NK4基因转染对人宫颈癌细胞CaSki生物学特性的影响

 目的 通过在人宫颈癌细胞CaSki中转染NK4 cDNA片段，研究NK4对人宫颈癌的潜在治疗作用。方法 将NK4和荧光霉素表达质粒分别稳定转染人宫颈癌细胞CaSki。采用Western blot检测细胞培养基中NK4蛋白以及经不同培养基（CaSki, CaSki/LUC 及CaSki/NK4培养上清）培养后CaSki细胞中c-Met和磷酸化c-Met的表达。采用MTT法绘制细胞生长曲线。采用细胞划痕试验检测NK4对细胞转移的作用。结果 在CaSki/NK4培养基中有NK4蛋白表达，对照细胞（CaSki, CaSki/LUC）中无表达。磷酸化c-Met的表达在CaSki/NK4培养基培养的CaSki细胞中被抑制，而在对照细胞（CaSki, CaSki/LUC）培养基培养的CaSki细胞中正常表达。各组c-Met表达无差异。3种细胞体外生长曲线差异无统计学意义（P >0.05）。细胞划痕实验结果表明，CaSki/NK4划痕区域的细胞个数明显少于CaSki和CaSki/LUC。结论 NK4能抑制人宫颈癌细胞c-Met受体磷酸化，并能抑制宫颈癌细胞体外活动能力。     

Safety and efficacy of allogeneic natural killer cell immunotherapy on human immunodeficiency virus type 1 immunological non-responders: a brief report

Background:Allogeneic natural killer (NK) cell immunotherapy is recognized as a promising anti-tumor strategy, but whether it plays a role in poor CD4 recovery among human immunodeficiency virus type 1 (HIV-1) infected patients is unknown. This study aimed to investigate the safety and effectiveness of allogeneic NK cells immunotherapy on HIV-1 immunological non-responders (INRs) receiving antiretroviral therapy (ART).Methods:From February to April 2018, a prospective, randomized, controlled, open-label clinical trial, which enrolled 20 HIV-1 INRs following specific inclusion criteria, was conducted at Nankai University Second People''s Hospital. Participants were randomly allocated (simple randomization 1:1) to either the combined treatment (NK + ART) group (n = 10) or the control (ART) group (n = 10). The allogenic highly activated NK cells from killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen (HLA)-Cw mismatched healthy donor were prepared (10⁸ cells in each injection) and intravenously infused to each recruited patient of NK+ART group in three courses. Key immune parameters (CD4 count, CD8 count, CD4/CD8 ratio), laboratory tests (count of blood cells, biochemistry panel) and symptoms at baseline and at month 1, 3, 6, 9, 12, and 24 were measured/collected to analyze the safety and efficacy of the therapy. Comparisons were between the seven time-points of both groups using repeated measurement analysis of variance (ANOVA) test. Generalized estimating equations (GEE) model was performed to evaluate the overall effect of the NK+ART group vs. the ART group.Results:From baseline to 24 months, we noted a mean CD4 count augmentation (139 to 243 cells/μL) in the NK + ART group and (144 to 176 cells/μL) in the ART group (difference, 67; 95% CI, 10 to 124; P = 0.024). Our estimations revealed that NK+ART group could improve CD4 level (β = 54.59, P = 0.006) and CD8 level (β = 322.47, P = 0.010) on average among the six measurements compared with the ART group. Only two (2/10, 20%) participants in the NK+ART group developed a transient mild fever after the first course.Conclusions:This preliminary study informs that HIV-1 INRs, allogenic NK cells immunotherapy is safe and could significantly improve CD4 recovery but not CD4/CD8 ratio. The practical effects, however, need long-term follow-up observations. Further study on the potential underlying mechanism is warranted.Registration info:www.chictr.org.cn/showproj.aspx?proj=34912 (No. ChiCTR1900020634).     

Interstitial lung diseases in children

Interstitial lung disease (ILD) in children (chILD) is a heterogeneous group of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. The pathogenesis of the various chILD is complex and the diseases share common features of inflammatory and fibrotic changes of the lung parenchyma that impair gas exchanges. The etiologies of chILD are numerous. In this review, we chose to classify them as ILD related to exposure/environment insults, ILD related to systemic and immunological diseases, ILD related to primary lung parenchyma dysfunctions and ILD specific to infancy. A growing part of the etiologic spectrum of chILD is being attributed to molecular defects. Currently, the main genetic mutations associated with chILD are identified in the surfactant genes SFTPA1, SFTPA2, SFTPB, SFTPC, ABCA3 and NKX2-1. Other genetic contributors include mutations in MARS, CSF2RA and CSF2RB in pulmonary alveolar proteinosis, and mutations in TMEM173 and COPA in specific auto-inflammatory forms of chILD. However, only few genotype-phenotype correlations could be identified so far. Herein, information is provided about the clinical presentation and the diagnosis approach of chILD. Despite improvements in patient management, the therapeutic strategies are still relying mostly on corticosteroids although specific therapies are emerging. Larger longitudinal cohorts of patients are being gathered through ongoing international collaborations to improve disease knowledge and targeted therapies. Thus, it is expected that children with ILD will be able to reach the adulthood transition in a better condition.     

Low doses of rIL2 after autologous bone marrow transplantation induce a “prolonged” immunostimulation of NK compartment in high-grade non-Hodgkin's lymphomas

Ten patients with high-grade non-HodgKin's lymphoma (HG-NHL) entered a subcutaneous (s.c.) recombinant interleukin 2 (rIL2) trial within 2 months of undergoing autologous bone marrow transplantation (ABMT). Immunological studies, consisting in T- and natural killer (NK)-cell subset assessment, together with functional assays, such as NK activity and CD16-mediated redirected killing assay, were performed before therapy, after 2 weeks, and then monthly. Phenotypic analysis showed a significant increase (p=0.01) of CD16 and CD56 NK cells, from 12% to 28% and from 17% to 37%, respectively. In particular, the CD56^bright NK cell population showed a tenfold increase, while CD56^dim NK cells remained unmodified compared with pretreatment values. The expression of IL2 receptors was also studied, and a significant increase (p=0.01) of CD122 (p75)-positive cells from 8% to 30% was found, while no significant increase was observed in CD25 (p55)-positive cells. Furthermore, rIL2 administration led to an increase of NK activity even at the lowest effectors: target ratio and to an increase of CD16-mediated redirected killing assay. These phenotypic and functional modifications lasted throughout the duration of rIL2 therapy and remained after completion of therapy. In addition, none of the ten patients relapsed, and two of them who started IL2 treatment while still showing residual disease experienced a complete disappearance of the disease after 10 and 7 months of therapy, respectively. Our data suggest that infusion of rIL2 s.c. after ABMT is safe, can selectively increase NK cell number and function, and may have a beneficial effect on the minimal residual disease.This work sas supported in part by CNR PF ACRO: 94.01214.PF39.     

Cytotoxic Chemotherapy Successfully Induces Durable Complete Remission in 2 Patients with Mosquito Allergy Resulting from Epstein-Barr Virus-Associated T-/Natural Killer Cell Lymphoproliferative Disease

Recent findings indicate that Epstein-Barr virus (EBV)-infected T-/natural killer (NK) cells play an important role in the pathogenesis of mosquito allergy, and most patients with mosquito allergy die early in life if not properly treated. Over the last 7 years, we have been using combination chemotherapy and allogeneic stem cell transplantation for the treatment of EBV-associated T-/NK cell lymphoproliferative disease (LPD) in which chronic active EBV infection and mosquito allergy were included. As of this writing, we have successfully treated 2 patients with mosquito allergy with chemotherapy in which EBV-infected T-/NK cells were eradicated. The findings suggest the possible role of chemotherapy in the treatment of EBV-associated T-/NK cell LPD.     

自然杀伤细胞抗肝癌的研究新进展

肝癌是最常见的恶性肿瘤之一,预后差,复发率高。免疫治疗已成为继手术、放疗、化疗后的第四种治疗模式,其中自然杀伤细胞可以在细胞、分子及基因水平抑制肿瘤转移及复发,在肝癌的免疫治疗中起关键性作用,在开展新的治疗模式以及提高肝癌患者生活质量方面具有很广阔的应用前景。     

Advances in immunotherapy for pediatric acute myeloid leukemia

Introduction: Achieving better disease control in patients diagnosed with acute myeloid leukemia (AML) has proven challenging. Overall survival has been impacted by addressing treatment related mortality with focused supportive care measures. Despite this improvement, it remains difficult to induce durable leukemia remissions despite aggressive chemotherapeutic regimens. The addition of hematopoietic stem cell transplants (HSCT) has allowed further treatment intensification and provided the benefit of graft-versus-leukemia (GVL) effect. However, HSCT carries the risk of transplant related morbidities, particularly GVHD, and anti-tumor responsiveness is still suboptimal. Thus, there is a need for alternate therapies. Immunotherapy has the potential to address this need.

Areas covered: This review highlights promises and challenges to immune-based therapies for AML. It aims to summarize immunotherapeutic strategies trialed in AML patients to date, inclusive of: antibodies, vaccines, and cellular therapy. It emphasizes those being used in the pediatric population, but also includes adult clinical trials and translational science that may ultimately extend to pediatric patients.

Expert opinion: The elusiveness of an ideal surface antigen target together with an immunosuppressive leukemic microenvironment add to the already difficult challenge in developing AML-targeted immunotherapies. Though many hurdles remain, recent translational discovery and progressive clinical advances anticipate exciting future developments.     

In vitro exposure of DE‐71, a penta‐PBDE mixture,on immune endpoints in bottlenose dolphins (Tursiops truncatus) and B6C3F1 mice

Polybrominated diphenyl ethers (PBDEs) are an emerging contaminant of concern with low level exposures demonstrating toxicity in laboratory animals and wildlife, although immunotoxicity studies have been limited. Bottlenose dolphin peripheral blood leukocytes (PBLs) and mouse splenocytes were exposed to environmentally relevant DE‐71 (a penta‐PBDE mixture) concentrations (0–50 µg ml^?1) in vitro. Natural killer (NK) cell activity and lymphocyte (B and T cell) proliferation were evaluated using the parallelogram approach for risk assessment. This study aimed to substantiate results from field studies with dolphins, assess the sensitivities between the mouse model and dolphins, and to evaluate risk using the parallelogram approach. In mouse cells, NK cell activity increased at in vitro doses 0.05, 0.5 and 25 µg DE‐71 ml^?1, whereas proliferation was not modulated. In dolphin cells, NK cell activity and lymphocyte proliferation was not altered after in vitro exposure. In vitro exposure of dolphin PBLs to DE‐71 showed similar results to correlative field studies; NK cell activity in mice was more sensitive to in vitro exposure than dolphins, and the parallelogram approach showed correlation with all three endpoints to predict risk in bottlenose dolphins. Copyright © 2014 John Wiley & Sons, Ltd.     

Diagnosis and treatment of epstein-barr virus-associated natural killer cell lymphoproliferative disease

Epstein-Barr virus (EBV) exhibits tropism for both lymphocytes and epithelial cells and can induce both replicative (productive/lytic) and latent (persistent) infections that result in a variety of human diseases. With regard to lymphocytes, latent EBV infection is linked to development of heterogeneous lymphoproliferative disease (LPD), such as B-cell LPD and T-cell/natural killer cell (T/NK cell) LPD. Unlike B-cell LPD, LPD derived from T-cells and NK cells sometimes has overlapping clinical symptoms, as well as histologic and immunophenotypic features, because both types of cells are derived from a common precursor. However, determination of cell lineage is important in classification of lymphoid neoplasms, and combined modern techniques allows us to distinguish NK cell LPD from T-cell LPD in most instances. Because NK cell LPD seems to be heterogeneous in terms of clinical features, prognosis, and diagnosis and has a monoclonal or polyclonal (or oligoclonal) nature, this review attempts to clarify recent research and clinical findings and to establish diagnostic and therapeutic strategies.     

人类白细胞抗原单倍体不合造血干细胞移植后自然杀伤性细胞和T淋巴细胞杀伤抑制性受体表达的研究

目的研究人类白细胞抗原（HLA）单倍体不合的造血干细胞移植后自然杀伤性细胞（NK细胞）和T淋巴细胞杀伤抑制性受体重建的规律。方法2004—04～2004—12对北京大学血液病研究所借助三色和四色荧光标记技术，用流式细胞仪对应用该所GIAC方案（即G：粒细胞集落刺激因子＋I：强免疫抑制＋A：抗胸腺细胞球蛋白＋C：外周血与骨髓联合）进行HLA单倍体不合造血干细胞移植的24例患者（移植前、移植后＋30、＋60、＋90、＋120、＋180d）及其供者外周血NK细胞及T细胞上杀伤免疫球蛋白类受体（KIR），包括CD158a（KIR2DL1）、CD158b（KIR2DL2）、CD158e（K11t3DL1）和Lectin样受体（CD94／NKG2A）的表达进行了测定。结果根据移植后NK细胞上受体重建规律不同可以分为两组：第1组在移植后＋30d，与供者表达水平相比，病人受体表达明显升高（CD94，P=0．013；CD94：NKG2A，P〈0．0001；CD158e，P=0．063），随后逐渐下降，至＋180d时重建为供者水平（CD158e、CD94）或仍明显高于供者水平（CD94：NKG2A、P=0．001）；第2组在移植后＋30d，与供者表达水平相比，病人受体表达明显降低（CD158a，P=0．016；CD158b，P〈0．003），随后逐渐升高，至正常供者水平。移植后患者外周血T淋巴细胞上杀伤抑制性受体的表达规律与NK细胞第1组相近，在＋30和（或）60d时，所有KIR及CD94：NKG2A的表达均明显高于正常供者水平，其后逐渐下降，＋180d时降至正常供者水平（CD158a、CD158a／CD158b及CD158e）或仍明显高于供者水平（CD94／NKG2A，CD158b）。结论移植后NK细胞上KIR受体的低表达及CD94：NKG2A的高表达可能是移植后早期NK细胞杀伤功能低下的主要影响因素；而移植后T细胞上KIR及CD94：NKG2A的高表达可能有利于移植后的移植物抗宿主疾病（GVHD）及移植物抗白血病（GVL）效应分离。