The Role of Follicular Regulatory T Cells/Follicular Helper T Cells in Primary Immune Thrombocytopenia.

INTRODUCTION: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by thrombocytopenia. Herein, we sought to identify potential immune-related therapeutic targets in ITP. METHODS: The differentially expressed genes (DEGs) between ITP patients and controls in GSE43177 and PRJNA299534 were analyzed. The intersections of the two DEG groups were screened as common genes, and enrichment analysis was performed. Additionally, differential analysis of immune cell levels between ITP and controls was performed. Changes in the proportions of T follicular helper (Tfh) and follicular regulatory T (Tfr) cells in peripheral blood samples from ITP patients, ITP patients responding to therapy, and healthy controls were identified. The expression changes in B-cell lymphoma (Bcl)-6 and interleukin (IL)-21 were further evaluated. RESULTS: A total of 76 common genes were identified, and enrichment analysis found that these genes were mainly associated with neutrophil-mediated immunity, the MAPK signaling pathway, and the FOXO signaling pathway. Furthermore, we found different levels of Tfh cells in patients with ITP and controls. The level of Tfh cells in the peripheral blood is significantly increased in ITP patients and declines after responding to therapy. The Tfr/Tfh ratio was reduced in ITP patients and increased after responding to therapy. IL-21 and Bcl-6 were more highly expressed in ITP patients than in controls. CONCLUSION: We identified abnormally expressed genes in ITP related to immune-related biological functions. We further identified the changes in Tfh and Tfr cells during ITP treatment. This provides a rationale for immunotherapy in ITP patients.

Relationships of platelet glycoprotein specific antibody with therapeutic efficacy of short-term high-dose dexamethasone and bleeding score in the newly diagnosed adult patients with primary immune thrombocytopenia.

Objectives: We aimed to investigate relationships of platelet glycoprotein (GP) specific antibody with therapeutic efficacy of high-dose dexamethasone (HD-DXM) and bleeding score in primary immune thrombocytopenia (ITP) adults. Methods: A retrospective study was carried out to analyze relationships of polymorphism of GP specific antibody with initial therapeutic efficacy of HD-DXM and bleeding score of newly diagnosed ITP adults between 1 June, 2016 and 31 January, 2020. Results: 59 patients were involved in the study, with 33 cases of responders and 26 cases of non-responders between June 2016 and January 2020. At admission, there were 31 (52.5%) GP antibody-positive patients. Initial therapy of HD-DXM was effective for 78.6% GP antibody-negative patients and 35.5% GP antibody-positive patients, with a better therapeutic efficacy in patients with anti-GP Ib/IX antibody or anti-GP IIb/IIIa antibody but not in those with anti-GP Ib/IX antibody plus anti-GP IIb/IIIa antibody. Notably, therapeutic efficacy is much worse for minority (Uyghur) patients compared with corresponding Han patients. Similarly, it was much lower in GP antibody-positive patients compared with corresponding negative ones at low and medium bleeding score, with no response in GP antibody-positive patients at high bleeding score. Furthermore, there was a moderate negative correlation between therapeutic efficacy and GP-specific antibody (p < 0.05), but no obvious linear relationship between clinical bleeding degree and GP-specific antibody (p > 0.05). Conclusion: Collectively, the newly diagnosed ITP adults with GP-specific antibody have a poor response to short-term HD-DXM, especially in minority (Uyghur) patients with GP-specific antibody in China.

Imbalance of follicular regulatory T (Tfr) cells/follicular helper T (Tfh) cells in adult patients with primary immune thrombocytopenia.

This study is to investigate the role of follicular regulatory T (Tfr) cells/follicular helper T (Tfh) cells imbalance in adult patients with primary immune thrombocytopenia (ITP). Totally, 40 cases of primary ITP patients and 30 healthy controls were enrolled. Blood samples were collected from ITP patients (pre- and post-therapy) and controls. Flow cytometry was used to detect the proportion of Tfr and Tfh cells in peripheral blood. Real-time quantitative polymerase chain reaction (PCR) was performed to detect the mRNA expression levels of FOXP3, BCL-6, and BLIMP-1. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect interleukin (IL)-10 and IL-21 levels. Spearman's correlation was used for correlation analysis. Compared with control, Tfr cell proportion, FOXP3 mRNA, and IL-10 were significantly decreased in the pre-therapy ITP group, but were significantly increased post-therapy. Tfh cell proportion, BCL-6 mRNA, and IL-21 were increased, while BLIMP-1 mRNA was decreased, in the pre-therapy ITP group than the control group. These effects were reversed in the post-therapy ITP group. Moreover, the Tfr/Tfh ratio was decreased in the pre-therapy ITP group than control group, whereas was increased in the post-therapy ITP group than the pre-therapy ITP group. Furthermore, Tfr cell proportion, FOXP3 mRNA, IL-10, and Tfr/Tfh ratio were positively correlated with the platelet count (PLT) in the ITP pre-therapy group. In addition, Tfh cell proportion, BCL-6 mRNA, and IL-21 were negatively correlated with the PLT, while BLIMP-1 mRNA was positively correlated with the PLT. Conclusively, Tfr cell proportion in peripheral blood is decreased and Tfh cell proportion is increased, leading to unbalanced Tfr/Tfh ratio in ITP patients pre-therapy. The imbalance of Tfr/Tfh is recovered post-therapy, suggesting that the Tfr and Tfh cells may be involved in ITP pathogenesis. The abnormal expression of FOXP3, BCL-6, and BLIMP-1 mRNA and the changes in IL-10 and IL-21 levels may be related to the imbalance of Tfr/Tfh.

Clinical report on 5 cases of advanced malignancies with blood stasis and toxin treated by collateral disease theory.

BACKGROUND: "Collateral disease theory", as an important theory of traditional Chinese medicine, is often used in the clinical treatment of tumors, showing a remarkable effect, especially in advanced malignancies with blood stasis and toxin. METHODS: In this study, we analyzed 5 cases of advanced malignancies, and discussed the efficacy of collateral disease theory in advanced malignancies with blood stasis and toxin. The 5 cases were suffered from right lung squamous cell carcinoma, left lung squamous cell carcinoma, stage IV endometrial carcinoma, right submandibular lymphatic follicular lymphoma and right lower lung cancer, respectively. Combining with the pathogenesis of collateral disease in traditional Chinese medicine dialectically and taking insect medicine as an example, traditional Chinese medicine was prescribed. Furthermore, the application effect of "collateral disease theory" in malignancy with blood stasis and toxin was explored. RESULTS: After treated with traditional Chinese medicine, the tumor lesions in the 5 cases were reduced to varying degrees. CONCLUSION: The treatment based on "collateral disease theory" is effective for advanced malignancy with blood stasis and toxin, but this finding needs to be verified by prospective studies.

Effects of Shu Bu Wenshen Guchang recipe on intestinal injury and the TLR4/NF-κB signaling pathways in mice with irinotecan-induced delayed-type diarrhea.

Background: Irinotecan (also known as CPT-11) is a topoisomerase I inhibitor that is primarily used for the treatment of advanced colorectal cancer. CPT-11 and its active metabolite SN-38 can directly damage intestinal mucosal cells. In addition, CPT-11 can activate the Toll-like receptor 4 (TLR4) inflammasome/nuclear factor kappa-B p65 (NF-κB p65) pathway, ultimately leading to intestinal inflammation-related injury. Shu Bu Wenshen Guchang recipe (SBWGR) has the spleen and kidneys. Herein, we investigated the effects of SBWGR on intestinal injury and the TLR4/NF-κB signaling pathways in mice with CPT-11-induced delayed-type diarrhea, aiming to provide evidence for the treatment of CPT-11-induced delayed-type diarrhea. Methods: Thirty tumor-bearing mice were divided into normal control, model control, octreotide, low dose SBWGR, and high dose SBWGR groups, with 6 mice in each group. After successful modelling of delayed diarrhea, the normal and model control groups were given equal amounts of saline for 5 consecutive days, and the other three groups gave the corresponding intra-drug administration. Body weight, tumor size, Chiu score, intestinal ischemia and reperfusion injury, and disease activity index (DAI) were recorded in each group. The levels of intestinal interleukin-1ß (IL-1ß), IL-18, and tumor necrosis factor-α (TNF-α) were measured by an enzyme-linked immunosorbent assay (ELISA). Intestinal TLR4 and NF-κB p65 levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and protein blotting. Results: The weight of octreotide and kidney was higher than the control group (P<0.05); The tumor volume comparison of the model control group, octreotide group, warm kidney intestine low dose group, and warm kidney intestine high dose group were not significantly different (P>0.05). Octreotide group, intestinal Chiu score, diarrhea score, DAI level, intestinal inflammatory cytokines, IL-1ß, IL-18 and TNF-α intestinal level, intestinal TLR4, NF-κB p65 mRNA protein expression levels were significantly lower than those of the model control group (P<0.05), and the amount of the treatment group was increased (P<0.05). Conclusions: SBWGR exerts a prominent protective effect on intestinal damage caused by CPT-11-induced delayed-type diarrhea, which may be achieved by inhibiting the activation of the intestinal TLR4/NF-κB signaling pathway.

Regulation of Th1/Th2 and Th17/Treg by pDC/mDC imbalance in primary immune thrombocytopenia.

This study investigates the regulatory effect of plasmacytoid dendritic cells (pDC)/myeloid dendritic cells (mDC) imbalance on balance of Th1/Th2 and Th17/Treg in primary immune thrombocytopenia (ITP). A total of 30 untreated ITP patients and 20 healthy controls were recruited. Compared with healthy control, the pDC proportion of ITP patients was significantly reduced (P = 0.004), while the mDC proportion was not significantly changed (P = 0.681), resulting in a decrease in the pDC/mDC ratio (P = 0.001). Additionally, compared with controls, serum levels of interleukin (IL)-6, IL-12, and IL-23 were increased in ITP patients (P < 0.001), and mRNA levels of IL-12p40, IL-12p35, and IL-23p19 were also increased (P =0.014, P = 0.043, P < 0.001). Compared with the healthy control, the proportion of Th1 and Th17 cells in ITP patients increased (P = 0.001, P = 0.031). Serum levels of interferon gamma (IFN-γ) and IL-17 in ITP patients also increased (P = 0.025, P = 0.005). Furthermore, T-bet and RORγt mRNA levels were increased in peripheral blood of ITP patients (P = 0.018, P < 0.001). Correspondingly, the proportion of Th2 and Treg cells decreased (P = 0.007, P < 0.001), along with a decrease in serum IL-4 and transforming growth factor beta (TGF-ß) (P = 0.028, P = 0.042), and an increase in GATA-3 mRNA (P < 0.001). However, there was no significant difference in Foxp3 mRNA levels (P = 0.587). Pearson correlation analysis showed that the proportion of total dendritic cells (DCs) was positively correlated with IL-12 (r = 0.526, P = 0.003) and IL-23 (r = 0.501, P = 0.005) in ITP patients. Th1/Th2 ratio, IFN-γ, and IL-12 levels were negatively correlated with platelet counts (r = -0.494, P = 0.009; r = -0.415, P = 0.028; r = -0.492, P = 0.032). However, IL-23 was positively correlated with IL-17 (r = 0.489, P = 0.006) and negatively correlated with platelet count (r = -0.564, P = 0.001). The ratio of IL-6 and Th17 cells was negatively correlated with platelet count (r = -0.443, P = 0.014; r = -0.471, P = 0.011). The imbalance of pDC/mDC and the increase of IL-6, IL-12, and IL-23 lead to the increased differentiation of CD4+ T cells into Th1 and Th17 cells, which might be the important mechanisms underlying the imbalance of Th1/Th2 and Th17/Treg in ITP patients.

Multifunctional homochiral lanthanide camphorates with mixed achiral terephthalate ligands.

Seven homochiral lanthanide camphorates with mixed achiral terephthalate ligands, namely, {Ln(2)(cam)(2)(bdc)(H(2)O)(2)}·DMF [Ln = Sm (1), Eu (2), Gd (3), Tb (4), Dy (5), Ho (6), Er (7); d-H(2)cam = d-(+)-camphoric acid; H(2)bdc = 1,4-benzenedicarboxylic acid; DMF = N,N'-dimethylformamide], have been solvothermally synthesized. Single-crystal X-ray diffraction analyses reveal that compounds 1-7 are isostructural and crystallize in orthorhombic, chiral space group P2(1)2(1)2(1). These structures feature three-dimensional open frameworks based on rodlike [Ln(2)(OCO)(6)(H(2)O)(2)](n) secondary building units, with the guest DMF molecules occupying the void space of the host {Ln(2)(cam)(3)(bdc)(H(2)O)(2)} framework. The photophysical and magnetic properties of some of these complexes have been investigated. Notably, the terbium compound 4 is highly emissive with a quantum yield of 63.68%. Additionally, thermogravimetric analyses, variable-temperature IR spectroscopy, and powder X-ray diffraction of 2 as a representative were performed to determine its thermal stability, which indicates that the framework still remains intact until 300 °C.

PD-1/PD-L1 pathway activation restores the imbalance of Th1/Th2 and treg/Th17 cells subtypes in immune thrombocytopenic purpura patients.

This study aims to investigate the changes of cytokines and the effect of programmed death ligand 1 (PD-L1) signaling pathway on T cell function in patients with immune thrombocytopenic purpura (ITP).Totally, 40 untreated ITP patients were recruited and 30 healthy people were recruited as the healthy control. Then whole blood of ITP patients and healthy control was collected, respectively. The sPD-L1/anti-PD-1 was used to activate or block the programmed death (PD-1)/PD-L1 signaling pathway. The expression of PD-1 and PD-L1 on peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry. PBMCs were treated with cluster of differentiation (CD3), cluster of differentiation 28 (CD28), and phytohaemagglutinin (PHA) for 48 hours. Serum levels of sPD-1, sPD-L1, and cytokines were measured by enzyme-linked immunosorbent assay (ELISA).Compared with the healthy control group, the percentages of PD-1+CD3+CD4+ T cells and PD-L1+HLA-DR+CD11c+ DC cells were increased in ITP patients. The levels of interferon-gamma (IFN-γ), interleukin-17 (IL-17), and sPD-1 in the serum of ITP patients were increased, while IL-4 and transforming growth factor-ß (TGF-ß) were decreased. Additionally, the level of sPD-1 was negatively correlated with the platelet count. Consistently, after treatment with CD3, CD28, and PHA, IFN-γ and IL-17 levels in culture supernatant of PBMCs from ITP patients were significantly higher than those from healthy controls whereas IL-4 and TGF-ß levels were significantly lower. Furthermore, IFN-γ and IL-17 levels secreted by PBMCs from ITP patients decreased after sPD-L1 administration, however, IL-4 and TGF-ß levels were increased. The level of IFN-γ in ITP group remained higher after anti-PD-1 blockage, but the levels of IL-4, TGF-ß, and IL-17 were not significantly influenced.sPD-1 may cause the dysfunction of PD-1/PD-L1 signaling pathway, and its level is related to the severity of ITP patients. Activation of PD-1/PD-L1 with sPD-L1 may restore the imbalance of Th1/Th2 and Treg/Th17 cell subtypes in ITP patients but anti-PD-1 may exacerbate disease by enhancing IFN-γ production.

Chemical and structural analysis on magnetic tunnel junctions using a decelerated scanning electron beam.

Current information technology relies on the advancement of nanofabrication techniques. For instance, the latest computer memories and hard disk drive read heads are designed with a 12 nm node and 20 nm wide architectures, respectively. With matured nanofabrication processes, a yield of such nanoelectronic devices is typically up to about 90%. To date the yield has been compensated with redundant hardware and software error corrections. In the latest memories, approximately 5% redundancy and parity bits for error corrections are used, which increase the total production cost of the devices. This means the yield directly affects the device costs. It is hence critical to increase the yield in nanofabrication. In this paper, we have applied our recently developed method to image buried interfaces in combination with chemical analysis to evaluate magnetic tunnel junctions and have revealed their different magnetoresistance ratios caused by the presence of materials formed at the junction edges. The formation of these materials can be avoided by optimising the junction patterning process to remove residual carbon introduced from resist. Our imaging method with chemical analysis have demonstrated a significant potential for the improvement of junction performance, resulting in higher yields. This can be used as a quality assurance tool in a nanoelectronic device production line.

Percentages of PD-1+CD4+T cells and PD-L1+DCs are increased and sPD-1 level is elevated in patients with immune thrombocytopenia.

The present study is to measure the expression of programmed death (PD)-1 / programmed death ligand-1 (PD-L1) negative costimulatory molecules, soluble format sPD-1 in patients with immune thrombocytopenia (ITP), and to investigate their correlation with the secretion of cytokines. A total of 35 patients with ITP were included in the present study. Twenty healthy subjects who received physical examination at our hospital were included as control group. Peripheral blood was collected from all ITP patients and healthy subjects. Flow cytometry was performed to determine the percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients and healthy subjects. Enzyme-linked immunosorbent assay was performed to measure the concentrations of interferon (IFN)-γ, interleukin (IL)-17 and sPD-1 in peripheral blood from ITP patients and healthy subjects. Percentages of PD-1+CD4+T cells and PD-L1+DCs in peripheral blood from ITP patients before treatment were significantly higher than that from healthy subjects, but were not different from those after treatment. Serum concentrations of IFN-γ, IL-17 and sPD-1 in ITP patients before treatment were significantly higher than those in healthy subjects, and these concentrations were significantly reduced after treatment. The concentration of sPD-1 was positively correlated with the concentration of IFN-γ, and negatively correlated with platelet count. Percentages of PD-1+CD4+T cells and PD-L1+DCs in ITP patients are higher than those in healthy subjects, but elevated sPD-1 concentration in the blood blocks PD-1/PD-L1 signaling pathway, leading to unaffected Th cell function. Elevated concentrations of IFN-γ and IL-17 in the blood may participate in the occurrence and development of ITP.

The effect of single course high dose dexamethasone on CD28/CTLA-4 balance in the treatment of patients with newly diagnosed primary immune thrombocytopenia.

To evaluate the effect of a single course of high dose dexamethasone (HD-DXM) on CD28 and CTLA-4 expression in patients with newly-diagnosed primary immune thrombocytopenia (ITP). Twenty-8 ITP patients (18 females and 10 males, age range 18-65 years, median age 38.5 years) enrolled in this study and 26 healthy volunteers (19 women and 7 men, age range 16-66 years, median age 37 years) served as a control group. The patients were treated with HD-DXM (40 mg/day) for 4 consecutive days. CD28 and CTLA-4 expression was assessed by flow cytometry once-monthly for 6 months. Plasma levels of the cytokines IFN-γ and IL-10 were determined by enzyme-linked immunosorbent assay. One month after treatment, a platelet response was observed in 23 (82%) of the patients. The response rates over the next 5 months were 71%, 57%, 53%, 46%, and 39%, chronologically. We observed a significant decrease in CD28 expression after the first month (34.7 ± 4.8% vs. 44.5 ± 4.4% before treatment), after which the CD28 levels gradually increased. In contrast, CTLA-4 expression increased after the first month (3.2 ± 0.5% vs. 0.8 ± 0.4 before treatment), after which the CTLA-4 levels gradually decreased. Similar dynamic changes were seen in the levels of IFN-γ and IL-10. The dynamic changes of CD28 and CTLA-4 were consistent with those of IFN-γ and IL-10 and with the effectiveness of HD-DXM in the treatment of ITP. Our results suggest that a disturbed CD28/CTLA-4 balance may contribute to the immunopathogenesis of ITP.

Exosome-mediated Let-7a inhibits malignant biological behaviors of triple-negative breast cancer cells by down-regulating MYC expression / 中国肿瘤生物治疗杂志

@#Objective: To investigate the role of exosome (EXO) transporting Let-7a to regulate MYC gene in the malignant biological behaviors of triple negative breast cancer (TNBC) cell, and to explore the underlying mechanism. Methods: After the completion of cell culture, the gene and protein expressions of MYC and Let-7a in TNBC MDA-MB-231cells were detected by qPCR and WB, respectively. Recombinant lenti-virus vector carrying Let-7a and Crisper/Cas-9 system with MYC knockdown were transfected into MDA-MB-231 cells; MTT assay, Transwell assay and Scratch healing assay were performed to examine the proliferation, invasion and migration of MDA-MB-231 cells. Luciferase activity assay was performed to validate the binding between MYC and Let-7a. EXO was isolated and identified by transmission electron microscopy and WB assay in wild-type and Let-7a over-expressed MDA-MB-231 cells, respectively. After co-incubation of two types of EXO and MDA-MB-231 cells, the effects of Let-7a on biological behaviors of MDAMB-231 cells via EXO were detected by qPCR, WB, MTT and Transwell etc. Results: Let-7a was negatively correlated with MYC in breast cancer tissues and cell lines (all P<0.05); MYC promoted while Let-7a inhibited the proliferation, migration and invasion of breast cancer cells (all P<0.01); Let-7a silenced MYC by acting on 3'UTR of MYC gene, thereby reducing the expression of MYC protein (P<0.05); Let-7a was enveloped by EXO and transported to cancer cells, there by inhibiting the proliferation, migration and invasion of MDA-MB-231 cells. Conclusion: EXO some mediated Let-7a silences MYC gene by acting on its 3'UTR region, thus inhibiting the proliferation, migration and invasion of MDA-MB-231 cells.