Clinical analysis of combined immunotherapy in patients with malignant pleural mesothelioma / 中华肿瘤杂志

Objective: To observe the present situation, efficacy and safety of immunotherapy in patients with malignant pleural mesothelioma (MPM). Methods: The data of 39 patients with MPM in two centers from 2016 to 2021 were collected and the efficacy and safety were evaluated. According to the application of immune checkpoint inhibitors (ICIs), these patients, whose median clinical follow-up amounting to 18.97 months, were divided into immunotherapy group (19 cases) and control group (20 cases). Kaplan-Meier method and Log-rank test were used for the survival analysis. Results: The objective response rate (ORR) and the disease control rate (DCR) in the immunotherapy group is 21.05% and 79.0% respectively, compared with 10.0% and 55.0% in the control group; and the difference was not statistically significant (P>0.05). The median overall survival (OS) in the immunotherapy group was significantly longer than that in the control group (14.53 months vs 7.07 months, P=0.015), but there was no significant difference in the median progression free survival (PFS) between two groups (4.80 months vs 2.03 months, P=0.062). Single factor survival analysis showed that the nature of pleural effusion, pathological subtype and the efficacy of immunotherapy were related to both PFS and OS of the patients with MPM (P<0.05). The incidence of adverse reactions in immunotherapy group was 89.5% (17 out of 19 cases), and the most common adverse event was hematological toxicity (9 cases), followed by nausea and vomiting (7 cases), fatigue (6 cases) and skin damage (6 cases). Five patients had immune checkpoint inhibitors (ICIs) related adverse reactions with grade 1-2. Conclusions: Patients with MPM have begun to receive immunotherapy in more than 2-line mainly combined chemotherapy in the real world, and the median treatment line is 2-line. Either combined with chemotherapy or anti-angiogenesis therapy, ICI inhibitors have significant efficacy, controllable adverse events and good clinical value.

Effect of Lymph Node Metastasis on Change of Positive Thyroglobulin Antibody in Differentiated Thyroid Carcinoma after Initial Treatment / 中国医学科学院学报

Objective To investigate the influence of lymph node metastasis on the change of positive thyroglobulin antibody(TgAb)in differentiated thyroid carcinoma after initial treatment.Methods We retrospectively analyzed the clinical data of 98 differentiated thyroid carcinoma patients with positive TgAb(≥115 IU/ml)before radioiodine(RAI)therapy.All of whom underwent total or near total thyroidectomy,neck lymph node dissection,and subsequent RAI therapy.Patients were divided into negative group(n=83)and non-negative group(n=15)according to the disappearance of positive TgAb or not after a mean follow-up of 21.0 months.Analysis of variance,χtest,and Mann-Whitney rank-sum test were applied to compare the basic clinical features including number of metastatic lymph nodes,lymph node metastasis rate and node stage,and dose of RAI ablation.The receiver operating characteristic curves were employed to evaluate the predictive values of TgAb levels(negative or positive)and optimal cut-off points.Multivariate analyses were further performed to explore the independent indicators for persistent positive TgAb. Results Compared with the negative group,the proportions of N1a and N1b in the non-negative group were significantly higher,with no N0 in the non-negative group(Fisher's Exact Test,P=0.032).The median metastatic lymph node rate was also significantly higher in the non-negative group(Mann-Whitney U=-3.498,P=0.000).The cut-off value for metastatic lymph node rate to predicting disappearance of positive TgAb was 24%,and its sensitivity was 71.4%.The multivariate analysis showed that only lymph node stage(OR=3.183,P=0.038)was the independent indicator for persistent positive TgAb. Conclusions Lymph node stage was an independent indicator for the disappearance of positive TgAb.A metastatic lymph node rate of higher than 24% may be predictive for the disappearance of positive TgAb.

Concurrent chemoradiotherapy followed by consolidation chemotherapy and sequential chemoradiotherapy for stage III non-small cell lung cancer: comparison in 93 patients / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the efficacy and toxicity of concurrent chemoradiotherapy followed by consolidation chemotherapy (CCRT-CT) and sequential chemoradiotherapy (SCRT) in the treatment of stage III non-small cell lung cancer.</p><p><b>METHODS</b>From February, 2007 to June, 2010, 93 patients with unresectable stage III non-small cell lung cancer were treated with SCRT or CCRT-CT. SCRT group (50 cases) received radiotherapy after 2-6 cycles of chemotherapy (median 2 cycles) followed by 0-4 cycles (median 2 cycles) of chemotherapy. CCRT-CT group (43 cases) received 2 cycles of chemotherapy every 3 weeks with concurrent radiotherapy followed by 2-4 cycles (median 2 cycles) of chemotherapy with the same drugs. The chemotherapy consisted of cisplatin plus gemcitabine, docetaxel or vinorelbine. Radiotherapy was administered using two-dimensional conformal irradiation (36-40 Gy/18-20f) followed by three-dimensional conformal boost to 56-70 Gy/28-35f (median DT64Gy) or using three-dimensional conformal irradiation 50-74 Gy/25-37f (median DT62Gy).</p><p><b>RESULTS</b>The response rates were 76.7% and 54.0% in CCRT-CT and SCRT group, respectively (P<0.05). The median progression-free time in the two groups was 16.0 and 10.0 months, with the overall survival time of 18.0 and 12.5 months, respectively. The 1-, 2- and 3-year overall survival rates were 83.7%, 48.8% and 20.9% in CCRT-CT group and 52.0%, 20.0%, and 2.0% in SCRT group, respectively (P<0.05). CCRT-CT group showed a significantly lower rate of distant metastasis than SCRT group (P<0.05), but the local recurrence rate was similar between the two groups. The main side effects included radiation pneumonitis, radiation esophagitis, nausea/vomiting and anemia/leucopenia/thrombocytopenia. CCRT-CT group had a significantly higher rate of III-IV grade nausea/vomiting and anemia/leucopenia/thrombocytopenia than SCRT group.</p><p><b>CONCLUSION</b>Compared to SCRT, CCRT-CT can improve the response rate, progression free survival and overall survival and decrease the rate of distant metastasis, but is associated with a higher toxicity.</p>

Detection of epidermal growth factor receptor and c-Met gene copies for prognostic evaluation of non-small cell lung cancer / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of epidermal growth factor receptor (EGFR) and c-Met in the oncogenesis, development and prognosis of non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The relative copy numbers of EGFR and c-Met mRNA were detected in 61 cases of NSCLC by fluorescent RT-PCR, and the correlation between EGFR and c-Met as well as their correlation to the clinicopathological data of the patients were analyzed. A survival analysis was also performed in relation to EGFR and c-Met expressions.</p><p><b>RESULTS</b>The relative copy numbers of EGFR and c-Met were positively correlated (r=0.352, P=0.005). The levels of these two genes in smokers were 0.15 and 0.14, respectively, significantly higher than those in non-smokers (P<0.05); their levels were 0.16 and 0.14 in adenocarcinoma, respectively, significantly higher than those in squamous carcinomas (P<0.05). In patients with squamous carcinomas, a higher level of EGFR and c-Met DNA copies was associated with poorer prognosis, and Log Rank analysis indicated a survival difference in relation to EGFR and c-Met DNA copies (P=0.015, P=0.046).</p><p><b>CONCLUSIONS</b>EGFR and c-Met may interact in a synergistic manner in the oncogenesis and development of NSCLC, and may help in the prognostic evaluation of squamous carcinomas.</p>

Clinicopathological features and prognostic implications of EGFR/c-Met gene copy number and protein expression in non-small cell lung cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the gene copy number and protein expression of EGFR/c-Met, so as to explore the relationship between them and the clinicopathological features and prognosis in non-small cell lung cancer (NSCLC) patients.</p><p><b>METHODS</b>The expression of EGFR/c-Met protein was detected by immunohistochemical staining in NSCLC tissues of 61 cases. The level of EGFR/c-Met gene copy number was detected by fluorescent RT-PCR.</p><p><b>RESULTS</b>The positive rates of EGFR and c-Met protein expression were 77.5% and 57.4%, respectively. These rates in well/moderately differentiated NSCLC tissues were 61.5% and 38.5%, all significantly lower than the data in poorly differentiated tissues (P < 0.05). The relative EGFR and c-Met gene copy number was 0.22 ± 0.22 and 0.20 ± 0.21 in smokers, respectively, all significantly higher than that in non-smokers (P < 0.05). The gene copy number of EGFR and c-Met in adenocarcinoma was 0.24 ± 0.26 and 0.23 ± 0.25, respectively, all higher than the data in squamous cell carcinoma (P < 0.05). There was a significant correlation in regard to the EGFR and c-Met protein expression, EGFR and c-Met gene copy number, and the protein expression vs. the gene copy number only in EGFR (P < 0.05). But there was no significant correlation between the c-Met protein expression and gene copy number (P = 0.259). There was a statistical significance between the postoperational median survival times (MST) of low EGFR gene copy number (48 months) and the high EGFR gene copy number (36 months) patients (P = 0.039). Similarly, there was also a significant difference between the MST of the low and high c-Met gene copy number patients (44 and 31 months, P = 0.022).</p><p><b>CONCLUSION</b>There is a correlation between the EGFR and c-Met protein expression and the differentiation of NSCLC. The relative gene copy number is correlated with pathologic types and smoking of NSCLC patients, and it can be used in the prediction of prognosis.</p>

Recombinant lentivirus-mediated gene transfer of NT4-p53(N15)-Ant inhibits the growth of hepatocellular carcinoma cells in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct a recombinant lentivirus vector containing fusion gene NT4-p53(N15)-Ant and transfer it into HepG2 cancer cells for gene therapy.</p><p><b>METHODS</b>The gene of p53(N15)-Ant was obtained by T-vector method. After restriction enzyme digestion, the interest gene of p53(N15)-Ant was inserted in pBV220/NT4 vector and fusion gene of NT4-p53(N15)-Ant was subcloned into the plasmid of lentivirus and cotransferred into HEK-293 cells with helper plasmid. The recombinant lentivirus was produced by homologous recombination of the above mentioned two plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of LV. NT4-p53-Ant in transfected HepG2 cells was finally confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of LV. NT4-p53(N15)-Ant on HepG2 cells was measured by a colorimetric 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibition effect on HepG2 cells of LV. NT4-p53(N15)-Ant and its potential mechanism was detected by light microscopy, electron microscopy, MTT, LDH-release assay and annexin V-PI double staining.</p><p><b>RESULTS</b>The gene of p53(N15)-Ant was confirmed by restriction enzyme digestion and DNA sequencing. High titer of the recombinant lentivirus was obtained by homologous recombination in HEK-293 cell lines (1 x 10(11) pfu/ml), and the expression of NT4-p53(N15)-Ant gene in HepG2 cells was confirmed by RT-PCR. The viability of HepG2 cells was decreased to 83.4%, 46.9% and 33.9%, at 24 h, 48 h and 72 h, respectively, after infection by LV. NT4-p53(N15)-Ant. Compared with the LV. EGFP control group, there were significant differences (P < 0.01). The LDH level in HepG2 cells infected by LV. NT4-p53(N15)-Ant at 48 h, 72 h and 96 h after infection was 682 IU/L, 815 IU/L and 979 IU/L, respectively, significantly increased than that in the LV. EGFP group (P < 0.01), indicating the cell membrane destruction.</p><p><b>CONCLUSION</b>The recombinant lentivirus vector encoding gene NT4-p53(N15)-Ant is successfully constructed in this experiment by molecular cloning and recombination in vitro techniques, and the results suggested that this fusion gene has an anti-tumor effect, which provides the basis for further research on recombinant adenovirus for cancer gene therapy.</p>

Adenovirus-mediated killing of hepatocellular carcinoma HepG2 cells by heterogeneous fusion gene NT4p53(N15)Ant / 南方医科大学学报

<p><b>UNLABELLED</b>OBJECTIVE To construct a recombinant adenovirus Ad.NT4p53(N15)Ant and explore its cytotoxic effect against hepatocellular carcinoma HepG2 cells in vitro.</p><p><b>METHODS</b>The recombinant adenovirus containing the fusion gene of neurotrophin 4 (NT4)signal peptide, N-terminal residues (12-26) of p53 and 17 amino acid Drosophila homeobox protein Antennapedia (Ant) was constructed by gene cloning protocol. The effect of this fusion gene on HepG2 cells was evaluated by MTT assay, PI staining and flow cytometry.</p><p><b>RESULTS</b>The fusion gene Ad.NT4p53(N15)Ant was successfully constructed, as verified by restriction endonuclease digestion and PCR. Ad.NT4p53(N15)Ant could strongly suppress the growth of HepG2 cells (with a growth inhibition rate of 63.3% 48 h after infection) without affecting NIH-3T3 cells. Flow cytometry showed that Ad.NT4p53(N15)Ant could induce obvious apoptosis of HepG2 cells.</p><p><b>CONCLUSION</b>The recombinant adenovirus containing NT4p53(N15)Ant fusion gene can inhibit the growth the of HepG2 cells in vitro partially by inducing cell apoptosis.</p>

Expression and purification of tissue factor pathway inhibitor in Pichia pastoris / 中国医学科学院学报

<p><b>OBJECTIVE</b>To generate recombinant human tissue factor pathway inhibitor (TFPI) in Pichia pastoris.</p><p><b>METHODS</b>To improve the expression of TFPI, a silent mutation was generated at the specific site of TFPI cDNA. Both wild-type TFPI cDNA and mutated TFPI cDNA were cloned into the expression vector pPic9. The constructed plasmids were subsequently transformed into Pichia pastoris cells GS115 and KM71, and the transformants were confirmed by polymerase chain reaction and DNA sequencing. The expression of recombinant protein was induced by addition of 0.5% methanol in the culture medium. The cell culture medium after induction was concentrated through ultra filtration. The recombinant protein was further purified by a three-step process (Heparin-sepharose CL-6B affinity chromatography, DEAE-Sepharose Fast Flow affinity chromatography, and Sephadex G75-gel filtration). The amount of the recombinant protein was quantified with gel imaging system. The activity of the recombinant protein was analyzed by the chromogenic substrate assay.</p><p><b>RESULTS</b>The amount of TFPI expressed in the mutated clone (1 mg/L) was much higher than that in the wild type clone (0.1 mg/L). The TFPI activity in the recombinant GS115 cells could be detected 12 hours after induction and reached the peak at 36 hours, while the TFPI activity in the recombinant KM71 cells started to show up at 24 hours after induction and reached the peak at 72 hours. The expression of recombinant protein in the silent mutant was significantly higher than those of wild type clone in both GS115 and KM71 host cells. The relative molecular mass of recombinant TFPI was approximately 42 000.</p><p><b>CONCLUSION</b>Introduction of the silent mutation at the specific site of TFPI cDNA can increase the recombinant protein expression in Pichia pastoris, which is much higher than that in insect cells or saccharomyces cerevisiae.</p>

Cellular uptake and cytotoxicity of modified chitosans as gene carriers / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the effects of arginine modified chitosan or hexadecylated modified chitosan as gene carriers on the cellular uptake by vascular smooth muscle cells and its in vitro cytotoxicity. METHODS Plasmid DNA was labeled with alpha-32P-dATP and complexed with the modified chitosans or unmodified chitosan to form nanoparticle complexes by complex coacervation method. Uptake of all kinds of chitosan/ DNA nanoparticle complexes (CNC) by A10 cells was measured by beta-liquid scintillation counting. The in vitro cytotoxicity of the CNC was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay.</p><p><b>RESULTS</b>The diameters of the CNC ranged from 55.9-174.9 nm and the zeta potentials were from 10. 8 mV for the arginine modified chitosan/DNA nanoparticle complexes (ACNC) to 1.8 mV for the hexadecylated chitosan/DNA nanoparticle complexes (HCNC). The cellular uptake of the modified chitosan/ DNA nanoparticle complexes (MCNC) by A10 cells increased significantly when compared with the unmodified chitosan/DNA nanoparticle complexes (UCNC) (P < 0.05), with the HCNC at N/P ratio of 1:1 and the ACNC at ratio of 8:1 showing the highest cellular uptake (1.3 fold higher than UCNC, P < 0.05). MCNC were much less cytotoxic when compared with Lipofectamine 2000-DNA nanoparticles.</p><p><b>CONCLUSION</b>DNA nanoparticle complexes prepared with either arginine or hexadecylated modified chitosan can improve the cellular uptake of the DNA, while the in vitro cytotoxicity of both of the modified chitosan is much less than that of Lipofectamine 2000.</p>

Inhibition of the expression of p42MAPK in HeLa cell line by RNA interference / 中华病理学杂志

<p><b>OBJECTIVE</b>To screen for siRNAs that inhibit the expression of p42(MAPK) in HeLa cell line.</p><p><b>METHODS</b>Three p42(MAPK) siRNAs and one random siRNA were synthesized using Silencer siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry.</p><p><b>RESULTS</b>Two siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42(MAPK) expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control.</p><p><b>CONCLUSION</b>Inhibition of p42(MAPK) expression with siRNA technique can inhibit the proliferation of HeLa cells.</p>

Hypoxia inducible factor-1alpha and leukemic cell differentiation / 生理学报

Arsenic trioxide (As2O3, ATO) is a recently developed drug for the effective treatment of acute promyelocytic leukemia (APL). Experimental studies showed that in vitro differentiation-inducing ability on APL cells of this drug is not significant compared with its in vivo activity. We unexpectedly found recently that hypoxia-mimetic agents and moderate real hypoxia triggered acute myeloid leukemic cells to undergo differentiation. Furthermore, intermittent hypoxia significantly prolonged the survival of the transplanted leukemic mice with inhibition of infiltration and induction of differentiation of leukemic cells. In the following works, molecular mechanisms of hypoxia-induced differentiation were investigated and some interesting results have been obtained. This review will shortly summarize the related progresses and discuss the questions remained to be further investigated.

Small interfering RNA-mediated MAPK p42 silencing induces apoptosis of HeLa cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of small interfering RNA (siRNA)-induced MAPK p42 silencing on the survival of HeLa cells.</p><p><b>METHODS</b>Two siRNAs targeting at the MAPK p42 gene and one random siRNA were synthesized respectively by Silencer siRNA Construction Kit and transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) in the transfected HeLa cells was analyzed by Western blotting and immunohistochemistry, and the morphology of cells were observed with electron microscope. TUNEL assay and Annexin V/PI staining were employed for detecting the cell apoptosis.</p><p><b>RESULTS</b>The expression of p42(MAPK) in the HeLa cells was remarkably suppressed after transfection with the two siRNAs, reduced by about 2.5 and 3.2 folds respectively in comparison with the negative control. Chromatin margination in the cell nuclei were observed in the transfected cells, and TUNEL assay and Annexin V/PI staining further confirmed the occurrence of cell apoptosis.</p><p><b>CONCLUSION</b>In vitro MAPK p42 siRNA-1 and siRNA-2 transfection can specifically silence the gene expression and induce apoptosis of HeLa cells.</p>

Influence of water-soluble additives on drug release kinetics from biodegradable poly(lactic-co-glycolic acid) matrix / 药学学报

<p><b>AIM</b>To evaluate the effects of an array of additives on drug release from double-layered poly(lactic-co-glycolic acid) (PLGA) matrices.</p><p><b>METHODS</b>Additives differing in molecular size, hydrophilicity and steric configuration were selected for this study. An anti-proliferative 2-aminochromone, U-86983 (U-86, Pharmacia and Upjohn), was used as a model agent because of our interest in investigating local drug delivery systems for the inhibition of restenosis.</p><p><b>RESULTS</b>In vitro release of U-86 PLGA matrices without additive showed a typical biphasic release kinetics, i.e. a slow diffusion release (Phase I) followed by a fast erosion-mediated release (Phase II). The water-soluble additives in PLGA matrices changed the biphasic release pattern to a near monophasic profile by increasing the release of the Phase I. Increasing the ratio of additives to PLGA in matrices caused a significant increase in U-86 release rates. A high molecular weight water-soluble additive, Pluronic F127, resulted in a matrix showing perfect zero-order release kinetics. The morphologic evaluation of matrices using scanning electron microscopy indicated that the water-soluble additives were leachable and thus generated a highly porous structure in the matrices. Conclusion Water-solubility, molecular size and steric configuration of additives are the important determinants in generating various types of pore structures in polymer matrix which in turn affect the release mechanism and release kinetics.</p>

Chemical coupling of anti-dNA antibody on collagen coating / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the feasibility and stability of chemically conjugating IgM on collagen films.</p><p><b>METHODS</b>IgM was labeled with 125I using the chloramine-T method. Six collagen films were randomly divided into two groups. In chemical coupling group 125I-labeled IgM was chemically coupled with the films through N-succinmiclyl-3- (2-pyridyl-dithio) propionate reaction. In control group 125I-labeled IgM was absorbed onto collagen films. The amount of IgM on the collagen films and the amount of IgM remained on the films after extensive rinsing with phosphate buffered saline were monitored by counting the radioactivity of 125I.</p><p><b>RESULTS</b>The amount of antibodies loaded onto collagen films in the chemical coupling group was 15 times higher than that on the control films, showing significant statistical difference (P < 0.01). And the stability of conjugation antibodies on collagen films was significantly better than the control films.</p><p><b>CONCLUSION</b>Chemical coupling is an effective approach to immobilize antibodies on collagen for further plasmid DNA tethering.</p>

Protein splicing and its application / 生物工程学报

Protein splicing is a newly discovered posttranslational editing process that removes an internal protein fragment from the protein precursor. During the splicing process the internal protein fragment, intein, triggered the self-excision from the precursor protein and the concomitant ligation of the flanking protein fragments, exteins. The self-catalysis requires neither auxiliary enzymes nor cofactors and only involves four intramolecular reactions. A number of key catalytic residues in inteins and flanking fragments have been identified, which led to the development of the protein splicing process as a protein engineering tool. Controllable cleavage of the peptide bond at either the N or the C terminus of an intein has allowed the design of novel strategies for manipulation of protein and peptides. Affinity purification of recombinant proteins can be facilitated by fusion the target protein with an intein. The fusion also creates C-terminal thioester, which expands the scope of chemical ligation in protein. Inteins can be engineered in a "split and inverted" configuration to form a cyclic polypeptide consisting of the sequence linking two intein subdomains. This article summarizes the recent advance in the mechanism of protein splicing and its applications in protein purification, protein ligation and protein cyclization.