Comparison of the characteristics of NK cells after two different methods of expansion and observation of the clinical efficacy in patients who relapsed post allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

Objective: To explore the differences in the biological effects of different expansion systems on natural killer (NK) cells, as well as the safety and preliminary clinical efficacy in the treatment of patients with recurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: Peripheral blood cells from healthy donors were stimulated with either CD3 combined with CD52 or K562 feeder cells loaded with IL-21/4-1BB to induce NK cell expansion. Changes in the NK cell phenotype, cytokine secretion, and cytotoxicity before and after expansion were detected. We also evaluated the safety and clinical efficacy of two different expansion strategies for patients received NK infusion. Results: Compared with the CD3/CD52 monoclonal antibody amplification system, the feeder cell expansion group had a higher purity of NK cells and higher expression ratios of NK cell surface activation receptors such as DNAM-1 and NKp30, while inhibitory receptor CTLA-4 expression was low and NKG2D/CD25/CD69/ Trail/PD-1/TIM-3/TIGIT had no statistically significant differences between the groups. Further functional results showed that the expression level of KI67 in NK cells after expansion in the two groups increased significantly, especially in the feeder cell expansion group. Simultaneously, the perforin and granzyme B levels of NK cells in the feeder cell expansion group were significantly higher than in the CD3/CD52 expansion group. A retrospective analysis of eight patients who received monoclonal antibody-expanded NK cell reinfusion and nine patients with trophoblast cell-expanded NK cell reinfusion was done. The disease characteristics of the two groups were comparable, NK cell reinfusion was safe, and there were no obvious adverse reactions. Clinical prognostic results showed that in the CD3/CD52 monoclonal antibody amplification group, the MRD conversion rate was 50% (2/4) , and the feeder cell expansion group was 50% (3/6) . After 5 years of follow-up from allo-HSCT, three patients in the monoclonal antibody expansion group had long-term survival without leukemia, and the remaining five patients had died; two patients died in the feeder cell expansion group, and the other six patients had long-term survival. Six cases had GVHD before NK cell reinfusion, and GVHD did not aggravate or even relieved after NK cell reinfusion. Conclusions: Preliminary results show that the biological characteristics of NK cells with diverse expansion strategies are significantly different, which may affect the clinical prognosis of patients with recurrence or persistent minimal residual disease after HSCT. The two groups of patients treated with NK cells from different expansion strategies had no obvious adverse reactions after NK cell infusion, but efficacy still needs to be further confirmed.

Bilateral papillary renal cell carcinoma following kidney transplantation: A case report / 北京大学学报(医学版)

With the continuous development of kidney transplantation technique, the survival time after kidney transplantation is gradually prolonged. Thus, the malignant tumor has been the important influencing factor on the long-term survival for kidney transplantation patients. Renal cell carcinoma is a relatively common tumor after kidney transplantation. Besides, clear cell renal cell carcinoma and papillary renal cell carcinoma are the relatively common pathological types for renal cell carcinoma following kidney transplantation. However, bilateral renal cell carcinoma following kidney transplantation is comparatively rare. In this article, we presented a case of bilateral papillary renal cell carcinoma, which occurred after kidney transplantation. And the diagnosis and treatment were introduced in detail. The patient was 37 years old, and he underwent kidney transplantation 13 years ago in our hospital, because of kidney failure. After kidney transplantation, he had regular medical check-up every year. In this year, his urological ultrasound results indicated bilateral renal tumors. And then, he received abdominal and pelvic computed tomography, and the result also showed bilateral renal tumors, which were likely to be malignant tumors. After adequate consultation, the patient chose surgical treatment. The patient received long-term immunosuppressive therapy, because of kidney transplantation. Considering this, the surgeon decided to choose a staging surgical treatment, in order to reduce the bad influence of one-stage surgery. Then, the patient first underwent retroperitoneal laparoscopic radical nephrectomy for right renal tumor in our hospital, and he had no complications after operation. The pathological results showed papillary renal cell carcinoma. He was discharged successfully. He underwent retroperitoneal laparoscopic radical nephrectomy for left renal tumor in our hospital one month later, and he had no complications after operation. The pathological results also showed papillary renal cell carcinoma. He was discharged successfully two days after surgery. In the 3-month follow-up, the patient was recovering well. To sum up, the incidence of bilateral renal cell carcinoma following kidney transplantation is relatively rare, and bilateral radical nephrectomy is effective and safe treatment. Above all, it is the patient's condition that determines the choice of staging surgery or simultaneous surgery.

Experimental study of enhanced anti-pancreatic cancer effects by combination treatment with HUVEC vaccine and gemcitabine / 中国药理学通报

Aim To investigate whether low dose gem- citabine ( GEM) could enhance the anti-pancreatic cancer effects of human umbilical vein endothelial cell (HUVEC) vaccine.Methods C57BL/6 mice were randomly divided into four groups; PBS control group, GEM group, HUVEC vaccine group and HUVEC vaccine combined GEM group ( HUVEC-GEM group).Mice were inoculated with Pan02 pancreatic cancer cells to establish a subcutaneous xenograft model to observe tumor growth and adverse reactions in tumor bearing mice.Whether GEM could enhance the immune responses induced by HUVEC vaccine was determined by ELISA analysis of the immune serum, splenic lymphocyte proliferation assay, cytotoxic T lymphocyte killing assay and IFN-7 content assay.Results The results of subcutaneous transplantation tumor model showed that the introduction of GEM into the HU-VEC vaccine treatment could enhance the therapeutic anti-pancreatic cancer effects of HUVEC vaccine.Enzyme-linked immunoassay results confirmed that GEM acted as an immune adjuvant could effectively increase the HUVEC antibodies and I FN-7 level in the immune serum of mice.The results of lymphocyte proliferation experiment and CTL killing activity assay indicated that GEM could effectively enhance the spleen lymphocyte transformation activity and CTL killing ability of HUVEC vaccine immunized mice.Conclusions Low dose GEM could enhance the immune responses induced by HUVEC vaccine and thus enhance the anti- Pan02 pancreatic cancer effects of HUVEC vaccine.

Monitoring and Prognostic Analysis of Peripheral Blood Circulating Tumor Cell Before and After Operation for Early and Middle Stage Colorectal Cancer / 中山大学学报(医学科学版)

@#【Objective】To study the relationship between circulation tumor cell（CTC）and clinicopathological characteristics in early and middlestage colorectal cancer，and to evaluate the diagnostic and prognostic value of peripheral blood CTC through dynamically monitoring the changes of peripheral blood CTC in the patients before and after operation.【Methods】 Prospectively 67 patients with early and middle stage colorectal cancer were included，and 20 healthy volunteers served as contrast during the same period. Peripheral venous blood（7.5 mL）was collected 24 h before radical operation，3 months after radical operation and in control group. SE- iFISH technique was used to isolate and identify CTC. The cutoff value of CTC in diagnosis of colorectal cancer was determined by the receiver operating curve（ROC）and Youden index. Kaplan-Meier and log-rank methods were used for survival analysis and multivariate COX regression analysis for multivariate correction. The value of CTC in the diagnosis of early and middle stage colorectal cancer was evaluated comprehensively，and then the relationship between CTC in peripheral blood and clinicopathological characteristics and prognosis was synthetically evaluated combined with clinicopathological characteristics and postoperative follow- up data. 【Results】The positive rate of CTC in patients with early and middle stage colorectal cancer was significantly higher than that in healthy people（91.0% vs. 5.0% ，P<0.01）. The CTC enumeration was significantly correlated with the depth of invasion and tumor location（P = 0.001，P = 0.044），but not with gender，age，tumor size，lymph node metastasis，TNM stage ，CEA level and CA-199 level. The preoperative CTC enumeration were not correlated with the above-mentioned clinicopathological parameters. Preoperative CTC had no predictive significance for disease free survival（DFS）and total survival（OS）（AUC = 0.359，P = 0.068；AUC = 0.428，P = 0.423），and postoperative CTC critical point of 3/7.5 mL had predictive significance for DFS and OS（AUC = 0.936 ，P < 0.001 ；AUC = 0.863 ，P < 0.001）. It was found that patients with early and middle stage colorectal cancer were divided into two groups ：good prognosis group（CTC < 3 after operation or the number of CTC after operation was equal or decreased with the number of CTC before operation）and bad prognosis group（CTC ≥ 3 after operation or the number of CTC after operation was higher than the number of CTC before operation）. The DFS of the good prognosis group was significantly longer than that of the poor prognosis group （43.7 months vs. 20.4 months，P < 0.001；48.7 months vs. 26.8 months，P < 0.001），and the OS was also significantly longer （54.7 months vs. 43.3 months，P < 0.001；54.8 months vs. 45.1 months，P < 0.001）. Multivariate analysis showed that CTC ≥ 3/7.5 mL was a bad independent factor of DFS and OS.【Conclusions】CTC has high clinical value in patients with early and middle stage colorectal cancer. Preoperative CTC values can not predict the prognosis of early and middle stage colorectal cancer ，but postoperative CTC values and dynamic detection for CTC changes before and after operation can independently predict the prognosis of early and middle stage colorectal cancer.

Detection of human papillomavirus in esophageal carcinoma tissues from Baoding City of Hebei Province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the human papillomavirus (HPV) and esophageal carcinoma in Baoding City of Hebei Province.</p><p><b>METHODS</b>We detected HPV DNA in 42 formalin-fixed and paraffin-embedded tissues from surgically resected esophageal carcinomas from Baoding City of Hebei Province, by PCR with the general primer set of GP5 + /6 + for HPV L1 gene and type-specific primer sets for HPV16 and 18 as well.</p><p><b>RESULTS</b>37 from 42 esophageal carcinoma samples were HPV positive and the rate was 88.1%. Among the samples detected, 19 were HPV16 E6 positive and rate was 45.2%, eight were HPV18 E6 positive and rate was 19.0%.</p><p><b>CONCLUSION</b>The high rate of HPV in the esophageal carcinoma samples suggested that HPV plays an etiologic role in the development of esophageal cancer in Baoding City of Hebei Province.</p>

Meta analysis on etiological relationship between human papillomavirus and esophageal carcinoma / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the relationship between human papillomavirus (HPV) and esophageal cancer development in China.</p><p><b>METHODS</b>We searched and collected the published articles in Chinese related to HPV and esophageal cancer, and selected the articles with the PCR approach to detect HPV in the esophageal cancer specimens.</p><p><b>RESULTS</b>We filtered our publication collection with standards as (1) PCR as the detection approach, (2) specimens as the paraffin-embedded sections, and (3) description of the primer in the experiments, and fifteen articles were enrolled for our meta-analysis. Among the articles, totally 980 specimens were tested, and 460 were HPV positive with the average HPV prevalence was 46.9% (95% CI: 43.8%-50.0%), varied from 8.3%-69.8% in the different locations. On the other hands, among 556 specimens whose HPV detection spectrum included HPV16, 139 showed the positivity of HPV16, the average prevalence was 25.0%, (95% CI: 21.4%-28.6%) varied from 4.4%-63.4% dependent on the locations; among 485 specimens whose HPV detection spectrum included HPV18, thirty-three specimens showed the positivity of HPV18, the average prevalence was 6.8% (95% CI: 4.6%-9.0%) varied from 0%-19.0% dependent on the locations. Third, among the fifteen articles enrolled in the meta-analysis, four articles used the same primer set for HPV detection in totally 406 paraffin-embedded specimens with the prevalence of 40.2% (95% CI: 36.0%-45.4%) varied from 20.3%-67.6% in different locations.</p><p><b>CONCLUSION</b>Our analysis result suggested the HPV prevalence in the esophageal cancer samples of China and clued the possible etiological relationships between HPV infection and the esophageal cancer development.</p>

Effects of glutamine-enriched enteral nutrition on nutritional status and prognosis of patients with severe head injury / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of glutamine-enriched enteral nutrition on the nutritional status and prognosis of patients with severe head injury.</p><p><b>METHODS</b>Thirty-three patients with severe head injury were randomly divided into control group (C, 15 cases) and glutamine-enriched group (Gln, 18 cases). Patients in both groups were given routine treatment and enteral nutrition with the same amount of nitrogen and calorie. Patients in Gln group were given glutamine 0.5 g x kg(-1) x d(-1) additionally added into the nutrient fluid. Vital signs and the occurrence of side effects of all patients were observed before and after nutrition support. Venous blood and urine sample of all patients were collected before and 7, 14 days after treatment to determine the parameters of blood, urine routine and hepatorenal function. At the same time points, body mass, skin fold thickness at the region of triceps brachii (TSF), upper arm circumference (AC), upper arm muscle circumference (AMC) and fasting blood glucose of all patients were detected and determined, Glasgow coma scale (GCS) scoring was performed. The length of hospital stay of all patients was recorded.</p><p><b>RESULTS</b>Vital signs and parameters of blood, urine routine and hepatorenal function of patients in 2 groups after nutrition treatment were close to those before treatment. Side effects, such as nausea and diarrhea occurred with spontaneous remission in a few patients. There was no statistical significant difference between 2 groups, and within each group before and after treatment, in respect of body mass and TSF (P > 0.05). Values of AC and AMC of patients in Gln group were obviously higher than those of C group (P < 0.01) on post-treatment day 14. Fasting blood glucose and GCS score of all patients before treatment were close to those on post-treatment day 14 (P > 0.05). Fasting blood glucose and GCS score of patients was respectively lower and higher in Gln group than that in C group on post-treatment day 7 (P < 0.05). Length of hospital stay of patients in Gln group (25 +/- 9) d was obviously shorter than that of C group (33 +/- 12) d (P < 0.05).</p><p><b>CONCLUSIONS</b>Glutamine-enriched enteral nutrition can control the blood glucose level, prevent the loss of lean tissue, improve nutrition status of patients,shorten hospital stay, and accelerate the recovery of patients to some extent.</p>

Detection of human papillomavirus in tissues of esophageal carcinomas by polymerase chain reaction / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the human papillomavirus (HPV) and esophageal carcinomas.</p><p><b>METHODS</b>We detected HPV DNA in 31 fresh tissue of esophageal carcinomas from Linzhou City, Henan Province, by PCR with the general primer set of GP5+/6+ for HPV L1 gene and type-specific primer sets for HPV16 and 18 as well.</p><p><b>RESULTS</b>29 from 31 esophageal carcinoma samples were HPV positive and the rate was 93.5%. Among the samples detected, 19 were HPV16E6 positive and rate was 61.3%, eight were HPV18 E6 positive and rate was 25.8%; our result also showed five were the multiple infection containing HPV16 and 18 as well and the rate was 71.0%.</p><p><b>CONCLUSION</b>The high rate of HPV in the esophageal carcinoma samples suggested that HPV plays an etiologic role in the development of esophageal cancer in Linzhou City, Henan Province.</p>

Suppressive effect of rhIL-24 protein on the human A375 cell melanoma in nude mouse / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the suppressive effect of purified and renaturalized rhIL-24 protein on the human A375 cell melanoma in nude mouse.</p><p><b>METHODS</b>Human A375 cells were injected into the nude mouse. After the volume of tumor attained, rhIL-24 was injected into the tumor. 2 weeks later, the tumors were resected for measurement of volume and weight, following with pathological and immunohistochemistry examination.</p><p><b>RESULTS</b>The volume and weight of tumors were decreased markedly after treatment of rhIL-24, when compared with those in controls. The expression of Bax gene upregulated, while Bcl-2, CD34 and VEGF gene downregulated. It indicated tumor growth inhibition and inducing of apoptosis of tumor cells.</p><p><b>CONCLUSIONS</b>rhIL-24 has a suppressive effect on the A375 cell melanoma in nude mouse. It can also induce the A375 cell apoptosis without side effect on nude mouse.</p>

Cloning, expression of fibrinolytic enzyme gene Efp-I from Eisenia fetida in Escherichia coli and activity analysis / 生物工程学报

Earthworm fibrinolytic enzyme (EFE) is a group of protease having fibrinolytic and plasminogen-activator activities isolated from earthworm. Molecular biology research showed that there were 21 EFE coding sequences, in which only one sequence, AY438624, whose translated protein had similar N-terminal amino-acid sequence to EfP-I purified from Eisenia fetida. To obtain coding sequence of EfP-I , we designed specific primers according to 5' and 3' sequences of AY438624. A new DNA sequence was obtained by RT-PCR, sequence analysis showed that the protein translated from the coding sequence had identical N-terminal amino-acid sequence with EfP-I purified from Eisenia fetida and Lumbricus rubellus. Analysis by using ScanProsite prediction programs proved that the sequence had high similarity to AY438624 and belonged to trypsin family of serine protease. But there was difference between two sequences, that was there was a domain of characteristic amino acids of N-glycosylation site Asn-Xaa-Ser/Thr(N-x-S/T)in the new sequence (DQ418454). Then the expressed vector pMAL-c2X-Efp-I was constructed by cloning the gene into the plasmid pMAL-c2X, and was transformed to E. coli TB1. After induction and expression of the recombinant, the product MBP-EfP-I was purified by MBP affinity chromatography. Western blotting analysis showed that the product reacted with both anti-MBP and anti-EfP-I -1 serum. Casein plate test and fibrin plate test showed that the protein expressed had fibrinolytic activity.

In silico cloning of Efp-0, a novel earthworm fibrinolytic enzyme gene and verification of its coding region by RT-PCR / 生物工程学报

There are four different types of N-terminal amino acid sequences (F-I-0, F-I, F-II, F-III) in the multicomponents of earthworm fibrinolytic enzymes (EFE). In GenBank 21 nucleic acid sequences of EFE have been reported. Among them, most of the N-terminal amino acid sequences belong to the F-III type,few belong to the F-II type. Only one is similar to the F-I type, but none to F-I-0. In this research we hoped to obtain the gene encoding component F-I-0 of EFE by the bioinformatics tools. Based on the N-terminal amino acid sequence VVGGSDTTIGQYPHQL of the F-I-0 type from Lumbricus rubellus, a nucleic acid sequence was obtained by in silico cloning from dbEST of Lumbricidae using the software DNAMAN. A new gene of EFE from Eisenia foetida was successfully obtained by RT-PCR using specific primers designed according to this sequence. The new gene named EfP-0 was cloned in pMAL-c2x and expressed as the fusion protein MBP-EfP-0 in the supernatant of lysate. The fusion protein MBP-EfP-0 purified by affinity chromatography had hydrolytic activity on casein plate. Sequencing result shows, EfP-0 has 678bp and encodes a protein of 225 amino acids. The protein is a serine protease belonging to trypsin family. It has similar amino acid composition to F-I-0. BLAST in GenBank shows that the similarity is lower than 40% between EJP-0 gene and other EFE genes. By this we conclude that EfP-0 gene of EFE is a novel gene and it is the first time to be reported, its accession number for Genbank is DQ836917.

A component of earthworm fibrinolytic enzyme having higher thrombolytic activity than total components in vivo / 药学学报

<p><b>AIM</b>To select higher thrombolytic and lower toxic single component of earthworm fibrinolytic enzymes (EFE).</p><p><b>METHODS</b>EFE containing total components were obtained by affinity chromatography from Eisenia fetida. Using ion-exchange chromatography to separate three main components EfP-0-2, EfP-I-1 and EfP-I-2 from EFE, their thrombolytic activity and toxicity were compared with EFE.</p><p><b>RESULTS</b>Among these components, EfP-I-1 had higher thrombolytic activity in vitro. When 4.5 mg x kg(-1) of these components were injected, the contents of fibrinogen in rat serum were not affected, but only EfP-I-1 exhibited distinct thrombolytic activity. When 6.0 mg x kg(-1) of them were injected intravenously, the bleeding time was not evidently delayed only by EfP-I-1. The acute toxicity test showed that the LD50 of EfP-I-1 was higher than EFE by 2. 17 times.</p><p><b>CONCLUSION</b>Because of distinct thrombolytic activity, lower toxicity in vivo, higher content in EFE and easy to purify, EfP-I-1 was adapted to be developed as a single component medicine for treating thrombus.</p>

Purification and renaturation of recombinant human Cu, Zn-SOD by metal-chelating affinity chromatography / 生物工程学报

Overexpression of recombinant Human Cu, Zn-Superoxide Dismutase (rhCu, Zn-SOD) in E. coli results in the form of insoluble inclusion body. Purity of rhSOD inclusion body was over 80% by isolation and purification. After preliminary renaturation by conventional dilution or dialysis, enzyme preparations was respectively purified by using Copper Metals-Chelating Affinity Chromatography (Copper-MCAC). RhSOD specific activity purified by MCAC (from the sample renatured partly by dialysis) was 2.2 times as much as that by dialysis and protein recovery was 64%. RhSOD specific activity purified by MCAC (from the sample renatured partly by dilution) was 5.3 times as much as that by dilution and protein recovery was 25%. The two rhSOD preparations purified by MCAC had specific activities about 5000 u/mg and activity recoveries were all over 130% of the enzyme activities in the samples renatured partly by dilution or dialysis. The above-mentioned results indicated that Copper-MCAC resulted in a purification and further renaturation of target protein. SDS-PAGE showed that the target protein rhSOD (19 kD) was purified homogeneously and NBT activity identification proved that the purified and renatured rhSOD had very strong SOD activity. In conclusion, Copper Metals-Chelaing Affinity Chromatography appears to be a simple, rapid and efficient procedure for purifying and further renaturing rhCu, Zn-SOD by dilution or dialysis. The method provided a new idea for purifying and renaturing recombinant proteins expressed in the form of inclusion body in E. coli.

The tendency of physiological and psychological change of the armored vehicle drivers working continuously in hot environment / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To probe the tendency of physiological and psychological change of armored vehicle drivers working continuously in hot environment.</p><p><b>METHODS</b>Ten cross-designed standard subjects were divided into control group and driver group. The driver group worked continuously in 32 degrees C approximately 38 degrees C and RH 50% approximately 80% environment. Heart rate, body temperature, systolic blood pressure and pulse pressure were synchronously and dynamically determined during work. Psychological parameters such as profile of mood states (POMS) questionnaire, serial addition/subtraction (SAS) and two-digit search (TDS) selected from neurobehavioral evaluation system (NES) before and after work were also tested.</p><p><b>RESULTS</b>The curves of the four main physiological parameters rapidly synchro-rose to a peak at (36 +/- 3) min, whereafter the curves descended smoothly. At (144 +/- 8) min, the curves climbed up to the second peak. The determination of the behavioral psychology revealed that the degree of fatigue of the drivers increased gradually when the working time prolonged, and the grip descended by great extent, excitement reduced markedly after work, but the negative mood, such as confusion and depression were increased, error of visual apperceive-operation was increased, right decreased, intelligence reduced, rapidity and delicacy of movement reduced.</p><p><b>CONCLUSION</b>The physiological parameters of armored vehicle drivers working continuously in the hot condition appeared double-peak effect, the 1st peak was at (36 +/- 3) min, and the body was in mobilizing stage. Whereafter, the body was in smooth stage. And at (144 +/- 8) min, when the 2nd peak appeared, the body then was in physiological tolerance limit stage.</p>