Efficacy and peripheral immunity analysis of allogeneic natural killer cells therapy in patients with hepatocellular carcinoma / 北京大学学报(医学版)

OBJECTIVE@#To evaluate the safety and efficacy of allogeneic natural killer (NK) cells in the treatment of primary hepatocellular carcinoma (HCC), and to elucidate the mechanism of NK cells therapy.@*METHODS@#Twenty-one patients with primary HCC treated with allogeneic NK cells at the Fifth Medical Center of the PLA General Hospital were followed up for 1 year. Peripheral blood mononuclear cells (PBMCs) were isolated from patient-related donors and cultured in vitro for 15 days and infused to the patients in two consecutive days. Clinical data and laboratory data were collected and analyzed, including survival, clinical features, imaging changes, hematology, immunology, and biochemical indicators to evaluate the safety and efficacy of allogeneic NK cell therapy. The changes of peripheral blood lymphocyte subsets after treatment were also analyzed to explore the possible anti-tumor mechanisms.@*RESULTS@#(1) Of the 21 patients with primary HCC, 11 patients were treated once, 5 patients were treated twice, and 5 patients were treated 3 times. After allogeneic NK cells infusion, 10 patients had fever, 1 patient had slight hepatalgia and 1 patient had slight headache, no other adverse events occurred including acute and chronic graft-versus-host disease (GVHD). They resolved spontaneously within 8 hours without other treatment. (2) The total disease control rate was 76.2% during one-year follow-up. Among them, the patients with Barcelona clinic liver cancer (BCLC) stage A had a disease control rate of 100%, stable disease (SD) in 10 cases; BCLC stage B patients had a disease control rate of 60%, partial response (PR) in 1 case, and SD 2 in cases; BCLC stage C patients had a disease control rate of 50%, complete response (CR) in 1 case, and 2 cases of PR. (3) The frequencies of NK cells and CD8+ T cells in peripheral blood were significantly lower than that before at 24 hours after treatment, and the frequencies of CD4+ T cells and CD4/CD8 were significantly higher than the baseline.@*CONCLUSION@#Allogeneic NK cells have good safety and efficacy in the treatment of primary HCC. The anti-tumor effect of the allogeneic NK cells may play an important role in the activation of the patient's natural immune system and delay disease progression, suggesting that allogeneic NK cells combined with sorafenib may be a very effective treatment for advanced HCC, and further large-sample multicenter randomized controlled clinical trials are needed to validate this result.

Regulatory effect of icariin and oleanolic acid combined with dexamethasone on JAKs/STATs signaling pathway in hormone-resistant CEM-C1 cells / 医学研究生学报

Objective The mechanisms of epimedium and Ligustrum Lucidum with glucocorticoid (GC) acting on asthma are closely related to the regulation of the JAKs / STATs pathway associated with the Th1/Th2 balance in the lung tissue of the asthmatic rats. This study aimed to investigate the synergistic effect of icariin and oleanolic acid with dexamethasone on the protein expressions of JAKs/STATs in GC-sensitive CEM-C7 and GC-resistant CEM-C1 cells.Methods We divided CEM-C7 and CEM-C1 cells into groups A (complete culture medium control), B (dexamethasone at 10-6mol/L), C (icarrin at 100 mg/mL), D (oleanolic acid at 100 mg/mL), E (icarrin+oleanolic acid both at 50 mg/mL), and F (icariin+oleanolic acid+dexamethasone at 50 mg/mL, 50 mg/mL and 10-6 mol/L, respectively), and treated them with corresponding agents for 24 hours. Then, we determined the protein expressions of JAKs (JAK1 and JAK2) and STATs (STAT1, STAT3, STAT5, and STAT6) in the CEM-C7 and CEM-C1 cells of different groups by Western blot.Results The protein expressions of JAK1 and JAK2 in the CEM-C1 cells were 0.22±0.01 and 0.23±0.01 in group A, 0.24±0.01 and 0.24±0.01 in group B, 0.23±0.01 and 0.22±0.01 in group C, 0.24±0.01 and 0.23±0.01 in group D, 0.22±0.01 and 0.21±0.01 in group E, and 0.18±0.01 and 0.19±0.01 in group F, both significantly lower in groups E and F than in B (P<0.01), and in groups C, D and F than in E (P<0.01). The expressions of STAT1 and STAT3 proteins were 0.23±0.01 and 0.23±0.01 in group A, 0.23±0.01 and 0.22±0.01 in group B, 0.23±0.01 and 0.22±0.01 in group C, 0.23±0.01 and 0.23±0.01 in group D, 0.18±0.01 and 0.20±0.02 in group E, and 0.17±0.01 and 0.16±0.01 in group F, both remarkably lower in groups E and F than in B (P<0.01), and that of STAT3 even lower in F than in E (P<0.01). The expressions of STAT5 and STAT6 were 0.24±0.01 and 0.24±0.01 in group A, 0.23±0.01 and 0.23±0.02 in group B, 0.23±0.01 and 0.24±0.01 in group C, 0.23±0.01 and 0.24±0.01 in group D, 0.19±0.01 and 0.19±0.01 in group E, and 0.16±0.01 and 0.20±0.02 in group F, both markedly lower in groups E and F than in B (P<0.01), and even lower in F than in E (P<0.01). The protein expressions of JAK1 and JAK2 in the CEM-C7 cells were 0.24±0.01 and 0.22±0.02 in group A, 0.12±0.01 and 0.49±0.01 in group B, 0.23±0.01 and 0.27±0.01 in group C, 0.25±0.01 and 0.25±0.02 in group D, 0.27±0.01 and 0.23±0.01 in group E, and 0.20±0.01 and 0.32±0.01 in group F, the former increased while the latter decreased significantly in groups B, C, D, E and F as compared with group A (P<0.01), the former even lower and the latter even higher in groups C and F than in E (P<0.01). The expressions of STAT1 and STAT3 were 0.23±0.01 and 0.23±0.01 in group A, 0.10±0.01 and 0.11±0.02 in group B, 0.27±0.01 and 0.26±0.01 in group C, 0.27±0.01 and 0.27±0.01 in group D, 0.28±0.01 and 0.27±0.01 in group E, and 0.21±0.01 and 0.23±0.02 in group F, both remarkably higher in groups C, D, E and F than in B (P<0.01), though lower in F than in E (P<0.01). The expressions of STAT5 and STAT6 were 0.24±0.01 and 0.24±0.01 in group A, 0.10±0.01 and 0.11±0.02 in group B, 0.23±0.01 and 0.23±0.02 in group C, 0.23±0.01 and 0.23±0.01 in group D, 0.24±0.01 and 0.24±0.01 in group E, and 0.20±0.01 and 0.21±0.05 in group F, both significantly upregulated in groups C, D, E and F as compared with B (P<0.01), though lower in F than in E (P<0.05).Conclusion In case of hormone resistance, icariin and oleanolic acid combined with dexamethasone may regulate the JAKs/STATs signaling pathway and improve the sensitivity to hormone action.

NVP-BEZ235 inhibits proliferation and colony-forming capability of CD34(+)CD38(-) human acute myeloid leukemia stem cells / 中国实验血液学杂志

This study was aimed to explore the effect of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on proliferation, cell cycle and colony forming capability of CD34(+)CD38(-) human acute myeloid leukemia (AML) KG1a cells. Flow cytometry was used to detect expression of CD34 and CD38 on the surface of human AML KG1a cells; Trypan blue assay was used to analyze the effect of NVP-BEZ235 at various concentrations on proliferation of KG1a cells; flow cytometry was performed to examine the cell cycle of KG1a cells after NVP-BEZ235 treatment; Soft agar colony-forming experiment was used to detect the colony forming ability of KG1a cells treated with NVP-BEZ235 at various concentrations. The results indicated that the percentage of CD34(+)CD38(-) AML KG1a cells was (98.02 ± 0.72)%. NVP-BEZ235 (0.125 - 1 µmol/L) inhibited the proliferation of KG1a cells in a time-and dose-dependent manner (P < 0.05) and the 50% inhibition concentrations (IC50) at 24 h and 48 h were 0.597 µmol/L and 0.102 µmol/L, respectively. KG1a cells were arrested at G0/G1 phase after treating with 0.5 µmol/L NVP-BEZ235 for 24 h, it was significantly higher than that of control group (83.2 ± 3.80)% vs (43.47 ± 9.60)% (P < 0.05). KG1a cells treated with NVP-BEZ235 (0 - 1 µmol/L) for 14 d and 21 d, the number of colony decreased respectively from (375.67 ± 21.46) per 2500 KG1a cells and (706.33 ± 87.31) per 2500 KG1a cells to 0, with statistical significance (P < 0.05). It is concluded that NVP-BEZ235 can inhibit proliferation and colony-forming capability of CD34(+)CD38(-) human AML KG1a cells.

Peptide mapping of H-2d restricted T-cell epitope against six antigens of HIV-1 subtype B'/C by ELISPOT assay / 病毒学报

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.

The non-replicating recombinant vaccinia virus expressing six genes of HIV-1 can be passaged stably in CEF / 病毒学报

To investigate the genetic stability (including the vector of vaccinia virus and six foreign genes: gp160, gag, pol, rev, tat and nef) of the HIV-1 non-replicating recombinant vaccinia virus (rNTV-C). rNTV-C was serially passaged to passage 25 (P25) in primary chicken embryo fibroblast (CEF). P9, P12, P15 and P25 were selected to study the genetic stability in four aspects, including the genetic stability of viral vector, the genetic stability of six foreign genes, the expressing stability of foreign genes and the genetic loss of foreign genes. The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably; foreign gene sequences matched with designed sequences, the insert sites were right, and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C; the target proteins could be expressed effectively, and the expression level was stable within different passages of rNTV-C; the genetic loss of gag and nef was less than 5% within different passages of rNTV-C. The above results provided important data for the vaccine production.

Expression and immunity of multi-HIV B'/C subype genes in replicating DNA vaccines / 病毒学报

To understand the effect of various gene structures of HIV B'/C subtype on the gene expression and immunity in DNA vaccine, replicating DNA vector pSCK2 was used to construct seven DNA vaccines carrying one or more of HIV B'/C subtype genes: gagpol, gp160 and rtn (rev, tat and nef fusion gene). Immunofluorescence staining indicated that Gag, Gp160, Rev, Tat and Nef could be expressed from the seven DNA vaccines. Stronger expression was observed with the gene in single-gene expression plasmid or with the gene located at upper-IRES in double- or multi-gene expression plasmid. ELISA test showed that Gag induced higher antibody response, but the antibody titers stimulated by Gp160, Pol, or RTN were very low. Both Gag single-gene expression plasmid and Gag-RTN double-gene expression plasmid separately inoculating induced stronger antibody response against Gag than Gag-Gp160 double-gene expression plasmid and Gagpol-Gp160-RTN multi-gene expression plasmid or combined inoculation of Gag and Gp160 single-gene expression plasmids did. ELISPOT detection showed that all the seven DNA vaccines could stimulate cellular immune response against Gag, Pol, Gp160, Tat, and Nef, respectively. Gagpol or Gp160 single-gene expression plasmid separately inoculating stimulated the strongest cellular immune response. Tat and Nef expressed in all the plasmids induced similar immune response. These results indicated that HIV B'/C subtype genes gagpol, gp160 and rtn could be efficiently expressed in the replicating DNA vaccine vector, single-gene expression plasmid had the higher gene expression level and induced stronger immune response; combined immunization of Gagpol and Gp160 had dramatically lower immunity than Gagpol or Gp160 separated immunization did. Immunity of RTN had no difference between combined and separated immunizations. Therefore, in case of immunization with DNA vaccines containing different HIV genes, it is necessary to optimize the combined immunization procedure, especially for the combination of Gag and Gp160-containing vaccines.

Vaccination with three HIV-1 cross neutralizing epitopes fused to HBV S antigen could induce robust antibody immune response in mice / 病毒学报

To enhance immunogenicity of HIV-1 cross neutralizing epitopes , three HIV-1 cross neutralizing epitopes (ELDKWA, NWFDIT, GPGRAFY) were fused to 3' end of HBV S gene by PCR cloning technology, respectively. Three vaccinia virus (Tiantan strain) recombinants expressing separately the three fusion genes were subsequently constructed, named as RVJ1175S-2F5 (ELDKWA), RVJ1175S-4E10 (NWFDIT) and RVJ1175S-447-52D (GPGRAFY), respectively. From the supernatants of CEF cells infected by these vaccinia recombinants, three subunit vaccines (PS-2F5, PS-4E10 and PS-447-52D) were prepared after purification. Biology and immunology characteristics of these fusion antigens in vaccinia recombinants and subunit vaccines were comparatively studied. It was confirmed by PCR and sequencing that the fusion genes were inserted into the TK locus of vaccinia virus (Tiantan strain) correctly. The Fusion proteins were expressed efficiently and secreted into supernatant of the infected cells, which was demonstrated by HBsAg ELISA test. Two typical HBsAg bands of 23kD and 27kD were detected in all the purified samples by SDS-PAGE. These two bands were reacted well to HBsAb and corresponding HIV-1 monoclonal antibodies 2F5, 4E10 and 447-52D. BALB/c mice were immunized with subunit and vaccinia recombinant vaccines by intraperitoneal injection. High levels of HBsAb and anti-HIV-1 cross neutralizing epitope antibody in peripheral blood of immunized mice were tested by ELISA, and all the antibody titers induced by three subunit vaccines were higher than that induced by correlated vaccinia recombinants in mice. This work provides a basis for future study on neutralizing activity of these immunized sera and enhancing immune effect through the combined immunization with different type of vaccines.