广东省两个地区2021-2022年百日咳鲍特菌分离株的抗菌药物敏感性和抗原基因型

目的了解广东省两个地区百日咳鲍特菌(Bordetella pertussis,Bp)抗菌药物敏感性和抗原基因型。方法通过MALDI-TOF-MS微生物质谱、荧光定量PCR和血清凝集试验对广州市和佛山市2021-2022年Bp分离株进行鉴定,采用E-test法进行10种抗菌药物的敏感性检测,利用二代测序平台和生物信息学分析软件进行全基因组测序和拼接,采用BLASTn软件分析抗原基因型和23 S rRNA基因突变。结果在15株Bp分离株中,12株(80.0%)对大环内酯类抗菌药物(红霉素、阿奇霉素和克拉霉素)耐药,且与Bp疫苗株相比存在23 S rRNA A2047 G突变,所有菌株对磺胺类、β-内酰胺类和喹诺酮类抗菌药物敏感;13株和2株分离株的抗原基因型分别为prn2/ptxA1/ptxC2/ptxP3/fim2-1/fim3-1/tcfA2和prn1/ptxA1/ptxC1/ptxP1/fim2-1/fim3-1/tcfA2。结论研究地区Bp分离株对大环内酯类抗菌药物的耐药率高,抗原基因型与疫苗株存在差异;需持续加强Bp流行株抗原基因型和药物敏感性监测。

HLA不全相合同胞脐血移植治疗儿童重型β珠蛋白生成障碍性贫血——附6例报告

目的:评价人类白细胞抗原(human leukocyte antigen,HLA)不全相合同胞脐血移植治疗儿童重型β珠蛋白生成障碍性贫血(重型β珠贫)的可行性。方法:对6例重型β珠贫患儿进行HLA2个位点不合的同胞脐血移植,输入脐血总有核细胞6.1×107/kg^15.6×107/kg(中位数9.7×107/kg),CD34+细胞0.3×105/kg^13.3×105/kg(2.4×105/kg)。预处理方案包括,应用羟基脲、氟达拉滨、白消安、环磷酰胺及抗胸腺细胞球蛋白,大量输注红细胞悬液,同时应用去铁胺。结果:5例患儿获得植入,其中3例为长期稳定植入并脱离红细胞输注,获得植入的5例患儿均发生Ⅰ~Ⅱ度急性移植物抗宿主病,无发生慢性移植物抗宿主病。1例短暂植入(移植后17~21d)后发生排斥,并于移植后75d死于颅内出血和大肠埃希菌败血症。结论:HLA不全相合同胞脐血移植治疗儿童重型β珠贫,虽然移植排斥率较高,但急性移植物抗宿主病严重程度低,为无HLA全相合供者的儿童重型β珠贫患者提供了新的治疗手段。

干扰TOX基因联合抗CD38 CAR-T细胞的构建及其对血液肿瘤细胞增殖和凋亡能力的影响

目的构建干扰胸腺细胞选择相关高迁移率群体盒(TOX)基因联合抗CD38 CAR-T细胞,观察其对血液肿瘤细胞增殖和凋亡能力的影响。方法采集健康志愿者外周血,分离单个核细胞(PBMC),提取原代T淋巴细胞。构建以shRNA技术靶向抑制TOX的抗CD38 CAR分子,分别命名为TOX-shRNA1-CD38 CAR和TOX-shRNA2-CD38 CAR,另设计一条不针对任何靶点的无意义RNA序列作为对照,命名为CD38 CAR分子。将分子酶切并连接逆病毒载体包装质粒pMFG,获得pMFG-TOX-shRNA1-MYC-CD38 CAR、pMFG-TOX-shRNA2-MYC-CD38 CAR和pMFG-MYC-CD38 CAR。将人原代T淋巴细胞分为CD38 TOX-shRNA1-CD38 CAR-T组、TOX-shRNA2-CD38 CAR-T组、CAR-T组,分别转导pMFG-TOX-shRNA1-MYC-CD38 CAR、pMFG-TOX-shRNA2-MYC-CD38 CAR和pMFG-MYCCD38 CAR质粒,获得CD38 CAR-T细胞、TOX-shRNA1-CD38 CAR-T细胞和TOX-shRNA2-CD38 CAR-T细胞。采用RT-QPCR法检测三组细胞中TOX mRNA的相对表达量;记录三组细胞0~10 d体外培养生长倍数,观察细胞增殖能力。选取CD38高表达的人多发性骨髓瘤荧光素酶标记细胞RPMI-Luc、人Burkitt’s淋巴瘤荧光素酶标记细胞Raji-Luc作为靶细胞,与三组CAR-T细胞分别共培养48 h;另取三组CAR-T细胞单独培养作为对照,不加肿瘤细胞。采用流式细胞术检测三组CAR-T细胞表面CD69的表达效率,评价CAR-T细胞活化情况;对三组细胞进行CFSE染色,采用流式细胞术观察CAR-T细胞增殖能力;荧光素酶化学发光法观察CAR-T细胞在不同效靶比(1∶2、1∶4、1∶8)时对肿瘤细胞的杀伤效率;ELISA法检测三组CAR-T细胞上清中IFN-γ释放量;流式细胞术检测三组细胞表面PD-1表达量,评估CAR-T细胞的耗竭程度。结果 CD38 CAR-T组、TOX-shRNA1-CD38 CAR-T组、TOXshRNA2-CD38 CAR-T组的转导效率分别为41.51%、41.28%、44.84%,均超过40%。与CD38 CAR-T组比较,TOXshRNA2-CD38 CAR-T组TOX mRNA表达水平降低(P <0.05),TOX-shRNA1-CD38 CAR-T组无明显变化(P>0.05)。三组CAR-T细胞体外培养0~10 d均稳定增殖,三组间生长倍数比较差异无统计学意义(P <0.05)。三组与肿瘤细胞共培养后,CD69表达效率均升高(P均<0.01)。与单独培养的CAR-T细胞相比,三组CAR-T细胞与Raji-Luc、RPMI-Luc细胞共培养后FITC信号左移,增殖加快,三组增殖能力比较无统计学差异(P> 0.05)。与CD38 CAR-T组相比,TOX-shRNA2-CD38 CAR-T组在不同效靶比时对肿瘤细胞的杀伤效率均提高,γ干扰素释放水平升高,细胞表面PD-1表达水平降低(P <0.05或<0.01),而TOX-shRNA1-CD38 CAR-T组与CD38 CAR-T组比较,肿瘤细胞的杀伤效率、γ干扰素释放水平、细胞表面PD-1表达水平均无统计学差异(P均>0.05)。结论成功构建了靶向抑制TOX基因联合抗CD38 CAR-T细胞,TOX-shRNA-CD38 CAR-T细胞能够有效抑制TOX表达;经肿瘤细胞激活后,其体外增殖活性及抗肿瘤能力显著增强,且TOX-shRNA-CD38 CAR-T细胞耗竭也得到改善。

B2型黑色素瘤抗原基因对肝细胞肝癌增殖的影响及其调控机制

目的探讨B2型黑色素瘤抗原基因（MageB2）对肝细胞肝癌（HCC）细胞增殖能力的影响及其可能的下游调控机制。方法Western blot检测HCC细胞株Huh7和正常肝脏细胞株HL7702内MageB2蛋白的表达。采用MageB2-小干扰RNA（siRNA）转染Huh7细胞，72h后应用细胞计数试剂盒（CCK-8）检测细胞活性，48h后流式细胞术检测细胞周期，反转录-聚合酶链反应（RT-PCR）检测细胞周期蛋白E1（Cyclin E1）和c-myc的mRNA表达水平。结果MageB2蛋白在Hub7和HL7702细胞中的相对表达量分别为0．80±0．13和0．34±0．07（P〈0．01）。MageB2-siRNA转染可成功抑制Huh7细胞中MageB2蛋白的表达，抑制率达（71．84±4．32）％（P〈0．01）。沉默MageB2后，Huh7细胞活性在72h后明显减弱，为空白对照组细胞活力的（62．43±3．95）％（P〈0．01）。48h后G0／G1期细胞比例升高，同时S和G2／M期细胞比例降低，分别为（70．87±2．08）％、（19．63±2．31）％和（9．49±0．56）％（P〈0．01）。此外CyclinE1和c-myc的mRNA表达均下降，分别降至（56．78±4．97）％和（39．38±6．72）％（P〈0．01）。结论MageB2在HCC细胞中表达较高，并参与其细胞周期的调控，沉默MageB2能抑制HCC细胞的增殖。

Relationship of human leukocyte antigen class II genes with the susceptibility to hepatitis B virus infection and the response to interferon in HBV-infected patients

AIM： To study the relationship of human leukocyte antigen （HLA）-DRB1 and -DQB1 alleles with the genetic susceptibility to HBV infection and the response to interferon （IFN） in HBV-infected patients. METHODS： Low-resolution DNA typing kit was used to determine HLA-DR-1 and -DQB1 genes in 72 patients with chronic hepatitis B （CHB） and HLA-DRB1 in 200 healthy people ready to donate their bone marrow in Shanghai. Among CHB patients, 35 were treated with IFNα-1b for 24 wk. RESULTS： The frequencies of HLA-DRBI＊06, DRBI＊08 and DRB1＊16 alleles in 72 patients were higher than in 200 healthy people （2.08% vs0%, OR = 3.837, P= 0.018; 11.11% vs5.50%, OR = 2.148, P= 0.034; and 6.94% vs 3.00%, OR = 0.625, P = 0.049, respectively）; whereas that of DRBI＊07 allele was lower （2.78% vs 7.75%, OR = 0.340, P= 0.046）. The frequency of HLA-DRBI＊ 14 allele was higher in 11 responders to IFN compared with 24 non-responders （18.18% vs2.08%, OR = 10.444, P = 0.031）, whereas that of DQBI＊07 allele was inverse （9.09% vs37.50%, OR = 0.167, P= 0.021）. CONCLUSION： The polymorphism of HLA class II may influence the susceptibility to HBV infection and the response to IFN in studied CHB patients. Compared with other HLA-DRB1 alleles, HLA-DRBI＊06, DRBI＊08, and DRB1＊16 may be associated with chronicity of HBV infection, HLA-DRBI＊07 with protection against HBV infection, and HLA-DRB1＊14 allele may be associated with a high rate of the response of CHB patients to IFN treatment. Compared with other HLA-DQB1 alleles, HLA-DQBI＊07 may be associated with low response rate to IFN. 2005 The WJG Press and Elsevier Inc. All rights reserved

Quantitation of HBsAg predicts response to entecavir therapy in HBV genotype C patients

AIM： To analysis the factors that predict the response to entecavir therapy in chronic hepatitis patients with hepatitis B virus （HBV） genotype C. METHODS： Fifty patients [hepatitis B e antigen （HBeAg）- negative：HBeAg-positive = 26：24] with HBV genotype C, who received nalve entecavir therapy for 〉 2 years, were analyzed. Patients who showed HBV DNA levels ≥ 3.0 log viral copies/mL after 2 years of entecavir ther- apy were designated as slow-responders, while those that showed 〈 3.0 log copies/mL were termed rapid- responders. Quantitative hepatitis B surface antigen （HBsAg） levels （qHBsAg） were determined by the Archi- tect HBsAg QT immunoassay. Hepatitis B core-related antigen was detected by enzyme immunoassay. Pre-C and Core promoter mutations were determined using by polymerase chain reaction （PCR）. Drug-resistance muta- tions were detected by the PCR-Invader method. RESULTS： At year 2, HBV DNA levels in all patients in the HBeAg-negative group were 〈 3.0 log copies/mL. In contrast, in the HBeAg-positive group, 41.7% were slow-responders, while 58.3% were rapid-responders. No entecavir-resistant mutants were detected in the slow-responders. When the pretreatment factors were compared between the slow- and rapid-responders; the median qHBsAg in the slow-responders was 4.57 log IU/mL, compared with 3.63 log IU/mL in the rapid- responders （P 〈 0.01）. When the pretreatment factors predictive of HBV DNA-negative status at year 2 in all 50 patients were analyzed, HBeAg-negative status, low HBV DNA levels, and low qHBsAg levels were signifi- cant （P 〈 0.01）. Multivariate analysis revealed that the low qHBsAg level was the most significant predictive factor （P = 0.03）. CONCLUSION： Quantitation of HBsAg could be a use- ful indicator to predict response to entecavir therapy.

Human leukocyte antigen class II DQB1*0301, DRB1*1101 alleles and spontaneous clearance of hepatitis C virus infection: A meta-analysis

AIM： To assess the associations of human leukocyte antigen （HI_A） class Ⅱ DQB1＊0301 and/or DRB1＊1101 allele with spontaneous hepatitis C virus （HCV） clearance by meta-analysis of individual dataset from all studies published till date. METHODS： To clarify the impact of HLA class Ⅱ polymorphisms on viral clearance, we performed a metaanalysis of the published data from 11 studies comparing the frequencies of DQB1＊0301 and DRB1＊1101 alleles in individuals with spontaneous resolution to those with persistent infection. As we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. RESULTS： Meta-analyses yielded summary estimatesodds ratio （OR） of 2.36 [95%CI （1.62, 3.43）, P〈0.00001] and 2.02 [95%CI （1.56, 2.62）, P〈0.00001] for the effects of DQB1＊0301 and DRB1＊1101 alleles on spontaneous clearance of HCV, respectively. CONCLUSION： These results support the hypothesis that specific HLA class Ⅱ alleles might influence the susceptibility or resistance to persistent HCV infection. Both DQB1＊0301 and DRB1＊1101 are protective alleles and present HCV epitopes more effectively to CD4^＋T lymphocytes than others, and subjects with these two alleles are at a lower risk of developing chronic HCV infection. Large, multi-ethnic confirmatory and welldesigned studies are needed to determine the host genetic determinants of HCV infection.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to chronic hepatitis B

AIM： To assess the three polymorphJsm regions within cytotoxic T-lymphocyte antigen 4 （CTLA-4） gene, a C/T base exchange in the promoter region-318 （CTLA-4 -318C/T）, an A/G substitution in the exon 1 position 49 （CTLA-4 49A/G）, a T/C substitution in 1172 （CTLA-4 -1172T/C） in patients with chronic hepatitis B. METHODS： Fifty-one patients with chronic hepatitis B virus infection and 150 healthy subjects were recruited sequentially as they presented to the hepatic clinic. Classification of chronic hepatitis B virus （HBV）-infected patients was as asymptomatic carrier state （26 patients） and chronic hepatitis B （25 patients）. Genomic DNA was isolated from anti-coagulated peripheral blood Bully coat using Miller＇s salting-out method. The presence of the CTLA-4 gene polymorphisms was determined using polymerase chain reaction amplification refractory mutation system （ARMS）. RESULTS： We observed a significant association between -318 genotypes frequency （T＋C-, T＋C＋, T-C＋） and susceptibility to chronic hepatitis B （P=0.012, OR=0.49, 95%CI： 0.206-1.162）. However, we did not observe a significant association for ＋49 genotype frequency （T＋C＋, T＋C- T-C＋） and -1172 genotype frequency （C＋T＋, T＋C- C＋T-） and state of disease. CONCLUSION： Our results suggest that CTLA-4 gene polymorphisms may partially be involved in the susceptibility to chronic hepatitis B.

Significance of hepatitis B virus surface antigen, hepatitis C virus expression in hepatocellular carcinoma and pericarcinomatous tissues

AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.

CART-19在血液恶性肿瘤治疗中的临床应用

嵌合型抗原受体基因修饰的T细胞(chimeric antigen receptor-modified T cells,CART)免疫疗法是通过基因工程方法改造宿主T细胞,使其能自主靶向结合并杀死肿瘤细胞的治疗方法。该疗法临床研究最多的是CART-19,其是以B细胞表面特异性蛋白CD19为靶抗原的CART细胞,主要用于治疗急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)、慢性淋巴细胞白血病(chronic lymphocytic leukemia,BLL)和B细胞系肿瘤。文章就CART-19在血液恶性肿瘤中治疗的临床应用进展作一综述。

Quantification of HBsAg:Basic virology for clinical practice

Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.

Isolation and Characterization of Exosomes Derived from Tumor Cells Genetically Expressing Model Antigen

Tumor cell-derived exosomes have been proposed as non-cellular nanomeric vaccine which could induce potent anti- tumor immune response in mice. In order to develop the protocols to prepare tumor cell-derived exosomes for basic research and clinical trail, we isolated exosomes from ovalbumin (OVA)-expressing thymoma cells EG.7-OVA by various preparation methods. We demonstrate the non-sedimentation method is simple, rapid, efficient with higher yield and purity of exosomes. EG.7-OVA-derived exosomes are 40-100 nm in diameter sequestered by lipid bi-layer, and contain rich heat shock protein (HSP) and OVA. The result of the size distribution determination is consistent with the calculation by the visual microscopic inspection, with 90.4% particles at the range of 50-90 nm. Moreover, as a model antigen of the EG.7 cells, OVA concentra- tion in EG.7-derived exosomes can be regarded as a good quality control parameter. Therefore, we have established a platform to efficiently prepare exosomes for tumor immunotherapy.

CTLA-4 gene A/G polymorphism associated with diabetes mellitus in Han Chinese

OBJECTIVE: To investigate the association of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) gene A/G polymorphism with susceptibility to diabetes mellitus in Han Chinese. METHODS: An A/G transition at position 49 of exon 1 was analyzed in 31 patients with type 1 diabetes, 31 patients with type 2 diabetes, and 36 controls were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: A highly significant increase in the frequency of the G allele was seen in patients with type 1 diabetes compared with controls (66.1 % vs. 34.7%, respectively; P

Genotyping and preparation of the recombinant nucleocapsid protein antigen of hantavirus

Objective To identify new recombinant antigens with potential for diagnosis of hemorrhagic fever with renal syndrom (HFRS) and establish reverse transcription-polymerase chain reaction-restriction fragments length polymorphism (RT-PCR-RFLP) for genotyping of hantavirus.Methods One group of primers was used to clone the full-length S genome segment and the partial S genorme segment of the N-terminal. The two cloned genes were both fusionally expressed and nonfusionally expressed in the T7 system. The other group of primers was used to establish a RT-PCR method to detect RNAs in 37 virus isolates, 2 positive standard virus strains of hantavirus and 5 negative controls.The method for typing RFLP was set up by digesting the PCR products of 20 virus isolates with Ras Ⅰ and Hind Ⅲ.Results The non-fusionally expressed products with a working concentration of 1:10 000 by chapping enzyme-linked immunosorbent assay (cELISA), presented good biological activity though yields were lower than that of the fusionally expressed products.The specific component of the hantavirus genome (299 bp or 577 bp) wes seen in all viral samples. The genotyping of hantavirus showed that 9 out of the total were hantann (HTN) viruses, 8 were seol (SEO) viruses and 3 were not determined.Conclusions The good working titrer of expressed recombinant antigen showed that it has the potential to replace the natural antigen for detecting hantavirus antibodies. On comparison with cELISA, the detection rates for these two methods were 100% and 84.6%, and the coincidence rate was 84.6%. The former had a 15.4% higher sensitivity than the latter. The typing efficiency of RT-PCR-RFLP and sero-typing method was 85% (17/20) and 55% (11/20), respectively, showing that the former was 30% higher than the latter, while their results were highly consistant.

Effect of Lichong Decoction on expression of IGF-I and proliferating cell nuclear antigen mRNA in rat model of uterine leiomyoma

OBJECTIVE:To study the effect of Lichong Decoction(Lichong Decoction for strengthening anti-pathogenic Qi and eliminating blood stasis) on the expression of insulin-like growth factor-I(IGF-I) and proliferating cell nuclear antigen(PCNA) mRNA in a rat model of uterine leiomyoma.METHODS:Fifty female Wistar rats were randomized into a normal control group,model group,Lichong Decoction group,Guizhifuling Capsule(Capsule containing Cassia Twig and Poria) group,and Mifepristone group.The uterine leiomyoma model was established by peritoneal injections of exogenous estrogen and progesterone hormone.The ultrastructural changes in cells of rat uterine tissues were observed with transmission electron microscopy,and the expression of IGF-I and PCNA mRNA was detected by real-time fluorescent quantitative PCR.RESULTS:Following treatment,cells in the Lichong Decoction group appeared to be arranged normally,the cellular morphology were almost in a normal state,hyperplasia and hypertrophy of the chondriosome was reduced,collagen fibers were arranged in a regular manner,without obvious hyperplasia,and the expression of IGF-I and PCNA mRNA was significantly decreased compared with the model group(P<0.01).CONCLUSIONS:The effect of Lichong Decoction on uterine leiomyoma is related to its function in reducing the expression of IGF-I and PCNA mRNA.