Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects / 中华血液学杂志

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.

Clinical Characteristics of 223 Chinese Patients with Hemophilia in A Medical Center of Gansu Province in China / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the clinical characteristics, diagonsis and treatment of patients with hemophilia in Gansu province of China.</p><p><b>METHODS</b>The clinical data of 223 cases of hemophilia in our center between January 2010 and May 2015 were collected and analyzed retrospectively, these 223 cases of hemophilia were from 14 cities in Gansu and neighboring provinces, including 203 cases of hemophili A (HA) and 20 cases of hemophili B (HB), among them 222 cases were male, only 1 female(HA), 177 cases were from Rural areas (79.4%).</p><p><b>RESULTS</b>The median age of first bleeding was 2 years old, and the average age of confirmed as hemophilia was 5.6±6.5 years, the delayed time of diagnoses of HA and HB was 2.50±4.91 and 2.07±4.76 years, respetively, among all the patients 168 caese complicated with joint hemorrhage (75.3%), 123 cases with joint deformities (55.2%). 91.6% of the patients were treated according to demand, the HBV and HCV infection rates were 1.7% and 6.2% respectively. The first-visited hospital of 86.9% patients was hospitalized below 3 grade of level, only 15.9% of these patients were considered to diagnose as hemophili.</p><p><b>CONCLUSION</b>The accurate level of diagnosis rate for hemophiliacs in Gansu province is low, the delay time of diagnosis is longer, the ratios of complicated joint hemorrhage, total accumulative joint deformity were high, HCV infection rate is also high.</p>

Study on effect of brain injury factor in patients with acute cerebral infarction by Ginkgetin injection / 中国生化药物杂志

Objective To explore effect of ginkgetin injection on brain damage factor in patients with acute cerebral infarction.Methods 180 cases of acute cerebral infarction patients were selected from June 2014 to February 2015, and divided into experiment group and control group.88 cases in control group were treated with clinical routine therapy, 92 cases in experimental group were treated on base of the control with ginkgetin injection, 2 weeks for a course.The clinical efficacy, MESS and serum HIF-1α, TNF-α, Caspase-3, were detected and compared.Results The clinical efficacy of the experiment group was better than that of control group(P<0.05),and the MESS of the experiment group was lower than the control group(P<0.05).Serum HIF-1α, Caspase-3, TNF-αlevels were lower than the control group(P<0.05).Conclusion Ginkgetin injection can effectively improve the patient’s neurological symptoms, it is of great significance to the clinical treatment of cerebral infarction.

Screening for EGFR mutations in lung cancer by a novel real-time PCR with double-loop probe and Sanger DNA sequencing / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes.</p><p><b>METHODS</b>A total of 208 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx's EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed.</p><p><b>RESULTS</b>The traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 (94.2%) lung cancer samples, and 22 samples (11.2%) showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases (100.0%), and 40 samples (19.2%) showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% (10/208) and 11.6% (23/208) of total mutations, respectively, and the remaining mutations were rare.</p><p><b>CONCLUSIONS</b>The success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing (P < 0.01). There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.</p>

Screening for K-ras mutations in colorectal and lung cancers by using a novel real-time PCR with ADx-K-ras kit and Sanger DNA sequencing / 中华病理学杂志

<p><b>OBJECTIVE</b>to map out the frequency and types of K-ras gene mutations present in colorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes.</p><p><b>METHODS</b>a total of 827 formalin-fixed paraffin-embedded (FFPE) blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx's K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed.</p><p><b>RESULTS</b>533 out of 583 (91.4%) colorectal cancer samples and 144 out of 244 lung cancer samples (59.0%) were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583 (100.0%) colorectal plus 244 out of 244(100.0%) lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 (32.9%) showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples (27.4%) with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26 (10.7%) showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples (8.3%) showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% (66/188) mutations, followed by GGC→GAC at codon 13 with 26.6% (50/188) and GGT→GTT at codon 12 with 18.6% (35/188), while GGT→GCT at codon12 was the most rare with only 1.6% (3/188) of the total mutation cases. In patients with lung cancer analyzed, GGT→GTT at codon 12 was the most common mutation, accounting for 40.9% (9/22), and GGT→GCT at codon 12 the most rare with only about 4.5% (1/22) of the total mutation cases.</p><p><b>CONCLUSIONS</b>K-ras gene mutations were present in colorectal cases, and significantly more frequent than that in lung cancer. There were significant statistical differences between the two methods. ADx-K-ras real-time PCR showed much higher successful detection rates and mutation ratios compared to Sanger sequencing. As a result, the real-time PCR with ADx-K-ras kit proves to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is a effective and reliable tool for clinical screening of somatic gene mutations in tumors.</p>