The importance of axonal damage in multiple sclerosis (MS) has been recently stressed in proton magnetic resonance spectroscopy and pathological studies, but the exact mechanism producing this damage ...is unknown. The aim of our study was to ascertain whether soluble mediators present in the cerebrospinal fluid (CSF) of patients with relapsing-remitting MS could induce neuron injury in culture. Different biochemical and cytochemical parameters were determined in primary embryonal rat neuron cultures following 8 days of exposure to CSF. Cytotoxic activity was evaluated with a blue formazan production colorimetric assay. Morphological and immunocytochemical studies performed with antibodies against β-tubulin revealed neuritic fragmentation, axonal damage and cellular shrinkage indicating apoptosis. Detection of apoptosis was carried out using the fluorescent DNA-binding dye Hoechst 33342, as well as by a Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay. We observed that soluble factors in CSF from patients with “aggressive” MS i.e, those with poor recovery after relapses, induced neurite breakdown and neuronal apoptosis in cultures. Neuron injury is not related with blood-brain barrier dysfunction nor with IgG index. Interestingly, CSF from patients with “non-aggressive” MS i.e., relapsing-remitting patients with a good recovery after relapses, did not induce any damage. In conclusion, we report that CSF from patients with aggressive MS bears soluble mediators that induce axonal damage and apoptosis of neurons in culture. These mediators can be present during the first attack of the disease, and the neuronal damage caused could be related to the functional deficit of these MS patients.
Neuronal apoptosis has recently been implicated in multiple sclerosis (MS). Apoptotic cell death of neurons is induced in cultures exposed to cerebrospinal fluid (CSF) from MS patients. Since ...caspases are essential in the regulation of apoptosis, direct evidence was sought linking caspases to CSF-induced neuronal death. Caspase activity was measured in cell extracts from MS CSF-treated cultured neurons by the cleavage of caspase-1 and caspase-3 substrates. Caspase-3 activity, but not caspase-1, was induced in neuronal cultures in response to MS CSF treatment. This caspase-3 activity was inhibited in vitro by Ac-YVAD-cmk and Ac-DEVD-cmk caspase inhibitors. Treatment of MS CSF-incubated neuronal cells with these caspase inhibitors completely preserved neuronal survival and largely attenuated DNA fragmentation detected in situ. These findings show that neuronal cells are rescued from MS CSF-induced death by caspase inhibitors and suggest ways to treat MS.
IL-15 is a proinflammatory cytokine which has recently been implicated in multiple sclerosis (MS) pathogenesis, where it may play a role in the initiation and/or progression of the disease. We have ...used reverse transcriptase-polymerase chain reaction (RT-PCR) to study IL-15 mRNA levels in peripheral blood mononuclear cells (PBMC) from healthy controls and relapsing–remitting MS (RRMS) patients in a stable phase of the disease and during a bout, both before and after corticosteroid treatment (CST). IL-15 mRNA expression was found to be similar in controls and stable patients. We have detected an increased level of IL-15 mRNA in PBMC of patients with a relapse, which was maintained after CST. We have also found an inverse correlation between PBMC IL-15 mRNA levels at the onset of the relapse and the time elapsed since the previous attack, as well as an absence of correlation between IL-15 mRNA levels and the patient demographic and clinical characteristics. Results in the present work further suggest a role for IL-15 in MS pathophysiology.
Clinical laboratory tests proving normal included an echocardiogram, chest x ray, repetitive nerve stimulation and single fibre electromyography, erythrocyte sedimentation rate, full blood count, ...serum electrolytes, fasting lipid profile, angiotensin converting enzyme, protein S, protein C, lupus anticoagulant, anticardiolipin antibodies (IgG and IgM), plasma homocysteine levels, C3 and C4 complement, anti-ENA (anti-Sm, SS-A/anti-Ro, SS-B/anti-La, and anti-RNP), antinuclear antibodies, antiacetylcholine antibodies, serum syphilis serology, and cerebrospinal fluid findings. The clinical features of bilateral medial medullary infarctions are flaccid quadriplegia sparing the face, bilateral disturbance of deep sensation, weakness of the tongue, and respiratory failure. 3, 4 The case here reported broadens the spectrum of the medial medullary syndrome.
To determine the frequency and the linkage distribution of seven mutations at the polymorphic gene coding for the arylamine N-acetyl transferase (NAT2; EC 2.3.1.5) in 121 unrelated patients with ...sporadic PD and in 121 unrelated healthy volunteers.
The study was performed with mutation-specific PCR using genomic DNA obtained from blood of the probands.
Comparison of the NAT2 genotypes of the overall PD patients and control subjects did not indicate statistically significant differences. However, patients with early-onset PD (onset before the age of 50 years, n=37) showed a higher frequency of slow-acetylation genotypes (78.4% patients) compared with both healthy control subjects (55.4%) and with late-onset (onset after 51 years of age, n=84) PD patients (54.8%). Such a difference was statistically significant (p < 0.015) and was the result of a homogeneous increase in the frequency of slow-acetylation alleles. All subgroups analyzed in the study were in Hardy-Weinberg equilibrium for mutations at the NAT2 gene.
Slow-acetylation-mutated alleles may be considered low-penetrance genes in early-onset PD pathogenesis, with a relative risk ratio for individuals with slow-acetylation genotype of 2.92 (95% CI, 1.26 to 6.78). This study provides evidence for the interaction of genetic and environmental factors in the etiology of sporadic PD.
We have studied the noxious effect of cerebrospinal fluids (CSF) from patients with primary-progressive multiple sclerosis (MS) on cultured neurons. Cells were exposed to CSF for 8 days and the ...possible neuronal damage was determined. Morphological studies with phase-contrast microscopy showed cellular shrinkage indicating apoptosis. CSF-induced apoptosis as evidenced by the fluorescent DNA-binding dye Hoechst 33342, as well as by the TUNEL-reaction, was only present in primary-progressive MS patients with a worsening disease. This neuron injury did not correlate with blood–brain barrier dysfunction nor with intrathecal IgG synthesis. On the contrary, CSF from either stable primary-progressive or other non-inflammatory neurological diseases, did not induce any culture damage. Undetectable or low similar tumor necrosis factor-alpha (TNF-
α) levels (range to 8.7 pg/ml) were found in the CSFs tested regardless they damage cultures or not. These results suggest that soluble factors, other than TNF-
α, molecules transudated from blood or IgG, present in the CSF of active primary-progressive patients with MS induce neuronal apoptosis.
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