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Objective Anti–citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). However, the molecular basis for ACPA production is still unclear. The purpose of this study was to determine if circulating plasmablasts from RA patients produce ACPAs and whether Porphyromonas gingivalis facilitates the generation of ACPAs. Methods Using a single-cell antibody cloning approach, we generated 217 and 110 monoclonal recombinant antibodies from circulating plasmablasts from 7 RA patients and 4 healthy controls, respectively. Antibody reactivity with citrullinated antigens was tested by a second-generation anti–cyclic citrullinated peptide (anti-CCP) kit and by enzyme-linked immunosorbent assays (ELISAs) against citrullinated human antigens. Antibody reactivity with P gingivalis was tested by ELISAs against outer membrane antigens (OMAs) and citrullinated enolase from P gingivalis. Results Approximately 19.5% of plasmablast-derived antibodies from anti-CCP–positive RA patients, but none from 1 anti-CCP–negative RA patient or the healthy controls, specifically recognized citrullinated antigens. The immunoglobulin genes encoding these ACPAs were highly mutated, with increased ratios of replacement mutations to silent mutations, suggesting the involvement of active antigen selection in ACPA generation. Interestingly, 63% of the ACPAs cross-reacted with OMAs and/or citrullinated enolase from P gingivalis. The reactivity of ACPAs against citrullinated proteins from P gingivalis was confirmed by immunoblotting and mass spectrometry. Furthermore, some germline-reverted ACPAs retained their reactivity with P gingivalis antigens but completely lost their reactivity with citrullinated human antigens. Conclusion These results suggest that circulating plasmablasts in RA patients produce ACPAs and that this process may be facilitated by anti–P gingivalis immune responses.

Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh) signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt)1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo) agonist purmophamine, which activates Shh signaling, increased the expression of growth-associated protein(GAP)-43, synaptophysin, Shh, Patched (Ptc)-1, Smo, glioma-associated oncogene homolog (Gli)-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1.

Chemokine ligand 12(CXCL12) mediates signaling through chemokine receptor 4(CXCR4), which is essential for the homing and maintenance of Hematopoietic stem cells (HSCs) in the bone marrow. FLT3-ITD mutations enhance cell migration toward CXCL12, providing a drug resistance mechanism underlying the poor effects of FLT3-ITD antagonists. However, the mechanism by which FLT3-ITD mutations regulate the CXCL12/CXCR4 axis remains unclear. We analyzed the relationship between CXCR4 expression and the FLT3-ITD mutation in 466 patients with de novo AML to clarify the effect of FLT3-ITD mutations on CXCR4 expression in patients with AML. Our results indicated a positive correlation between the FLT3-ITD mutant-type allelic ratio (FLT3-ITD MR) and the relative fluorescence intensity (RFI) of CXCR4 expression in patients with AML (r = 0.588, P ≤ 0.0001). Moreover, the levels of phospho(p)-STAT5, Pim-1 and CXCR4 proteins were positively correlated with the FLT3-ITD MR, and the mRNA levels of CXCR4 and Pim-1 which has been revealed as one of the first known target genes of STAT5, were upregulated with an increasing FLT3-ITD MR(P < 0.05). Therefore, FLT3-ITD mutations upregulate the expression of CXCR4 in patients with AML, and the downstream signaling intermediates STAT5 and Pim-1 are also involved in this phenomenon and subsequently contribute to chemotherapy resistance and disease relapse in patients with AML. However, the mechanism must be confirmed in further experiments. The combination of CXCR4 antagonists and FLT3 inhibitors may improve the sensitivity of AML cells to chemotherapy and overcome drug resistance.

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