Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

白血病抑制因子受体细胞内区对HL-60细胞表达CD14和CD15的影响

观察白血病抑制因子 (LIF)受体gp190亚基完整的细胞内区和gp190胞内区C末端片段(190CT)对人白血病系HL 6 0表达CD14、CD15的影响 ,进一步了解LIF引发白血病细胞增殖抑制和分化的关系 .用基因重组技术将LIF另一亚基gp130的细胞内区换成gp190的细胞内区 ,用PCR技术扩增gp190细胞内区C末端的一个多肽的编码序列 ,构成嵌合体受体基因 130 /190及 190CT片段 ,并分别在HL 6 0细胞表达 .用免疫组化和流式细胞术检测分析在LIF的诱导下 ,HL 6 0细胞表达CD14、CD15的水平 .转染pcDNA130 /190的HL 6 0细胞 ,CD15表达量明显增高 ;转染pcDNA190CT的细胞 ,CD15的表达量降低 ;但 2组细胞的CD14表达量均较低且水平接近 .LIF可能诱导HL 6 0细胞向粒细胞而不向单核细胞分化 ,该效应是由gp190亚基细胞内区介导的 ,而gp190C末端片段可干扰LIFα受体介导的信号传导效应 .     

Neuroimmunomodulative properties of dipeptidyl peptidase IV/CD26 in a TNBS-induced model of colitis in mice

Causal connections between dipeptidyl peptidase IV, also known as CD26 molecule (DPP IV/CD26) and inflammatory bowel disease (IBD) have been shown, but mechanisms of these interactions are unclear. Our hypothesis was that DPP IV/CD26 could affect the neuroimmune response during inflammatory events. Therefore, we aimed to evaluate its possible role and the relevance of the gut-brain axis in a model of IBD in mice. Trinitrobenzenesulfonic acid-induced (TNBS) colitis was induced in CD26-deficient (CD26(-/-) ) and wild-type (C57BL/6) mice. Pathohistological and histomorphometrical measurements were done. Concentrations and protein expressions of DPP IV/CD26 substrates neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined. Concentrations of IL-6 and IL-10 were evaluated. Investigations were conducted at systemic and local levels. Acute inflammation induced increased serum NPY concentrations in both mice strains, more enhanced in CD26(-/-) mice. Increased NPY concentrations were found in colon and brain of C57BL/6 mice, while in CD26(-/-) animals only in colon. VIP and IL-6 serum and tissue concentrations were increased in both mice strains in acute inflammation, more pronouncedly in CD26(-/-) mice. IL-10 concentrations, after a decrease in serum of both mice strains, increased promptly in CD26(-/-) mice. Decreased IL-10 concentration was found in brain of C57BL/6 mice, while it was increased in colon of CD26(-/-) mice in acute inflammation. DPP IV/CD26 deficiency affects the neuroimmune response at systemic and local levels during colitis development and resolution in mice. Inflammatory changes in the colon reflected on investigated parameters in the brain, suggesting an important role of the gut-brain axis in IBD pathogenesis.     

蛋白激酶C对血管内皮细胞锚蛋白、CD44表达及亚细胞分布的影响

通过外源性底物对[γ-32P]-ATP的摄入量来测定豆蔻酰佛波醇乙酯(phorbol-myristate-acetate,PMA)处理后的人脐静脉内皮细胞(humanumbilicalveinendothelialcells,HUVECs)膜蛋白激酶C(proteinkinaseC,PKC)的活性;利用间接免疫荧光标记和Western印迹方法分析蛋白激酶C活性对锚蛋白及CD44的亚细胞分布及蛋白质表达的影响。结果发现HUVECs的锚蛋白及CD44表达水平趋势与PKC活性变化相吻合;PKC活化导致CD44在细胞膜上呈聚集状,而锚蛋白则移位并聚集于CD44处;PKC抑制剂能抑制PKC活化所带来的上述作用。结果表明PKC活化通过磷酸化作用能上调锚蛋白及CD44表达,并同时导致二者发生一致性运动及共分布。     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

羟基化修饰对大鼠胶原热稳定性的影响研究

采用差示扫描量热仪(DSC)研究羟脯氨酸(Hyp)含量对胶原热稳定性的影响。以不同周龄BN大鼠皮肤为原料制备了胶原,分析制备胶原中Hyp的含量;采用DSC测定不同Hyp含量胶原的临界变性温度及焓变;采用圆二色光谱(CD)检测提取胶原的二级结构。结果表明,提取胶原在41.3℃发生三螺旋解聚,CD光谱分析结果表明,当样品经临界变性温度处理后,部分三螺旋结构转化为无规则线圈结构。胶原变性过程中所需热量与羟脯氨酸含量呈正相关,实验建立了胶原热变性过程中焓变与Hyp含量的关系。该研究表明胶原中脯氨酸羟基化修饰是影响胶原热稳定性的关键因素。     

Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8 + T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8⁺ T cells responses in the elderly has come back into focus since the COVID‐19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8⁺ T cells in aging. We focused on the different subpopulations and time‐resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8⁺ T cells from elderly mice are much faster than those of CD8⁺ T cells from adult mice. This is true not only in the total CD8⁺ T cell population but also for their effector (T_EM) and central memory (T_CM) T cell subpopulations. TIRF experiments reveal that CD8⁺ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8⁺ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell‐intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.     

HIV/AIDS患者机会性感染与CD4+ T淋巴细胞间的关联性

目的 研究人免疫缺陷病毒（HIV）感染者及艾滋病（AIDS）患者发生机会性感染的概率与自身CD4+ T淋巴细胞之间的关系,为HIV患者机会性感染的防治提供参考。方法 以2016年6月至2017年6月我院400例HIV患者为研究对象,回顾性分析不同CD4+T淋巴细胞计数HIV患者发生机会性感染的情况。结果 400例HIV患者发生机会性感染178例,总感染率为44.5%。CD4+T淋巴细胞计数≤50个/μL的患者机会性感染发生率（86.67%）最高,与其他各组比较差异有统计学意义（P<0.05）。随着CD4+ T淋巴细胞计数的减少,HIV患者机会性感染率升高。178例机会性感染者中,单一感染82例,2部位感染52例,3部位感染28例,4部位以上感染16例。感染病原体检测显示,细菌感染84例（47.19%）,结核杆菌感染36例（20.22%）,病毒感染30例（16.85%,包括巨细胞病毒感染18例、单纯疱疹病毒感染12例）,真菌感染77例（43.25%,包括假丝酵母感染35例,肺孢子菌感染20例,马尔尼菲青霉菌感染12例,新型隐球菌感染10例）,未明确病原体性质34例（19.10%）,复合感染多见。结论 CD4+ T淋巴细胞水平与HIV患者继发机会性感染的概率关系密切。HIV患者CD4+ T淋巴细胞水平的监测对其继发机会性感染的防控具有重要临床意义。     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M−1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.