Expression of serum inflammatory cytokines and oxidative stress markers and their correlation with coronary artery calcium score in patients with coronary heart disease.

Introduction: The present study was conducted to explore the expression of serum inflammatory cytokines and oxidative stress markers in patients with coronary heart disease (CHD), with an attempt to analyze their relationship with the coronary artery calcium score (CACS) by coronary computed tomography angiography (CCTA). Material and methods: It total 81 patients with coronary heart disease and 81 healthy adults were included as the observation group and the control group, respectively. The levels of serum interleukin (IL)-6 and IL-12 of the two groups were detected by ELISA, and serum superoxide dismutase (SOD) was detected by the hydroxylamine oxidation method. Micro-RNA-497-5p (miR-497-5p) was screened out as a possible new CHD biomarker and its serum level was measured by real-time fluorescence quantitative PCR. The CACS of patients in the observation group was calculated by the Agatston method to analyze the correlation between the abovementioned indexes and CACS. Results: With increase in the number of CHD lesions, the levels of IL-6, IL-12 and miR-497-5p rose gradually while the level of SOD decreased gradually. In the observation group, IL-6, IL-12 and miR-497-5p were positively correlated with CACS while SOD was negatively correlated with CACS. Conclusions: Abnormal expression levels of serum IL-6, IL-12, SOD and miR-497-5p may be able to reveal the severity of the disease, and the combination with CACS is of potential value in terms of evaluating the condition of patients harboring coronary heart disease.

Extracellular vesicle-mediated bone metabolism in the bone microenvironment.

Extracellular vesicles (EVs) are phospholipid membrane-enclosed entities containing specific proteins, RNA, miRNA, and lncRNA. EVs are released by various cells and play a vital role in cell communication by transferring their contents from the host cells to the recipient cells. The role of EVs has been characterized in a wide range of physiological and pathophysiological processes. In this context, we highlight recent advances in our understanding of the regulatory effects of EVs, with a focus on bone metabolism and the bone microenvironment. The roles of EVs in cell communication among bone-related cells, stem cells, tumor cells, and other cells under physiological or pathological conditions are also discussed. In addition, promising applications for EVs in treating bone-related diseases are proposed.

Development of a novel anti-human aspartyl-(asparaginyl) ß-hydroxylase monoclonal antibody with diagnostic and therapeutic potential.

Human aspartyl-(asparaginyl)-ß-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.

Mechanism and Anticancer Activity of the Metabolites of an Endophytic Fungi from Eucommia ulmoides Oliv.

Backgroud: Pinoresinol (Pin) and pinoresinol monoglucoside (PMG) are plant-derived lignan molecules with multiple functions. We showed previously that an endophytic fungus from Eucommia ulmoides Oliv., Phomopsis sp. XP-8 is able to produce Pin and PMG. OBJECTIVE: This study was carried out to test the anti-tumor capability of the culture of XP-8 and identify the major effective compounds. METHOD: The fungal culture was added in the culture of HepG2 and K562 cells, and the viabilities of these cells were detected and the possible mechanism was analyzed. RESULT: The fungal culture showed significant capaiblity in decreasing the viability of tumor cells and induce apoptosis via up-regulation of the expression of apoptosis-related genes. It also significantly inhibited the adhesion and migration of HepG2 cells by blocking MMP-9 expression. Pin and PMG were isolated from the growth culture and shown to be the major effective components for inhibition. CONCLUSION: The study indicated the potential application of XP-8 in the production of anti-tumour products by the bioconversion of glucose.

Advances in paper-based sample pretreatment for point-of-care testing.

In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.

Heat shock protein 90 inhibitors induce functional inhibition of human natural killer cells in a dose-dependent manner.

Heat shock protein 90 (Hsp90) is a ubiquitously expressed ATP-dependent molecular chaperone across all species that helps to the correct the folding of many proteins related to important signaling pathways. Tumor cells expressing Hsp90 have more ATP-binding affinity than normal cells. Many correlative inhibitors have been developed to promising anti-tumor strategies and have been evaluated in clinical trials. However, the effect of Hsp90 inhibitors on immunocytes cannot be ignored. Natural killer (NK) cells are key components of the innate immune system that play a pivotal role in tumor surveillance. The present study has investigated the potential effect of four Hsp90 inhibitors (NVP-AUY922, BIIB021, 17-DMAG, and SNX-2112) on human primary NK cells. The viability, cytotoxicity, apoptosis, phenotype, and cytokine secretion of NK cells after inhibitor treatment were assessed. The results of this study demonstrated that the inhibitors had negative effects on NK cell activity in a dose-dependent manner. The four inhibitors significantly reduced the cytotoxicity of the NK cells by decreasing viability, inducing apoptosis and down-regulating the expression of cytokines and functional receptors. These findings suggest that more attention should be given to the effect of Hsp90 inhibitors on NK cell function during clinical trials and also represent a potential immunosuppressant strategy.

Carbon Monoxide Releasing Molecule Accelerates Reendothelialization after Carotid Artery Balloon Injury in Rat.

OBJECTIVE: This study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model. METHODS: Male rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis. RESULTS: CORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2. CONCLUSION: CORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.

Multiple effects of IL-21 on human NK cells in ex vivo expansion.

Natural killer (NK) cells (CD56(+)CD3(-)) are large, granular immunocytes that play a very pivotal role in the anti-inflammatory response and tumor surveillance. As an ideal cytotoxic lymphocyte (CTL), NK cells have attracted much attention in clinical trials. However, an insufficient number and their limited life span are bottlenecks that limit the application of NK cells in adoptive immunotherapy. Interleukins such as IL-2, IL-15 and IL-18 are recognized as factors that stimulate NK cells and have been used in NK cells ex vivo expansion. Similar to IL-2 and IL-15, IL-21 is a common γ-chain cytokine that is important in NK cell activation, maturation and proliferation. The present study aims to assess the effects of membrane-bound and soluble IL-21 on primary human NK cells during ex vivo expansion. IL-21 was found to have multiple effects on NK cells, increasing their cytotoxicity in a concentration-dependent manner by up-regulating IFN-γ and Granzyme-B expression. Nevertheless, at a high concentration (50 ng/mL), IL-21 curtailed the life span of NK cells by significantly inducing apoptosis. Moreover, when treated with IL-21, the number of NKT (CD56(+)CD3(+)) cells increased among peripheral blood mononuclear cells (PBMCs) during ex vivo expansion in a concentration-dependent manner. IL-21 also promoted expanded cells to enter into S phase of the cell cycle during the first to second weeks of culture. All these results suggest that IL-21 has multiple effects on NK cell development and functions. More attention should be given to the dosage and multiple effects of IL-21 when it was applied to NK cells in ex vivo expansion.

Concentration-dependent biphasic effects of resveratrol on human natural killer cells in vitro.

Resveratrol (RES) is a polyphenol phytoalexin from plants, which has been reported to possess a variety of biological effects. The properties of RES on human natural killer (NK) cells were assessed in this study. Results showed that RES has concentration-dependent biphasic effects on NK cells. In high concentration (50 µM), RES can inhibit viability and promoted apoptosis of NK cells and human lymphoblastoid T (Jurkat) cells, which may affect the caspase signaling pathway. The Jurkat cells were more sensitive than NK cells on the RES caused cell death. However, when the concentration range reduced from 3.13 to 1.56 µM, RES showed the positive effects on NK cells by increasing the NK cells cytotoxicity via up-regulating the expression of NKG2D and IFN-γ (in mRNA and protein levels). These results indicated that one needs to pay more attention to the dosage and biphasic effects when RES was applied as antitumor drugs or health products.

Inhibition of human natural killer cell functional activity by human aspartyl ß-hydroxylase.

Natural killer (NK) cells are a key component of the innate immune system and play pivotal roles as inflammatory regulators and in tumor surveillance. Human aspartyl ß-hydroxylase (HAAH) is a plasma membrane and endoplasmic reticulum protein with hydroxylation activity, which is over-expressed in many malignant neoplasms and can be detected from the sera of tumor patients. HAAH is involved in regulating tumor cell infiltration and metastasis. Escaping from immune surveillance may help tumor cell infiltration and metastasis. However, the effects of HAAH on tumor immune surveillance have not yet been investigated carefully. The present study investigated the potential use of HAAH as an immune regulator of human NK cells. We assessed the effects of recombinant HAAH (r-HAAH) on primary human NK cell morphology, viability, cytotoxicity, apoptosis, receptors expression and cytokine/cytolytic proteins production. Our results demonstrated that r-HAAH negatively affects NK cell activity in a time and dose-dependent manner. It noticeably reduces the viability of the NK cells by increasing apoptosis and necrosis via caspase signaling pathways. Moreover, r-HAAH reduces the NK cell cytotoxicity by inhibiting surface expression of NKG2D, NKp44 and IFN-γ secretion. These findings suggest that one of the ways by which HAAH actively promotes tumor formation and proliferation is by inhibiting NK cell-surveillance activity.

Protective effect of polysaccharides on simulated microgravity-induced functional inhibition of human NK cells.

Polysaccharides are believed to be strong immunostimulants that can promote the proliferation and activity of T cells, B cells, macrophages and natural killer (NK) cells. This study aimed to investigate the effects of five polysaccharides (Grifola frondosa polysaccharide (GFP), lentinan (LNT), G. lucidum polysaccharide (GLP), Lycium barbarum polysaccharide (LBP) and yeast glucan (YG)) on primary human NK cells under normal or simulated microgravity (SMG) conditions. Our results demonstrated that polysaccharides markedly promoted the cytotoxicity of NK cells by enhancing IFN-γ and perforin secretion and increasing the expression of the activating receptor NKp30 under normal conditions. Meanwhile polysaccharides can enhance NK cell function under SMG conditions by restoring the expression of the activating receptor NKG2D and reducing the early apoptosis and late apoptosis/necrosis. Moreover, the antibody neutralization test showed that CR3 may be the critical receptor involved in polysaccharides induced NK cells activation. These findings indicated that polysaccharides may be used as immune regulators to promote the health of the public and astronauts during space missions.

Monoclonal antibodies against human aspartyl (asparaginyl) beta-hydroxylase developed by DNA immunization.

We newly cloned the gene encoding the human aspartyl (asparaginyl) beta-hydroxylase (HAAH) from the surgical tissue of a patient with hepatocellular carcinoma. This study was designed to generate HAAH-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were co-immunized with naked plasmid DNA containing N-terminal domain of encoding HAAH gene and recombinant HAAH polypeptide. Hybridomas were developed by the electrofusion of the splenocytes from mice immunized with plasmid DNA to Sp2/0 myeloma cells in vitro. Three hybridoma cell lines (designated G3, G9, and F11, respectively) stably secreting HAAH-specific MAbs were obtained. The specificity and sensitivity of MAbs were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results showed that the three MAbs belong to IgG1 kappa isotype, the titer of MAbs reached was 5 x 10(4) - 1 x 10(5), and the affinity constant (k(aff)) of MAbs ranged between 2.5 x 10(8) - 1.1 x 10(9). MAb G3 was preliminarily applied to detection expression of HAAH for seven tumor tissues, including hepatocellular carcinoma, lung cancer, kidney cancer, cholangiocarcinoma, prostate cancer, breast cancer, and glioblastoma by immunohistochemical stain. Our studies demonstrated that co-immunization of naked DNA containing encoding gene of target antigen and recombinant target protein, and combined with in vitro electrofusion, is an effective and simple method to raise MAbs.

[Effect of autophagy in MTB's infection and the expression of its related gene].

AIM: To study the effect of autophagy in MTB's infection and the expression of its related gene. METHODS: The formation of autophagy was induced by Rapamycin and observed by the transmission electron microscope. The cleaning role of autophagy to the MTB H37Rv virulent strain after its formation was detected by clone forming unit (CFU). Realtime PCR was used to detect the mRNA of the autophagy related gene was expressed. RESULTS: The RAW264.7 cell could form autophagosome under the induction of the Rapamycin, and it had the determinate cleaning role to the H37Rv strain in the cell after which formed. The mRNA of atg5, atg8 and atg12 which participated the formation of autophagy were expressed more, but the expression of atg7 had no change. CONCLUSION: Autophagy participated the process of immune response of anti-MTB. Atg5, atg8 and atg12 were the important molecule which control the formation of autophagy when MTB infected.

[Expansion of human natural killer cells ex vivo].

AIM: To obtain sufficient numbers of pure and activated human primary NK cells for potential clinical application. METHODS: PBMC based ex vivo expansion of natural killer cells was set up. The expansion was initiated by co-culture of PBMC and stimulator cells (irradiate genetic modified K562 cells) in RPMI1640 medium with 1 000 U/mL IL-2. Genetic modified K562 cells 'K562D3' were prepared by expressing IL-15, 4-1BBL and IL-18 on K562 cell membrane. RESULTS: An average of 500 fold expansion of CD56(+)CD3(-) cells was observed after 3 weeks of co-culture. The NK cells population could reach 93% after expansion, comparing with 7% before expansion. The expanded NK cells lysed 95% of K562 targets in a 5:1 effector to target ratio. IL-15/4-1BBL bound K562 stimulatory cells could expand same fold NK cells as K562D3, but the cytotoxicity of NK cells expanded by 'K562D3' was 10% higher. CONCLUSION: The described method is a simple and efficient way for the expansion of human NK cells.

[Preparation of specific monoclonal antibody against nucleocapsid protein of SARS coronavirus].

AIM: To express nucleocapsid(N) protein of SARS coronavirus and produce monoclonal antibody(mAb) to N protein. METHODS: N protein gene was amplified by RT-PCR. After being confirmed by DNA sequencing, the gene was subcloned into prokaryotic expression vector. N protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb. RESULTS: DNA sequencing confirmed that the amplified fragment was N protein gene. SDS-PAGE analysis showed that the M(r) of the expressed protein was approximately 43 kd. This expressed protein could be further confirmed in Western blot by using serum from a convalescent SARS patient as primary antibody. Western blot analysis proved that three mAbs obtained could react specifically to the recombinant N protein. CONCLUSION: The prepared recombinant N protein and mAbs against N protein lay the foundation for further development of early diagnosis assays for SARS coronavirus infection.

[DNA family shuffling of tissue-type plasminogen activator genes].

AIM: To probe the possibility of screening tPA with higher activity through DNA shuffling. METHODS: Human, rat and rhesus monkey tPA cDNAs were used as the templates of "DNA family shuffling". The diverse tPA library was expressed in CHO cells, then the screening was carried out. RESULTS: Two shuffled clones, t9 and t17 were selected. The activity of tPA expressed by t9 clone was 4-fold higher than the activity of human tPA. tPA expressed by t17 clone was found having a 88-amino-acid deletion, but still showed the same activity as human tPA. DNA sequencing demonstrated that the sequences of t9 and t17 were mainly derived from human and monkey tPA cDNAs. CONCLUSION: Above work has laid the foundation for further experiments in the screening for higher activity of tPA.

[Expression of Japanese encephalitis virus E protein in methylotrophic yeast Pichia pastoris].

AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris. METHODS: The gene coding for JEV E protein was sub-cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot. RESULTS: The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000. CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.