Tongue sole creatine kinases function as DAMP and activate antimicrobial immunity via TLR2.

Creatine kinase (CK) is an enzyme that regulates adenosine triphosphate (ATP) metabolism to maintain energy homeostasis. Although CK has been reported to be involved in pathogen infection, the immune function of CK remains elusive. In this study, we identified two muscle-type CK from the teleost tongue sole Cynoglossus semilaevis (designated CsCKM-1 and CsCKM-2). Bacterial infection modulated CsCKM-1/2 expression in tongue sole tissues and induced the release of CsCKM-1/2 into serum. Recombinant CsCKM-1/2 (rCsCKM-1/2) exhibited robust kinase activity and bound to bacterial pathogens and pathogen-associated molecular patterns. rCsCKM-1/2 also bound to tongue sole peripheral blood leukocytes (PBLs) and promoted PBLs to uptake bacterial pathogens, inhibit bacterial proliferation, and express proinflammatory cytokines. When co-expressed in HEK293T cells, CsCKM-1/2 were found to interact with the leucine rich domain of toll-like receptor 2 (TLR2). The presence of TLR2 antagonist significantly reduced CsCKM-1/2-induced immune response and antibacterial effect. Taken together, these results indicated that tongue sole creatine kinases function as damage-associated molecular pattern (DAMP) molecules and play an important role in antimicrobial immunity via TLR2.

Effects of Long-Term Supplementation of Bovine Colostrum on the Immune System in Young Female Basketball Players. Randomized Trial.

An intensive physical exercise program could lead to a decrease in immune system function. Effects of long-term supplementation of bovine colostrum on the response of immune function on physical exercise test in athletes were examined. Twenty-seven elite female basketball players (age 16-19) were randomly assigned to either an experimental group or a control group. Eventually, n = 11 athletes completed intervention in the experimental group (3.2 g bovine colostrum orally twice a day for 24 weeks), while n = 9 athletes in the control group were given a placebo. Before the supplementation, after 3 and 6 months, subjects performed the physical exercise stress test. Before, just after, and 3 h after physical exercise testing, blood was drawn and immune system indicators were examined. Plasma interleukin (IL)-1alpha, IL-2, IL-10, IL-13, tumor necrosis factor (TNF) alpha, creatine kinase (CK MM), immunoglobulin G (IgG), insulin-like growth factor 1 (IGF1), and WBC, lymphocyte (LYM), monocyte (MON), and granulocyte (GRA) were measured. A statistically significant change in IL-10 in response to the exercise program during the supplementation period in both groups was observed (p = 0.01). However, the results of the rest of the comparisons were statistically insignificant (p > 0.05). Contrary to our initial hypothesis, there were no significant effects of bovine supplementation on the dynamics of immune system function indicators.

Role of creatine kinase as an allergen in immediate selective allergy to pork meat

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Roles of macrophage migration inhibitory factor in polymyositis: Inflammation and regeneration.

Objective To elucidate the clinical significance of macrophage migration inhibitory factor (MIF) serum concentration in patients with polymyositis. Methods Thirty-six patients with polymyositis were enrolled. Serum samples were obtained and stored to detect MIF and interleukin (IL)-6 using commercially available enzyme-linked immunosorbent assay kits. The relationships between these cytokines and clinical data were analyzed. Results The serum MIF concentration was significantly lower in patients in remission (34.74 ± 17.75) and in healthy controls (38.87 ± 9.30 ng/ml) than that in patients with active polymyositis (50.04 ± 23.84 ng/ml). There were no significant differences between healthy controls and patients in remission. The serum IL-6 concentration in patients with active polymyositis (19.67 ± 7.16 pg/ml) was significantly higher than that in patients in remission (15.81 ± 4.00 pg/ml) and controls (8.14 ± 3.71 pg/ml). The serum IL-6 concentration was negatively correlated with the serum MIF concentration (r = -0.283). No relationship was found between the serum MIF concentration and glucocorticoid dose. The MIF concentration peaked twice during treatment when the creatine kinase concentration was decreasing. Conclusion MIF and IL-6 play important roles in the inflammation associated with polymyositis. MIF might also be involved in the early stage of regeneration in polymyositis. MIF may thus serve as a biomarker of disease activity and outcome.

[Development and application of CK-MB specific monoclonal antibodies].

The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche's CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche's was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche's.

Diagnosis and Management of Immune-Mediated Myopathies.

Immune-mediated myopathies (IMMs) are a heterogeneous group of acquired muscle disorders characterized by muscle weakness, elevated creatine kinase levels, and myopathic electromyographic findings. Most IMMs feature the presence of inflammatory infiltrates in muscle. However, the inflammatory exudate may be absent. Indeed, necrotizing autoimmune myopathy (NAM), also called immune-mediated necrotizing myopathy, is characterized by a necrotizing pathologic process with no or minimal inflammation in muscle. The recent discovery of antibodies associated with specific subtypes of autoimmune myopathies has played a major role in characterizing these diseases. Although diagnostic criteria and classification of IMMs currently are under revision, on the basis of the clinical and muscle histopathologic findings, IMMs can be differentiated as NAM, inclusion body myositis (IBM), dermatomyositis, polymyositis, and nonspecific myositis. Because of recent developments in the field of NAM and IBM and the controversies around polymyositis, this review will focus on NAM, IBM, and dermatomyositis.

Serum levels of adipokines in patients with idiopathic inflammatory myopathies: a pilot study.

Adipokines are cytokines not only regulating metabolic and endocrine activities, but also modulating inflammatory and immune responses in several clinical settings, including autoimmunity. This study was aimed to evaluate whether serum adipokine levels may be useful as markers of disease activity in patients with idiopathic inflammatory myopathies (IIM). Adiponectin, leptin, chemokine C-C motif ligand-2 (CCL2), interleukin (IL)-6, and tumor necrosis factor (TNF) were measured in the serum of all participants. For each adipokine, we evaluate the area under the ROC curve (AUC) and its correlation with creatine kinase (CK) levels. Thirteen patients with IIM and 13 age- and gender-matched healthy individuals were studied. In patients, the levels of CK (273 ± 321 versus 54 ± 29 U/L; P < 0.0001), leptin (1994 ± 1355 versus 818 ± 738 pg/mL; P = 0.024), and IL-6 (32.4 ± 24.1 versus 13.9 ± 3.5 pg/mL; P = 0.003) were significantly higher than in controls. As a result, CK (AUC = 0.929, 0.833-1.00; P = 0.0002), leptin (AUC = 0.783, 0.588-0.977; P = 0.025), and IL-6 (AUC = 0.846, 0.680-1.00; P = 0.005) significantly discriminated between patients and controls. Neither CCL2 (3256 ± 4585 versus 1118 ± 399 pg/mL; P = 0.319) nor TNF (85.1 ± 83.3 versus 58.2 ± 16.8 pg/mL; P = 0.809) levels were different. Additionally, only serum levels of CCL2 were significantly correlated with CK titers (Spearman´s rho coefficient 0.620, 0.087-0.877; P = 0.023). The levels of CCL2 are in parallel with CK activity in the serum of patients with IIM, suggesting a potential utility as markers of disease activity. Elevated levels of leptin and IL-6 also support a role for adipokines in IIM.

Designing an Antibody-Based Chaperoning System through Programming the Binding and Release of the Folding Intermediate.

The protein folding pathway consists of sequential intramolecular interactions, while chaperones exert their functions either by stabilizing folding intermediates or by preventing nonspecific intermolecular interactions, which are often associated with aggregation involving exposed hydrophobic residues in folding intermediates. As chaperones do not possess specificity for individual client proteins, we designed an antibody-based chaperoning system to mimic the sequential binding and release of client proteins undergoing folding. The single-chain variable fragment of antibody (scFv) A4 binds to human muscle creatine kinase (HCK) and prevents it from aggregating. The slow dissociation of HCK from A4 resulted in delayed but eventually high-quality refolding, as reflected by the higher recovery of enzymatic activity as well as abolished aggregation. Peptide P6, a sequence in HCK involved in A4 binding, competes with HCK, promotes its dissociation from A4, and accelerates the rate of high-quality refolding. The sequential addition of A4 and P6 is essential for the chaperoning effect. The programmed binding/release method can also be applied to refold HCK from inclusion bodies. Because the association/dissociation of the folding intermediate with the antibody is highly specific, the method can be used to design tailored refolding systems and to investigate chaperoning effects on protein folding/aggregation in a sequence-specific manner.

Paraneoplastic cerebellar degeneration associated with an onconeural antibody against creatine kinase, brain-type.

Onconeural immunity, a cancer-stimulated immune reaction that cross-reacts with neural tissues, is considered to be the principal pathological mechanism for paraneoplastic neurological syndromes (PNS). A common PNS is paraneoplastic cerebellar degeneration (PCD). We had encountered a PCD patient with urothelial carcinomas (UC) of the urinary bladder who was negative for the well-characterized PNS-related onconeural antibodies. In the present study, we aimed to identify a new PCD-related onconeural antibody, capable of recognizing both cerebellar neurons and cancer tissues from the patient, and applied a proteomic approach using mass spectrometry. We identified anti-creatine kinase, brain-type (CKB) antibody as a new autoantibody in the serum and cerebrospinal fluid from the patient. Immunohistochemistry indicated that anti-CKB antibody reacted with both cerebellar neurons and UC of the urinary bladder tissues. However, anti-CKB antibody was not detected in sera from over 30 donors, including bladder cancer patients without PCD, indicating that anti-CKB antibody is required for onset of PCD. We also detected anti-CKB antibody in sera from three other PCD patients. Our study demonstrated that anti-CKB antibody may be added to the list of PCD-related autoantibodies and may be useful for diagnosis of PCD.

Identification of New Potential Allergens From Nile Perch (Lates niloticus) and Cod (Gadus morhua)

Antecedentes: La globalización de la industria alimentaria proporciona la exposición a nuevos pescados no domésticos y se hace necesaria la identificación de los alérgenos potenciales para diagnosticar las reacciones alérgicas. Objetivo: El objetivo de este estudio fue estudiar las proteínas fijadoras de IgE que constituyen los alérgenos de la perca del Nilo (L. niloticus). Métodos: Mediante electroforesis 2D en gel se separaron las proteínas del músculo del L. niloticus y G. morhua y se enfrentaron al suero de 12 pacientes con historia de reacción inmediata a pescado, así como al suero de pacientes atópicos y controles sanos. Las proteínas reactivas a IgE fueron identificadas mediante espectrofotometría de masas. Results: En los resultados, el paciente mostraba un índice bajo de fijación de IgE a parvalbúminas, sin embargo mostraba fijación de IgE a 8 alérgenos diferentes a la parvalbúmina de L. niloticus y 5 a la G. morhua. Observamos una sensibilización cruzada de 7/12 (58%) de los individuos alérgicos a pescado a la enolasa-3 del L. niloticus, mientras que 11/12 (92%) de los pacientes estaban sensibilizados a la enolasa-3 del G. morhua. Conclusión: La identificación de los alérgenos especie-específicos o de la sensibilización individual podría en el futuro mejorar las estrategias de evitación (AU)

Background: Globalization of the food industry has led to widespread exposure to new nondomestic fish species; therefore, identification of potential allergens is necessary in order to diagnose allergic reactions. Objective: Contact with a patient who was allergic to Nile perch (Lates niloticus) prompted us to investigate the immunoglobulin (Ig) E– reactive proteins that could be allergens of this species. Methods: 2D gel electrophoresis was used to separate the muscle proteins of L niloticus and Gadus morhua. Immunoblotting was performed with sera from 12 patients with a history of immediate-type allergic reaction to fish and from atopic and nonatopic controls. IgE-reactive proteins were detected and identifi ed using mass spectrometry. Results: The index patient had low levels of IgE binding to parvalbumins. However, 8 putative allergens other than parvalbumin from L niloticus and 5 from G morhua were identified. Further investigation revealed cross-sensitivity to enolase 3 from L niloticus in 7 of the 12 fish-allergic individuals (58%), whereas 11 of the 12 patients (92%) were sensitized to enolase 3 from G morhua. However, atopic control patients were also sensitized to enolase 3 from L niloticus and G morhua. Conclusion: Identification of species-specific allergens and individual sensitization could help us to improve avoidance strategies (AU)

Antibody cross-reactivity and the viral aetiology of type 1 diabetes.

Type 1 diabetes (T1D) is caused by the destruction of insulin-producing pancreatic ß cells by the patient's immune system. While the underlying genetics and immunopathology are fairly well characterized, the environmental trigger remains unidentified. Numerous studies have centred on the role of enteroviruses as aetiological factors that could initiate or accelerate T1D development. The most convincing evidence to date consists of an array of reports documenting the presence of enteroviral nucleic acids in peripheral blood at diagnosis. A prominent hypothesis is that enteroviruses may infect the pancreatic islets and thus be responsible for the islet-specific up-regulation of MHC class I that is commonly observed, possibly enabling T cell recognition and cytotoxicity. Past immunohistochemical studies have indeed shown that antibodies binding the enteroviral capsid protein VP1 preferentially stain the pancreatic ß cells from diabetic individuals. New data now indicate that the VP1 antibody used in these studies cross-reacts with mitochondrial proteins.

Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross-reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins.

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.

Effects of dehydration on immune functions after a judo practice session.

We investigated the effects of dehydration after a judo practice session on player muscle and immune functions. Subjects included 25 female university judoists. Investigations were performed before and after 2.5 h of regular judo practice. Body composition, serum enzymes (myogenic enzymes, immunoglobulins and complements), neutrophils counts, reactive oxygen species (ROS) production capability, and phagocytic activity (PA) were measured. Subjects were divided into two groups according to level of dehydration after practice (mild dehydration and severe dehydration groups) and results were compared. Creatine kinase was found to increase significantly after practice. In addition, neutrophil count also increased significantly after practice in both groups. The changing ratios of IgA, IgG and C3 observed in the mild dehydration group were significantly higher than those in the severe dehydration group. In the severe dehydration group, post-practice PA/neutrophil had decreased significantly. Significant positive correlations were found between severity of dehydration and changing ratios of IgA, IgG, IgM, C3, C4 and ROS production capabilities, whereas no significant association was seen with PA and/or serum SOD activity. These results suggest that dehydration resulted in immunosuppression, including decreased neutrophil function.

Correlation of anti-signal recognition particle autoantibody levels with creatine kinase activity in patients with necrotizing myopathy.

OBJECTIVE: Anti-signal recognition particle (anti-SRP) autoantibodies are associated with severe acquired necrotizing myopathies. The role of these autoantibodies remains elusive, and the evolution of anti-SRP levels over time is unknown. In this study, we developed an addressable laser bead immunoassay (ALBIA) technique to investigate a correlation between anti-SRP levels, serum creatine kinase (CK) levels, and muscle strength in patients with necrotizing myopathy. METHODS: The diagnostic value of the ALBIA assay was determined by comparing serum levels of anti-SRP autoantibodies in 31 anti-SRP immunodot-positive patients to those in 190 healthy blood donors and 199 control patients with different inflammatory/autoimmune conditions or polyclonal hypergammaglobulinemia. Among the 31 anti-SRP-positive patients, serum samples from 8 patients were monitored over time for levels of anti-SRP autoantibodies and levels of CK (determined at least 3 times, consecutively, over a mean followup period of 783 days). The relationship between levels of anti-SRP autoantibodies and levels of CK was tested using a linear mixed model. RESULTS: The assay yielded positive results for anti-SRP in all anti-SRP immunodot-positive serum samples tested, while all control sera tested negative. The 8 anti-SRP-positive patients who were followed up longitudinally were found to have normalized CK levels and improved muscle strength. There was a striking correlation between the degree of myolysis, as measured by CK levels, in patients receiving therapy and the anti-SRP54 autoantibody levels in these same patients (P = 0.002). CONCLUSION: Anti-SRP-positive myositis appears to be one of the few autoimmune diseases in which specific autoantibody levels are correlated with surrogate disease activity markers. These results reveal the usefulness of monitoring anti-SRP autoantibody levels in patients receiving therapy, and may also suggest a possible pathogenic role for anti-SRP autoantibodies in the necrotizing myopathies.

AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.

In a clinical trial for adeno-associated virus serotype 1 (AAV-1)-mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-gamma enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4(+) and CD8(+) T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti-AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.

Ginsenoside Rb1 preconditioning protects against myocardial infarction after regional ischemia and reperfusion by activation of phosphatidylinositol-3-kinase signal transduction.

BACKGROUND: Ginsenoside Rb1, a major bioactive component of Panax ginseng, bears various beneficial effects on the cardiovascular system. This study investigated whether ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia-reperfusion injury and its potential mechanism. METHODS: Rats subjected to 45 min of myocardial ischemia followed by 120 min of reperfusion were assigned to the following groups: sham-operated, ischemia-reperfusion (I/R), ginsenoside Rb1+I/R, wortmannin(a specific PI3K inhibitor)+I/R, wortmannin drug vehicle (dimethyl sulfoxide, DMSO), wortmannin+sham, ginsenoside Rb1+ wortmannin +I/R. Infarct size was assessed by triphenyltetrazolium chloride staining. Plasma creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and troponin T levels were also measured. Akt phosphorylation expression was assessed by immunoblotting. RESULTS: Ginsenoside Rb1 preconditioning reduced infarct size compared with that in the I/R group: 30 +/- 2.6% versus 51 +/- 2.7% (p < 0.01). Ginsenoside Rb1 preconditioning also markedly reduced the plasma CK, CK-MB, LDH and troponin T levels in blood. Akt phosphorylation expression increased after ginsenoside Rb1 preconditioning. These effects of ginsenoside Rb1 preconditioning were significantly inhibited by wortmannin. CONCLUSION: This is the first study to demonstrate that ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia and reperfusion injury, partly by mediating the activation of the PI3K pathway and phosphorylation of Akt.