Decrease in lymphokine-activated killer sensitivity of a human renal-cell carcinoma cell line after cytokine treatment

Recent studies have shown that cytokine treatment of tumor cells alters the sensitivity of these cells to lymphokine-activated killer (LAK) cells, depending on the cell line. In this study, we analyzed the decrease in LAK sensitivity of a human renal-cell carcinoma cell line (SMKT-R-3). The LAK sensitivity of SMKT-R-3 was decreased by treatment with a combination of interferon (IFN) and tumor necrosis factor (TNF). However, the cytokine treatment increased the expression of intercellular adhesion molecule-1 (ICAM-1) on the renal-cell carcinoma cell surface. The conjugate-formation assay also confirmed a slight increase in the binding rate of LAK cells to the renal-cell carcinoma cells. When actinomycin D (a protein synthesis inhibitor) was added to the culture medium prior to treatment with IFN and TNF, the LAK sensitivity of SMKT-R-3 recovered to the level demonstrated by the cells that had not received any cytokine treatment. These results suggest that the effect of cytokines in reducing LAK sensitivity of SMKT-R-3 is mediated by protein synthesis occurring when LAK cells are bound to SMKT-R-3 cells.     

Allogeneic dendritic cell vaccination against metastatic renal cell carcinoma with or without cyclophosphamide

In this phase I/II study, we evaluated the feasibility, safety and efficacy of allogeneic dendritic cells (DCs) with or without cyclophosphamide in the treatment of patients with metastatic renal cell carcinoma (RCC). Immunomagnetic beads were used to isolate CD14⁺ monocytes from healthy donor leukapheresis products, and CD83⁺ antigen-pulsed monocyte-derived DCs (moDCs) loaded with tumor lysate and keyhole limpet hemocyanin (KLH) were generated. Twelve patients were treated with allogeneic moDCs alone, while ten patients also received cyclophosphamide on days 4 and 3 prior to vaccination. Of the 22 patients enrolled, 20 received full treatment consisting of at least three vaccinations at monthly intervals. Two mixed responses with substantial tumor regression were observed. In 3 patients, disease stabilization occurred, in 13 patients disease progressed and 4 patients were lost to follow-up. Overall, immune responses against KLH and tumor lysate were weak or absent; however, the strongest increases in antigen-independent and KLH-specific responses were observed in the 2 patients with mixed responses. In addition, 1 of them showed a substantial increase in oncofetal antigen (OFA)-specific IFN- production. Importantly, the 2 mixed responders and 1 patient with stable disease belonged to the cyclophosphamide group. Median overall survival in the cyclophosphamide group was 23.2 and 20.3 months in the group that received allogeneic moDCs alone. Allogeneic immunotherapy with moDCs is feasible and well tolerated. However, the immunogenicity of allogeneic moDCs is clearly less pronounced than that of autologous moDC immunotherapy. Cyclophosphamide may have the capacity to augment DC-induced antitumor immunity.     

Frequency of regulatory T cells in renal cell carcinoma patients and investigation of correlation with survival

Background Regulatory T cells are important in maintaining immune homeostasis, mediating peripheral tolerance and preventing autoimmunity. Increased frequencies of CD4⁺CD25^high T regulatory (T_Reg) cells have been documented in the peripheral blood of patients with several types of cancer consistent with a role in tumour escape from immunological control. We have investigated the presence of T_Reg cells systemically and in situ in previously untreated patients with renal cell carcinoma (RCC). Results We have shown that there is a significant increased frequency of CD4⁺CD25^high T cells in RCC patients (n = 49) compared to normal donors (n = 38), respectively, 2.47% versus 1.50%; P < 0.0001. We confirmed these data using the FOXP3 marker of T_Reg cells in a subset of these patients and normal donors. The population of T_Reg cells identified showed the expected phenotype with CD4⁺CD25^high population in both RCC patients and normal donors contained higher proportions of CD45RO and GITR than CD4⁺CD25^−/low populations and exhibiting suppressive activity in an anti-CD3 and anti-CD28 induced proliferation assay. CD4⁺FOXP3⁺ T cells were detected in the tumour microenvironment by immunofluorescence and the numbers enumerated in lymphocytes recovered following enzymatic disaggregations of biopsies; their frequency was higher in the tumour than the peripheral blood of the same patients. The early follow up data show an association between higher peripheral blood regulatory T-cell count and adverse overall survival. Conclusion These data confirm the increase of T_Reg cells in RCC patients and provide impetus to further investigate modulation of T_Reg activity in RCC patients as part of therapy. Richard W. Griffiths and Eyad Elkord have equally contributed to the study.     

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study

Purpose Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (γδ) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated γδ T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. Experimental design Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous γδ T cells every week or every 2 weeks, 6–12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. Results Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of γδ T cells in peripheral blood and also a high response to the antigen in vitro. Conclusions The results indicated that adoptive immunotherapy using in vitro-activated autologous γδ T cells was well tolerated and induced anti-tumor effects.     

Activity of a short course of interferon α for metastatic renal cell carcinoma — a phase-2 study

Twelve patients with metastatic renal cell carcinoma were entered into a phase-2 study of an 8-week course of interferon (INF) therapy. INF was given subcutaneously at a dose of 3 mu, three times per week. The patients were WHO performance status 0–2. A complete response was obtained in two patients (17% response rate), which has been maintained at 23 and 45 months. One of these patients presented with cranial and lung metastases and received cranial irradiation and decradron concurrent with INF. The toxicity of INF has been low. The optimal duration of INF therapy warrants further evaluation.     

Patient-derived renal cell carcinoma cells fused with allogeneic dendritic cells elicit anti-tumor activity: in vitro results and clinical responses

Renal cell carcinoma (RCC) has been shown to be susceptible to immunotherapeutic treatment strategies. In the present study, patient-derived tumor cells were fused with allogeneic dendritic cells (DC) to elicit anti-tumor activity against RCC. DC from HLA-A2+ healthy donors were fused with primary RCC cells from ten patients. Phenotype of fusion cells were characterized by flow cytometer and confocal microscopy. In vitro, T cell proliferation, IFN-γ secretion and cytotocic T lymphocytes (CTL) activity elicited by allogeneic DC/RCC fusion cells were assessed. Clinically, ten patients were vaccinated with allogeneic DC/RCC fusion vaccine. The adverse effects and toxicity were observed. The clinical response was evaluated by CT scans. After fusion, the created hybrids expressed both tumor associated antigen and DC-derived molecules and could stimulate the proliferation and IFN-γ secretion of T cells as well as elicit strong CTL activity against RCC cells in vitro. In vivo, no serious adverse effects, toxicity, or signs of autoimmune disease were observed after vaccination therapy. Percentage of T lymphocyte subsets in peripheral blood of patients was increased significantly. One of ten patients exhibited a partial response with regression of lung metastases. Six patients showed stable disease with stabilization of previously progressive disease (follow up 1.5 years). The PR and SD responses, exhibited by 7/10 patients who received the allogeneic DC/RCC fusion vaccine treatment, suggest that this approach is safe and can elicit immunological responses in a significant portion of patients with RCC. J. Zhou and D. Weng contributed equally.     

一株新的人肾癌原代细胞系的建立及其生物学特性鉴定

目的:取肾癌病人标本建立一株新的人肾癌细胞系,初步对该细胞系进行鉴定,为进一步的肾癌基础研究提供实验模型.方法:2008-2009年间共采集20例肾癌新鲜手术标本,于手术后1 h内将每例标本切取四块0.5cm× 0.5 cm× 0.5 cm组织块,分别包埋于2只裸鼠的右后肢皮下及背部皮下,连续传代3次,取移植瘤体外培养,记录细胞株的生长曲线,测定细胞集落形成率,对细胞进行DNA含量测定,进行染色体分析及病理学检查.结果:其中1只裸鼠皮下移植瘤生长,继续传代,肿瘤生长速度明显加快.取移植瘤标本体外培养得到肾癌细胞系XJG-9201.形态结构,分化程度与原发瘤一致,染色体众数为65.细胞群体倍增时间为38.2h,细胞周期分析G1期62.7%,G2期11.2%,S1期25.3%,集落形成率为70%.结论:肾癌细胞系XJG-9201与原发癌保持相同的生物学特性,体外连续传代1年以上传115代.细胞形态不变,生长周期恒定,已成为一个稳定的细胞系.     

Vaccination strategies in patients with renal cell carcinoma

Although new treatment options for patients with advanced renal cell cancer (RCC) have been developed within recent years, vaccination is still a promising emerging treatment option. An increasing number of tumor-associated antigens (TAA) available for RCC are currently used and analyzed for their efficacy for antigen-specific vaccine strategies. Recently, antigen-specific vaccination with dendritic cells in patients with metastatic RCC was shown to induce cytotoxic T cell response associated with objective clinical responses in some of the patients. Furthermore, current studies focus on the development of more effective vaccine regimes, such as the application of polyvalent, HLA-independent RNA coding for multiple TAA and adjuvants. First results demonstrate promising clinical and immunological efficacy. The efficacy of antigen-specific vaccination might be improved by a combination of tyrosine kinase inhibitors, since sunitinib was shown to promote T cell induction following vaccination in a mouse model and elimination of regulatory T cells. “Immunotherapy: from basic research to clinical applications” symposium of the Collaborative Research Center (SFB) 685 held in Tübingen, Germany, 6–7 March 2008.     

MiR-24-3p as a prognostic indicator for multiple cancers: from a meta-analysis view

A growing number of researches suggest that microRNAs (miRNAs) as oncogene or tumor suppressor genes play a fundamental role in various kinds of cancers. Among them, miR-24-3p, as a star molecule, is widely studied. However, the prognostic value of miR-24-3p is unclear and controversial. We conducted this meta-analysis to evaluate the prognostic value of miR-24-3p in a variety of cancers by integrated existing articles from four databases. PubMed, Embase, Web of Science, and Cochrane Library (last update in March 2020) were searched for approach literature. Hazard ratios (HRs) and odds ratios (ORs) were used to evaluate the association between miR-24-3p expression levels and prognostic value or clinicopathological characteristics, respectively. A total of 15 studies from 14 literature were finally qualified and concluded in the present meta-analysis. A significantly worse overall survival was observed in higher expression of miR-24-3p cancer group for OS (overall survival) of log-rank tests and Cox multivariate regression by fixed effects model. Also, we found a significant correlation between elevated miR-24-3p levels to RFS (recurrence-free survival) and DFS (disease-free survival). In addition, the pooled odds ratios (ORs) showed that evaluated miR-24-3p was also associated with the larger tumor size (≥5 cm) and advanced TNM stage (III and IV). Built on the above findings, elevated expression levels of miR-24-3p may serve as a promising biomarker used to predict the worse prognosis of cancer patients.     

Design,synthesis and biological evaluation of 4-aniline-thieno[2,3-d]pyrimidine derivatives as MNK1 inhibitors against renal cell carcinoma and nasopharyngeal carcinoma

MAP Kinase Interacting Serine/Threonine Kinase 1 (MNK1) play important roles in the signaling transduction of MAPK pathways. It is significantly overexpressed in renal clear cell carcinoma and head-neck squamous cell carcinoma tissues in both mRNA and protein levels. Based on the crystallographic structure of MNK1 protein and binding modes analysis of known MNK inhibitors, we have designed and synthesized a series of 4-aniline-thieno[2,3-d]pyrimidine derivatives as potential MNK1 inhibitors. These synthetic compounds are tested in biochemical and cell proliferation assays, and six of them display potent inhibitory capacity against MNK1 kinase and cancer cell lines. Compound 12dj with strongest inhibitory capacity is transferred to molecular mechanism studies, and the results indicated that 12dj remarkably suppresses the phosphorylation of EIF4E, a substrate of MNK1. And the expression levels of MNK1, ERK1/2 and pERK1/2 are not affected by compound 12dj incubation in SUNE-1 and 786-O cells. In summary, our works suggested that these novel 4-aniline-thieno[2,3-d]pyrimidine based MNK1 inhibitors might be attractive lead compounds for targeted therapy of renal cell carcinoma and nasopharyngeal carcinoma.     

血清miRNAs作为食管鳞癌新的生物标志物

食管鳞癌是我国最常见的恶性肿瘤之一。由于缺乏有效的早期诊断方法,大多数食管鳞癌患者在确诊时已到中晚期并预后不良。MicroRNAs(miRNAs)是一类可通过抑制其特异性靶基因表达从而调控食管鳞癌发生发展的非编码内源性小RNA。相比于传统的生物标志物(例如mRNA和蛋白质),miRNAs更加稳定并易于筛选及精确地定量分析,从而成为理想的新一代癌症早期诊断和预后评估的生物标志物。近来的研究结果显示,食管鳞癌病人血清中的一些miRNAs表达水平的变化与病情诊断及预后的结果显著相关。文章综述了食管鳞癌病人血清中miRNAs的变化规律,讨论了检测这些miRNAs的表达水平变化作为一种新的方法应用于食管鳞癌的早期诊断和预后评估的可能性。值得注意的是,不同的血清miRNAs的检测方法所产生的结果是不完全一致的,文章还对这些差异产生的原因进行了讨论。     

Flocculation in Saccharomyces cerevisiae: a review

The present work reviews and critically discusses the aspects that influence yeast flocculation, namely the chemical characteristics of the medium (pH and the presence of bivalent ions), fermentation conditions (oxygen, sugars, growth temperature and ethanol concentration) and the expression of specific genes such as FLO1, Lg‐FLO1, FLO5, FLO8, FLO9 and FLO10. In addition, the metabolic control of loss and onset of flocculation is reviewed and updated. Flocculation has been traditionally used in brewing production as an easy and off‐cost cell‐broth separation process. The advantages of using flocculent yeast strains in the production of other alcoholic beverages (wine, cachaça and sparkling wine), in the production of renewal fuels (bio‐ethanol), in modern biotechnology (production of heterologous proteins) and in environmental applications (bioremediation of heavy metals) are highlighted. Finally, the possibility of aggregation of yeast cells in flocs, as an example of social behaviour (a communitarian strategy for long‐time survival or a means of protection against negative environmental conditions), is discussed.     

Pinin acts as a poor prognostic indicator for renal cell carcinoma by reducing apoptosis and promoting cell migration and invasion

Pinin (PNN) was originally characterized as a desmosome-associated molecule. Its function and the mechanism of its regulation in renal cell carcinoma (RCC) are still undefined. Data on PNN expression, clinicopathological features, and prognosis of patients with RCC were obtained from The Cancer Genome Atlas (TCGA) database. Immunohistochemistry revealed high PNN expression in tumour cells. PNN expression showed negative correlation with survival in patients with RCC, acting as an independent prognostic factor in RCC. PNN up-regulation might be attributed to epigenetic alterations in RCC. Immunofluorescence revealed PNN expression mainly in the nucleus of RCC cells. The transfection of siRNA targeting the PNN gene resulted in enhanced apoptosis, which was detected by flow cytometry, and reduced cell migration and invasion, which were assessed using wound healing and transwell migration assay. Gene set enrichment analysis revealed associations between PNN expression and several signalling pathways involved in cancer progression, as a potential mechanism underlying the carcinogenicity of PNN. The analyses of the Tumor Immune Estimation Resource platform showed significant positive associations between high PNN expression and tumour immune infiltrating cells. PNN may function as an oncogenic factor by reducing apoptosis and promoting cell migration and invasion in RCC.     

肾癌预后分子生物标记物研究进展

肾癌发病率约占全身恶性肿瘤的3%。肾癌组织学行为多变,预后有不确定性。外科手术可以治疗局限性肾癌,但有将近20%的局限性肾癌患者原发肿瘤切除后出现转移,而且肾癌对化疗及放疗均不敏感。基于此临床上开展了许多辅助试验的研究,并建立了许多模型来研究肾癌术后的预后,而模型的精准度一般都需要依据肾癌的生物标记物监测。有很多分子生物标记物已经证实和肾癌预后相关,如VHL、P53、Ki-67等,本文综述了肾癌预后的分子生物标记物的最新进展。     

哮喘患者外周血Treg和Th1/Th2的变化及其与哮喘病情的关系

目的：探讨哮喘患者外周血调节性T细胞（Treg）以及辅助性T细胞（Th1/Th2）的比例的变化,探讨其在哮喘的临床治疗中的作用。方法：80例哮喘患者（哮喘组）按临床表现分为急性发作期组（54例）和缓解期组（26例）,同时选择50例健康体检者。应用流式细胞仪检测上述各组外周血CD4＋CD25＋Foxp3＋Treg、CD4＋IFN-γ＋Th1和CD4＋IL-4＋Th2细胞水平,并进行统计学分析。结果：哮喘组CD4＋CD25＋Foxp3＋Treg水平亦明显低于正常对照组（P〈0.05。其中急性发作期组Treg水平明显低于缓解期组和正常对照组（P〈0.05）。而哮喘组Th1/Th2比值显著低于对照组（P〈0.05）,且在哮喘急性发作组中Th1/Th2比值显著低于缓解期组和正常对照组（P〈0.05）。结论：提示Treg和Th在哮喘的发生和发展中起着重要的作用。     

A 3-year experience of quality control and quality assurance in the multisite delivery of a lymphocyte-based cellular therapy for renal cell carcinoma

Autolymphocyte therapy (ALT) is outpatient-based adoptive immunotherapy using ex vivo-activated memory T-cells. To support the safe and reproducible delivery of ALT at three cell processing facilities (Boston, MA; Atlanta, GA; Orange, CA) we created a comprehensive quality assurance/quality control program compliant with recent FDA guidance relevant to activated lymphocytes and somatic cell therapies. Each facility performed extensive QC testing to ensure sterility, viability, and proper cell yield. Additonally, several QC tests were performed at Cellocr's centralized reference laboratory to monitor cell potency and identity of the ex vivo-processed lymphocytes. We report here the successful implementation of this QA/QC program for ALT which has resulted in the safe preparation and delivery of cell infusion products amounting to over 3600 treatments at seven clinical sites nationwide. We believe this program will serve as a model for other cellular therapies.     

Expression and regulation of IL-22 in the IL-17-producing CD4＋ T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.     

Serum adenosine deaminase,catalase, and carbonic anhydrase activities in patients with renal cell carcinoma

Objectives: To determine whether serum levels of adenosine deaminase (AD), catalase (CAT), and carbonic anhydrase (CA) enzymes may be useful biomarkers in the diagnosis of renal tumors and may lead to early diagnosis of renal tumors.

Material and methods: The study included 33 patients with renal cell carcinoma (RCC) and 31 healthy controls. The activity of serum AD, CA, and CAT was determined and analyzed using the Giusti spectrophotometric method, H₂O₂ substrate, and C0₂ hydration, respectively.

Results: Serum AD and CA activity were significantly higher in patients with RCC than in controls. However, serum CAT activity was significantly lower in patients with RCC than in controls.

Conclusion: These markers might be potentially important as an additional biochemical tool for diagnosing RCC. We believe multidisciplinary studies are needed to plan patients’ preoperative and postoperative treatment and to create follow-up protocols.