The ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor targets

Cancers are able to grow by subverting immune suppressive pathways, to prevent the malignant cells as being recognized as dangerous or foreign. This mechanism prevents the cancer from being eliminated by the immune system and allows disease to progress from a very early stage to a lethal state. Immunotherapies are newly developing interventions that modify the patient's immune system to fight cancer, by either directly stimulating rejection-type processes or blocking suppressive pathways. Extracellular adenosine generated by the ectonucleotidases CD39 and CD73 is a newly recognized “immune checkpoint mediator” that interferes with anti-tumor immune responses. In this review, we focus on CD39 and CD73 ectoenzymes and encompass aspects of the biochemistry of these molecules as well as detailing the distribution and function on immune cells. Effects of CD39 and CD73 inhibition in preclinical and clinical studies are discussed. Finally, we provide insights into potential clinical application of adenosinergic and other purinergic-targeting therapies and forecast how these might develop in combination with other anti-cancer modalities.     

Decreased Frequency and Secretion of CD26 Promotes Disease Progression in Indian Post Kala-azar Dermal Leishmaniasis

Purpose

Leishmania, the causative organisms for leishmaniasis, reside in host macrophages and survive by modulating the microbicidal pathways via attenuation of the oxidative burst and/or suppression of cell-mediated immunity. As post kala-azar dermal leishmaniasis (PKDL), the dermal sequela of visceral leishmaniasis, has no animal model, the underlying mechanism(s) that nullify the microbicidal effector mechanisms remain poorly understood. This study was aimed at assessing the status of dipeptidyl peptidase CD26, a co-stimulatory molecule that is essential for T-cell signal activation.

Methods

The frequency/expression of CD26 and CD45RO/RA was evaluated by flow cytometry, while levels of soluble CD26 (sCD26), CXCL-10, RANTES, IL-10 and TGF-β along with adenosine deaminase (ADA) activity were measured using ELISA.

Results

In patients with PKDL vis-à-vis healthy individuals, there was a significant decrease in the frequency and expression of CD26 on CD3⁺CD8⁺ T-cells, which was accompanied by a significant lowering of plasma levels of sCD26. Furthermore, these patients showed a significant decrease in the frequency of CD45RO⁺/CD8⁺ T-cells, concomitant with a significant increase in the proportion of CD45RA⁺/CD8⁺ T-cells. This could collectively translate into reduced formation of the immunological synapse of CD26, CD45RO, and ADA, and lead to an attenuation of the Th1 responses. The decreased levels of CD26 and sCD26 correlated negatively with raised levels of Th2 cytokines, IL-10, and TGF-β along with the lesional parasite load, indicating disease specificity.

Conclusions

Taken together, the decreased expression and secretion of CD26 in patients with PKDL resulted in impairment of the CD26-ADA interaction, and thereby possibly contributed to T-cell unresponsiveness, emphasizing the need to develop immunomodulatory therapies against PKDL and by extension, the leishmaniases.

     

Selective Activation of Adenosine A2A Receptors on Immune Cells by a CD73-Dependent Prodrug Suppresses Joint Inflammation in Experimental Rheumatoid Arthritis

Adenosine A(2A) receptor (A(2A)R) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A(2A)R agonists (prodrugs) that require the presence of ecto-5'-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5'-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 ((19)F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A(2A)R. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A(2A)R expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A(2A)R agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.     

Susceptibility of aging mice to listeriosis: Role of anti-inflammatory responses with enhanced Treg-cell expression of CD39/CD73 and Th-17 cells

Foodborne Listeria monocytogenes (Lm) causes serious illness and death in immunosuppressed hosts, including the elderly population. We investigated Lm susceptibility and inflammatory cytokines in geriatric mice. Young-adult and old mice were gavaged with a Lm strain Lmo-InlA^m. Tissues were assayed for Lm burden and splenocytes were analyzed for Th1/Th2/Th17/Treg responses and expression of CD39 and CD73. Old Lm-infected mice lost body-weight dose-dependently, had higher Lm colonization, and showed higher inflammatory responses than Lm-infected young-adult mice. After infection, IL-17 levels increased significantly in old mice whereas IFN-γ levels were unchanged. Levels of IL-10 and Treg cells were increased in infected old mice as compared to infected young-adult mice. Age-dependent enhanced expression of CD39/CD73 was observed in purified Treg prior to infection, suggesting increased baseline adenosine production in old mice. Lm lysate-treated splenocytes from older mice produced significantly higher levels of IL-10, IL17, and IL-1β, produced less IFN-γ and IL-2, and proliferated less than splenocytes from young-adult mice. Data suggests that older mice maybe more susceptible to Lm infection due to an imbalance of Th cell responses with disproportionate and persistent anti-inflammatory responses. Lm infection enhanced differentiation of proinflammatory Th17 cells, which may also exacerbate pathological responses during listeriosis.     

CD39和CD73在CD4~+CD25~(high)Foxp3~+调节性T细胞发挥免疫抑制功能中的作用研究

本研究旨在检测正常人CD4~+CD25~(high)Foxp3~+Treg表面腺苷代谢分子的表达,并探索该分子在CD4~+CD25~(high)Foxp3~+Treg发挥抑制功能中的作用。采用流式细胞术检测10例健康献血者外周CD4~+CD25~(high) Foxp3~+Treg表面腺苷代谢分子CD39、CD73的表达,免疫组化染色检测了5例健康人皮肤中腺苷代谢分子CD39、CD73及Foxp3的表达。对5例健康献血者,流式细胞仪分选得到CD4~+CD25~(high)、CD4~+CD25~(high) CD39~+、CD4~+CD25~(high) CD39~+CD73~+共3部分细胞,采用3H-TdR掺入方法检测这3部分T细胞对CD4~+CD25-T细胞增殖的抑制作用。同样采用流式细胞术对10例斑块型银屑病患者外周CD4~+CD25~(high)Foxp3~+Treg表面CD39和CD73分子的表达进行分析。结果显示,正常人外周血CD4~+CD25~(high) Foxp3~+Treg与CD4~+CD25mid和CD4~+CD25-T细胞相比,CD39明显高表达(P<0.01),CD73低表达(P<0.05)。正常人皮肤中,CD39主要表达在表皮角质形成细胞,而CD73则主要分布于真皮中。CD39~+CD73~+Treg对CD4~+CD25-T细胞增殖的抑制最强(P<0.01)。银屑病患者外周血CD73~+CD4~+CD25~(high)Foxp3~+Treg比例较正常人明显降低(P<0.01)。由此推测腺苷代谢分子是CD4~+CD25~(high)Foxp3~+Treg发挥抑制功能的重要物质,并可能参与银屑病的发生。     

CD73 and adenosine generation in the creation of regulatory microenvironments

Extracellular adenosine 5′‐triphosphate (ATP) acts on many immune cells to promote inflammation. Conversely, the ATP metabolite adenosine is mainly an anti‐inflammatory molecule. The ecto‐enzymes CD39 and CD73 can dephosphorylate extracellular ATP to adenosine, thereby controlling this important pathway of immune modulation. Despite their established roles in the immune system, little is known of how CD39 and CD73 are themselves regulated. Recent data have shown that CD73 expression and adenosine generation are up‐regulated by transforming growth factor‐β, depending on the cytokine content of the local microenvironment. We review here these recent findings and discuss their implications in disease.     

IFN-beta regulates CD73 and adenosine expression at the blood-brain barrier

IFN-beta treatment reduces the relapse rate in MS but its mechanism of action remains incompletely understood. Our aim was to clarify the beneficial effect of IFN-beta in the treatment of MS. We assessed the influence of IFN-beta treatment on (i) CD73 expression on the surface of primary cultures of human blood-brain barrier endothelial cells (BBB-EC) and human astrocytes using immunofluorescence staining and flow cytometry, (ii) transmigration of CD4+ T lymphocytes using an in vitro model of BBB and (iii) CD73 enzyme activity, i.e. ecto-5'-nucleotidase activity in the serum of MS patients using a radiochemical assay. IFN-beta increases the expression of ecto-5'-nucleotidase both on BBB-EC and astrocytes. As a consequence, lymphocyte transmigration through BBB-EC is reduced. Importantly, this reduction can be reversed using alpha,beta-methyleneadenosine-5'-diphosphate, a specific inhibitor of ecto-5'-nucleotidase. CD73 is strongly expressed in microvasculature in samples of postmortem MS brain and, moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto-5'-nucleotidase activity and skin microvascular CD73 expression after IFN-beta treatment. Upregulation of ecto-5'-nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFN-beta on MS via enhancing the endothelial barrier function.     

Vaccination with Live Leishmania major and CpG DNA Promotes Interleukin-2 Production by Dermal Dendritic Cells and NK Cell Activation

Cutaneous leishmaniasis due to Leishmania major is an emerging, chronic parasitic disease that causes disfigurement and social stigmatization. Drug therapy is inadequate, and there is no vaccine. Inoculation of virulent parasites (leishmanization) is the only intervention that has ever provided protection, because it mimics natural infection and immunity, but it was discontinued due to safety concerns (uncontrolled vaccinal lesions). In an effort to retain the benefits (immunity) while avoiding the side effects (lesions) of leishmanization, we immunized C57BL/6 mice with L. major and CpG DNA (Lm/CpG). This combination prevented lesions while inducing immunity. Also, the vaccination with live parasites and the Toll-like receptor 9 agonist enhanced innate immune responses by activating dermal dendritic cells (DCs) to produce cytokines. Here we report that the Lm/CpG vaccine induced dermal DCs, but not bone marrow-derived DCs, to produce interleukin-2 (IL-2). The release of this unusual DC-derived cytokine was concomitant with a peak in numbers of NK cells that produced gamma interferon (IFN-γ) and also enhanced activation of proliferation of IFN-γ⁺ CD4⁺ T cells. Parasite growth was controlled in Lm/CpG-vaccinated animals. This is the first demonstration of the ability of dermal DCs to produce IL-2 and of the activation of NK cells by vaccination in the context of leishmaniasis. Understanding how the Lm/CpG vaccine enhances innate immunity may provide new tools to develop vaccines against L. major, other chronic infectious diseases, or other conditions, such as cancer.The leishmaniases are among the most important emerging parasitic diseases, second only to malaria in terms of the number of affected people. The prevalence of cutaneous leishmaniasis due to Leishmania major, a chronic disease leading to disfigurement, functional impairment, and social stigmatization, is estimated to be 2 million cases (4); recent data demonstrate that this number is greatly underestimated (1). Naïve individuals are very susceptible, leading to dramatic outbreaks. Current drugs are inadequate due to toxicity, resistance, cost, and adverse effects, and there is no vaccine. Thus, there is a clear need for both prophylactic and therapeutic control measures. Inoculation of virulent L. major (leishmanization), practiced in areas of leishmaniasis endemicity for more than 1,000 years, is the only strategy that has ever provided lifelong protection, because it mimics the natural infection, enabling parasite persistence and the subsequent concomitant immunity. It was widely applied but was discontinued due to the development of large vaccinal lesions in about 10% of the immunized patients (14).Because no vaccine other than leishmanization has been successful with humans, we work to understand its mechanism of action. In an effort to retain the benefits (immunity) while avoiding the side effects (lesions) of leishmanization, we immunized C57BL/6 mice with L. major alone or in combination with CpG DNA (Lm/CpG). Lm/CpG prevented vaccinal lesions while achieving L. major persistence and immunity (13, 29). Mechanistically, we found that Lm/CpG causes early activation of dermal dendritic cells (DCs) to produce interleukin-6 (IL-6), reducing the accumulation of regulatory T cells (Tregs) in the vaccination site (29). Activated DCs also produced IL-12, promoting activation and proliferation of gamma interferon (IFN-γ)-producing CD4⁺ T cells (13, 29). The lack of suppressors and the increase in effectors resulted in parasite killing and a lack of vaccinal lesions. Interestingly, Treg numbers recovered in the skin of mice vaccinated with Lm/CpG, enabling concomitant immunity and lifelong protection (13), as previously demonstrated by us (3).While continuing the study of the mechanism of action of the Lm/CpG vaccine, we detected a rapid increase in IL-2 expression in the vaccination site. IL-2 is a survival factor for B and T lymphocytes, but it is also necessary for DC-dependent NK cell activation (7). Thus, NK cells may be critical in the early control of parasite growth in the vaccinated mice. In this paper, we report the unusual IL-2 production by CD11c⁺ DCs in the skin of mice vaccinated with Lm/CpG. This unusual secretion is concomitant with a peak in IFN-γ-producing NK cells and CD4⁺ T cells and the control of parasite growth. Thus, vaccination with live parasites and Toll-like receptor 9 agonists appears to enhance not only adaptive immunity but also the response of innate immune cells to decrease parasite growth and the development of vaccinal pathology.     

Extracellular Hydrolytic Enzymes of Rabbit Dermal Tuberculous Lesions and Tuberculin Reactions Collected in Skin Chambers

To evaluate extracellular hydrolytic enzymes in an in vivo system, plastic chambers were glued over rabbit dermal BCG lesions in various stages of development, after the central epithelium was removed with a scalpel. They were filled with tissue culture medium and left in place 2 days. The following enzymes in the fluid were assayed: collagenase (an enzyme secreted but not stored in macrophages); lysozyme (both secreted and stored); DNase and RNase (released on cell death and possibly regurgitated but not secreted); and, as a control, lactic dehydrogenase (released only on cell death). Tissue sections were prepared and studied histologically for the type of cell infiltrate, for β-galactosidase (our marker enzyme for macrophage activation), and for necrosis. At 11 and 18 days of age the BCG lesions were largest and the number of activated macrophages in the chamber beds was highest. At this time the levels of the five enzymes assayed in the chamber fluids reached their peaks, tuberculin hypersensitivity was well developed, and the bacilli components would still be plentiful. In general, the chamber fluids from 11- and 18-day BCG lesions contained higher enzyme levels than chamber fluids from tuberculin reactions. Active collagenase was only detected in fluids from such BCG lesions. Evidently, the serum in the chamber fluids was sufficient to inhibit the lower amounts of collagenase probably released from smaller BCG lesions and tuberculin reactions (and from the 2-week polystyrene lesions that were also evaluated). These studies demonstrate that in chronic inflammatory reactions, both acid-acting and neutral-acting hydrolytic enzymes are released extracellularly. Tissue components would be hydrolyzed locally wherever the acid-acting hydrolytic enzymes encounter a drop in pH and wherever the concentration of neutral-acting hydrolytic enzymes exceeds the concentration of their inhibitors.     

Fibrinogen Cleavage by the Streptococcus pyogenes Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.     

Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.     

Overexpression of CD39 and high tumoral CD39+/CD8+ ratio are associated with adverse prognosis in resectable gastric cancer

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39⁺/CD8⁺ ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39⁺/CD8⁺ ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.     

Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-β expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-β-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.     

Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells

Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.     

Crosstalk between Suppression of Tumorigenicity 2 and Transforming Growth Factor-β Receptor Signaling Promotes Renal Fibrosis

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CD73对骨髓间充质干细胞增殖与衰老的影响

目的探讨CD73对骨髓间充质干细胞(BMSCs)增殖与衰老的影响。方法培养小鼠BMSCs,构建慢病毒CD73过表达载体和CD73 RNAi干扰载体,分别转染BMSCs,实验分组:CD73过表达组(OE组)、CD73RNAi干扰组(RNAi组)和对照组(CON组)。衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞的衰老情况;MTT法检测细胞增殖能力变化;流式细胞术检测细胞周期变化;透射电子显微镜观测转染后细胞的超微结构;非损伤细胞功能分析仪检测细胞生长曲线。结果慢病毒转染BMSCs后72 h和168 h,衰老细胞数量RNAi组CON组OE组(P0.05);转染后3~4d BMSCs的增殖能力为OE组较RNAi组和CON组细胞增殖速度快(P0.05);转染后3d各组细胞周期的变化是:OE组、RNAi组和CON组的G1期细胞所占比例分别为25.9%、44.1%和51.7%;S期细胞所占比例分别为48.8%、30.9%和26.9%;G2期细胞所占比例分别为25.2%、25.0%和21.4%;透射电子显微镜观察结果显示,OE组细胞质内粗面内质网和核糖体增多;细胞生长曲线显示OE组高于CON组和RNAi组。结论 CD73促进BMSCs增殖且抑制BMSCs衰老。     

Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

Recently,serum Golgi protein 73(GP73) levels have been found to be elevated in patients with hepatocellular carcinoma(HCC),and GP73 has been proposed as a novel marker for HCC.However,GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent assay(ELISA).Therefore,an anti-GP73 antibody with high specificity was highly demanded.In the present study,by hybridoma screening,we generated an anti-GP73 monoclonal antibody(mAb) designated as 6A2 using recombinant GP73 protein produced by prokaryotic expression.The specificity of 6A2 was evaluated by Western blotting,immunohistochemistry and immunoprecipitation.The results showed that 6A2 recognized GP73 in both native and denatured forms.In addition,we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures,and measured the serum GP73 level of patients using this assay.Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls(P = 0.0036).Furthermore,for the first time,GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls(P = 0.0172).     

小鼠CD86基因启动子的构建及活性分析

为构建小鼠CD86启动子,并对其特异性调控能力进行鉴定。首先自C57BL/6J小鼠基因组中采用高保真PCR扩增小鼠CD86基因的启动子,经测序鉴定后,插入pCDNA3.1-EGFP重组质粒。然后分别转染小鼠来源细胞株:RAW264.7、NIH/3T3、P815、SP2/0、EL4细胞,观察各细胞内绿色荧光蛋白的表达。结果显示,小鼠CD86基因启动子的序列与GenBank报道一致;双酶切鉴定证实小鼠CD86启动子序列已经克隆入重组载体;小鼠CD86启动子能调控EGFP在巨噬细胞系RAW264.7细胞中表达,不能调控EGFP在NIH/3T3、P815、SP2/0和EL4非抗原提呈细胞内表达。说明成功构建小鼠CD86基因启动子,且该启动子具有特异性调控目的基因表达的活性。