Franz Blaes

Autoimmunity against the β2 adrenergic receptor and muscarinic-2 receptor in complex regional pain syndrome

Summary Complex regional pain syndrome patient sera contain functional active autoantibodies against the β2 adrenergic receptor and muscarinic‐2 receptor, supporting the concept of an autoimmune etiology in CRPS. ABSTRACT Complex regional pain syndrome (CRPS) is a painful condition affecting one or more extremities of the body, marked by a wide variety of symptoms and signs that are often difficult to manage because the pathophysiology is incompletely understood. Thus, diverse treatments might be ineffective. A recent report revealed the presence of autoantibodies against differentiated autonomic neurons in CRPS patients. However, it remained unclear how the antibodies act in the development of CRPS. We therefore aimed to characterize these antibodies and identify target antigens. Functional properties of affinity‐purified immunoglobulin G of control subjects or CRPS patients were assessed using a cardiomyocyte bioassay. Putative corresponding receptors were identified using antagonistic drugs, and synthesized peptide sequences corresponding to segments of these receptors were used to identify the target epitopes. Chinese hamster ovary cells were transfected with putative receptors to ensure observed binding. Further, changes in the intracellular Ca2+ concentration induced by agonistic immunoglobulin G were measured using the Ca2+‐sensitive fluorescent dye fura‐2 assay. Herein, we demonstrate the presence of autoantibodies in a subset of CRPS patients with agonistic‐like properties on the β2 adrenergic receptor and/or the muscarinic‐2 receptor. We identified these autoantibodies as immunoglobulin G directed against peptide sequences from the second extracellular loop of these receptors. The identification of functionally active autoantibodies in serum samples from CRPS patients supports an autoimmune pathogenesis of CRPS. Thus, our findings contribute to the further understanding of this disease, could help in the diagnosis in future, and encourage new treatment strategies focusing on the immune system.

Surface-binding autoantibodies to cerebellar neurons in opsoclonus syndrome

Childhood opsoclonus‐myoclonus syndrome can occur with or without associated neuroblastoma. An autoimmune pathogenesis has been discussed for both forms. We show here that the majority of children with opsoclonus‐myoclonus syndrome (10/14) have autoantibodies binding to the surface of isolated rat cerebellar granular neurons. In some patients, these antibodies are masked by IgG binding to ubiquitous surface antigens, which could be removed by preincubation with the nonneuronal control cell line HEK 293. A newly introduced competitive binding assay showed that the surface binding is directed against the same autoantigen in different patients. Therefore, we hypothesize that opsoclonus‐myoclonus syndrome may be the result of an autoimmune process against a neuronal surface protein. Ann Neurol 2005

Autoantibodies in complex regional pain syndrome bind to a differentiation-dependent neuronal surface autoantigen.

ABSTRACT Complex regional pain syndrome, which is characterised by pain and trophic disturbances, develops frequently after peripheral limb trauma. There is an increasing evidence of an involvement of the immune system in CRPS, and recently we showed that CRPS patients have autoantibodies against nervous system structures [6]. Therefore we tested the sera of CRPS patients, neuropathy patients and healthy volunteers for surface‐binding autoantibodies to primary cultures of autonomic neurons and differentiated neuroblastoma cell lines using flow cytometry. Thirteen of 30 CRPS patients, but none of 30 healthy controls and only one of the 20 neuropathy sera had specific surface binding to autonomic neurons (p < 0.001). The majority of the sera reacted with both sympathetic and myenteric plexus neurons. Interestingly, 6/30 CRPS sera showed binding to undifferentiated SH‐SY5Y neuroblastoma cells. However, differentiation of SH‐SY5Y into a cholinergic phenotype induced a surface antigen, which is recognised by 60% of CRPS sera (18/30), but not by controls (p < 0.001). Our data show that about 30–40% of CRPS patients have surface‐binding autoantibodies against an inducible autonomic nervous system autoantigen. These data support an autoimmune hypothesis in CRPS patients. Further studies must elucidate origin and function of these autoantibodies in CRPS.

IgG from "seronegative" myasthenia gravis patients binds to a muscle cell line, TE671, but not to human acetylcholine receptor.

Antibodies to acetylcholine receptor (AChR) are found in 85% of patients with myasthenia gravis (seropositive MG [SPMG]) and are thought to be pathogenic; but in 15% of MG patients, the standard immunoprecipitation test for anti‐AChR is negative (seronegative MG [SNMG]). Here, we used a novel approach, fluorescence‐activated cell sorting analysis, to measure binding of SPMG and SNMG IgG antibodies to rhabdomyosarcoma cell lines that express human adult (TE671‐ε) or fetal (TE671‐γ) AChR, and to human embryonic kidney (HEK) fibroblasts that express adult human AChR (HEK‐AChR). We found that whereas most SPMG antibodies bind to all three cell lines, IgG from 8 of 15 SNMG sera/plasmas bind to the surface of both TE671 cell lines but not to HEK‐AChR cells. These results indicate that SNMG antibodies bind to a muscle surface antigen that is not the AChR, which strongly supports previous studies that suggest that SNMG should be considered a distinct subtype of MG. Ann Neurol 2000;47:504–510.

Anemia as a risk factor for cerebral venous thrombosis? An old hypothesis revisited. Results of a prospective study.

BackgroundSeveral case reports have linked iron deficiency anemia with the occurrence of cerebral venous thrombosis (CVT) or stroke, yet, it is unclear whether this is a chance association.MethodsIn a case-control design data of whole blood count and screening for thrombophilic coagulation abnormalities of 121 prospectively identified patients with CVT and 120 healthy controls were compared. Anemia was defined as a hemoglobin (Hb) concentration of <120 g/l in females, and <130 g/l in males, severe anemia as a Hb <90 g/l. Adjusted odds ratios (OR) were calculated based on a logistic regression model treating variables with a level of significance of p ≤0.2 on univariate analysis as potential confounders.ResultsThrombophilia (OR 1.22, 95% CI 1.07-1.76, p < 0.01), severe anemia (OR 1.10, 95% CI 1.01-2.22, p < 0.05), and hypercholesterinemia (OR 1.21, 95% CI 1.04-2.57, p < 0.05) were the only independent variables associated with CVT on multivariate analysis.ConclusionSevere anemia is significantly and independently associated with CVT.

Antineural and antinuclear autoantibodies are of prognostic relevance in non-small cell lung cancer

BACKGROUND Autoantibodies against nervous system structures have been proven to be a prognostic factor in small cell lung cancer. However, little is known about humoral autoimmunity in non-small cell lung cancer (NSCLC) and its prognostic significance. METHODS We examined antineural antibodies (AnAb) and antinuclear antibodies (ANA) in the sera of 61 patients with NSCLC (histologically: 29 adenocarcinoma, 32 squamous cell carcinoma). Twenty-one patients had stage I NSCLC, 11 stage II, and 29 patients stage III. Autoantibody detection was done by immunofluorescence test; Western blotting was used as a confirmation test. RESULTS Of the NSCLC patients, 27.8% were antineural antibody positive, and 32.7% had ANA. No differences were found between the histological groups. AnAb-positive patients showed a better survival in all patients (p = 0.005). There was also a higher survival of ANA-positive patients, but this was only significant in stage III (p = 0.0025). Cox regression analysis showed that antineural and antinuclear antibodies are a stage-independent prognostic factor in NSCLC. CONCLUSIONS Antineural and antinuclear autoantibodies are a stage-independent prognostic factor in patients with NSCLC and may represent an effective immune response to the tumor.

Functional characterisation of autoantibodies from patients with pediatric opsoclonus–myoclonus-syndrome

Paraneoplastic opsoclonus-myoclonus-syndrome (OMS) both in children and adults is suspected to be the result of an autoimmune response directed against cross-reactive proteins of tumor and neuronal cells. We here characterised the binding and functional activities of anti-neuroblastoma antibodies in IgG fractions from 11 OMS children with and without neuroblastoma. IgG fractions from neuroblastoma without OMS (NB) and healthy children served as controls. Indirect immunofluorescence and Western blot revealed IgG binding to intracellular autoantigens in all OMS patients, but in only one of the controls (p<0.001). Using flow cytometry, we could demonstrate surface binding of IgG fractions in all OMS patients, but only in one of control (p<0.001). Moreover OMS IgG exhibited a significant anti-proliferative and a cytotoxic effect on neuroblastoma cells compared to control IgG (p<0.001 and p<0.01). TUNEL assay revealed increased apoptotic cell death of the neuroblastoma cells after exposure to OMS IgG, but not to NB or control IgG (p<0.01). Preabsorption of membrane binding abandoned the anti-proliferative effect of OMS IgG. These findings indicate that surface-binding autoantibodies are present in OMS patients and these autoantibodies cause inhibition of cell proliferation and induce apoptosis.

Experimental stroke: ischaemic lesion volume and oedema formation differ among rat strains (a comparison between Wistar and Sprague–Dawley rats using MRI)

Investigating focal cerebral ischaemia requires animal models that are relevant to human stroke. This study was designed to evaluate the influence of early reperfusion and choice of rat strains on infarct volume and oedema formation. Thirty-six Wistar and Sprague–Dawley rats were subjected to temporary middle cerebral artery occlusion (MCAO) for 90 min (groups I and II) or to permanent MCAO (groups III and IV) using the suture technique. Ischaemic lesion volume and oedema formation were quantified 24 h after MCAO using 7T-magnetic resonance imaging (MRI). Impact of rat strains: Reperfusion led to significant larger ischaemic lesion volumes in Wistar rats as compared to Sprague–Dawley rats (P<0.0005). Oedema formation was similar in both rat strains. Permanent MCAO led to significantly larger ischaemic lesion volumes in Sprague–Dawley rats (P<0.05). Oedema formation, however, was significantly more accentuated in Wistar rats (P<0.005). Impact of reperfusion: Reperfusion did not cause any changes in ischaemic lesion volume in Wistar rats. Oedema formation, however, was significantly reduced (P<0.0005). In Sprague–Dawley rats, reperfusion caused a significant reduction of ischaemic lesion volume (P<0.00005), but did not modify oedema formation. These findings emphasize the critical importance of rat strain differences in experimental stroke research.

Intravenous immunoglobulins in the therapy of paraneoplastic neurological disorders

Abstract The treatment of paraneoplastic neurological syndromes (e.g., tumor therapy, immunosuppressive therapy, plasmapheresis) rarely leads to an improvement in the neurological symptoms. We treated four patients suffering from paraneoplastic neurological syndromes with intravenous immunoglobulins. All four had high titers of antineuronal antibodies in serum and CSF. Two of the patients, one suffering from paraneoplastic cerebellar degeneration and the other from paraneoplastic brain stem encephalitis and polyneuropathy, received intravenous immunoglobulin treatment within 3 weeks of the onset of neurological symptoms. Both patients showed clinical improvement within 2 weeks after the initiation of therapy. They also showed a decline in the intrathecal antibody synthesis of the antineuronal antibody. Two other patients, who had suffered from paraneoplastic neuropathy for 3 and 6 months showed no improvement with the intravenous immunoglobulin therapy. In these cases there was no effect on intrathecal antibody synthesis. When started early, intravenous immunoglobulins may be of therapeutical value in treating paraneoplastic neurological syndromes. Specific intrathecal antibody synthesis may be a better measure of clinical course that autoantibody serum titers.

Evaluation of Methods to Predict Early Long-Term Neurobehavioral Outcome After Coronary Artery Bypass Grafting

Postoperative cognitive decrease (POCD) represents the most frequent complication in modern cardiac surgery. The application of easily assessable surrogate parameters that predict long-term POCD at early time points is tempting. The aim of the present study was to analyze the predictive value of cerebral biomarkers, diffusion-weighted magnetic resonance imaging (DWI), and cognitive bedside testing after coronary artery bypass grafting (CABG). From 106 patients who underwent elective CABG, blood samples were drawn for the measurement of protein S100B and neuron-specific enolase release at baseline, at the end of surgery, and 48 hours afterward. Cerebral DWI was carried out before and 2 to 4 days after surgery. Cognitive functioning was assessed before, 2 to 4 days (bedside testing) after, and 3 months after CABG. On DWI, lesions were detected in 15.1% of patients. Biomarker levels and the presence of acute ischemic lesions on DWI were not associated with long-term POCD. Early POCD was correlated with 3-month POCD (r = 0.46, p <0.001). Ninety-one percent of patients who had shown moderate to severe POCD (<-1.5 z scores) in the early phase still had decreased memory functioning at 3 months compared to baseline (likelihood ratio 5.23). Early POCD was asserted as the only predictor for long-term POCD in a stepwise multiple linear regression model (R(2) = 0.20, p <0.001), excluding age, length of surgery, aortic clamping and cardiopulmonary bypass duration, the number of anastomoses, and postoperative neuron-specific enolase and S100B levels. In conclusion, the results show that in contrast to biomarkers, DWI, age, or intraoperative variables, early neuropsychological bedside testing predicts long-term POCD after CABG with acceptable accuracy.