpine.

Tryptophan is critical for toxin activity [36], rising the question if a tryptophan restricted diet may be beneficial for some patients.

Antibodies to the toxin: (anti-toxin). Frequently asked question by patients and physicians is if anti-toxin should be given? The present practice guidelines allow the toxin to be given only in the first 72 hours after exposure, since once BoNT/A reaches the nerve terminals the anti-toxin becomes ineffective. However, the possibility of longer lasting local toxin micro-depots presence has not been explored yet.

Suggested algorithm for safer toxin use:

• Inform yourself, your colleagues and your patients about the existence of the INCS and its consequences.
• Advise the patients who develop a reaction that it is likely not safe to continue injecting.
• If possible, restrain from vaccinating during the period of symptoms activity. Be aware that tetanus toxoid carries 35% amino-acid similarity to botulinum [55].
• Avoid giving serotonin re-uptake inhibitors and other mediator modulating drugs or steroids since worsening may occur.
• Advise not to push trough fatigue, do massages or use rigorous detoxifying methods, since crises may occur.
• Consider skin biopsy to diagnose small fiber polyneuropathy if persistent pain and tingling are present.
• Consider that injecting botulinum for cosmetic reasons, most likely, does not weight against the side effect profile of the drug.
• Report all reactions to the toxin to The Food and Drug Administration or its equivalents in other countries and publish the results in peer review literature to increase awareness.
• Try to send serum for lethal mouse bioassay when possible at early or later stages of the disease to establish the assay sensitivity in such cases.
• Know the difference between INCS and botulinum induced autoimmune encephalitis and polyneuropathy since there is a difference in the therapy.
• We suggest establishment of a separate reporting and monitoring system for botulinum related side effects to increase safety.

Conclusion

We conclude that Impaired Neuronal Communication Syndrome (INCS) is a serious and long lasting complication to BoNT/A therapy. The syndrome has specific features which distinguish it from the foodborne botulism by the rare involvement of the pupillary reaction and the rare presence of true weakness. The syndrome, as well, differs from the autoimmune side effects to BoNT/A described in the literature, such as autoimmune encephalitis, Guillain- Barre syndrome and others, by having, most of the time normal examination, laboratory results and imaging. There is no current wide-spread awareness of the syndrome among the medical society. There are no readily available markers to establish connection to the toxin making the clinical knowledge highly important and often the only tool for correct diagnosis. Further research is urgently needed.

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