A multianalyte assay for the detection of dermatomyositis-related autoantibodies based on immunoprecipitation combined with immunoblotting.

OBJECTIVE: To develop a multianalyte assay for the detection of dermatomyositis (DM)-related autoantibodies using immunoprecipitation (IP) combined with immunoblotting (IB). METHODS: Sera from 116 DM patients were subjected to RNA and protein immunoprecipitation assays as well as commercial enzyme-linked immunosorbent assays (ELISAs) for anti-aminoacyl transfer RNA synthetase, anti-melanoma differentiation antigen 5 (MDA5), anti-Mi-2, anti-transcriptional intermediary factor-1γ (TIF-1γ), and anti-U1 ribonucleoprotein antibodies. The IP/IB assay was developed by immunoprecipitation of autoantigens from HeLa cell extracts using patient sera, followed by immunoblotting with an antibody against Mi-2, TIF-1γ, OJ, nuclear matrix protein (NXP)-2, MDA5, PM/Scl, small ubiquitin-like modifier activating enzyme (SAE), or Ku. A multianalyte assay was designed by mixing primary antibodies in the IP/IB assay. RESULTS: IP assays identified any DM-related autoantibodies in 100 patients (86%), of which 82% were covered by commercial ELISAs, with a false-positive result in two sera and a false-negative result in one serum. The results obtained from the multianalyte IP/IB assay and 'gold-standard' IP assays were concordant in terms of the presence or absence of anti-MDA5, anti-TIF-1γ, anti-OJ, anti-NXP-2, anti-PM/Scl, anti-SAE, anti-Mi-2, and anti-Ku antibodies. CONCLUSION: This multianalyte IP/IB assay combined with commercial ELISAs is an alternative to 'gold-standard' IP assays for the detection of DM-related autoantibodies.

Lipid profiling reveals the presence of unique lipid mediators in human milk from healthy and mastitic subjects.

Milk lipids are an important energy source for infants, but the composition of milk lipids has not yet been clarified in detail. In this study, we analyzed free fatty acids and their metabolites in milk from humans and cows. In comparison to cow milk, human milk showed a higher content of free fatty acids including polyunsaturated fatty acids, especially ω-3 fatty acids and their metabolites. Polyunsaturated fatty acids were enriched at an early period of lactation, while saturated fatty acids did not change significantly over the period. Moreover, human milk contained high levels of ω-3 fatty acid metabolites, particularly 18-hydroxyeicosapentaenoic acid, an eicosapentaenoic acid-derived metabolite with anti-inflammatory activity. In comparison with human normal milk, thromboxane B2 and protectin D1 levels were significantly elevated in milk from individuals with mastitis, suggesting that these lipid mediators could be potential biomarkers of obstructive mastitis. Overall, the unique lipid profile of human milk supports the efficacy of breast-feeding for supply of more nutritional and bioactive lipids in comparison to artificial or cow milk to infants, in whom digestive and absorptive functions are still immature.

Antiviral proinflammatory phenotype of monocytes in anti-MDA5 antibody-associated interstitial lung disease.

OBJECTIVE: To evaluate upstream and downstream regulators leading to macrophage activation and subsequent cytokine storm in patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-associated interstitial lung disease (ILD). METHODS: We conducted an integrated miRNA-mRNA association analysis using circulating monocytes from 3 patients with anti-MDA5-associated ILD and 3 healthy controls and identified disease pathways and a regulator effect network by Ingenuity Pathway Analysis (IPA). The expression of relevant genes and proteins was verified using an independent validation cohort, including 6 patients with anti-MDA5-associated ILD, 5 with anti-aminoacyl tRNA synthetase antibody-associated ILD, and 6 healthy controls. RESULTS: IPA identified 26 matched pairs of downregulated miRNA and upregulated mRNAs and revealed that canonical pathways mediated by type I IFN signalling and C-C motif ligand 2 (CCL2) were responsible for the pathogenic process (P < 0.05 for all pathways). The regulatory network model identified IFN-ß; Toll-like receptors 3, 7, and 9; and PU.1 as upstream regulators, while the downstream effect of this network converged at the inhibition of viral infection. mRNA and protein expression analysis using validation cohort showed a trend towards the increased expression of relevant molecules identified by IPA in patients with anti-MDA5-associated ILD compared with those with anti-aminoacyl tRNA synthetase antibody-associated ILD or healthy controls. The expression of all relevant genes in monocytes and serum levels of CCL2 and IFN-ß declined after treatment in survivors with anti-MDA5-associated ILD. CONCLUSION: An antiviral proinflammatory network orchestrated primarily by activated monocytes/macrophages might be responsible for cytokine storm in anti-MDA5-associated ILD.

Clinical impact of myositis-specific autoantibodies on long-term prognosis of juvenile idiopathic inflammatory myopathies: multicentre study.

OBJECTIVES: This study aimed to investigate the clinical characteristics, treatment and prognosis of juvenile idiopathic inflammatory myopathies (JIIM) in Japan for each myositis-specific autoantibody (MSA) profile. METHODS: A multicentre, retrospective study was conducted using data of patients with JIIM at nine paediatric rheumatology centres in Japan. Patients with MSA profiles, determined by immunoprecipitation using stored serum from the active stage, were included. RESULTS: MSA were detected in 85 of 96 cases eligible for the analyses. Over 90% of the patients in this study had one of the following three MSA types: anti-melanoma differentiation-associated protein 5 (MDA5) (n = 31), anti-transcriptional intermediary factor 1 alpha and/or gamma subunits (TIF1γ) (n = 25) and anti-nuclear matrix protein 2 (NXP2) (n = 25) antibodies. Gottron papules and periungual capillary abnormalities were the most common signs of every MSA group in the initial phase. The presence of interstitial lung disease (ILD) was the highest risk factor for patients with anti-MDA5 antibodies. Most patients were administered multiple drug therapies: glucocorticoids and MTX were administered to patients with anti-TIF1γ or anti-NXP2 antibodies. Half of the patients with anti-MDA5 antibodies received more than three medications including i.v. CYC, especially patients with ILD. Patients with anti-MDA5 antibodies were more likely to achieve drug-free remission (29 vs 21%) and less likely to relapse (26 vs 44%) than others. CONCLUSION: Anti-MDA5 antibodies are the most common MSA type in Japan, and patients with this antibody are characterized by ILD at onset, multiple medications including i.v. CYC, drug-free remission, and a lower frequency of relapse. New therapeutic strategies are required for other MSA types.

Motion sickness-susceptible participants exposed to coherent rotating dot patterns show excessive N2 amplitudes and impaired theta-band phase synchronization.

Visually induced motion sickness (VIMS) can occur via prolonged exposure to visual stimulation that generates the illusion of self-motion (vection). Not everyone is susceptible to VIMS and the neural mechanism underlying susceptibility is unclear. This study explored the differences of electroencephalographic (EEG) signatures between VIMS-susceptible and VIMS-resistant groups. Thirty-two-channel EEG data were recorded from 12 VIMS-susceptible and 15 VIMS-resistant university students while they were watching two patterns of moving dots: (1) a coherent rotation pattern (vection-inducing and potentially VIMS-provoking pattern), and (2) a random movement pattern (non-VIMS-provoking control). The VIMS-susceptible group exhibited a significantly larger increase in the parietal N2 response when exposed to the coherent rotating pattern than when exposed to control patterns. In members of the VIMS-resistant group, before vection onset, global connectivity from all other EEG electrodes to the right-temporal-parietal and to the right-central areas increased, whereas after vection onset the global connectivity to the right-frontal area reduced. Such changes were not observed in the susceptible group. Further, the increases in N2 amplitude and the identified phase synchronization index were significantly correlated with individual motion sickness susceptibility. Results suggest that VIMS susceptibility is associated with systematic impairment of dynamic cortical coordination as captured by the phase synchronization of cortical activities. Analyses of dynamic EEG signatures could be a means to unlock the neural mechanism of VIMS.

Immune Checkpoint Inhibitor-Induced Myositis: a Case Report and Literature Review.

PURPOSE OF THE REVIEW: We clarify clinical characteristics of patients with immune checkpoint inhibitor (ICI)-induced myositis. RECENT FINDINGS: In 13 of 15 cases with ICI-induced myositis, the type of malignancy was melanoma. Eight, 4, and 3 patients received anti-PD-1 alone, anti-CTLA4 alone, and a combination of those, respectively. The mean period to the onset of ICI-induced myositis from the initiation of ICI was 4 weeks. Myocarditis was a complication in five patients. Seven of the patients died. The causes of death were myocarditis in three patients, respiratory muscle paralysis in two patients, and cancer progression in two patients. In patients without myocarditis or respiratory muscle paralysis, the prognosis for myositis was favorable with normalization of the CK levels occurring upon the cessation of ICI and the administration of immunosuppressive agents. Myocarditis and respiratory muscle paralysis are the major causes of death as immune-related adverse events in patients with ICI-induced myositis.

Phase-Amplitude Coupling of Neural Oscillations Can Be Effectively Probed with Concurrent TMS-EEG.

Despite the widespread use of transcranial magnetic stimulation (TMS), knowledge of its neurophysiological mode of action is still incomplete. Recently, TMS has been proposed to synchronise neural oscillators and to thereby increase the detectability of corresponding oscillations at the population level. As oscillations in the human brain are known to interact within nested hierarchies via phase-amplitude coupling, TMS might also be able to increase the macroscopic detectability of such coupling. In a concurrent TMS-electroencephalography study, we therefore examined the technique's influence on theta-gamma, alpha-gamma, and beta-gamma phase-amplitude coupling by delivering single-pulse TMS (sTMS) and repetitive TMS (rTMS) over the left motor cortex and right visual cortex of healthy participants. The rTMS pulse trains were of 5 Hz, 11 Hz, and 23 Hz for the three coupling variations, respectively. Relative to sham stimulation, all conditions showed transient but significant increases in phase-amplitude coupling at the stimulation site. In addition, we observed enhanced coupling over various other cortical sites, with a more extensive propagation during rTMS than during sTMS. By indicating that scalp-recorded phase-amplitude coupling can be effectively probed with TMS, these findings open the door to the technique's application in manipulative dissections of such coupling during human cognition and behaviour in healthy and pathological conditions.

Improved quantification of a commercial enzyme-linked immunosorbent assay kit for measuring anti-MDA5 antibody.

OBJECTIVES: To compare the quantitative performance for measuring anti-MDA5 antibody titer of two enzyme-linked immunosorbent assay (ELISA) systems: an in-house ELISA and the commercial MESACUPTM anti-MDA5 test. METHODS: Anti-MDA5 antibody titer was measured in sera from 70 patients with dermatomyositis using an in-house ELISA and the MESACUPTM anti-MDA5 test side-by-side. For the commercial ELISA kit, serum samples diluted 1:101 were used according to the manufacturer's protocol, but serial dilutions of sera were also examined to identify the optimal serum dilution for quantification. RESULTS: The anti-MDA5 antibody titers measured by the in-house and commercial ELISAs were positively correlated with each other (r = 0.53, p = .0001), but the antibody titer measured by the commercial ELISA was less sensitive to change after medical treatment, and 37 (80%) of 46 anti-MDA5-positive sera had antibody titer exceeding the quantification range specified by the manufacturer (≥150 index). Experiments using diluted serum samples revealed that diluting the sera 1:5050 improved the quantitative performance of the MESACUPTM anti-MDA5 test, including a better correlation with the in-house ELISA results and an increased sensitivity to change. CONCLUSION: We improved the ability of the commercial ELISA kit to quantify anti-MDA5 antibody titer by altering its protocol.

ß2-Glycoprotein I-specific T cells are associated with epitope spread to lupus-related autoantibodies.

Systemic lupus erythematosus (SLE) is a prototypic model for B cell epitope spread in autoimmunity. Autoantibodies to numerous and molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting the apoptotic cell-binding protein ß2-glycoprotein I (ß2GPI). Notably, mice immunized with ß2GPI and LPS display a remarkably similar pattern of autoantibody emergence to that seen in human SLE. Here, we used this model to investigate whether epitope spread to SLE-related autoantibodies is associated with a unique or limited ß2GPI-specific T cell response. We ask whether MHC class II haplotype and its associated T cell epitope restriction impact epitope spread to SLE-related autoantibodies. We found that ß2GPI/LPS-immunized mice produced similar SLE-related autoantibody profiles regardless of their ß2GPI T cell epitope specificity or MHC class II haplotype. Although ß2GPI T cell epitope specificity was clearly determined by MHC class II haplotype, a number of different ß2GPI T cell epitopes were associated with epitope spread to SLE-related autoantibodies. Notably, one ß2GPI T cell epitope (peptide 23, NTGFYLNGADSAKCT) was also recognized by T cells from an HLA-DRB1*0403(+) autoimmune patient. These data suggest that the generation of a ß2GPI-reactive T cell response is associated with epitope spread to SLE-related autoantibodies, independent of epitope specificity or MHC class II restriction. On the basis of these findings, we propose that factors enabling a ß2GPI-reactive T cell response may predispose individuals to the development of SLE-related autoantibodies independent of their MHC class II haplotype.

Real-time MEG neurofeedback training of posterior alpha activity modulates subsequent visual detection performance.

It has been demonstrated that alpha activity is lateralized when attention is directed to the left or right visual hemifield. We investigated whether real-time neurofeedback training of the alpha lateralization enhances participants' ability to modulate posterior alpha lateralization and causes subsequent short-term changes in visual detection performance. The experiment consisted of three phases: (i) pre-training assessment, (ii) neurofeedback phase and (iii) post-training assessment. In the pre- and post-training phases we measured the threshold to covertly detect a cued faint Gabor stimulus presented in the left or right hemifield. During magnetoencephalography (MEG) neurofeedback, two face stimuli superimposed with noise were presented bilaterally. Participants were cued to attend to one of the hemifields. The transparency of the superimposed noise and thus the visibility of the stimuli were varied according to the momentary degree of hemispheric alpha lateralization. In a double-blind procedure half of the participants were provided with sham feedback. We found that hemispheric alpha lateralization increased with the neurofeedback training; this was mainly driven by an ipsilateral alpha increase. Surprisingly, comparing pre- to post-training, detection performance decreased for a Gabor stimulus presented in the hemifield that was un-attended during neurofeedback. This effect was not observed in the sham group. Thus, neurofeedback training alters alpha lateralization, which in turn decreases performances in the untrained hemifield. Our findings suggest that alpha oscillations play a causal role for the allocation of attention. Furthermore, our neurofeedback protocol serves to reduce the detection of unattended visual information and could therefore be of potential use for training to reduce distractibility in attention deficit patients, but also highlights that neurofeedback paradigms can have negative impact on behavioral performance and should be applied with caution.

Comparison of radioimmunoprecipitation versus antigen-specific assays for identification of myositis-specific autoantibodies in dermatomyositis patients.

BACKGROUND: To confirm the antigen specificities of autoantibodies that precipitate 140-kDa (anti-p140) or 155/140-kDa polypeptides (anti-p155/140) previously identified by radioimmunoprecipitation in Korean patients with dermatomyositis (DM) and to look into the relationship between each MSA and clinical features of DM. METHODS: Seventeen serum samples of classic DM patients who had been found to have either anti-p140 antibodies (n = 9) or anti-p155/140 (n = 8) antibodies in our previous study were examined using enzyme-linked immunosorbent assay (for anti-MDA5 antibodies) and immunoblotting (for anti-MJ/NXP-2 and anti-TIF-1γ antibodies). RESULTS: Seven out of nine anti-p140 antibody positive patients were found to have anti-MDA5 antibodies. Two out of nine had anti-MJ/NXP-2 antibodies with no interstitial lung disease (ILD). All eight anti-p155/140 antibody positive patients were found to have anti-TIF-1γ antibodies. Anti-TIF-1γ and anti-MDA5 antibodies were simultaneously detected in one patient with anti-p155/140 antibody, who suffered HIV infection and non-Hodgkin's lymphoma. The associations between anti-MDA5 antibody and rapidly progressive ILD and between anti-TIF-1γ antibody and cancer-associated DM were confirmed to be significant. CONCLUSIONS: Although radioimmunoprecipitation still looks to be a good screening tool, confirmation with antigen-specific assays seems mandatory. The associations between anti-MDA5 and rapidly progressive ILD and between anti-TIF-1γ and cancer-associated DM were confirmed in Korean patients with DM.

Clinical phenotyping in patients with anti-synthetase antibodies using cluster analysis.

Objectives: To characterize clinically distinct subgroups among unselected patients with anti-synthetase antibodies using cluster analysis. Methods: This study evaluated patients with anti-synthetase antibodies registered to two independent cohorts; 106 consecutive patients from a prospective, single-centre cohort of the Scleroderma/Myositis Centre of Excellence (SMCE) were used as a derivation cohort and 125 patients from the Multicentre Retrospective Cohort of Japanese Patients with Myositis-Associated Interstitial Lung Disease (JAMI) were used as a validation cohort. Anti-synthetase antibodies were identified by RNA immunoprecipitation. A multiple correspondence analysis followed by hierarchical clustering was performed to aggregate the patients into homogeneous subgroups. Subsequently, a simple-to-use classification tree was generated using classification and regression tree analysis. Results: Three clusters were identified in the SMCE cohort: cluster 1 (n = 48), the interstitial pneumonia with autoimmune features/amyopathic dermatomyositis cluster, associated with older age at diagnosis and a higher frequency of malignancy; cluster 2 (n = 46), the DM cluster, corresponded to a younger age at diagnosis with a higher prevalence of myositis, arthritis, DM pathognomonic rashes, mechanic's hands and fever; and cluster 3 (n = 12), the SSc cluster, characterized by chronic interstitial lung disease. There was no significant difference in overall survival or progression-free survival between the clusters. A simple classification tree using myositis and RP was created in the SMCE cohort. Clusters 1 and 2 were successfully reproduced and the classification tree demonstrated favourable performance in the JAMI cohort. Conclusion: Patients with anti-synthetase antibodies were classified into three distinct phenotypes, indicating substantial heterogeneity within this patient group.

Motion sickness resistant people showed suppressed steady-state visually evoked potential (SSVEP) under vection-inducing stimulation.

Visual stimulation can generate illusory self-motion perception (vection) and cause motion sickness among susceptible people, but the underlying neural mechanism is not fully understood. In this study, SSVEP responses to visual stimuli presented in different parts of the visual field are examined in individuals with different susceptibilities to motion sickness to identify correlates of motion sickness. Alpha band SSVEP data were collected from fifteen university students when they were watching roll-vection-inducing visual stimulation containing: (1) an achromatic checkerboard flickering at 8.6 Hz in the central visual field (CVF) and (2) rotating dots pattern flickering at 12 Hz in the peripheral visual field. Rotating visual stimuli provoked explicit roll-vection perception in all participants. The motion sickness resistant participants showed reduced SSVEP response to CVF checkerboard during vection, while the motion sickness susceptible participants showed increased SSVEP response. The changes of SSVEP in the presence of vection significantly correlated with individual motion sickness susceptibility and rated scores on simulator sickness symptoms. Discussion on how the findings can support the sensory conflict theory is presented. Results offer a new perspective on vection and motion sickness susceptibility. Supplementary Information: The online version contains supplementary material available at 10.1007/s11571-023-09991-7.

Dissociating Autoantibody Responses against Ro52 Antigen in Patients with Anti-Synthetase or Anti-MDA5 Antibodies.

We aimed to dissociate the autoantibody response against the Ro52 protein in patients with anti-synthetase or anti-melanoma differentiation-associated gene 5 (MDA5) antibodies to explore the potential roles of different anti-Ro52 autoantibody responses in disease subclassification. This study used a single-center, prospective myositis cohort involving 122 consecutive patients with anti-synthetase antibodies identified by RNA immunoprecipitation (RNA-IP) and 34 patients with anti-MDA5 antibodies detected using enzyme immunoassay (EIA). Anti-Ro52 antibodies were measured using commercial EIA kits, while anti-Ro/SSA antibodies were identified using RNA-IP. Clinical features and outcomes were stratified according to two different patterns of autoantibody responses against Ro52, including "isolated anti-Ro52", defined by positive anti-Ro52 and negative anti-Ro/SSA antibodies, and "anti-SSA-Ro52", defined by positive anti-Ro52 and anti-Ro/SSA antibodies. Isolated anti-Ro52 positivity was the most prevalent autoantibody response in patients with both anti-synthetase (40/122; 32.8%) and anti-MDA5 antibodies (8/34; 23.5%). Isolated anti-Ro52 or anti-SSA-Ro52 positivity was associated with Gottron's sign in patients with anti-synthetase antibodies, while in patients with anti-MDA5 antibodies, isolated anti-Ro52 positivity was associated with respiratory insufficiency at initial presentation and poor overall survival. Isolated anti-Ro52 positivity could be a potential biomarker for patient stratification; however, the clinical significance of dissociating isolated anti-Ro52 positivity from overall anti-Ro52 positivity was not evident.

Intracranial transplantation of monocyte-derived multipotential cells enhances recovery after ischemic stroke in rats.

Cell transplantation has emerged as a potential therapy to reduce the neurological deficits caused by ischemic stroke. We previously reported a primitive cell population, monocyte-derived multipotential cells (MOMCs), which can differentiate into mesenchymal, neuronal, and endothelial lineages. In this study, MOMCs and macrophages were prepared from rat peripheral blood and transplanted intracranially into the ischemic core of syngeneic rats that had undergone a left middle cerebral artery occlusion procedure. Neurological deficits, as evaluated by the corner test, were less severe in the MOMC-transplanted rats than in macrophage-transplanted or mock-treated rats. Histological evaluations revealed that the number of microvessels that had formed in the ischemic boundary area by 4 weeks after transplantation was significantly greater in the MOMC-transplanted rats than in the control groups. The blood vessel formation was preceded by the appearance of round CD31(+) cells, which we confirmed were derived from the transplanted MOMCs. Small numbers of bloodvessels incorporating MOMC-derived endothelial cells expressing a mature endothelial marker RECA-1 were detected at 4 weeks after transplantation. In addition, MOMCs expressed a series of angiogenic factors, including vascular endothelial growth factor, angiopoetin-1, and placenta growth factor (PlGF). These findings provide evidence that the intracranial delivery of MOMCs enhances functional recovery by promoting neovascularization in a rat model for ischemic stroke.

Quantification of circulating endothelial progenitor cells in systemic sclerosis: a direct comparison of protocols.

BACKGROUND: It has been proposed that dysfunctional endothelial progenitor cells (EPCs) play a role in pathogenic vasculopathy in systemic sclerosis (SSc). However, there is some debate as to whether the EPC count is reduced in SSc. The European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group recently proposed recommendations for evaluating EPCs. OBJECTIVE: To validate the proposed EUSTAR recommendations by a side-by-side comparison of methods for quantifying EPCs. METHODS: Peripheral blood samples were obtained from 11 patients with SSc and 11 age-matched healthy controls. EPCs were simultaneously quantified by two methods: flow cytometry combined with immunomagnetic CD34+ cell enrichment or rosette-based lineage-negative (Lin-) cell enrichment. EPCs, defined as CD34+CD133+VEGFR2+ cells, were counted with and without fluorosphere calibration. RESULTS: EPC counts measured with fluorosphere calibration correlated well with each other, regardless of the enrichment procedure used. In contrast, EPC counts from protocols that did not use fluorospheres correlated poorly with results from other protocols. CONCLUSIONS: The EUSTAR recommendations are valid when they are combined with fluorosphere calibration.

Heparin-dependent and -independent anti-platelet factor 4 autoantibodies in patients with systemic lupus erythematosus.

OBJECTIVE: Antibodies that recognize complexes formed by platelet factor 4 (PF4) and heparin are involved in the pathogenesis of heparin-induced thrombocytopenia (HIT). This study was undertaken to investigate the prevalence and clinical correlations of anti-PF4 autoantibodies in patients with SLE. METHODS: We studied 118 patients with SLE, 78 with primary immune thrombocytopenia (ITP), 27 with primary APS, 2 with HIT (as positive controls) and 47 healthy controls. Heparin-dependent and -independent anti-PF4 antibodies were measured with an ELISA. Antibody binding was confirmed to be heparin-dependent when inhibited by the presence of a high concentration of heparin. Pathogenic anti-PF4 antibody was assessed by serotonin-release assay. RESULTS: Heparin-dependent anti-PF4 antibodies were detected in 11 SLE (9%) and 2 primary ITP (3%) patients, but at much lower levels than in HIT patients. In serotonin-release assays, only the HIT sera induced platelet activation in vitro. Heparin-independent anti-PF4 antibodies were detected in 17 SLE patients (14%). There was no correlation between the levels of heparin-dependent and -independent anti-PF4 antibodies. Cross-reactivity between these two antibodies was not detectable by ELISA competitive assay. Heparin-dependent anti-PF4 antibodies were associated with thrombocytopenia and IgM aCLs (P = 0.007 for both comparisons), while heparin-independent anti-PF4 antibody levels were correlated with SLE disease activity index (P = 0.0005). None of the SLE patients with anti-PF4 antibodies had previous heparin exposure. CONCLUSION: PF4 is an autoimmune target in SLE patients. Heparin-dependent and -independent anti-PF4 autoantibodies may be involved in different aspects of pathophysiology of SLE.

Fibronectin binding is required for acquisition of mesenchymal/endothelial differentiation potential in human circulating monocytes.

We previously reported monocyte-derived multipotential cells (MOMCs), which include progenitors capable of differentiating into a variety of mesenchymal cells and endothelial cells. In vitro generation of MOMCs from circulating CD14(+) monocytes requires their binding to extracellular matrix (ECM) protein and exposure to soluble factor(s) derived from circulating CD14(-) cells. Here, we investigated the molecular factors involved in MOMC generation by examining the binding of monocytes to ECM proteins. We found that MOMCs were obtained on the fibronectin, but not on type I collagen, laminin, or poly-L-lysine. MOMC generation was followed by changes in the expression profiles of transcription factors and was completely inhibited by either anti-α(5) integrin antibody or a synthetic peptide that competed with the RGD domain for the ß(1)-integrin binding site. These results indicate that acquisition of the multidifferentiation potential by circulating monocytes depends on their binding to the RGD domain of fibronectin via cell-surface α(5)ß(1) integrin.

Effect of Carbazochrome Sodium Sulfonate in Addition to Tranexamic Acid in Bleeding Trauma Patients.

BACKGROUND: It is important to evaluate the effects of drugs considered to control hemorrhage. Tranexamic acid (TXA) has been shown to reduce the risk of death in bleeding trauma patients. Carbazochrome sodium sulfonate (CSS) is often used in combination with TXA; however, it is unknown whether CSS additionally improves the control of bleeding in trauma patients. METHODS: The aim of this study was to examine whether CSS reduces blood transfusion and death in addition to TXA by improving the control of bleeding. We retrospectively analyzed medical records of trauma patients from 2011 to 2019. We included patients aged ≥16 years, with significant hemorrhage, and who received TXA within eight hours from injury as per CRASH-2 (Clinical Randomisation of an Antifibrinolytic in Significant Haemorrhage) study. The primary outcome was the total amount of red blood cells (RBC), fresh frozen plasma (FFP), and platelet concentrate (PC) received within the first 24 hours from injury. Secondary outcomes were death in hospital within four weeks after injury, vascular occlusive events, and treatment. RESULTS: During this retrospective evaluation period, 5764 admissions with trauma were registered. A total of 326 cases met the selection criteria: 259 cases who received CSS in addition to TXA (CSS group; n=259) and 67 cases who received only TXA (no-CSS group; n=67). The mortality rate was 6% in the no-CSS group and 15.1% in the CSS group. There was no significant difference in mortality and vascular occlusive events between the two groups. We performed multiple regression analyses, with the amount of blood transfusion for each type as explanatory variables. The administration of CSS was an independent factor for the reduction of RBC transfusion (standard partial regression coefficient -0.1, 95% CI [-3.1 to -0.1], p=0.04), but not for transfusion of FFP or PC. We also performed multiple logistic regression analysis, with death as an explanatory variable. CSS was not an independent factor for any cause of death. CONCLUSION: CSS decreased RBC transfusion in trauma patients, without increasing the risk of vascular occlusion. However, CSS did not decrease mortality. This study can contribute to managing bleeding with trauma, but further research aimed at clarifying the effect of CSS is needed.

Abatacept downregulates Fcγ receptor I on circulating monocytes: a potential therapeutic mechanism in patients with rheumatoid arthritis.

BACKGROUND: Abatacept is a recombinant fusion protein composed of the extracellular domain of cytotoxic T-lymphocyte antigen 4 and the Fc portion of immunoglobulin (Ig) G. The mechanism of action of abatacept in rheumatoid arthritis (RA) is believed to be competitive inhibition of T cell costimulation mediated by the binding of CD28 to CD80/CD86 on antigen-presenting cells, and recent studies have shown that abatacept induces reverse signaling in macrophages and osteoclast precursors in a T cell-independent manner. This study aimed to investigate the therapeutic effects of abatacept on circulating monocytes that contribute to RA pathogenesis. METHODS: Purified circulating monocytes derived from RA patients and controls were cultured in the absence or presence of abatacept or CD28-Ig for 24 h. The recovered cells were subjected to flow cytometry to evaluate the expression levels of cell surface molecules, and cytokines and chemokines in the culture supernatant were measured by multiplex bead arrays. The expression of candidate molecules was further examined by immunoblotting using total cellular extracts of the cultured monocytes. Finally, the effects of abatacept on cytokine production in monocytes stimulated with the immune complex of anti-citrullinated peptide antibodies (ACPAs) were examined. RESULTS: CD64/FcγRI was identified as a monocyte-derived molecule that was downregulated by abatacept but not CD28-Ig. This effect was observed in both RA patients and controls. The abatacept-induced downregulation of CD64/FcγRI was abolished by treatment with anti-CD86 antibodies but not anti-CD80 antibodies. Abatacept suppressed the production of interleukin (IL)-1ß, IL-6, C-C motif chemokine ligand 2, and tumor necrosis factor-α in cultured monocytes stimulated with the ACPA immune complex. CONCLUSIONS: The therapeutic effects of abatacept on RA are mediated, in part, by the downregulation of CD64/FcγRI on circulating monocytes via direct binding to CD86 and the suppression of immune complex-mediated inflammatory cytokine production.