Blockade of CD300A enhances the ability of human NK cells to lyse hematologic malignancies

Objective: The human cluster of differentiation(CD)300A, a type-I transmembrane protein with immunoreceptor tyrosine-based inhibitory motifs, was investigated as a potential immune checkpoint for human natural killer(NK) cells targeting hematologic malignancies(HMs).Methods: We implemented a stimulation system involving the CD300A ligand, phosphatidylserine(PS), exposed to the outer surface of malignant cells. Additionally, we utilized CD300A overexpression, a CD300A blocking system, and a xenotransplantation model to evaluate the impact of CD300A on NK cell efficacy against HMs in in vitro and in vivo settings. Furthermore, we explored the association between CD300A and HM progression in patients.Results: Our findings indicated that PS hampers the function of NK cells. Increased CD300A expression inhibited HM lysis by NK cells. CD300A overexpression shortened the survival of HM-xenografted mice by impairing transplanted NK cells. Blocking PS–CD300A signals with antibodies significantly amplified the expression of lysis function-related proteins and effector cytokines in NK cells, thereby augmenting the ability to lyse HMs. Clinically, heightened CD300A expression correlated with shorter survival and an “exhausted” phenotype of intratumoral NK cells in patients with HMs or solid tumors.Conclusions: These results propose CD300A as a potential target for invigorating NK cell-based treatments against HMs.

Identification of prognostic molecular subtypes and model based on CD8+ T cells for lung adenocarcinoma

Background:Cytotoxic T lymphocytes(CD8+T)cells function critically in mediating anti-tumor immune response in cancer patients.Characterizing the specific functions of CD8+T cells in lung adenocarcinoma(LUAD)could help better understand local anti-tumor immune responses and estimate the effect of immunotherapy.Methods:Gens related to CD8+T cells were identified by cluster analysis based on the single-cell sequencing data of three LUAD tissues and their paired normal tissues.Weighted gene co-expression network analysis(WGCNA),consensus clustering,differential expression analysis,least absolute shrinkage and selection operator(LASSO)and Cox regression analysis were conducted to classify molecular subtypes for LUAD and to develop a risk model using prognostic genes related to CD8+T cells.Expression of the genes in the prognostic model,their effects on tumor cell invasion,and interactions with CD8+T cells were verified by cell experiments.Results:This study defined two LUAD clusters(CD8+0 and CD8+1)based on CD8+T cells,with cluster CD8+0 being significantly associated with the prognosis of LUAD.Three heterogeneous subtypes(clusters 1,2,and 3)differing in prognosis,genome mutation events,and immune status were categorized using 42 prognostic genes.A prognostic model created based on 11 significant genes(including CD200R1,CLEC17A,ZC3H12D,GNG7,SNX30,CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2,and KRT81)was able to independently estimate the death risk for patients in different LUAD cohorts.Moreover,the model also showed general applicability in external validation cohorts.Low-risk patients could benefit more from taking immunotherapy and were significantly related to the resistance to anticancer drugs.The results from cell experiments demonstrated that the expression of CD200R1,CLEC17A,ZC3H12D,GNG7,and SNX30 was significantly downregulated,while that of CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2 and KRT81 was upregulated in LUAD cells.Inhibition of CD200R1 greatly increased the invasiveness of the LUAD cells,but inhibiting CDCP1 expression weakened the invasion ability of LUAD cells.Conclusion:This study defined two prognostic CD8+T cell clusters and classified three heterogeneous molecular subtypes for LUAD.A prognostic model predictive of the potential effects of immunotherapy on LUAD patients was developed.

nCD11b、mCD14和mCD86表达对新生儿败血症病情严重程度的预测价值

目的 探讨中性粒细胞CD11b (nCD11b)、单核细胞CD14 (mCD14)和单核细胞CD86 (mCD86)表达对新生儿败血症病情严重程度的预测价值。方法 选取2018年2月至2022年2月南阳市中心医院收治的73例新生儿败血症作为疾病组,根据患儿是否发生休克分为休克组26例和非休克组47例,另选取同期体检的足月健康新生儿73例为健康组。采用流式细胞法检测所有新生儿的外周血n CD11b、m CD14和m CD86表达水平,比较各组新生儿外周血n CD11b、mCD14和m CD86表达水平,并采用受试者工作特征曲线(ROC)分析其对新生儿败血症病情程度的预测价值。结果 疾病组新生儿的外周血n CD11b、mCD86表达水平分别为(220.00±12.58) MFI、(62.89±7.69) MFI,明显高于健康组的(186.69±10.98) MFI、(41.27±5.09) MFI,而外周血m CD14表达水平为(38.85±6.27) MFI,明显低于健康组的(54.03±6.15) MFI,差异均有统计学意义(P<0.05);休克组新生儿的外周血nCD11b、m CD86表达水平分别为(227.69±11.62) MFI、(67.96±6.18) MFI,明显高于非休克组的(215.74±12.95) MFI、(60.08±8.29) MFI,而外周血m CD14表达水平为(34.99±5.83) MFI,明显低于非休克组的(40.98±6.54) MFI,差异均有统计学意义(P<0.05);经ROC分析结果显示,外周血nCD11b、m CD14、mCD86单独及其联合检测对新生儿败血症病情程度的曲线下面积(AUC)分为0.850、0.804、0.815和0.930,联合检测AUC均高于其单独检测(P<0.05)。结论 外周血n CD11b、m CD14、m CD86检测可用于新生儿败血症病情严重程度评估,且联合检测可提高新生儿败血症病情严重程度预测价值。

儿童急性呼吸道感染者PA、PSP、CD64、sCD14-ST的变化及其诊断价值

目的 探讨儿童急性呼吸道感染者血前白蛋白(PA)、血清胰石蛋白(PSP)、免疫球蛋白G Fc段受体I(CD64)、可溶性白细胞分化抗原14亚型(sCD14-ST)的变化及其临床诊断价值。方法 回顾性选取2019年9月至2023年3月眉山市四所医院收治的498例儿童急性呼吸道感染患儿作为研究组,根据咽拭子细菌培养结果将其分为细菌感染组232例和非细菌感染组266例;另选取同期体检的健康儿童506例作为对照组。检测所有儿童的血清PA、PSP、外周血sCD14-ST水平、CD64指数。比较三组儿童的临床资料,同时比较研究组和对照组以及细菌感染组和非细菌感染组患儿的血清PA、PSP、外周血sCD14-ST水平、CD64指数;采用受试者工作特征曲线(ROC)分析血清PA、PSP、外周血sCD14-ST水平、CD64指数单独及联合检测对儿童急性呼吸道感染的诊断价值。结果 研究组患儿的血清PA水平明显低于对照组,而血清PSP、外周血sCD14-ST水平、CD64指数则明显高于对照组,差异均有统计学意义(P<0.05);细菌感染组患儿的血清PA水平明显低于非细菌感染组,而血清PSP、外周血sCD14-ST水平、CD64指数则明显高于非细菌感染组,差异均有统计学意义(P<0.05);绘制ROC获得血清PA、PSP、外周血sCD14-ST水平、CD64指数单独及联合检测诊断儿童急性呼吸道感染患儿的AUC分别为0.885、0.780、0.818、0.848、0.934。其中,联合诊断的AUC高于各项单独检测(P<0.05),且联合检测的敏感度和特异度为89.20%,81.40%,诊断价值均较高。结论 儿童急性呼吸道感染者血清PA水平呈低表达,血清PSP、外周血sCD14-ST水平、CD64指数呈高表达;且细菌感染较非细菌感染的儿童急性呼吸道感染患儿血清PA水平降低,而血清PSP、外周血sCD14-ST水平和CD64指数则升高;联合检测血清PA、PSP、外周血sCD14-ST水平、CD64指数有助于诊断儿童急性呼吸道感染。

CD14基因启动子区-159C/T多态性在H型高血压患者中的分布

目的:调查H型高血压患者中CD14基因启动子区-159C/T基因多态性分布情况。方法:用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析技术对H型高血压组、普通高血压组和对照组的CD14基因启动子区-159C/T多态性分布进行研究,同时检测血清同型半胱氨酸(HCY)及其他生化指标。结果:H型高血压组CC型、CT型、TT型分布频率分别为10%、43%、47%,C和T等位基因频率分别为31.5%、68.5%;普通高血压组CC型、CT型、TT型分布频率分别为10%、50%、40%,C和T等位基因频率分别为35%、65%;对照组CC型、CT型、TT型分布频率分别为13%、53%、34%,C和T等位基因频率分别为39.5%、60.5%,三组基因分型和等位基因频率均无统计学意义(P>0.05)。与对照组相比,H型高血压组、普通高血压组高密度脂蛋白(HDL-C)水平降低,差异有统计学意义(P<0.05)。结论:H型高血压患者CD14基因启动子区-159C/T多态性分布无明显差异。

外周血CD4^(+)PD-1^(+)Tcells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性分析

目的 探讨外周血CD4^(+)程序性细胞死亡受体-1(PD-1)^(+)T cells及CD4^(+)T淋巴细胞三磷酸腺苷(ATP)含量与复发性卵巢癌疗效的相关性。方法 选取30例复发性卵巢癌患者为复发组,30例未复发卵巢癌患者为非复发组;另选取30名同期体检健康者作为对照组。评估外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性。结果 复发组和非复发组的CD4^(+)PD-1^(+)T cells较对照组明显升高(P<0.05)。复发组和非复发组的CD4^(+)T淋巴细胞ATP含量较对照组明显降低(P<0.05)。复发组治疗后CD4^(+)PD-1^(+)Tcells显著低于治疗前(P<0.05),治疗后CD4^(+)T淋巴细胞ATP含量显著高于治疗前(P<0.05)。CD4^(+)PD-1^(+)T cells与复发性卵巢癌疗效成负相关(r=-0.393,P=0.039),CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效成正相关(r=0.449,P=0.031)。结论 复发性卵巢癌患者外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与疗效密切相关。

Donor-derived CD 19 CAR-T Cells versus Chemotherapy Plus Donor Lymphocyte Infusion for Treatment of Recurrent CD 19-positive B-ALL after Allogeneic Hematopoietic Stem Cell Transplantation

Objective:This study aimed to compare the efficacy of anti-CD19 chimeric antigen receptor T cells(CAR-T cells)versus chemotherapy plus donor lymphocyte infusion(chemo-DLI)for treating relapsed CD 19-positive B-cell acute lymphoblastic leukemia(B-ALL)after allogeneic hematopoietic stem cell transplantation(allo-HSCT).Methods:Clinical data of 43 patients with B-ALL who relapsed after allo-HSCT were retrospectively analyzed.Twenty-two patients were treated with CAR-T cells(CAR-T group),and 21 with chemotherapy plus DLI(chemo-DLI group).The complete remission(CR)and minimal residual disease(MRD)-negative CR rates,leukemia-free survival(LFS)rate,overall survival(OS)rate,and incidence of acute graft-versus-host disease(aGVHD),cytokine release syndrome(CRS)and immune effector cell-associated neurotoxicity syndrome(ICANS)were compared between the two groups.Results:The CR and MRD-negative CR rates in the CAR-T group(77.3%and 61.5%)were significantly higher than those in the chemo-DLI group(38.1%and 23.8%)(P=0.008 and P=0.003).The 1-and 2-year LFS rates in the CAR-T group were superior to those in the chemo-DLI group:54.5%and 50.0%vs.9.5%and 4.8%(P=0.0001 and P=0.00004).The 1-and 2-year OS rates in the CAR-T versus chemo-DLI group were 59.1%and 54.5%vs.19%and 9.5%(P=0.011 and P=0.003).Six patients(28.6%)with grade 2-4 aGVHD were identified in the chemo-DLI group.Two patients(9.1%)in the CAR-T group developed grade 1-2 aGVHD.Nineteen patients(86.4%)developed CRS in the CAR-T group,comprising grade 1-2 CRS in 13 patients(59.1%)and grade 3 CRS in 6 patients(27.3%).Two patients(9.1%)developed grade 1-2 ICANS.Conclusion:Donor-derived anti-CD19 CAR-T-cell therapy may be better,safer,and more effective than chemo-DLI for B-ALL patients who relapse after allo-HSCT.

CD14反向辅助设门法在淋巴细胞亚群检测中的应用

目的建立CD14反向辅助设门法(简称CD14反向法),并探讨其在淋巴细胞亚群检测中的应用。方法在常规6色流式细胞术检测方案(常规法)中加入CD14-BV421抗体,建立CD14反向法。采用常规法和CD14反向法分别检测质控品、不同放置时间(4、24、48、72 h)的样本和不同疾病(系统性红斑狼疮、病毒性肺炎和肾移植术后)患者的淋巴细胞亚群,比较2种方法检测结果的差异和偏移情况。结果CD14反向法检测细胞低值质控品的淋巴细胞百分比(LYMPH%)和自然杀伤(NK)细胞百分比(NK%)低于常规法(P<0.05);细胞中值质控品2种方法之间所有项目差异均无统计学意义(P>0.05)。与放置4 h比较,CD14^(+)单核细胞与淋巴细胞的区分度随样本放置时间的延长而变小。与全血样本室温放置4 h比较,放置24 h后CD14反向法和常规法各项目的相对偏移均<5%,差异无统计学意义(P>0.05);放置48 h,CD14反向法各项指标相对偏移均<5%,常规法细胞毒性T淋巴细胞(CTL)(CD3^(+)CD8^(+))、B细胞(CD3-CD19^(+))百分比及其绝对数的相对偏移均>5%;放置72 h,2种方法所有项目的相对偏移均>10%,为临床不可接受。采用CD14反向法检测系统性红斑狼疮、病毒性肺炎、肾移植术后患者外周血淋巴细胞亚群,可较好地区分淋巴细胞和单核细胞,同时减少非典型单核细胞对NK%和NK细胞绝对数的影响。结论CD14反向法可减少流式细胞术检测中淋巴细胞门的误圈,有助于提高检测结果的准确性。

血清sTREM-1、suPAR和sCD14水平对妊娠期肾病综合征妊娠结局不良的预测效能

目的观察血清可溶性髓系细胞触发受体-1(sTREM-1)、可溶性尿激酶型纤溶酶原激活物受体(suPAR)和可溶性CD14(sCD14)水平对妊娠期肾病综合征(NSP)患者妊娠结局不良的预测效能。方法选择2020年1月至2022年12月在该院诊断为NSP的患者98例作为NSP组,根据NSP患者24 h尿蛋白定量将其分为轻度蛋白尿组(32例)、中度蛋白尿组(22例)和重度蛋白尿组(44例)。另选择同期在本院定期产检的健康孕妇45例作为正常妊娠组。观察各组血清sTREM-1、suPAR和sCD14水平。采用二元Logistic回归分析影响妊娠结局不良的因素;采用受试者工作特征(ROC)曲线分析血清sTREM-1、suPAR和sCD14水平对妊娠结局不良的诊断效能。结果NSP组血清sTREM-1、suPAR和sCD14水平均明显高于正常妊娠组(P<0.05);重度蛋白尿组血清sTREM-1、suPAR和sCD14水平均明显高于中度蛋白尿组和轻度蛋白尿组,而中度蛋白尿组血清sTREM-1、suPAR和sCD14水平均明显高于轻度蛋白尿组,差异均有统计学意义(P<0.05)。根据妊娠结局的不同,将NSP患者分为妊娠结局不良组(25例)和妊娠结局良好组(73例)。妊娠结局不良组尿蛋白、同型半胱氨酸、sTREM-1、suPAR和sCD14水平均明显高于妊娠结局良好组(P<0.05)。二元Logistic回归分析结果发现血清sTREM-1、suPAR和sCD14水平升高是发生妊娠结局不良的危险因素(P<0.05)。血清sTREM-1、suPAR和sCD14水平联合检测NSP患者发生妊娠结局不良的灵敏度为88.0%,特异度为95.9%,曲线下面积(AUC)为0.924,明显高于sTREM-1(z=2.039,P=0.041)、suPAR(z=2.143,P=0.032)和sCD14(z=2.417,P=0.016)单独检测,但血清sTREM-1、suPAR和sCD14单独检测的AUC比较,差异无统计学意义(P>0.05)。结论血清sTREM-1、suPAR和sCD14可以作为评估NSP严重程度的检测指标,且三项联合检测有助于提高NSP患者发生妊娠结局不良的诊断效能。

Subgroups of peripheral immune effector cells in cervical cancer patients are more sensitive to radiation therapy than chemotherapy

Background:CD8 positive T lymphocytes and natural killer(NK)cells in the peripheral blood of cervical cancer patients exhibit varying sensitivities to radiotherapy and chemotherapy.Methods:A total of 50 healthy peoples and 60 cervical cancer patients were recruited.The patients with cervical cancer were separated into two groups:radiation and chemotherapy,and blood sample were collected before and after treatment.Data on the proportion of CD8 positive T lymphocytes and NK cells were gathered for analytical evaluation.Results:Compared to healthy individuals,patients with cervical cancer exhibit a reduced proportion of CD8 positive T cells within their peripheral blood.And for patients with cervical cancer,radiation therapy has been found to be more effective than chemotherapy in increasing the proportion of CD8 positive T lymphocytes and NK cells.Conclusions:These results suggest that radiation therapy increases the levels of CD8 positive T lymphocytes and NK cells within the peripheral blood of patients with cervical cancer.The study hypothesis that the changes in the percentage of CD8 positive T lymphocytes may serve as a potential indicator for predicting treatment efficacy.

TNFSF15 facilitates the differentiation of CD11b^(+) myeloid cells into vascular pericytes in tumors

Objective:Immature vasculature lacking pericyte coverage substantially contributes to tumor growth,drug resistance,and cancer cell dissemination.We previously demonstrated that tumor necrosis factor superfamily 15(TNFSF15)is a cytokine with important roles in modulating hematopoiesis and vascular homeostasis.The main purpose of this study was to explore whether TNFSF15 might promote freshly isolated myeloid cells to differentiate into CD11b^(+) cells and further into pericytes.Methods:A model of Lewis lung cancer was established in mice with red fluorescent bone marrow.After TNFSF15 treatment,CD11b^(+) myeloid cells and vascular pericytes in the tumors,and the co-localization of pericytes and vascular endothelial cells,were assessed.Additionally,CD11b^(+) cells were isolated from wild-type mice and treated with TNFSF15 to determine the effects on the differentiation of these cells.Results:We observed elevated percentages of bone marrow-derived CD11b^(+)myeloid cells and vascular pericytes in TNFSF15-treated tumors,and the latter cells co-localized with vascular endothelial cells.TNFSF15 protected against CD11b^(+)cell apoptosis and facilitated the differentiation of these cells into pericytes by down-regulating Wnt3a-VEGFR1 and up-regulating CD49e-FN signaling pathways.Conclusions:TNFSF15 facilitates the production of CD11b^(+) cells in the bone marrow and promotes the differentiation of these cells into pericytes,which may stabilize the tumor neovasculature.

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

Peripheral CD4^(+)CD8^(+) double positive T cells:A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus

Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

Repetitive administration of cultured human CD34+cells improve adenine-induced kidney injury in mice

BACKGROUND There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease(CKD).AIM To examine the efficacy of cultured human CD34+cells with enhanced proliferating potential in kidney injury in mice.METHODS Human umbilical cord blood(UCB)-derived CD34+cells were incubated for one week in vasculogenic conditioning medium.Vasculogenic culture significantly increased the number of CD34+cells and their ability to form endothelial progenitor cell colony-forming units.Adenineinduced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice,and cultured human UCB-CD34+cells were administered at a dose of 1×106/mouse on days 7,14,and 21 after the start of adenine diet.RESULTS Repetitive administration of cultured UCB-CD34+cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group.Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group(P<0.01).Microvasculature integrity was significantly preserved(P<0.01)and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group(P<0.001).CONCLUSION Early intervention using human cultured CD34+cells significantly improved the progression of tubulointerstitial kidney injury.Repetitive administration of cultured human UCB-CD34+cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

The Mechanisms of CD8+ T Cells Exhaustion in the Tumor Microenvironment and Immune Therapy

In the tumor immune microenvironment, CD8+ T cells differentiate towards functional failure. The exhaustion of CD8+ T cells (Tex) showed varying degrees of effect dysfunction, loss of proliferation ability, and sustained high expression of a variety of inhibitory receptors, with metabolic and epigenetic changes. Tex cells are heterogeneous, including several subsets with different characteristics at different stages of differentiation. Immune checkpoint inhibitors (ICIs) can restore the effect or function of Tex cells, indicating that this T cell subset plays a key role in tumor immunotherapy. The understanding of the mechanism of CD8+ T cell exhaustion will be helpful to the implementation of tumor immunotherapy. This article reviews the production, differentiation and functional characteristics of Tex cells and their relationship with tumor immunotherapy.

Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease

AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM)or the reverse transcription polymerase chain reaction (RTPCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.RESULTS: In ethanol-fed group, the percentages of FITCCD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 %and 5.38 %), the difference was significant (P＜0.05). The expressions of CD14 mRNA were 7.56±1.02 and 8.74±1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77±0.21 and 1.98±0.23)(P＜0.05). Plasma endotoxin levels at 4 wk and 8 wk increased dramatically in ethanol-fed rats (112±15 IU/L and 147±22 IU/L) than those in the control animals (31±12 IU/L and 33±9 IU/L) (P＜0.05). In ethanol-fed rats, the levels of wk, respectively which were significantly higher than those fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.CONCLUSION: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.

A novel,rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+monocytes within 48 hours of in vitro culture

AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h of in vitro culture (FastDC).METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/1 L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1 μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/1 polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritornas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS).Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy.RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas.CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.

YKL40 expression in CD14^+ liver cells in acute and chronic injury

AIM:To demonstrate that CD14 + cells are an important source of the growth factor YKL40 in acute and chronic liver damage.METHODS:Rats were inoculated with one dose of CCl4 to induce acute damage.Liver biopsies were obtained at 0,6,12,24,48 and 72 h.For chronic damage,CCl4 was administered three days per week for 6 or 8 wk.Tissue samples were collected,and cellular populations were isolated by liver digestion and purified by cell sorting.YKL40 mRNA and protein expression were evaluated by realtime polymerase chain reaction and western blot.RESULTS:Acute liver damage induced a rapid increase of YKL40 mRNA beginning at 12 h.Expression peaked at 24 h,with a 26fold increase over basal levels.By 72 h however,YKL40 expression levels had nearly returned to control levels.On the other hand,chronic damage induced a sustained increase in YKL40 expression,with 7and 9fold higher levels at 6 and 8 wk,respectively.The pattern of YKL40 expression in different subpopulations showed that CD14+cells,which include Kupffer cells,are a source of YKL40 after acute damage at 72 h[0.09 relative expression units(REU)]as well as after chronic injury at 6 wk(0.11 REU).Hepatocytes,in turn,accounted for 0.06 and 0.01 REU after 72 h(acute)or 6 wk(chronic),respectively.The rest of the CD14cells(including T lymphocytes,B lymphocytes,natural killer and natural killer T cells) yielded 0.07 and 0.15 REU at 72 h and 6 wk,respectively.YKL40 protein expression in liver was detected at 72 h as well as 6 and 8 wk,with the highest expression relative to controls(11fold;P≤0.05)seen at 6 wk.Macrophages were stimulated by lipopolysaccharide.We demonstrate that under these conditions,these cells showed maximum expression of YKL40 at 12 h,with P<0.05 compared with controls.CONCLUSION:Hepatic CD14 + cells are an YKL40 mRNA and protein source in acute and chronic liver injury,with expression patterns similar to growth factors implicated in inflammationfibrogenesis.

In vitro expression of CD14 protein and its gene in Kupffer cells induced by lipopolysaccharide

OBJECTIVES: To observe expression of CD14 protein and its gene in Kupffer cells induced bylipopolysaccharide (LPS) and explore the role of CD14 in LPS-induced Kupffer cell activation.METHODS: Kupffer cells were isolated from Wistar rat livers by in situ collagenase digestion, followedby culture and incubation with 100μg/ml LPS for 0, 30, 60 and 120 min, respectively. CD14 proteinexpressed on the membrane of Kupffer cells was examined using confocal microscopy and Western blottinganalysis. Expression of CD14 mRNA in Kupffer cells was determined by reverse transcription polymerasechain reaction (RT-PCR). Tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the supernatantwere measured using enzyme-linked immunosorbent assay (ELISA) kit.RESULTS: Expression of CD14 mRNA and CD14 protein in isolated Kupffer cells was upregullatedquickly after LPS stimulation and increased with time. Likewise, there was a time-dependent increase ofTNF-α and IL-6 in the supernatant with upregulation of CD14 expression. There were significantdifferences between normal and LPS-stimulated Kupffer cells (P【0. 05).CONCLUSIONS: LPS can upregulate expression of CD14 protein and its gene in isolated Kupffer cells.CD14 may play an important role in the activation of LPS-induced Kupffer cells.

CD14启动子C-159T基因型多态性与广东地区尘螨过敏儿童及血清IgE水平的相关性

目的:研究广东地区尘螨过敏儿童脂多糖受体基因(CD14)多态性和等位基因频率的分布情况,探讨其多态性与儿童尘螨过敏及与血浆IgE水平的关系。方法:对161例尘螨过敏患儿(尘螨过敏组)、186例疾病对照组和100名健康儿童(正常对照组)用散射比浊法测定血浆总IgE水平,采用酶联免疫捕获法进行过敏原特异性IgE(sIgE)检测,采用聚合酶链反应一限制性片段长度多态性(PCR-RFLP)法进行基因型分析,调查多态位点的分布特征,比较不同基因型的血浆尘螨特异性IgE水平,比较3组儿童多态位点的基因差异。结果:3组儿童均发现CDl4-159T/C存在不同的基因型,均以CC、CT和TT基因型多见,尘螨过敏组频率分别为55.9%、33.5%和10.6%,疾病对照组频率分别为10.2%、34.9%和54.8%,正常对照组分别为35%、47%和18%。尘螨过敏组、疾病对照组和正常对照组间基因型差异具有统计学意义(P<0.05),疾病对照组与正常对照组间差异有统计学意义(P<0.05),其他组间差异无统计学意义。与正常对照组相比,尘螨过敏组的CD14 CC基因型携带频率增高,TT基因型携带频率较低。疾病对照组同组内T等位基因携带者IgE水平高于非携带者(P<0.05);同一基因型中,尘螨过敏组和疾病对照组IgE水平高于正常对照组(P=0.02)。结论:广东地区儿童尘螨过敏患儿存在CD14基因启动子C-159T多态性,C等位基因与血浆IgE升高相关。