Demethylating agents in combination with CD7-targeted CAR-T for the successful treatment of a case with mixed-phenotype acute leukemia relapsed after allogeneic hematopoietic stem cell transplantation: A Case Report.

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has cured many patients with malignant hematologic diseases such as mixed phenotype acute leukemia (MPAL), while those relapsing after allo-HSCT still exhibit high mortality, poor prognosis, and no standard treatment modalities. It is necessary to explore more therapeutic modalities for patients with post-transplant relapse to obtain a better prognosis. Case presentation: In this case report, a young male with MPAL received allo-HSCT after reaching complete remission (CR) by induction chemotherapy. Unfortunately, relapse of both myeloid and T lineages occurred nine months later. After receiving demethylating chemotherapy, myeloid lineage measurable residual disease (MRD) turned negative. T-lineage MRD turned negative after CD7-targeted chimeric antigen receptor (CAR)-T cell therapy. The bone marrow remained MRD-negative for 4 months. This case preliminarily demonstrated a long-lasting CR with CD7-targeted CAR-T cell therapy, allowing a better prognosis. Conclusion: Demethylating drugs combined with CD7-targeted CAR-T cell therapy is feasible in treating MPAL patients with relapse after transplantation, with good efficacy and safety, which will be a promising treatment option for MPAL.

Relationship between gut microbiota and lymphocyte subsets in Chinese Han patients with spinal cord injury.

This study is to investigate the changes of lymphocyte subsets and the gut microbiota in Chinese Han patients with spinal cord injury (SCI). We enrolled 23 patients with SCI and 21 healthy controls. Blood and fecal samples were collected. The proportion of lymphocyte subsets was detected by flow cytometry. 16S rDNA sequencing of the V4 region was used to analyze the gut microbiota. The changes of the gut microbiota were analyzed by bioinformatics. Correlation analysis between gut microbiota and lymphocyte subsets was performed. CD4 + cells, CD4 + /CD8 + ratio and CD4 + CD8 + cells in peripheral blood of SCI patients were significantly lower than those of the control group (P < 0.05). There was no significant difference in B cells and CIK cells between the SCI group and the control group. The gut microbiota community diversity index of SCI patients was significantly higher than that of healthy controls. In SCI patients, the relative abundance of Lachnospiraceae (related to lymphocyte subset regulation), Ruminococcaceae (closely related to central nervous system diseases), and Escherichia-Shigella (closely related to intestinal infections) increased significantly, while the butyrate producing bacteria (Fusobacterium) that were beneficial to the gut were dramatically decreased. Correlation analysis showed that the five bacterial genera of SCI patients, including Lachnospiraceae UCG-008, Lachnoclostridium 12, Tyzzerella 3, Eubacterium eligens group, and Rumencocciucg-002, were correlated with T lymphocyte subsets and NK cells. In the SCI group, the flora Prevotella 9, Lachnospiraceae NC2004 group, Veillonella, and Sutterella were positively correlated with B cells. However, Fusobacterium and Akkermansia were negatively correlated with B cells. Moreover, Roseburia and Ruminococcaceae UCG-003 were positively correlated with CIK cells. Our results suggest that the gut microbiota of patients with SCI is associated with lymphocyte subsets. Therefore, it is possible to improve immune dysregulation in SCI patients by modulating gut microbiota, which may serve as a new therapeutic method for SCI.

Ibuprofen-mediated potential inhibition of biofilm development and quorum sensing in Pseudomonas aeruginosa.

AIMS: Pseudomonas aeruginosa is one of the leading causes of opportunistic and hospital-acquired infections worldwide, which is frequently linked with clinical treatment difficulties. Ibuprofen, a widely used non-steroidal anti-inflammatory drug, has been previously reported to exert antimicrobial activity with the specific mechanism. We hypothesized that inhibition of P. aeruginosa with ibuprofen is involved in the quorum sensing (QS) systems. MAIN METHODS: CFU was utilized to assessed the growth condition of P. aeruginosa. Crystal violent staining and acridine orange staining was used to evaluate the biofilm formation and adherence activity. The detection of QS virulence factors such as pyocyanin, elastase, protease, and rhamnolipids were applied to investigation the anti-QS activity of ibuprofen against P. aeruginosa. The production of 3-oxo-C12-HSL and C4-HSL was confirmed by liquid chromatography/mass spectrometry analysis. qRT-PCR was used to identify the QS-related gene expression. Furthermore, we explored the binding effects between ibuprofen and QS-associated proteins with molecular docking. KEY FINDINGS: Ibuprofen inhibits P. aeruginosa biofilm formation and adherence activity. And the inhibitory effects of ibuprofen on C4-HSL levels were concentration-dependent (p < 0.05), while it has no effect on 3-oxo-C12-HSL. Moreover, ibuprofen attenuates the production of virulence factors in P. aeruginosa (p < 0.05). In addition, the genes of QS system were decreased after the ibuprofen treatment (p < 0.05). Of note, ibuprofen was binding with LuxR, LasR, LasI, and RhlR at high binding scores. SIGNIFICANCE: The antibiofilm and anti-QS activity of ibuprofen suggest that it can be a candidate drug for the treatment of clinical infections with P. aeruginosa.

Inhibition of siglec-1 by lentivirus mediated small interfering RNA attenuates atherogenesis in apoE-deficient mice.

BACKGROUND: Siglec-1 is highly expressed on circulating monocytes and plaque macrophages in atherosclerotic patients, but the exact role of Siglec-1 in atherosclerosis has not been elucidated. METHODS: Lentiviral vector containing small interfering RNA targeting Siglec-1 (Lv-shSiglec-1) or control vector (Lv-shNC) were injected intravenously into 6-week old Apoe-/- mice. Then onset of atherosclerosis was observed. RESULTS: Siglec-1 was highly expressed in aortic plaques and it can be down-regulated by Lv-shSiglec-1 injection. The plaque area and serum pro-inflammatory cytokine (IL-1ß, IL-6, TNF-α and IL-17A) levels in Lv-shSiglec-1 mice were significantly lower than Lv-shNC mice, whereas IL-10 was higher. Moreover, plaque macrophages accumulation in aortic wall in Lv-shSiglec-1 mice was diminish, partly by decreased secretion of MCP-1/CXCL2 and CCR2/CXCR2 of aortas and monocytes, respectively. Furthermore, silencing of Siglec-1 can attenuate oxLDL uptake by peritoneal macrophages. CONCLUSIONS: Inhibition of Siglec-1 can prevent atherosclerotic lesion formation by suppress monocytes-endothelial cells adhesion and macrophages accumulation.

APOC3 induces endothelial dysfunction through TNF-α and JAM-1.

BACKGROUND: The fatality rate for cardiovascular disease (CVD) has increased in recent years and higher levels of triglyceride have been shown to be an independent risk factor for atherosclerotic CVD. Dysfunction of endothelial cells (ECs) is also a key factor of CVD. APOC3 is an important molecule in lipid metabolism that is closely associated with hyperlipidemia and an increased risk of developing CVD. But the direct effects of APOC3 on ECs were still unknown. This study was aimed at determining the effects of APOC3 on inflammation, chemotaxis and exudation in ECs. METHODS: ELISA, qRT-PCR, immunofluorescence, flow cytometry and transwell assays were used to investigate the effects of APOC3 on human umbilical vein endothelial cells (HUVECs). SiRNA-induced TNF-α and JAM-1 silencing were used to observe how APOC3 influenced the inflammatory process in the ECs. RESULTS: Our results showed that APOC3 was closely associated with the inflammatory process in ECs, and that this process was characterized by the increased expression of TNF-α. Inflammatory processes further disrupted the tight junctions (TJs) between HUVECs by causing increased expression of JAM-1. JAM-1 was involved in maintaining the integrity of TJs, and it promoted the assembly of platelets and the exudation of leukocytes. Changes in its expression promoted chemotaxis and the exudation of ECs, which contributed to atherosclerosis. While the integrity of the TJs was disrupted, the adhesion of THP-1 cells to HUVECs was also increased by APOC3. CONCLUSIONS: In this study, we describe the mechanism by which APOC3 causes inflammation, chemotaxis and the exudation of ECs, and we suggest that controlling the inflammatory reactions that are caused by APOC3 may be a new method to treat CVD.

Increased expression of Siglec-1 on peripheral blood monocytes and its role in mononuclear cell reactivity to autoantigen in rheumatoid arthritis.

OBJECTIVES: Elevated expression of Siglec-1 on circulating monocytes has been reported in some inflammatory and autoimmune diseases, but its expression and role in RA has not been elucidated. The aims of this study were to determine the expression of Siglec-1 in peripheral blood and to explore its role in mononuclear cell reactivity to autoantigen in RA. METHODS: Siglec-1 protein and mRNA levels in 42 RA patients, 39 OA patients, 28 SLE patients and 42 normal controls were determined by flow cytometry and quantitative RT-PCR, respectively. In addition, 10 patients with active RA received DMARDs for 12 weeks and the frequencies of Siglec-1-positive cells and the 28-joint DAS (DAS28) were assessed before and after therapy. Furthermore, TNF-α, IFN-γ and type II collagen were used to up-regulate Siglec-1. Peripheral blood mononuclear cells (PBMCs) from different groups were stimulated with mitogens or antigens and cell proliferation and cytokine production were determined. RESULTS: The protein and mRNA levels of Siglec-1 on PBMCs and monocytes in RA patients were significantly higher than those in OA patients and healthy controls. Moreover, the expression of Siglec-1 protein on PBMCs was positively correlated with DAS28, ESR, high-sensitivity CRP and IgM-RF, but not with anti-CCP antibody. Interestingly, Siglec-1 expression was decreased in parallel with the decrease in the DAS28 after 12 weeks of anti-rheumatic treatment. Furthermore, TNF-α, IFN-γ and type II collagen can up-regulate Siglec-1 in PBMCs. Elevated PBMC proliferation and proinflammatory cytokine production to collagen stimulation in RA patients decreased when Siglec-1 was inhibited by anti-Siglec-1 antibodies. CONCLUSION: Elevated Siglec-1 expression in PBMCs and monocytes can potentially serve as a biomarker for monitoring disease activity in RA. Siglec-1 may also play a proinflammatory role in stimulating lymphocyte proliferation and activation in RA.

The role of Siglec-1 and SR-BI interaction in the phagocytosis of oxidized low density lipoprotein by macrophages.

BACKGROUND: Macrophages play a proatherosclerotic role in atherosclerosis via oxLDL uptake. As an adhesion molecular of I-type lectins, Siglec-1 is highly expressed on circulating monocytes and plaque macrophages of atherosclerotic patients, but the exact role of Siglec-1 has not been elucidated. METHODS: In this study, oxLDL was used to stimulate Siglec-1 and some oxLDL receptors (SR-BI, CD64, CD32B, LOX-1 and TLR-4) expression on bone marrow-derived macrophages, whereas small interfering RNA was used to down-regulate Siglec-1. Meanwhile, an ELISA-based assay for Siglec-1-oxLDL interaction was performed, and co-immunoprecipitation (co-IP) and laser scanning confocal microscopy (LSCM) were used to determine the role of Siglec-1 in oxLDL uptake by macrophages. RESULTS: We found that oxLDL could up-regulate the expression of various potential oxLDL receptors, including Siglec-1, in a dose-dependent manner. Moreover, down-regulation of Siglec-1 could attenuate oxLDL uptake by Oil red O staining. LSCM revealed that Siglec-1 and CD64/SR-BI may colocalize on oxLDL-stimulated macrophage surface, whereas co-IP showed that Siglec-1 and SR-BI can be immunoprecipitated by each other. However, no direct interaction between Siglec-1 and oxLDL was found in the in vitro protein interaction system. CONCLUSIONS: Thus, Siglec-1 can interact with SR-BI in the phagocytosis of oxLDL by macrophages, rather than act as an independent receptor for oxLDL.

Contribution of monocytes Siglec-1 in stimulating T cells proliferation and activation in atherosclerosis.

OBJECTIVE: Atherosclerosis (AS) is widely accepted as an inflammatory disease and monocytes are particularly important in inflammatory immune responses. As an important biomarker of monocytes activation, Siglec-1 is highly expressed on circulating monocytes and atherosclerotic plaques of coronary artery disease (CAD) patients, but the exact role of Siglec-1 has not been elucidated. METHODS: M-CSF, INF-α, IFN-γ, TNF-α and ox-LDL alone or in combination were used to stimulate Siglec-1 expression on monocytes, whereas small interfering RNA (si-RNA) or blocking antibody was used to down-regulate Siglec-1. Meanwhile, the role of Siglec-1 in chemokines secretion was determined. Then monocytes from CAD patients or healthy controls were cocultured with CD4+ or CD8+ T cells from a third healthy individual, and lymphocyte proliferation and activation were determined. RESULTS: All the stimuluses could enhance Siglec-1 expression on monocytes in a dose-dependent manner, and M-CSF could synergistically stimulate Siglec-1 expression with ox-LDL. Moreover, the secretion of MCP-1, MIP-1α and MIP-2 were enhanced when Siglec-1 was up-regulated and down to normal level when Siglec-1 was blocked. More importantly, increased Siglec-1 expression on monocytes was related to the increased T cell proliferation and pro-inflammatory cytokines secretion in CAD patients. However, down-regulation of Siglec-1 could attenuate proliferation and activation of cocultured CD4+ and CD8+ T cells. CONCLUSION: Siglec-1 can promote chemokines and pro-inflammatory cytokines secretion and influence the inflammatory process of AS.

Siglec-1 on monocytes is a potential risk marker for monitoring disease severity in coronary artery disease.

OBJECTIVES: Siglec-1 has long been considered as an important biomarker of the activation of monocyte/macrophage and a type I interferon-specific imprint, but its role in atherosclerosis has not been elucidated. METHODS: We examined the expression of Siglec-1 by flow cytometry and RT-PCR in 83 CAD patients and 38 healthy controls. In addition, the levels of serum lipids, Gensini score, hs-CRP and homocysteine were determined. RESULTS: The transcriptional and protein levels of Siglec-1 on monocytes in CAD patients were significantly increased compared with healthy controls [3.17 versus 1.0, P<0.01; (11.5+/-3.9)% versus (1.8+/-2.0)%, P<0.01], but the increased Siglec-1 had no correlation with the level of native serum lipids. Interestingly, the expression of Siglec-1 was positively correlated with Gensini score (r=0.338, P=0.015), hs-CRP (r=0.316, P=0.016) and homocysteine level (r=0.224, P=0.042). CONCLUSION: Siglec-1 may be considered as a potential non-invasive indicator for monitoring disease severity and a biomarker for predicting the relative risk of cardiovascular events.