Parkinsonism caused by Lipopolysaccharides
of Salmonella Minnesota (a Case Report)

Ines Niehaus

(contact: Ines_Niehaus@gmx.de)

Poster presentation at the XIV International Congress on Parkinson's Disease

Helsinki, Finland, 27 July - 1 August 2001

Abstract

A laboratory worker aged 22 was accidentally contaminated with 10 microgram Salmonella minnesota lipopolysaccharides (LPS) through an open wound on the left thumb. A strong sepsis-like inflammatory reaction to LPS started with fever, flu-like-symptoms, cough with dyspnoea and vomiting. Three weeks after the contamination she suffered from generalized cramps, disturbances of sensibility and symptoms of parkinsonism (bradykinesia, rigidity, tremor, stiffness). These symptoms were increasing over the years due to the chronic inflammation of the central nervous system caused by LPS (6600 pg LPS / ml cerebrospinal fluid measured 6 years after the contamination with LPS). The progression of the degeneration of dopaminergic neurons was being tried to reduce with a dose of 3 mg of the pineal hormone melatonin. The natural sleeping hormone melatonin is able to protect cells including neurons against oxidative damage. Positron emission tomographies showed damages to the cerebral cortex and dopaminergic striatal system. LPS of E. coli or Salmonella minnesota injected in the substantia nigra or striatum of rats induces inflammation-mediated degeneration of the nigral and striatal dopaminergic system. The first years of illness, the symptoms of parkinsonism were only treated with the amino acid DL-phenylalanine (450 mg daily). This racemate of the amino acid phenylalanine is able to restore the balance between the phenethylamine-dopamine and the acetylcholine-serotonine-tryptamine side. Later 50 mg dehydroepiandrosterone (DHEA) was added every other morning. In 2001 a treatment with low doses of L-Dopa (3 x 75 mg) and amantadine (2 x 100 mg) was started. Dopamine agonists were not tolerated in generally but a treatment with low doses of pramipexole started in 2007 was sucessfully.

More information on DHEA and parkinsonism in this case report!

Introduction

Parkinson's disease (PD) is a specific degeneration of dopaminergic neurons in the midbrain. ¹ Many cases of PD are accompanied by general brain inflammation with a dramatic proliferation of reactive amoeboid macrophages and microglia in the substantia nigra (SN).¹ At the same time in the SN of PD patients it has been described an increased density of glial cells expressing different pro-inflammatory cytokines as TNF-alpha, IL-6, and IFN-y.¹ Microglia are considered to be the resident immune cells in the brain and are the most responsive to immunological challenges.² SN has the highest concentration of microglia in the brain.¹ Microglial activation involves a dramatic morphological change from a ramified resting form to fully activated amoeboid appearance.² Intranigral injections of LPS into rat SN result in a rapid activation of microglia followed by the loss of tyrosine hydroxylase (TH)-immunoreactive neurons.² A single injection of LPS produces a strong inflammatory response in SN which leads to permanent damage of the dopaminergic neurons without affecting other types of neurons.¹ This special sensitivity of dopaminergic neurons to the inflammatory response produced by intranigral injection of LPS suggests that inflammation cou ld be involved in some degenerative processes of the nigral dopaminergic system, i.e., PD.¹

Bacterial endotoxins (lipopolysaccharides) are part of the outer cell wall of Gram negative bacteria.³ Lipopolysaccharides (LPS) elicit multiple acute pathophysiological effects such as fever, lethality, Shwartzman reactivitiy, macrophage and B-lymphocyte activation, and other activities by animals and humans.^{4, 5} LPS are highly bioharzardous in very low doses. Small doses of LPS (100 ng of E. coli LPS) injected intravenously induce sepsislike symptoms and activate important inflammatory mediators whereas large doses of LPS precipitate life-threatening circulatory collapse and multiple organ failure.⁶ Encephalopathy and critical illness polyneuropathy (CIP) occur in 70% of septic patients.⁷ LPS increases the permeability of the BBB.⁸

Melatonin is a hormone produced by the pineal gland and exerts a potent antioxidant action on the nigrostriatal dopaminergic system. Melatonin may be of potential use in treating neurological disorders associated with oxidative stress (e. g. PD). ¹³

The activity of D-phenylalanine, in contrast to its L-isomer, may be explained by the fact that D-phenylalanine will not follow the catecholamine pathway not admitting the action of hydroxylase specific for the L-form. In this way only the ß-phenethylamine concentration would be increased in the brain structures related to extrapyramidal motility.¹¹

DHEA, in its sulphated form DHEA-S, which is generally metabolically inactive, is the steroid precursor of estrogens circulating in the human bloodstream. As cells take DHEA-S from the blood, they reconvert it into DHEA. DHEA is produced in the adrenal cortex.¹⁶ High IL-6 levels are implicated as a causal factor in PD. Decreased DHEA serum concentrations during aging or inflammatory diseases will be paralleled by a significant increase in IL-6 production.¹⁷ Furthermore DHEA is able to protect mice from endotoxin shock.

Aims

This is the first case describing toxic parkinsonism in consequence of lipopolysaccharides in the CNS and an effective treatment with the amino acid DL-phenylalanine and the hormones melatonin and DHEA.

Case Report

The author Ines Niehaus reports about her own case report:

In March 1995 during my education as a biologically technical assistant at the age of 22 the left thumb of my left hand was injured. 1 ml solution of RPMI-medium with 10 microgram Salmonella minnesota S-LPS leaked out of a tube over the wound. One hour later I showed first signs of illness typically for a contamination with LPS (fever, flu-like-symptoms, dyspnoea, headache, nausea, vomiting). These symptoms continued the next three weeks with different intensity. Three weeks after my accident I got neurological problems (convulsions, sensory disturbances, myalgia, tremor, stiffness, rigidity, difficulties in learning and seeing etc.). Three weeks after my accident I went to hospital. The physicians thinking I was suffering from an influenca did not treat this sepsis-like condition in spite of high temperature (39°C-40°C), generalized cramps, vomiting etc. because no viral or bacterial infection was found. One week later I was discharged afebrile.
Six weeks after my accident I was diagnosed with PD. For a few days I was treated with L-dopa, which was very helpful. Then I was misdiagnosed as suffering from restless legs syndrome, stiff-man-syndrome and essential tremor.
The LPS which has disrupted the blood-brain barriere (BBB) causes a chronic inflammation of the CNS which was getting acute in spring-time. The symptoms of parkinsonism turned to the worse in acute phases of inflammation, i. e., the left arm swing during walking has been missing since spring-time 1998. Furthermore I had dystonic cramps caused by the lack of dopamine, myalgia in the legs and back, headaches caused by the rigidity of the neck, coordination disturbances, hyperaesthesia and hyperreflexia (every noise, vibration or touch led to muscle cloni).
In March 1997 I began to treat myself with 450 mg pure phenylalanine once a day. In summer 1998 I started to treat myself with 1 mg melatonin in the evening, half a year later with 3 mg. The deterioration of the parkinsonism have been reduced by the melatonin I hope although I had further acute inflammations of the CNS. In March 2001 I began to treat myself with 50 mg dehydroepiandrosterone (DHEA) every other morning. It is very helpful especially against the stiffness and rigidity and I can move my left arm when I am walking.
In July 1998 great damages of my cerebral cortex were shown by positron emission tomography with [Fluorine-18] fluoro-2-deoxy-D-glucose. In February 2001 damages of the dopaminergic system was shown in the dopaminergic strial system by PET with fluorodopa.
In May 2001 parkinsonism was diagnosed in a clinic specialized for PD. A treatment was started with drugs for PD. I began to take 100 mg amantadine two times a day and 75 mg L-Dopa 3- to 4 times a day. I could not tolerate any kind of dopamine agonists but fortunately DHEA seems to be a drug with a long-lasting dopamine stimulating effect.

Neurological state in May 2001
Light hypomimia, language inconspicuous, walking alone possible, no difficulties in starting, length of stride light shortened, dragging of the left leg. No movement of the left arm during walking. Rigidity in the extremities and especially in the neck, cogwheel phenomenon in the elbow-joints and wrists. No spasticity, no contractures. Resting tremor on the left side, no postural or intention tremor. Coordination experiments sure. Diadochokinesia especially on the left side. No hyperkinesias, no dystonias. Cerebral nerves intact. Muscle reflexes on both sides middle lively. No pyramidal signs. Power of muscles normal. No trophic disturbances of the muscles. No sensory disturbances. Fine motor skills restricted, general retardation of moevements. Specimen of handwriting: light artefacts of tremor, tendency of micrography. EEG inconspicuous.

Materials and Methods

1) PET with FDG

Cerebral glucose metabolism was determined with [Fluorine-18] fluoro-2-deoxy-D-glucose (FDG) using positron emission tomography. The human brain depends on glucose as its only energy source. The majority of glucos e is used for maintaining the membrane potentials and restoring ion gradients. When clinically measuring glucose metabolism, the subject lies still for roughly 40 minutes after having FDG intravenously injected into their bloodstream. During this 40-minut e time frame, the FDG is taken up by cells and will reach equilibrium. The distribution of the Fluorine-18 is then measured in multiple tomographic sections, and this distribution will be based on cerebral glucose utilization under the specific pathological or mental condition of the brain.¹⁰

2) LAL test

Preparation of plasma for the limulus assay has been described in. CSF for the limulus assay was collected into LPS-free tubes without centrifugation and stored at -70°C.⁹ The chromogenic limulus lysate assay was used: Endotoxin induces coagulation of the lysate of hemolymph of Limulus polyphemus (horseshoe crab). Endotoxin induces gel-clot formation in the Limulus test. A synthetic chromogenic compound as the substrate for clotting enzyme is added to the lysate derived from L. polyphemus. Paranitroaniline (pNA) released from the substrate by the enzyme is then diazocoupled to make red azodye. Endotoxin content can be measured photometrically (545nm).¹²

Figure 1.Limulus test

3) PET with F-Dopa

18F-dopa PET provides a means of monitoring loss of dopaminergic function in PD objectively and is free from the confounding effects of symptomatic therapy.¹⁴ The loss of striatal dopaminergic function allows 18F-dopa PET to discriminate 100% of patients diagnosed as having PD from healthy subjects using clinial criteria. PD patients show, on average, a 50% loss of specific putamen 18F-dopa uptake. As putamen dopamine levels are reduced by over 90% in end-stage PD. A loss of 30% of nigral dopaminergic cells may be the threshold for the onset of symptoms.¹⁴ It was shown in patients with PD that at various stages of the disease the presynaptic uptake of 18F-dopa in the striatum is significantly diminished.¹⁵

Figure 2. positron emission tomograph: http://www.alra.de

Results Positron emission tomography patient: Ines Niehaus born: 17.08.1972 date of examination: 10.07.1998

Table 1. reduction of glucose utilization in x% of the maximum activity of the cerebral cortex (100%)

 n.c.: no comment

Normally there are 100% activity in all regions of the cerebral cortex.

Figure 3. Positron emission tomogram

annotation: red colour: normal; ochre-green: little damages; blue: moderate damages; black: strong damages

Computer tomography (CT) and nuclear spin tomography (NMR) of the brain are O.K.

2) LAL test

On 03.04.2000 the result of a LAL test of the CSF is 6600 pg LPS / ml. The result of a LAL test of the serum is negative.

3) Positron emission tomogram with F-Dopa

Substantial reduction of the decarboxylase activity in the nucl. caudatii without difference of sides. Moderate reduction of the functionality in the putamen on both sides. These results have to be interpreted as a disturbance of the dopaminergic system.

The semiquantitative analysis showed following results under calculation of the quotients:
Nucl. caud. right / Occ: 1,52
Nucl. Caud. left / Occ: 1,54
Putamen right / Occ: 1,78
Putamen left / Occ: 1,71

Discussion

The dose of 10 µg Salmonella minnesota S-LPS is sufficient to cause some of the first symptoms (fever, flu-like-symptoms, dyspnoea, headache, nausea, vomiting). Robert Burell reports similar symptoms for doses of about 3-4 ng/kg which were administered IV to healthy volunteers.⁵ Many humans are exposed to 10 µg LPS or higher doses during sepsis and septic shock. Encephalopathy and CIP occur in 70% of septic patients.⁷

The LPS has damaged and disrupted the BBB and is still in the CNS (see the LAL test). There it caused the damages of the cerebral cortex (see the PET) and of the dopaminergic neurons in the substantia nigra.

Figure 4. Chemical structure of a Salmonella LPS³

Abbreviations: Glc: D-glucose; Gal: D-galactose; GlcNAc: N-acetyl-D-glucosamine; Hep: heptose; Kdo: 2-keto-3-deoxyoctonic acid; Abe: Abequose; Man: D-Mannose; Rha: rhamnose; n: the number of repeating units may vary from 0 to approximately 50.

In the brain the LPS stuck to the neurons and anti-LPS antibodies hence activated complement etc. eliciting a chronic inflammation of the CNS because the nervous system was unable to destroy the LPS sticking on the neurons. Perhaps the LPS was even inserted into the lipid layer of the cellular membrane of the neurons. The reactions of the immune system against the LPS were getting stronger and stronger in the course of time, which led to a continuous worsening of the complaints due to the increasing brain damages.

Some cases of PD are associated to head trauma and encephalitis, suggesting an inflammatory component of this disease. Inflammatory stimulus, such as LPS, has diverse effects on the cells of the CNS. Thus, astrocytes and microglia have been shown to secrete cytokines.¹

The neuronal damage process induced by LPS is specific for dopaminergic neurons and it is triggered in the neuronal body in the SN.¹ LPS induced significant activation of microglia and microglial activation preceded the apparent neuronal degeneration.² Dopamine neurons in the SN appeared to be more vulnerable to injury than those in other brain areas perhaps due to a reduced antioxidant capacity.²

The symptoms of parkinsonism are treated with the racemate DL-phenylalanine. L-phenylalanine increases the concentration of dopamine in the brain and D-phenylalanine the concentration of ß-phenylethylamine so that this treatment restores the balance between the phenethylamine-dopamine and the acetylcholine-serotonine-tryptamine side. DL-phenylalanine has no side effects like L-dopa and it is not toxic for the dopaminergic neurons.

Figure 5.Synthesis of dopamine and ß-phenylethylamine

D-phenylalanine showed a therapeutic activity in PD, especially against rigiditiy, walking disability, speech difficulties and psychic depression, whereas its effect on tremor was not important. A combination of the amino acid with anticholinergic agents should be tried.¹¹

The neuroprotective effects of melatonin appear to be mediated by the antioxidant capacity of this pineal hormone. In this case melatonin prevents the dopaminergic neurons against the oxidative damage as it has been shown in several models of PD.

DHEA is effective against the stiffness and rigidity for two days. Dopamine release from primary hypothalamic cell cultures was stimulated by DHEA-S.¹⁸ Estrogens provide neuroprotection of CNS cells implicated in the etiology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Drugs with estrogen activity in the brain may have therapeutic potential either by modulating brain neurotransmitter transmission or through neuroprotective activitiy.¹⁹DHEA increases the synthesis of estrogen, which stimulates the production of dopamine.

Figure 6.Structure of melatonin and DHEA

Conclusions

LPS intranigrally injected into rats induces inflammation-mediated degeneration of the substantia nigra dopaminergic system. This case report shows that the animal model is transferable to humans.

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This page was published on 06-01-2001.

Last actualized: 10-23-2008.

Send an e-mail to Ines_Niehaus@gmx.de for questions or comments.