Flow cytofluorimetric analysis of anti-LRP4 (LDL receptor-related protein 4) autoantibodies in Italian patients with Myasthenia gravis

Background: Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoanti-bodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4(LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG. Methods: The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system. Results: We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals. Conclusion: Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis

Magnetic resonance imaging contribution for diagnosing symptomatic neurovascular contact in classical trigeminal neuralgia: A blinded case-control study and meta-analysis

Although classical trigeminal neuralgia (CTN) is frequently caused by neurovascular contact (NVC) at the trigeminal root entry zone (REZ), both anatomical and MRI studies have shown that NVC of the trigeminal nerve frequently occurs in individuals without CTN. To assess the accuracy of MRI in distinguishing symptomatic from asymptomatic trigeminal NVC, we submitted to high-definition MRI the series of CTN patients referred to our outpatient service between June 2011 and January 2013 (n = 24), and a similar number of age-matched healthy controls. Two neuroradiologists, blinded to the clinical data, evaluated whether the trigeminal nerve displayed NVC in the REZ or non-REZ, whether it was dislocated by the vessel or displayed atrophy at the contact site, and whether the offending vessel was an artery or a vein. Our data were meta-analyzed with those of all similar studies published from January 1970 to June 2013. In our sample, REZ contact, nerve dislocation and nerve atrophy were independently associated with CTN (P =.027; P =.005; P =.035 respectively). Compared to a rather low sensitivity of each of these items (alone or in combination), their specificity was high. When REZ contact and nerve atrophy coexisted, both specificity and positive predictive value rose to 100%. Meta-analysis showed that REZ NVC was detected in 76% of symptomatic and 17% of asymptomatic nerves (P <.0001), whereas anatomical changes were detected in 52% of symptomatic and 9% of asymptomatic nerves (P <.0001). In conclusion, trigeminal REZ NVC, as detected by MRI, is highly likely to be symptomatic when it is associated with anatomical nerve changes. © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved

Anti-LRP4 detection by immunoprecipitation of pools of sera.

<p>Supernatants from LRP4ecto HEK293T cells were immune-precipitated with the indicated pools (p) of sera that scored positive at FACS analysis: pool#1 and #2 from dSN-MG, Musk-MG pool and AChR-MG pool. Immuno-complexes were subdued to western blotting and probed with anti-c-Myc. Aliquots of total supernatants from EGFP-HEK293T and from LRP4ecto HEK293T cells were blotted alongside with immune-precipitates as negative and positive control, respectively. The specific, uppermost band is pointed by the arrow.</p

Anti-LRP4 detection by FACS analysis.

<p>Each of 101 MG s and 85 control sera was tested on both parental untransfected (shaded area) and on LRP4fl-HEK293T transfected cells (full line). None of the 45 NHS but only 14 MG sera showed a clear cut shift of mean fluorescence value with a ratio transfected/untransfected > 1.5. We show the immunoreactivity of one NHS, one dSN-MG (sample#8), one AChR-MG (sample#9) and one MuSK-MG serum (sample#14) (A,B,C,D respectively) as representative plots.</p

Anti-LRP4 immunoreactivity in 101 Italian myasthenic patients (MG) and controls.

<p>*:CI: Confidence Interval</p><p>Anti-LRP4 immunoreactivity in 101 Italian myasthenic patients (MG) and controls.</p

Expression of recombinant LRP4.

<p>(A) Flow cytofluorimetric analysis of parental untransfected HEK293T cells labeled with the rabbit anti-LRP4 antiserum (shaded area), compared to LRP4fl-HEK293T transfected cells (full line). (B) Precipitated supernatants from EGFP-HEK293T LRP4ecto-HEK293T cells were analyzed by anti-c-Myc immunoblotting; the band corresponding to the ecto-LRP4-myc tag fusion protein is indicated by arrow.</p