José Boucraut

TWEAK stimulation of astrocytes and the proinflammatory consequences

Astrocytes exert many active roles in brain homeostasis, potentially including the regulation of immune reactions. They possess a substantial aptitude for plasticity and, indeed, functional and phenotypic changes are frequently encountered in reactive gliosis observed in brain injuries. The significance of reactive astrocytes is still poorly defined, but it is clear that these cells are an important source of cytokines in inflamed brain. How tumor necrosis factor (TNF) and TNF‐receptor family members contribute to this reaction is an interesting issue that is currently being explored. It was previously shown that reactive astrocytes express high levels of Fas (CD95) and respond to Fas ligand (CD95L) by apoptosis or IL‐8 production. TWEAK (Apo‐3 ligand) is a recently identified member of the TNF family that is produced mainly by leukocytes that can infiltrate the inflamed brain and thus influence astrocyte behavior. Here we show that human astrocytes derived from different regions of the brain specifically bind TWEAK and are totally resistant to TWEAK mediated apoptosis. In addition, high amounts of IL‐8 and IL‐6 were secreted by astrocytes after TWEAK exposure. Finally, expression of cell surface molecules involved in the propagation and/or maintenance of brain inflammation was determined. TWEAK significantly increased ICAM‐1 expression on astrocytes, whereas no modification was detected in the expression of Fas, TNFRI, B7‐1, or MHC molecules. In conclusion, the proinflammatory effects induced by TWEAK on astrocytes in culture recapitulate many characteristics of reactive astrocytes observed in vivo, suggesting that TWEAK could play a significant role in brain inflammation. GLIA 32:102–107, 2000.

TWEAK is expressed by glial cells, induces astrocyte proliferation and increases EAE severity

TWEAK is a new TNF family member with proinflammatory and proliferative effects on different cell types, mediated by the recently identified Fn14 receptor. TWEAK expression was analyzed on mouse microglial cells and astrocytes. Both cell types express TWEAK mRNA. Astrocytes expressed Fn14 and proliferated in the presence of rTWEAK. TWEAK mRNA is expressed in normal CNS and its steady state level increases in spinal cord during EAE. Finally, EAE severity is enhanced in soluble TWEAK-overexpressing transgenic mice. These results support the contention that TWEAK is involved in CNS inflammation.

Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease

Background: The envelope protein from multiple sclerosis (MS) associated retroviral element (MSRV), a member of the Human Endogenous Retroviral family ‘W’ (HERV-W), induces dysimmunity and inflammation. Objective: The objective of this study was to confirm and specify the association between HERV-W/MSRV envelope (Env) expression and MS. Methods: 103 MS, 199 healthy controls (HC) and controls with other neurological diseases (28), chronic infections (30) or autoimmunity (30) were analysed with an immunoassay detecting Env in serum. Env RNA or DNA copy numbers in peripheral blood mononuclear cells (PBMC) were determined by a quantitative polymerase chain reaction (PCR). Env was detected by immunohistology in the brains of patients with MS with three specific monoclonals. Results: Env antigen was detected in a serum of 73% of patients with MS with similar prevalence in all clinical forms, and not in chronic infection, systemic lupus, most other neurological diseases and healthy donors (p<0.01). Cases with chronic inflammatory demyelinating polyneuropathy (5/8) and rare HC (4/103) were positive. RNA expression in PBMC and DNA copy numbers were significantly elevated in patients with MS versus HC (p<0.001). In patients with MS, DNA copy numbers were significantly increased in chronic progressive MS (secondary progressive MS vs relapsing–remitting MS (RRMS) p<0.001; primary progressive MS vs RRMS –<0.02). Env protein was evidenced in macrophages within MS brain lesions with particular concentrations around vascular elements. Conclusion: The association between MS disease and the MSRV-type HERV-W element now appears quite strong, as evidenced ex-vivo from serum and PBMC with post-mortem confirmation in brain lesions. Chronic progressive MS, RRMS and clinically isolated syndrome show different ELISA (Enzyme-Linked Immunosorbent Assay) and/or PCR profiles suggestive of an increase with disease evolution, and amplicon sequencing confirms the association with particular HERV-W elements.

Long term follow up of multifocal motor neuropathy with conduction block under treatment.

Eighteen patients (15 men, three women; age range 30 to 71 years, mean 45.8 years) with multifocal motor neuropathy treated with high dose intravenous immunoglobulin (IVIg) were evaluated for nine to 48 months (mean follow up 25.3 months). The median time between onset of multifocal motor neuropathy and treatment was 5.8 years. The dose of IVIg was 0.4 g/day for three to five days. The interval between each treatment was determined for each patient by the evaluation of the effect of the first course. Muscle strength was evaluated by a computerised analyser. Clinical improvement was seen in 12 patients treated with IVIg (67%). Isometric strength increased from 32% to 97% (mean 54.5%) of the initial value. Functional scales corroborated these findings. No clear predictive factors of response to IVIg was found except the presence of high titres of IgM anti-GM1 antibodies. Often, patients needed repeated courses of IVIg to maintain the improvement. In two patients, IVIg infusions were stopped without signs of relapse after one year. Four patients were initially treated with prednisone (1 mg/kg/day), without any clear improvement. Five patients with no response to IVIg or who were IVIg dependent were treated with cyclophosphamide, but only one showed improvement. These results show the long term benefits and safety of IVIg in multifocal motor neuropathy but also the transient effect of this expensive treatment in most patients.

Lewis-Sumner syndrome and multifocal motor neuropathy.

We compared the clinical, electrophysiological, laboratory, and pathological features of 13 patients with Lewis–Sumner syndrome (LSS) with those of 20 patients with multifocal motor neuropathy (MMN). LSS and MMN patients have several common clinical features: age at onset, weakness in the distribution of individual peripheral nerves, mild wasting, cramps and fasciculations, partial areflexia, and frequent stepwise disease course. Cerebrospinal fluid protein level was normal or slightly elevated, but always less than 100 mg/dl. Conduction blocks are the electrophysiological hallmarks of these two neuropathies, and no differences in distribution and number of blocks were found. Contrary to MMN, lower‐limb involvement at onset was frequent in LSS but extension to the upper limbs was a frequent later feature of the disease. Cranial nerve involvement was noted in 4 LSS patients during relapses and absent in all MMN patients. The major distinguishing features were the clinical and electrophysiological sensory involvement in LSS, and the lack of anti‐GM1 antibodies in LSS, whereas IgM anti‐GM1 were found in 40% of MMN patients. Some LSS patients responded to steroid therapy, whereas this was ineffective in MMN. From these features, LSS can be considered an entity distinct from MMN, with its own clinical, laboratory, and electrophysiological characteristics, and as an intermediate link between chronic inflammatory demyelinating polyneuropathy and MMN. Muscle Nerve, 2005

Lithostathine and pancreatitis-associated protein are involved in the very early stages of Alzheimer's disease

According to one of the theories formulated to explain the etiology of Alzheimers disease (AD), amylosis may reflect a specific inflammatory response. Two inflammatory proteins, lithostathine and PAP, were evidenced by immunohistochemistry in senile plaques and neurofibrillary tangles of patients with AD. In addition, lithostathine and PAP were significantly increased in the cerebrospinal fluid of patients with AD when compared to patients with multiple sclerosis, another inflammatory disease, and to normal control subjects. However, no correlation was observed with age of occurrence. Furthermore, lithostathine and PAP were increased even at the very early stages of AD, and their level remained elevated during the course of the AD unlike TNFalpha whose level, very high at very early stages, regularly decreased. Finally, if part of lithostathine and PAP are synthesized in the brain, a large part comes from serum by passage over the blood-brain barrier. These results indicate (i) the existence of an acute phase response followed by a chronic inflammation in AD, and (ii) that lithostathine and PAP are involved even at the first pre-clinical biochemical events of AD. In addition, because lithostathine undergoes an autolytic cleavage leading to its precipitation and the formation of fibrils, we believe that it may be involved in amyloidosis and tangles by allowing heterogeneous precipitation of other proteins.

Cryptogenic Partial Epilepsies with Anti-GM1 Antibodies: A New Form of Immune-Mediated Epilepsy?

Summary: Purpose: We wished to study immune system dysfunction which has been proposed as a potential cause of epilepsy. Epileptogenic action of antibodies directed against GM1 gangliosides was demonstrated in rats, but the potential role of anti‐GMI antibodies in human epilepsy has not yet been studied.

Chronic ataxic neuropathies associated with anti-GD1b IgM antibodies: response to IVIg therapy

Objective: To determine the responses to treatment of patients with chronic sensory ataxic neuropathy associated with anti-GD1b IgM antibodies. Methods: Patients with chronic sensory ataxic neuropathy associated with anti-GD1b IgM antibodies followed in our department for at least 12 months between 2001 and 2008 were identified and studied retrospectively. Patients were tested at regular intervals using the INCAT disability score. Patients whose disability scores improved by at least one point were taken to have responded to the treatment. Intravenous immunoglobulin (IVIg; 2 g/kg) was administered for 3 to 5 days once every 6 weeks or corticosteroids at an initial daily dose of 1 mg/kg. Results: 13 patients treated during the 8-year period of interest were included in this study. Seven of 13 patients displayed IgM anti-GQ1b, GT1b and GD3 antibodies suggesting reactivity against disialosyl epitope. IgM gammopathy was detected in four of six of serum with anti-disialosyl antibodies and two of the seven other sera. Nine of the 13 patients improved in response to IVIg. Oral corticosteroid treatment was attempted on four patients prior to IVIg treatment, and partial recovery occurred in one, who became steroid-dependent and showed little benefit in the long term. Conclusions: Screening for anti-GD1b IgM antibodies should be carried out on all patients with chronic ataxic sensory neuropathies. In 69% of the cases studied, the patients’ condition improved in response to IVIg. This study shows the short-term efficiency of this treatment. Sustained responses were obtained in the long term by continuing the infusions.

A protein chip membrane-capture assay for botulinum neurotoxin activity

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC(50)s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC(50) of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

Quantification of Immunoglobulin Free Light Chains in CerebroSpinal Fluid by Nephelometry

Oligoclonal free light chains (FLC) banding has been described in multiple sclerosis (MS) and should be correlated with disease activity. However, discrepancies between studies have been reported because of differences in methods. A new quantitative, rapid, and automated method using nephelometry is now available. Our objective was to investigate the interest of this method for the diagnosis and prognosis of MS. For this purpose, FLC index was determined in paired samples of CSF and serum from consecutive and unselected patients from the same department of neurology. We enrolled 89 patients (33 MS, 15 “possible MS”, and 41 controls) and correlated with IgG index, IgG oligoclonal banding, and clinical MS progression criteria. The main results were (1) FLC kappa index was more sensitive but less specific than IgG index for the diagnosis of MS, (2) two MS patients were negative for oligoclonal banding but exhibited a positive kappa index, (3) no relation between FLC kappa indices, MS clinical criteria, and disease progression was found. In conclusion, FLC kappa index should be considered as a useful complementary test for MS diagnosis. Its pronostic interest remains to be determined on a larger cohort of possible MS patients.