Being Eaten Alive: How Energy-Deprived Cells Are Disposed of, Mediated by C-Reactive Protein-Including a Treatment Option.

In medicine, C-reactive protein (CRP) has become established primarily as a biomarker, predicting patient prognosis in many indications. Recently, however, there has been mounting evidence that it causes inflammatory injury. As early as 1999, CRP was shown to induce cell death after acute myocardial infarction (AMI) in rats and this was found to be dependent on complement. The pathological effect of CRP was subsequently confirmed in further animal species such as rabbit, mouse and pig. A conceptual gap was recently closed when it was demonstrated that ischemia in AMI or ischemia/hypoxia in the severe course of COVID-19 causes a drastic lack of energy in involved cells, resulting in an apoptotic presentation because these cells cannot repair/flip-flop altered lipids. The deprivation of energy leads to extensive expression on the cell membranes of the CRP ligand lysophosphatidylcholine. Upon attachment of CRP to this ligand, the classical complement pathway is triggered leading to the swift elimination of viable cells with the appearance of an apoptotic cell by phagocytes. They are being eaten alive. This, consequently, results in substantial fibrotic remodeling within the involved tissue. Inhibiting this pathomechanism via CRP-targeting therapy has been shown to be beneficial in different indications.

[Journal clubs in nursing practice: An online survey of German university hospitals]. / Journal Clubs in der Pflegepraxis.

Journal clubs in nursing practice: An online survey of German university hospitals Abstract. Background: Journal clubs (JCs) are an evidence-based method for enabling nursing professionals to develop a scientifically based practice. Research results can be made available to nurses to directly influence their nursing practice. The level of implementation in German university hospitals is currently unknown. Aim: This study surveyed the current state of JCs at German university hospitals. The aim is to develop recommendations for the implementation of JCs for nursing practice. Methods: The online survey of all 27 representatives of the "Network of Nursing Science and Practice Development" of the VPU e.V. was conducted from October to November 2020. The questionnaire was developed based on literature. The data analysis was descriptive. Results: 15 of the 19 clinics surveyed currently implement, or have previously implemented, a JC (response rate 70,4%). In approximately half of the clinics, JC is an established structure that is also offered as part of advanced training and continuing education (46,6%) and is used to develop and expand methodological, scientific competencies (86,6%). Positive experiences, but also structural problems in the implementation of a JC were reported. Approximately one quarter of the clinics evaluate their JC regularly (26,7%). Conclusions: JCs at German university hospitals are currently being established. In order to establish JCs on a permanent basis, EBN knowledge must be continuously imparted in further education and training; in addition, JCs should be an essential component of academic role profiles.

[Attitudes Towards COVID-19 Vaccination Compared to Influenza Vaccination Among Hospital Staff]. / Einstellungen gegenüber der COVID-19-Impfung im Vergleich zur Grippeschutzimpfung bei Krankenhauspersonal.

This cross-sectional study compared hospital staff who had received influenza or COVID-19 vaccination or who had refused COVID-19 vaccination in terms of attitudes towards each vaccination, uptake of influenza vaccination and reasons for refusing COVID-19 vaccination. COVID-19 vaccine refusers rated the risk of infection for themselves and in general and the effectiveness of the vaccination lowest and the vaccination risk highest compared to the other two groups. They also reported the lowest past uptake of influenza vaccination. Perceived pressure to vaccinate proved to be a relevant barrier. Future vaccination campaigns should maintain a balance between information on vaccines, the need for vaccination, and voluntary uptake.

C-Reactive Protein (CRP) Blocks the Desensitization of Agonistic Stimulated G Protein Coupled Receptors (GPCRs) in Neonatal Rat Cardiomyocytes.

Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) signaling by CRP can be observed by using CRP in combination with different GPCR agonists on spontaneously beating cultured neonatal rat cardiomyocytes. All used agonists (isoprenaline, clenbuterol, phenylephrine, angiotensin II and endothelin 1) affected the beat rate of cardiomyocytes significantly and after washing them out and re-stimulation the cells developed a pronounced desensitization of the corresponding receptors. CRP did not affect the basal beating-rate nor the initial increase/decrease in beat-rate triggered by different agonists. However, CRP co-incubated cells did not exhibit desensitization of the respective GPCRs after the stimulation with the different agonists. This lack of desensitization was independent of the GPCR type, but it was dependent on the CRP concentration. Therefore, CRP interferes with the desensitization of GPCRs and has to be considered as a novel regulator of adrenergic, angiotensin-1 and endothelin receptors.

C-Reactive Protein Triggers Cell Death in Ischemic Cells.

C-reactive protein (CRP) is the best-known acute phase protein. In humans, almost every type of inflammation is accompanied by an increase of CRP concentration. Until recently, the only known physiological function of CRP was the marking of cells to initiate their phagocytosis. This triggers the classical complement pathway up to C4, which helps to eliminate pathogens and dead cells. However, vital cells with reduced energy supply are also marked, which is useful in the case of a classical external wound because an important substrate for pathogens is disposed of, but is counterproductive at internal wounds (e.g., heart attack or stroke). This mechanism negatively affects clinical outcomes since it is established that CRP levels correlate with the prognosis of these indications. Here, we summarize what we can learn from a clinical study in which CRP was adsorbed from the bloodstream by CRP-apheresis. Recently, it was shown that CRP can have a direct effect on blood pressure in rabbits. This is interesting in regard to patients with high inflammation, as they often become tachycardic and need catecholamines. These two physiological effects of CRP apparently also occur in COVID-19. Parts of the lung become ischemic due to intra-alveolar edema and hemorrhage and in parallel CRP increases dramatically, hence it is assumed that CRP is also involved in this ischemic condition. It is meanwhile considered that most of the damage in COVID-19 is caused by the immune system. The high amounts of CRP could have an additional influence on blood pressure in severe COVID-19.

Comparison of the '4-item assessment test' and 'nursing delirium screening scale' delirium screening tools on non-intensive care unit wards: A prospective mixed-method approach.

BACKGROUND: In elderly patients following surgery, postoperative delirium (POD) is the most frequent complication and is associated with negative outcomes. The 2017 European Society of Anaesthesiology guideline on POD aims to improve patient care by implementing structured delirium prevention, diagnosis and treatment. However, these recommendations, especially systematic delirium screening, are still incompletely adopted in clinical practice. The aim of this study was to evaluate the feasibility and acceptance of validated delirium screening tools and to identify barriers to their implementation on nonintensive care unit wards. METHODS: Screening rates, as well as practicability, acceptance and the interprofessional handling of positive results, were assessed for each group. Screening rates were calculated as a percentage of the total potential testing episodes completed (up to 15 per patient). Patients were considered eligible when aged 65 years and above. Barriers and motivating factors were assessed in a mixed method approach by utilising questionnaires and focus group discussions. INTERVENTION: In a 3-month phase, a guideline-compliant screening protocol involving screening for POD three times daily for 5 days following surgery was introduced in five wards: both the 4-item assessment test (4AT) and the nursing delirium screening scale (NuDESC) were used. Before commencing the study and again after 6 weeks, medical staff of the respective wards underwent a 45 min training session. RESULTS: Of a total of 3183 potential testing episodes, 999 (31.4%) were completed, with more NuDESC observational tests (43%) than 4AT bedside tests completed (20%). The 4AT was considered more difficult to integrate into daily working routines, it took longer to administer, and nurses felt uncomfortable conducting the screening (53 vs. 13%). Screening results indicating delirium were often not discussed within the team (47%), and nurses felt that often such results were not taken seriously by physicians (54%). CONCLUSION: The observational NuDESC showed a higher completion rate than the bedside 4AT, although overall testing rates were low. The necessary time needed to conduct the screening, the negative reactions by patients, insufficient team communication and a lack of initiation of any therapy were identified as major barriers in the implementation of the guideline-compliant screening protocol. For all staff, further education and awareness of the importance of POD diagnosis and treatment might improve the screening rates. The NuDesc received better results concerning acceptance, practicability and introduction into daily work routine, leading to higher screening rates compared with the 4AT. The latter instrument, which was intended to be used rather selectively or when POD is suspected, might therefore not be suitable for guideline-compliant regular and repeated screening for POD.

C-Reactive Protein Causes Blood Pressure Drop in Rabbits and Induces Intracellular Calcium Signaling.

Systemic diseases characterized by elevated levels of C-reactive protein (CRP), such as sepsis or systemic inflammatory response syndrome, are usually associated with hardly controllable haemodynamic instability. We therefore investigated whether CRP itself influences blood pressure and heart rate. Immediately after intravenous injection of purified human CRP (3.5 mg CRP/kg body weight) into anesthetized rabbits, blood pressure dropped critically in all animals, while control animals injected with bovine serum albumin showed no response. Heart rate did not change in either group. Approaching this impact on a cellular level, we investigated the effect of CRP in cell lines expressing adrenoceptors (CHO-α1A and DU-145). CRP caused a Ca2+ signaling being dependent on the CRP dose. After complete activation of the adrenoceptors by agonists, CRP caused additional intracellular Ca2+ mobilization. We assume that CRP interacts with hitherto unknown structures on the surface of vital cells and thus interferes with the desensitization of adrenoceptors.

[Dementia-specific characteristics of patients with fall-related femur fractures : A case study based on routine hospital data of patients with unspecified dementia]. / Demenzspezifische Kennzeichen bei Patienten mit sturzbedingter Fraktur des Femurs : Eine Case Study basierend auf Krankenhausroutinedaten von Patienten mit nicht näher bezeichneter Demenz.

BACKGROUND: A fall-related femur fracture is one of the most frequent reasons for hospital admission of patients with dementia. The aim of the study was to identify dementia-specific characteristics of patients with fall-related femur fractures and additionally an unspecified dementia in order to gain further knowledge about this particular patient group. METHODS: A descriptive explorative case study with a single case-embedded design based on routine hospital data from a university hospital was performed. A total number of 34 patient records from 2015 were analyzed. In the embedded unit of analysis I the data of selected items of the nursing assessment AcuteCare (ePA-AC©) were descriptively analyzed for the time of admission and discharge. In the embedded unit of analysis II a summarizing inductive content analysis was conducted. RESULTS: The results of the embedded unit of analysis I showed that this patient group is characterized by a high need for care, changes of cognition and behavioral symptoms. The status of cognition, behavior and reciprocity as well as the self-care index stagnated or deteriorated in many patients from admission to discharge. The embedded unit of analysis II identified observable (social)behavior, (non)compliance and orientation as central dementia-specific characteristics. CONCLUSION: The results reflect the complexity of the care situation of patients with unspecified dementia in acute care hospitals. The results showed that a higher awareness for this particular patient group is needed to improve outcomes.

PentraSorb C-Reactive Protein: Characterization of the Selective C-Reactive Protein Adsorber Resin.

C-reactive protein (CRP) is well known as a general marker of inflammation. It furthermore represents a reliable risk factor for cardiac events and mediates tissue damage in acute myocardial infarction (AMI). It has been demonstrated that selective CRP depletion by extracorporeal apheresis in a porcine AMI model had beneficial effects on the infarcted area and the cardiac output. We therefore developed a novel adsorber for CRP apheresis from human plasma (PentraSorb CRP). It is intended for use in the clinic as therapy for patients suffering from AMI or other acute inflammatory diseases with elevated CRP plasma levels. The PentraSorb resin specifically bound CRP from human blood plasma and almost no other proteins as determined via Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). The resin further efficiently and selectively depleted CRP from plasma with low as well as high CRP concentrations (10-100 mg/L) at different flow rates, ranging from 17 to 40 mL/min. The resin was regenerable for up to 200 times without losing its CRP binding capacity or affecting biocompatibility. The depletion of CRP from plasma was comparable between the utilized small-scale column (0.5 mL resin) and the PentraSorb CRP adsorber (20 mL resin volume). The established features can therefore be applied to the clinical setting. In summary, PentraSorb CRP provides a novel, specific, and efficient CRP-binding resin that could be used in apheresis therapy for patients suffering from inflammatory diseases such as AMI, stroke, acute pancreatitis, and Crohn's disease.

[Prevalence of dementia in university hospitals : Analysis of ICD-10 coding in 5 university hospitals in Germany over a 2-year period]. / Prävalenzen von Demenz in Universitätskliniken : Analyse von ICD-10-Kodierungen in fünf Universitätskliniken in Deutschland über zwei Geschäftsjahre.

BACKGROUND: Demographic changes result in a higher prevalence of patients suffering from dementia in hospital. In Germany, epidemiological data of this target group are scarce and prevalence rates from university hospitals (UH) are not available. The prevalence rates and distribution were analyzed on the basis of ICD-10-GM (German modification) routine data METHOD: A secondary analysis on ICD-10-GM main and secondary diagnoses of dementia from 2014 and 2015 from 5 UH was performed. All patients admitted to hospital for at least 24 h and ≥18 years old (2014 n = 187,168; 2015 n = 189,040) were included. A descriptive analysis for the >69-year-old group was carried out (2014, n = 67,111; 2015; n = 67,824). RESULTS: The 1­year prevalence (2014/2015) for all 5 UH for patients ≥18 years old was 1.3%/1.4% and for the >69-year-old group, 3.3%/3.5%. The prevalence rates between the five UH varied: for patients ≥18 years the range was 0.44-2.16% (2014) and 0.44-2.77% (2015) and for >69-year-olds 1.16-5.52% (2014) and 1.16-7.06% (2015). Most cases were correlated with major diagnostic categories of traumatology, cardiology, gastroenterology and neurology. CONCLUSION: Analysis of ICD-10-GM routine data can provide an indication of the prevalence of dementia in UH. Results of the >69-year-olds varied greatly between participating UH. The reasons for this might be different healthcare tasks, especially with respect to geriatric patients; however, it is also possible that assessment procedures are not standardized and unreliable and therefore the coding is invalid. A standardized procedure for the identification of people suffering from dementia is necessary.

Selective apheresis of C-reactive protein: a new therapeutic option in myocardial infarction?

BACKGROUND: There is substantial evidence that C-reactive protein (CRP) mediates secondary damage of the myocardium after acute myocardial infarction (AMI). The aim of this animal trial in pigs was to specifically deplete CRP from porcine plasma after AMI and to study possible beneficial effects of the reduced CRP concentration on the infarcted area. METHODS: Ten pigs received balloon catheter-induced myocardial infarction. CRP was depleted from five animals utilizing a new specific CRP-adsorber, five animals served as controls. The area of infarction was analyzed by cardiovascular magnetic resonance imaging on day 1 and day 14 after AMI. Porcine CRP levels were determined by ELISA. RESULTS: CRP-apheresis resulted in a mean reduction of the CRP levels up to 48.3%. The area of infarction was significantly reduced by 30 ± 6% (P = 0.003) within 14 days in the treatment group, whereas it increased by 19 ± 11% (P = 0.260) in the controls. Fourteen days after infarction, the infarcted area revealed compact, transmural scars in the controls, whereas animals receiving CRP-apheresis showed spotted scar morphology. In the interventional group, a significantly higher left ventricular ejection fraction (LVEF) was observed after 14 days as compared to the controls (57.6 ± 2.4% vs. 46.4 ± 2.7%; P = 0.007). CONCLUSIONS: In a pig model for AMI, we observed that selective CRP-apheresis significantly reduces CRP levels and the volume of the infarction zone after AMI. Additionally, it changes the morphology of the scars and preserves cardiac output (LVEF).

The structure of carbon monoxide adsorbed on the NaCl(100) surface­a combined LEED and DFT-D/vdW-DF study.

The structure of the first layer CO adsorbed on NaCl(100) is investigated experimentally by means of quantitative low-energy electron diffraction at 25 K, and theoretically by means of density functional theory. Consistent with earlier helium atom diffraction results, the monolayer structure has p(2×1) symmetry with a glide-plane along the longer axis of the unit cell. The structure analysis confirms the binding of CO via the carbon end to the NaCl(100) surface. The vertical distance of carbon above Na(+) is 2.58 ± 0.08 Å, in good agreement with geometry optimizations based on dispersion-corrected density functional theory, and 0.15 Å lower than predicted in calculations based on the nonlocal van der Waals density functional.

CRP and SAP from different species have different membrane ligand specificities.

Human C-reactive protein (CRP) and serum amyloid component P (SAP) are well-characterised ligands for dying and dead cells, while facets of their physiological function still need to be unravelled. We partially characterised CRP and SAP from different species with similar acute-phase systems. Human, rabbit and porcine CRP bound phosphocholine (PC) and phosphoethanolamine (PEt). Human and porcine SAP bound PEt while rabbits seem to have very low levels of SAP or rabbit SAP does not bind PEt. Porcine serum additionally contained other ligands for PC and PEt. Some of them were immunoglobulins. Therefore, rabbits, pigs and humans cover the ability to bind PC and PEt with different extents.

Detection of low level cryoglobulins by flow cytometry.

Several patients with cryoglobulin (CG) associated symptoms are seronegative for CG and other potentially causative biomarkers. We analyzed whether it is possible to detect cryoprecipitates by flow cytometry and whether the sensitivity of their demonstration can be increased as compared to visual inspection. Sera from 91 patients with suspected CG associated symptoms and 33 healthy controls were examined for the presence of CG by conventional visual testing and by flow cytometry for small diffracting particles. For calibration purposes we tested lipid micelle dilutions (positive controls) by both methods. The minimum concentrations of lipid micelles to be detected by visual inspection and flow cytometry were 128.5 and 2.0 pg ml(-1), respectively. Among the 91 patients and 33 controls, only 1 patient serum was positive for CG by conventional testing. This sample was also positive on flow cytometry. In the serum of a patient known to be positive for CG, laser diffracting particles were quantified by flow cytometry after keeping serum at 4°C for 3 days. Of the 91 patients, 14 additional samples displayed cold precipitates which redissolved after rewarming during flow cytometry. All 15 (1 + 14) patients positive for CG on flow cytometry suffered from symptoms usually associated with CG. Some precipitates were labeled with anti IgG and IgM antibodies confirming that the particles detected by flow cytometry contained immunoglobulins. No small diffracting particles were detected in the sera of the 33 healthy controls. Flow cytometry is equally specific but much more sensitive in the detection of CG than visual inspection.

Turning the 'mustard oil bomb' into a 'cyanide bomb': aromatic glucosinolate metabolism in a specialist insect herbivore.

Plants have evolved a variety of mechanisms for dealing with insect herbivory among which chemical defense through secondary metabolites plays a prominent role. Physiological, behavioural and sensorical adaptations to these chemicals provide herbivores with selective advantages allowing them to diversify within the newly occupied ecological niche. In turn, this may influence the evolution of plant metabolism giving rise to e.g. new chemical defenses. The association of Pierid butterflies and plants of the Brassicales has been cited as an illustrative example of this adaptive process known as 'coevolutionary armsrace'. All plants of the Brassicales are defended by the glucosinolate-myrosinase system to which larvae of cabbage white butterflies and related species are biochemically adapted through a gut nitrile-specifier protein. Here, we provide evidence by metabolite profiling and enzyme assays that metabolism of benzylglucosinolate in Pieris rapae results in release of equimolar amounts of cyanide, a potent inhibitor of cellular respiration. We further demonstrate that P. rapae larvae develop on transgenic Arabidopsis plants with ectopic production of the cyanogenic glucoside dhurrin without ill effects. Metabolite analyses and fumigation experiments indicate that cyanide is detoxified by ß-cyanoalanine synthase and rhodanese in the larvae. Based on these results as well as on the facts that benzylglucosinolate was one of the predominant glucosinolates in ancient Brassicales and that ancient Brassicales lack nitrilases involved in alternative pathways, we propose that the ability of Pierid species to safely handle cyanide contributed to the primary host shift from Fabales to Brassicales that occured about 75 million years ago and was followed by Pierid species diversification.

Specific removal of C-reactive protein by apheresis in a porcine cardiac infarction model.

BACKGROUND: C-reactive protein (CRP) is a possible causative factor of the destructive processes observed during the weeks after myocardial infarction. METHODS: We developed a clinically relevant animal model including the removal of CRP from blood plasma utilizing a specific CRP adsorber and the visualization of the infarct scar in the living animal by cardiovascular magnetic resonance imaging as a tool to investigate the impact of CRP after acute myocardial infarction. RESULTS: We describe the facets of this model system and kinetics of clinical blood parameters like CRP and troponin. In addition, we demonstrate the potency of CRP apheresis reducing CRP levels by ~70% in the established treatment system. CONCLUSION: We showed for the first time that it is possible to conduct apheresis at the following 2 days after acute myocardial infarction in a porcine infarction model and to analyze the infarct by cardiovascular magnetic resonance imaging at day 1 and 14.

CRP and the disposal of dying cells: consequences for systemic lupus erythematosus and rheumatoid arthritis.

C reactive protein (CRP) levels directly correlate with the disease activity of many inflammatory diseases, e.g. sepsis, infection, and various autoimmunopathies such as rheumatoid arthritis (RA). In contrast, insufficient CRP levels are implicated in the development of systemic lupus erythematosus (SLE). This article reports on the level-depended effects of CRP in various diseases. In detail we show that increased and decreased levels of CRP, as demonstrated in patients with RA and SLE, respectively can contribute to disease progression.

The influence on the immunomodulatory effects of dying and dead cells of Annexin V.

Apoptotic and necrotic cells expose phosphatidylserine (PS). This membrane modification ensures a swift recognition and uptake by phagocytes of the dying and dead cells. Annexin V (AxV) preferentially binds to anionic phospholipids and thereby, modulates the clearance process. First, we analyzed the influence of AxV on the immunogenicity of apoptotic cells. The addition to apoptotic cells of AxV prior to their injection into mice increased their immunogenicity significantly. Next, we studied the influence of endogenous AxV on the allogeneic reaction against apoptotic and necrotic cells. To preserve heat-labile, short-lived "danger signals," we induced necrosis by mechanical stress. Wild-type mice showed a strong, allogeneic delayed-type hypersensitivity (DTH) reaction. In contrast, AxV-deficient animals showed almost no allogeneic DTH reaction, indicating that endogenous AxV increases the immune response against dead cells. Furthermore, AxV-deficient macrophages had a higher immunosuppressive potential in vitro. Next, we analyzed the influence of AxV on chronic macrophage infection with HIV-1, known to expose PS on its surface. The infectivity in human macrophages of HIV-1 was reduced significantly in the presence of AxV. Finally, we show that AxV also blocked the in vitro uptake by macrophages of primary necrotic cells. Similar to apoptotic cells, necrotic cells generated by heat treatment displayed an anti-inflammatory activity. In contrast, mechanical stress-induced necrotic cells led to a decreased secretion of IL-10, indicating a more inflammatory potential. From the experiments presented above, we conclude that AxV influences the clearance of several PS-exposing particles such as viruses, dying, and dead cells.

Efficient in vitro transduction of naive murine B cells with lentiviral vectors.

The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.

Intracellular capture of B7 in antigen-presenting cells reduces costimulatory activity.

CTLA-4 gene constructs were designed to express CTLA-4 exclusively in the endoplasmic reticulum (ER). Four different CTLA-4 gene constructs were transfected into HEK 293 (human embryonic kidney) and A20 (Balb/c mouse B lymphoma) cells. All constructs contained an ER retention signal and coded for CTLA-4 expression in the ER. One of the constructs, which contained the membrane part of CTLA-4, coded for an expression both on the cell surface and in the ER. Three of the expressed CTLA-4 types (including the ER-membrane-expressed form) caused a reduced surface expression of B7 in the A20 cells. Only constructs which allow dimerization of CTLA-4 showed this effect. It is assumed that intracellular CTLA-4 bound B7 and inhibited therefore the transport of B7 to the surface. The binding obviously caused also an enhanced degradation of the complexes because both proteins showed a low concentration in the transfected cell lines. CTLA-4-transfected and B7-reduced A20 cells showed a diminished costimulating activity upon T cells. This was demonstrated by a reduced proliferation of T cells from ovalbumin-immunized Balb/c mice, incubated with ovalbumin peptide-primed CTLA-4-transfected A20 cells.