Search

Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage., Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction., Methods and Results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G + (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils ( Cd177 , Lcn2 , Fpr1 ), and putative activity of transcriptional regulators involved in hypoxic response ( Hif1a ) and emergency granulopoiesis ( Cebpb ). At 3 and 5 days, 2 major subsets of Siglecf hi (enriched for eg, Icam1 and Tnf ) and Siglecf low ( Slpi, Ifitm1 ) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4 , heart infiltrating neutrophils acquired a unique SiglecF hi signature. SiglecF hi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF hi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF hi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF hi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation., Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF hi signature.

Aim: To investigate the effects of affinity-purified rabbit anti-β2GP1, and anti-β2GP1 purified from patients with systemic lupus erythematosus (SLE), on adenosine diphosphate (ADP)-induced aggregation., Methods: Whole blood was collected and processed to obtain platelet poor plasma (PPP) from normal controls (n = 15) and SLE patients (n = 15). Using PPP, anti-β2GP1 titres were determined using an ELISA and IgG fractions isolated using a HiTrap protein G column. Anti-β2GP1 was purified from two SLE patients using purified β2GP1 coupled to a HiTrap NHS-activated HP column., Results: The effect of rabbit and human derived anti-β2GP1 (0-100 μg/mL), on ADP (2.5, 5 μM) induced platelet aggregation were investigated using light transmission aggregometry. Rabbit anti-β2GP1 significantly inhibited all parameters of 5 μM ADP-induced platelet aggregation; %Max (p = 0.028), %AUC (p = 0.014) and slope (p < 0.001). In contrast, anti-β2GP1 purified from SLE patients significantly enhanced the %Max (p = 0.031) and %AUC (p = 0.007) in a concentration dependent manner, but inhibited the slope (p < 0.05) of 5 μM ADP-induced platelet aggregation., Conclusion: Our data suggest anti-β2GP1 purified from different species have variable effects on in vitro platelet aggregation. The disparity between rabbit and human anti-β2GP1 may be due to the heterogeneous nature of anti-β2GP1, varying avidity or different antibody binding specificities between species.

Objective: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%-15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK-immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK., Methods: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq-/- mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice., Results: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dose-dependent block of MuSK binding to ColQ by but not to LRP4. Passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the size and density of AChR and MuSK., Conclusions: As lack of ColQ compromises agrin-mediated AChR clustering in Colq-/- mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox.

Background: Preeclampsia is a prevalent life-threatening hypertensive disorder of pregnancy. The circulating antiangiogenic factor, soluble endoglin (sEng), is elevated in the blood circulation of women with preeclampsia and contributes to disease pathology; however, the underlying mechanisms responsible for its induction in preeclampsia are unknown., Methods and Results: Here, we discovered that a circulating autoantibody, the angiotensin receptor agonistic autoantibody (AT(1)-AA), stimulates sEng production via AT(1) angiotensin receptor activation in pregnant mice but not in nonpregnant mice. We subsequently demonstrated that the placenta is a major source contributing to sEng induction in vivo and that AT(1)-AA-injected pregnant mice display impaired placental angiogenesis. Using drug screening, we identified tumor necrosis factor-alpha as a circulating factor increased in the serum of autoantibody-injected pregnant mice contributing to AT(1)-AA-mediated sEng induction in human umbilical vascular endothelial cells. Subsequently, among all the drugs screened, we found that hemin, an inducer of heme oxygenase, functions as a break to control AT(1)-AA-mediated sEng induction by suppressing tumor necrosis factor-alpha signaling in human umbilical vascular endothelial cells. Finally, we demonstrated that the AT(1)-AA-mediated decreased angiogenesis seen in human placenta villous explants was attenuated by tumor necrosis factor-alpha-neutralizing antibodies, soluble tumor necrosis factor-alpha receptors, and hemin by abolishing both sEng and soluble fms-like tyrosine kinase-1 induction., Conclusions: Our findings demonstrate that AT(1)-AA-mediated tumor necrosis factor-alpha induction, by overcoming its negative regulator, heme oxygenase-1, is a key underlying mechanism responsible for impaired placental angiogenesis by inducing both sEng and soluble fms-like tyrosine kinase-1 secretion from human villous explants. Our results provide important new targets for diagnosis and therapeutic intervention in the management of preeclampsia.

Background: Accumulating evidence established a positive association of anti-heat shock protein 60 (HSP60) autoantibodies and the presence of atherosclerosis in humans. However, whether these autoantibodies play a causal role in the development of atherosclerosis is unknown., Methods and Results: In the present study, anti-HSP60 autoantibodies from blood of patients with coronary heart disease were isolated by affinity chromatography and injected into the tail vein of apolipoprotein E-deficient mice. Atherosclerotic lesions in aortas were significantly increased 8 weeks after injection. Furthermore, administration of a specific mouse monoclonal antibody (II-13) recognizing amino acid residues 288 to 366 of HSP60 effectively induced atherosclerotic lesions in apolipoprotein E-deficient mice. II-13 injection resulted in endothelial cell damage, followed by increased leukocyte attachment and accumulation of macrophages and smooth muscle cells in lesions. Interestingly, II-13-induced atherosclerosis was blocked by pretreatment of animals with F(ab)2 segments derived from the antibody, but not mouse IgG F(ab)2., Conclusions: Autoantibodies recognizing amino acid residues 288 to 366 of HSP60 induce atherosclerosis via the mechanisms of autoimmune reactions to HSP60 expressed on arterial endothelial cells, which can be prevented by F(ab)2 segments derived from these antibodies.

Background: Preeclampsia is a serious disorder of pregnancy characterized by hypertension, proteinuria, edema, and coagulation and vascular abnormalities. At the cellular level, abnormalities include increased calcium concentration in platelets, lymphocytes, and erythrocytes. Recent studies have shown that antibodies directed against angiotensin II type I (AT1) receptors are also highly associated with preeclampsia., Methods and Results: We tested the hypothesis that AT1 receptor-agonistic antibodies (AT1-AAs) could activate AT1 receptors, leading to an increased intracellular concentration of free calcium and to downstream activation of Ca2+ signaling pathways. Sera of 30 pregnant patients, 16 diagnosed with severe preeclampsia and 14 normotensive, were examined for the presence of IgG capable of stimulating intracellular Ca2+ mobilization. IgG from all preeclamptic patients activated AT1 receptors and increased intracellular free calcium. In contrast, none of the normotensive individuals had IgG capable of activating AT1 receptors. The specific mobilization of intracellular Ca2+ by AT1-AAs was blocked by losartan, an AT1 receptor antagonist, and by a 7-amino-acid peptide that corresponds to a portion of the second extracellular loop of the AT1 receptor. In addition, we have shown that AT1-AA-stimulated mobilization of intracellular Ca2+ results in the activation of the transcription factor, nuclear factor of activated T cells., Conclusions: These results suggest that maternal antibodies capable of activating AT1 receptors are likely to account for increased intracellular free Ca2+ concentrations and changes in gene expression associated with preeclampsia.

Background: In a rat model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) has been reported to prevent allograft rejection. However, the molecular identities of immunosuppressive factors, which are in the early stage of post-OLT, remain elusive. This study was aimed to identify immunodominant suppressive factors present in early post-OLT serum., Methods: The immunosuppressive activities of post-OLT serum, immunoglobulin (Ig) G-depleted serum, and purified IgG were evaluated in vitro by inhibition of the mixed lymphocyte reaction (MLR). Autoantigens recognized by the MLR-inhibitory IgG in early post-OLT serum were identified by the internal protein sequencing., Results: Recipient post-OLT serum inhibited MLR, and its immunosuppressive activity vanished by means of the elimination of OLT-inducible IgG. IgG from post-OLT sera (2-3 weeks) specifically reacted to 31-, 34-, and 73-kDa autoantigens on splenic cells. The internal sequences of the doublet 31- and 34-kDa antigens coincided completely with those of histone H1 molecules. The levels of histone H1-specific antibodies were transiently increased to a plateau around 2 to 3 weeks after OLT but decreased in the later tolerogenic phase. Immunodepletion of antihistone H1 autoantibodies from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal antibody-directed histone H1 not only suppressed MLR but also prolonged allograft survival., Conclusions: In this article, the authors provide evidence that autoreactive antibodies against histone H1, which are transiently induced at the early stage by liver transplantation, are a major OLT-induced graft survival factor.

Background and Purpose: Moyamoya disease (MMD) is a rare entity that results in progressive occlusion of the arteries of the circle of Willis, but the pathogenesis of MMD is unknown., Methods: MMD sera (n=32) were tested for anti-endothelial cell antibodies by enzyme-linked immunoassays and flow cytometric analysis. Apoptosis was induced in human umbilical vein endothelial cells by tumor necrosis factor-alpha., Results: We found that a high proportion of MMD sera had anti-endothelial cell antibodies with apoptotic stimuli. Prominent reactivities of MMD sera (72%) with recombinant human alpha-fodrin were observed., Conclusions: Our study demonstrates that MMD sera contain a high incidence of anti-alpha-fodrin autoantibodies, providing new insight into the mechanisms of occlusion of MMD arteries.

Introduction: Antiribosomal P autoantibodies are detectable in 12-16% of patients with systemic lupus erythematosus., Aim: To assess whether antiribosomal P autoantibodies could be useful as a novel form of immunotherapy for pancreatic cancer., Methodology: Three pancreatic cancer cell lines were incubated with antiribosomal P or normal human immunoglobulin. Viability was assayed with MTT, and apoptosis was detected by TUNEL. MIA PaCa-2 cells were injected into athymic mice. Animals were treated with intraperitoneal antibody or control immunoglobulin. Serum antibody levels were measured by ELISA. Tumor nodule size was measured weekly. Binding of the antibody to tumors was demonstrated with fluorescent microscopy., Results: Antiribosomal P antibody inhibited pancreatic cancer cell proliferation up to 54.6% (p < 0.01) and was associated with a threefold increase in the rate of apoptosis (p < 0.05). Tumor volume after 4 weeks of treatment was 23.2 mm3, versus 141.5 mm3 for the control group (p < 0.05). Apoptosis rate within the nodules was increased twofold, to 11.4%, in comparison with control (p < 0.05)., Conclusion: Antiribosomal P autoantibody at levels similar to those that can exist in SLE inhibits the growth of pancreatic cancer cells, in vitro and in vivo. The mechanism involves surface binding and apoptosis.

Background: We recently identified agonistic autoantibodies directed against the angiotensin AT1 receptor (AT1-AA) in the plasma of preeclamptic women. To elucidate their role further, we studied the effects of AT1-AA on reactive oxygen species (ROS), NADPH oxidase expression, and nuclear factor-kappaB (NF-kappaB) activation., Methods and Results: We investigated human vascular smooth muscle cells (VSMC) and trophoblasts, as well as placentas. AT1-AA were isolated from sera of preeclamptic women. Angiotensin II (Ang II) and AT1-AA increased ROS production and the NADPH oxidase components, p22, p47, and p67 phox in Western blotting. We next tested if AT1-AA lead to NF-kappaB activation in VSMC and trophoblasts. AT1-AA activated NF-kappaB. Inhibitor-kappaBalpha (I-kappaBalpha) expression was reduced in response to AT1-AA. AT1 receptor blockade with losartan, diphenylene iodonium, tiron, and antisense against p22 phox all reduced ROS production and NF-kappaB activation. VSMC from p47phox-/- mice showed markedly reduced ROS generation and NF-kappaB activation in response to Ang II and AT1-AA. The p22, p47, and p67 phox expression in placentas from preeclamptic patients was increased, compared with normal placentas. Furthermore, NF-kappaB was activated and I-kappaBalpha reduced in placentas from preeclamptic women., Conclusions: NADPH oxidase is potentially an important source of ROS that may upregulate NF-kappaB in preeclampsia. We suggest that AT1-AA through activation of NADPH oxidase could contribute to ROS production and inflammatory responses in preeclampsia.

Background: In Guillain-Barré syndrome (GBS), the destruction or malfunction of blood-nerve barrier (BNB) has been considered to be the beginning of the disease process. It is unclear whether sera from patients with GBS can open the BNB, and which component of patient sera is most important in the dysregulation of the BNB., Methods: The authors evaluated the effect of sera from patients with GBS on permeability of an in vitro BNB model using bovine endoneurial microvascular endothelial cells (PnMEC) cultured on the luminal side of a collagen-coated culture insert (pore size: 0.4 micro m)., Results: PnMEC monolayers challenged by GBS sera showed significantly lower transendothelial electrical resistance and higher clearance of [carboxyl-(14)C]-inulin with or without complement. Sera with anti-GM1 antibody showed greater loosening of the barrier than others. This effect decreased significantly after incubation with pure GM1 antigen, suggesting the importance of anti-GM1 antibody in BNB dysregulation. Serial analyses of [carboxyl-(14)C]-inulin clearance in four patients disclosed a favorable effect of plasmapheresis in restoring BNB function in some cases., Conclusions: The authors found an unfavorable effect of sera from patients with GBS on BNB function, supporting involvement of humoral factors causing BNB derangement in the acute stage. Serial evaluation of permeability change using the authors' in vitro system might be useful for the clinical assessment of BNB derangement in individual patients.

Background: Immunoadsorption capable of removing circulating autoantibodies represents an additional therapeutic approach in dilated cardiomyopathy (DCM). The role played by autoantibodies belonging to the immunoglobulin (Ig) subclass G-3 in cardiac dysfunction remains to be elucidated., Methods and Results: Patients with DCM (left ventricular ejection fraction <30%) participated in this case-control study. Nine patients underwent immunoadsorption with protein A (low affinity to IgG-3), and 9 patients were treated with anti-IgG, which removes all IgG subclasses. Immunoadsorption was performed in 4 courses at 1-month intervals until month 3. In the 2 groups, immunoadsorption induced comparable reduction of total IgG (>80%). IgG-3 was effectively eliminated only by anti-IgG adsorption (eg, during the first immunoadsorption course; protein A, -37+/-4%; anti-IgG, -89+/-3%; P<0.001 versus protein A). The beta1-receptor autoantibody was effectively reduced only by anti-IgG (P<0.01 versus protein A). Hemodynamics did not change in the protein A group. In the anti-IgG group during the first immunoadsorption course, cardiac index increased from 2.3+/-0.1 to 3.0+/-0.1 L x min(-1) x m(-2) (P<0.01 versus protein A). After 3 months, before the last immunoadsorption course, cardiac index was 2.2+/-0.1 L x min(-1) x m(-2) in the protein A group and 3.0+/-0.2 L x min(-1) x m(-2) in the anti-IgG group (P<0.01 versus protein A). Left ventricular ejection fraction increased only in the anti-IgG group (P<0.05 versus protein A)., Conclusions: Autoantibodies belonging to IgG-3 may play an important role in cardiac dysfunction of DCM. The removal of antibodies of the IgG-3 subclass may represent an essential mechanism of immunoadsorption in DCM.

Mice were injected i.p. with IgG samples of different patients to test whether IgG from amyotrophic lateral sclerosis (ALS) can initiate an immune/inflammatory reaction targeting motor neurons. All IgG samples of five ALS patients and none of the disease controls recruited activated microglia cells in the ventral horn of the spinal cord. CD3 lymphocytes were not accumulated in the same tissue. Similar reaction was evoked by injection of IgG from guinea pigs with experimental autoimmune gray matter disease (EAGMD) induced by immunization with the homogenate of the ventral horn of bovine spinal cord. The results indicate that ALS IgG and anti-motoneuron IgG induce microglia reaction targeting motor neurons without initiating T cell response in the recipient mice.

Background: Miller Fisher syndrome (MFS), a variant of the Guillain-Barré syndrome, is associated with the presence of neuromuscular blocking antibodies, some of which may be directed at the ganglioside GQ1b., Materials and Methods: The authors investigated the in vitro effects of serum and purified immunoglobulin (Ig) G in a total of 11 patients with typical MFS during active disease, and in three of those patients after recovery. From one patient's serum, we prepared an IgG fraction enriched in anti-GQ1b antibodies by affinity chromatography. For combined pre- and postsynaptic analysis, endplate currents were recorded by a perfused macro-patch clamp electrode. Postsynaptic nicotinic acetylcholine receptor channels were investigated by an outside-out patch clamp technique in cultured mouse myotubes., Results: AllMFS-sera depressed evoked quantal release and reduced the amplitude of postsynaptic currents. Five of the 11 sera were additionally examined by outside-out patch clamp analysis and caused a concentration-dependent and reversible decrease in acetylcholine-induced currents. The time course of activation and desensitization of nicotinic acetylcholine receptor channels was not altered by MFS-IgG. Nine patients (82 %) were positive for anti-GQ1b antibodies in ELISA and dot-blot. The enriched anti-GQ1b antibody fraction had a similar effect as whole serum. After recovery from MFS, blocking activity was lost and sera originally positive for anti-GQ1b antibodies became negative., Conclusion: Circulating IgG antibodies induce both pre- and postsynaptic blockade and may play a pathogenic role in acute MFS.

Objective: To clarify the mechanism of block of nicotinic receptor channels by myasthenic antibodies., Background: Nicotinic acetylcholine receptor (nAChR) channel currents are functionally blocked by purified immunoglobulin G (IgG) of patients with myasthenia gravis (MG)., Methods: The molecular mechanism of block of IgG fractions containing antibodies to nAChR channels was tested with the patch-clamp technique in combination with a system for ultrafast solution exchange. For the experiments, outside-out patches from cultured mouse myotubes that express embryonic-type nAChR channels at their surface were used., Results: Incubation of outside-out patches with purified IgG from four myasthenic patients blocked nAChR channel currents activated by the application of 1.0 mM ACh reversibly. The peak current amplitude and the time course of block of nAChR channels decreased with increasing concentrations of IgG. The block became at least partly irreversible if incubation time of outside-out patches exceeded 2 minutes. For the block of nAChR channel currents with a-bungarotoxin, a similar mechanism of block was found., Conclusions: The reversibility of functional block of nAChR channel currents by myasthenic IgG depended strongly on the incubation time of the receptors with antibodies. Interaction of myasthenic antibodies with nicotinic receptors may proceed in several stages from a low-affinity reversible to a high-affinity irreversible binding.

Our previous studies have shown that Fc gamma receptor (FcgammaR)-mediated uptake of LDL-containing immune complexes (oxLDL-ICs) by human monocyte-derived macrophages leads to not only transformation of macrophages into foam cells but also macrophage activation and release of cytokines. It has been shown that cross-linking of FcgammaR triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we determined whether engagement of FcgammaR by oxLDL-ICs leads to activation of mitogen-activated protein (MAP) kinase pathway, a signaling cascade serving many important functions, including the regulation of gene expression, in THP-1 macrophage-like cells. Our results from immunoblotting, using specific anti-phosphorylated MAP kinase antibodies, showed that oxLDL-ICs induced extracellular signal regulated kinase 2 (ERK2) MAP kinase phosphorylation in THP-1 macrophage-like cells in time- and dose-dependent manners. Cholesterol loading before stimulation led to a longer phosphorylation of ERK2. Nuclear translocation of phosphorylated ERK was markedly increased after the stimulation. Moreover, our data showed that oxLDL-IC induction of MAP kinase was prevented by human monomeric IgG1, suggesting that the specific engagement of type I FcgammaR by oxLDL-IC is responsible for the MAP kinase activation. Finally, we showed that human anti-oxLDL autoantibody-containing immune complexes immobilized on type I collagen induced MAP kinase activation in THP-1 cells. These results strongly suggest that oxLDL-IC, which has been detected in atherosclerotic plaques, may play an important role in macrophage activation and atherogenesis.

Background: Autoantibodies directed against the beta1-adrenergic receptor have been described in patients with dilated cardiomyopathy. These autoantibodies exert an agonistic, chronotropic effect on spontaneously beating cultured neonatal rat cardiomyocytes. We studied the effect of such antibodies on beta1-adrenergic receptor expression., Methods and Results: Cardiomyocytes were incubated with either the beta-adrenergic agonist isoproterenol or autoantibodies for 72 hours. beta-Adrenergic receptor expression was studied on the mRNA level with semiquantitative reverse transcription-polymerase chain reaction and on the protein level with immunoblotting. Isoproterenol downregulated both mRNA and beta1- and beta2-adrenergic receptor protein subtypes, whereas the anti-beta1-adrenergic receptor autoantibodies decreased only the beta1-adrenergic receptor mRNA and protein. Long-term incubation of cultured cardiomyocytes with isoproterenol or the anti-beta1-adrenergic receptor autoantibodies reduced the acute stimulatory effect of isoproterenol on the myocytes. These effects were prevented by incubating the cells with isoproterenol in the presence of propranolol or with anti-beta1-adrenergic receptor autoantibodies in the presence of bisoprolol. Bisoprolol also abolished the reduction of the beta1-adrenergic receptor expression caused by longer-term incubation with isoproterenol and the autoantibodies., Conclusions: We conclude that after longer-term treatment with the anti-beta1-adrenergic receptor autoantibodies, the rat cardiomyocytes showed a beta-adrenergic receptor expression similar to that observed in failing hearts from patients with dilated cardiomyopathy.

Objective: To examine the presence of anti-L-type calcium channel antibodies in the serum of ALS patients., Background: Autoimmunity has been hypothesized as one of the mechanisms underlying the pathogenesis of sporadic ALS. Previous studies reported that sera from patients with sporadic ALS contain antibodies against voltage-gated calcium channels (L-type and P-type), but others do not support these findings., Methods: Regulated secretion of tritiated dopamine ([3H]DA) in PC12 cells is mediated exclusively by calcium entry through L-type calcium channels. To examine whether purified ALS immunoglobulin G (IgG) inhibits [3H]DA release by interfering with calcium entry through L-type calcium channels, evoked release in PC12 cells was determined in the presence of ALS IgG. This functional assay provides a sensitive way to examine L-type calcium channel interaction with IgG from ALS patients., Results: A significant inhibition of depolarization-evoked [3H]DA release (32+/-4%) was observed by purified IgG from ALS patients compared with control subjects (11+/-2%; p < 0.01). Significant inhibition by IgG occurred in 79% (15/19) of the ALS patients compared with only 29% (5/17) in the control group (p < 0.01). The level of calcium channel inhibition by ALS IgG correlated positively with disease duration (r = 0.68; p < 0.01) and correlated negatively with age (r = -0.48; p < 0.05)., Conclusions: These results confirm the presence of antibodies against the L-type calcium channel in the majority of sera from ALS patients, supporting their role in the pathogenesis of ALS.

An important advance in the description and understanding of congenital heart block (CHB) came in the 1970s with the observation that mothers of affected infants frequently had autoimmune diseases and, in particular, that many maternal sera contained antibodies to SSA/Ro and SSB/La ribonucleoproteins. Although the molecular biology of the candidate antigens has been extensively defined, the arrhythmogenic and electrophysiological effects of their cognate antibodies on the human fetal heart are unknown. In the present study, we provide evidence that IgG-enriched fractions and anti-52-kD SSA/Ro antibodies affinity-purified from sera of mothers whose children have CHB induce complete atrioventricular (AV) block in the human fetal heart perfused by the Langendorff technique and inhibit L-type Ca2+ currents at the whole-cell and single-channel level. Immunization of female BALB/c mice with recombinant 52-kD SSA/Ro protein generated high-titer antibodies that crossed the placenta during pregnancy and were associated with varying degrees of AV conduction abnormalities, including complete AV block, in the pups. These findings strongly suggest that anti-52-kD SSA/Ro antibodies are causally related to the development of CHB.

We investigated the heterogeneity of anti-voltage-gated calcium channel (VGCC) antibodies in the Lambert-Eaton myasthenic syndrome (LEMS) using a small-cell lung carcinoma line (MB) derived from an LEMS patient. Four of 13 LEMS patients had raised titers of anti-125I-omega-conotoxin-labeled (N-type) VGCCs, measured by radioimmunoassay using line MB as the source of antigen. Antagonists for L-type (nitrendipine and nifedipine) and N-type (omega-conotoxin) VGCCs inhibited K(+)-stimulated (voltage-dependent) Ca2+ flux into this line--by 22% for L-type and 2% for N-type at maximum concentration. Inhibition by the LEMS IgGs, by contrast, ranged from 46 to 78% at a concentration of 2 mg/ml. These differing effects on Ca2+ flux inhibition by LEMS IgGs on the one hand and by L- and N-type channel antagonists on the other, taken together with the observation that many of the sera failed to react with omega-conotoxin-labeled (N-type) channels in the immunoprecipitation assay, suggest that in many LEMS patients the autoantibodies target other VGCC subtypes besides L- or N-types, and that these are important in inducing the myasthenic disorder.

Patients with multiple sclerosis and matched controls were tested for lymphocyte stimulation response and induction of suppressor cell activity in response to concanavalin A (Con A) and antigens from axolemma or myelin. Of 17 stable patients, 6 failed to have a suppressor cell response activated by one of these brain cell antigens. Among the patients who lacked these suppressor responses, five had lymphocyte stimulation responses to the same antigens. All matched controls except for one had suppressor cell responses to these antigens and none responded with a positive cellular immune reaction. We found no difference in lymphoproliferative responses to Con A in patients and controls. The level of suppressor cell activity induced by Con A in the stable MS patients varied but did not differ significantly from that of controls.