Antagonistic effects of IL-4 and TGF-beta1 on Langerhans cell-related antigen expression by human monocytes

In this study, we analyzed the specific effects of transforming growth factor beta (TGF-beta1) and/or IL-4 on monocyte-derived cells. Monocytes were cultured with GM-CSF, GM-CSF/TGF-beta1, GM-CSF/IL-4, or GM-CSF/IL-4/TGF-beta1 before cell morphology, phenotype, and function were assessed. As expected, interleukin-4 is mandatory for monocyte differentiation into potent allostimulatory DC. In its absence, monocyte-derived cells share many phenotypic and functional features with macrophages. However, it is interesting that the cells express E-cadherin, independent of exogenous TGF-beta1, and addition of the cytokine induced CCR6 expression. Most importantly, a subset of monocytes cultured with GM-CSF/TGF-beta1 expresses Langerin, as confirmed by electron microscopy analysis. Langerin engagement with specific monoclonal antibodies induces its internalization and the formation of typical Birbeck granules. Monocytes cultured in GM-CSF/IL-4 did not express the LC markers E-cadherin, CCR6, or Langerin. The simultaneous addition of TGF-beta1 allows most of the cells to express E-cadherin but rarely CCR6 and Langerin. Taken together, the results add further evidence that LC can derive from monocytes and demonstrate an antagonistic effect of IL-4 and TGF-beta1 on monocyte differentiation toward the LC pathway.     

IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-beta1 and IL-4

Regulatory cytokines mediate the participation of oral mucosal epithelial cells (OMEC) in local immune responses. The aim of this study was to characterize the isoforms of IL-1 receptor antagonist (IL-1ra) in cultured human primary OMECs and to compare its production with that of IL-1 alpha (IL-1alpha) and IL-1 beta (IL-1beta). Western blot analysis showed that IL-1ra was 22 kDa in size hence slightly smaller than monocyte IL-1ra (25 kDa). A minor form of 20 kDa was also found in unstimulated cell culture lysates. In culture supernatants, IL-1 bioactivity increased after IL-1ra neutralization, indicating that the baseline production of IL-1ra is biologically relevant. Immunohistochemistry showed a relation between IL-1ra and involucrin expressions, suggesting that intracytoplasmic IL-1ra may be involved in cell terminal differentiation. In unstimulated culture lysates, there was far more IL-1ra than IL-1alpha and IL-1beta. TGF-beta1 markedly increased the IL-1ra/IL-1beta ratio from 93.6 : 1 to 300 : 1. IL-4, which is generally described as an anti-inflammatory cytokine, increased IL-1 but not IL-1ra production. TNF-alpha increased intracellular production of the three IL-1 members. IL-1ra levels were lower in supernatants than in lysates of cultured cells. Our results show that human OMECs constitutively produce significant amounts of a biologically active form of IL-1ra. TGF-beta1 mu(p)-regulation points to a positive amplification loop and IL-4 to a down-regulation loop, both including Th2 cells and OMECs. They may be important in oral tolerance and IgA production, respectively.     

Human papillomavirus-16-E7 oncoprotein enhances the expression of adhesion molecules in cervical endothelial cells but not in human umbilical vein endothelial cells.

OBJECTIVES: E7 is one of the oncoproteins encoded by human papillomavirus-16 (HPV-16), the major etiologic factor responsible for cervical cancer. Human papillomavirus-16-E7 expressed by human uterine cervix carcinoma cells is also released in the extracellular compartment where it induces immune suppression. We investigated whether E7 was also responsible for the enhanced endothelial adhesiveness required in cancer progression. STUDY DESIGN/METHODS: We treated cervical microvascular endothelial cells (CrMVEn) and human umbilical vein endothelial cells (HUVEC) with E7, tumor necrosis factor-alpha (TNF-alpha), and hydrogen peroxide (H2O2) and measured the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by fluorescent-activated cell sorter analysis. RESULTS: E7 strongly induced the expression of E-selectin, ICAM-1, and VCAM-1 in CrMVEn, but not in HUVEC. Tumor necrosis factor-alpha further increased the endothelial expression of adhesion molecules in CrMVEn. Hydrogen peroxide pre-treatment resulted in an enhanced ICAM-1 and a decreased E-selectin and VCAM-1 expression. We also show indirect effects when endothelial cells were stimulated with the supernatant of E7-pretreated macrophages. CONCLUSIONS: These results show that HPV-16-E7 oncoprotein strongly induces adhesion molecules expression in organ-specific endothelial cells.     

Distribution of human papillomavirus genotypes in women with high-grade cervical intraepithelial lesions and cervical carcinoma and analysis of human papillomavirus-16 genomic variants

AimTo analyze the distribution of high-risk human papillomavirus (HR-HPV) genotypes and the diversity of HPV-16 genomic variants in Croatian women with high-grade squamous intraepithelial lesions (HSIL) and cervical carcinoma.MethodsTissue biopsy specimens were obtained from 324 women with histopathologically confirmed HSIL or cervical carcinoma, 5 women with low-grade SIL, and 49 women with negative histopathology. HR-HPV DNA was detected with Ampliquality HPV-type nucleic-acid hybridization assay, which identifies 29 different HPV genotypes. HPV-16 genomic variants were analyzed by an in-house sequencing.ResultsThe most common HPV type in women with HSIL was HPV-16, detected in 127/219 (57.9%) specimens. HPV-16 was also the dominant type in squamous cell cervical carcinoma (46/69 or 66.7%) and in adenocarcinoma (18/36 or 50.0%). Out of 378 patients, 360 had HR-HPV (282 single infections and 79 multiple infections), 3 (0.8%) patients had low-risk HPV, and 15 (4%) tested negative. HPV-16 variants were determined in 130 HPV-16 positive specimens, including 74 HSIL and 46 carcinoma specimens. In HSIL specimens, 41 distinct variants were found, 98.6% belonging to the European branch and 1.4% belonging to the African branch. In cervical carcinoma specimens, 95% isolates grouped in 41 variants belonging to the European branch, one isolate (2.5%) belonged to the North American, and one (2.5%) to the Asian-American branch.ConclusionHPV-16, mainly belonging to the European branch, was the most frequent HPV genotype in women from Croatia with histologically confirmed HSIL and cervical cancer.

Cervical cancer is the second leading cause of death in women in low-income countries (1). Persistent infection with particular human papillomavirus (HPV) genotypes is a necessary but not a sufficient requirement for the development of cervical cancer (2). HPV DNA is detected worldwide in nearly all specimens of invasive cervical cancer, including squamous cell carcinomas, adenocarcinomas, and the majority (>95%) of immediate cervical cancer precursors (3). An epidemiological study by Bosch et al (4) has shown that the most common HPV genotypes in HSIL and squamous cell carcinomas were HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-45, HPV-52, and HPV-58, with a combined worldwide relative contribution of 91% and the predominant role of HPV-16, HPV-18, and HPV-45 in cervical adenocarcinoma.HPV genomic variants are defined as the viruses that vary by 2% or less in specified regions of the genome, and some display different oncogenicity (5). HPV-16 heterogeneity has been extensively investigated (6-12), and HPV-16 genomic variants have been identified to belong to five main branches: European, Asian-American, two African branches, and an Asian branch (13). Two subsequent studies expanded these classifications and reported a new branch: North American 1 (14,15).Epidemiological studies have shown that non-European HPV-16 variants may promote viral persistence and disease progression (16-19). HPV-16 E6 variants, including the European HPV-16 T350G variant in the E6 gene, were detected up to 20 times more often in patients with high-grade cervical disease compared with controls. A novel HPV-16 variant, identified in Croatia, harboring a 63-bp in-frame duplication in the E1 gene, was presumed to be of reduced oncogenicity (11).According to the several national or regional studies in women with normal and abnormal cytology, HPV-16 is the most common high-risk genotype in Croatian women (20-27). However, none of these studies involved HPV genotyping in tissue specimens, and the majority were performed in general population with a small number of women with histologically confirmed HSIL or cervical cancer. The genomic diversity of high-risk HPV genotypes in Croatia has not been studied to date. On the other hand, recommended, non-mandatory, free-of-charge, nine-valent HPV vaccine is available in Croatia and is intended for vaccination of both women and men aged 14 to 25 years (28).The aims of this study were to analyze the distribution of high-risk HPV genotypes (HR-HPV) in women with histologically confirmed HSIL and cervical carcinoma and to analyze the genomic diversity of HPV 16 in HSIL in comparison with invasive cervical cancer.     

乳头瘤病毒16型E6,P53,RB,PCNA在宫颈癌中的表达状态及相关性

应用核酸原位杂交和免疫组织化学技术，检测人子宫颈癌中人乳头瘤病毒（ＨＰＶ）１６型Ｅ６ＯＲＦ与抑癌基因产物Ｐ５３，ＲＢ和增殖细胞核抗原（ＰＣＮＡ）。在４４例宫颈癌石蜡切片中，原位杂交检测出ＨＰＶ１６Ｅ６ＯＲＦ阳性２７例（６１．３６％），其中免疫组化检测出Ｐ５３、ＲＢ、ＰＣＮＡ阳性分别为８例（２９．６３％），１４例（５２．８５％）、２０例（７４．０７％），而在１７例ＨＰＶ１６Ｅ６阴性标本中Ｐ５３、ＲＢ、ＰＣＮＡ阳性分别为７例（４１．１７％），９例（５２．９４％）、１２例（７０．５８％）。而在５例正常宫颈组织中未测出ＨＰＶ１６Ｅ６ＯＲＦ，ＰＣＮＡ只在宫颈组织上皮基底层细胞中表达。统计学分析表明，ＨＰＶ１６Ｅ６与宫颈癌密切相关（Ｐ＜０．０５），ＰＣＮＡ在宫颈癌与正常宫颈组织中有显著性差异（Ｐ＜０．０５）。未能发现宫颈癌组织中ＨＰＶ１６Ｅ６ＯＲＦ与Ｐ５３蛋白相关性（Ｐ＞０．０５）。     

p16INK4A expression in cervical premalignant and malignant lesions

p16^INK4a is a cyclin-dependent kinase (CDK) inhibitor which decelerates cell cycle by inactivating CDKs that phosphorylate pRb. Human Papillomavirus persistent infection plays an important role on cervical carcinogenesis, mainly by the action of two viral oncoproteins, E6 and E7, which interact with p53 and pRb, respectively. Increasing expression of E6 and E7 in dysplastic cervical cells might thus be reflected by increased expression of p16^INK4a. Recent studies revealed that p16^INK4a expression could be a marker for dysplastic and neoplastic cervical cells. The aim of this study was to analyze p16^INK4a expression in cervical preneoplastic and neoplastic lesions and correlate with lesion grade. Expression of p16^INK4a was analyzed by immunohistochemistry. A total of 6 low-grade squamous intraepithelial lesion (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 27 cancer samples were studied. In HPV-positive cervical samples (n = 48), p16^INK4a expression was observed in 1 of 3 LSIL, in 18 of 19 HSIL and in all 26 cancer cases. These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16^INK4a expression in cervical lesions. In our study, a statistically significant association was observed between cervical lesion grade and p16^INK4a expression (P < 0.001).     

乳头瘤病毒16型E6,P53,RB,PCNA在宫颈癌中的表达…

应用核酸原位杂交和免疫组织化学技术，检测人子宫颈癌中人乳头瘤病毒（ＨＰＶ）１６型Ｅ６ＲＦ与抑癌基因产物Ｐ５３，ＲＢ和增殖细胞核抗原（ＰＣＮＡ）。在４４例宫颈癌石蜡切片中，原位杂交检测出ＨＰＶ１６Ｅ６ＯＲＦ阳性２７例（６１．３６％），其中免疫组化检测出Ｐ５３、ＲＢ、ＰＣＮＡ一分别为８例（２９．６３＃），１４例（５２．８５％）、２０例（７４．０７％），而在１７例ＨＰＶ１６Ｅ６阴性 本中Ｐ５３、ＲＢ、Ｐ     

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4(+) and CD8(+) T cells from the cervix responded to the HPV-16 antigens and that interferon-gamma (IFN-gamma) production was HPV type-specific. Comparing HPV-specific T-cell IFN-gamma responses at the cervix with those in the blood, we found that while CD4(+) and CD8(+) T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-gamma responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4(+) and CD8(+) T-cell IFN-gamma responses to E7 were of similar magnitude in both compartments and CD8(+) responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.     

Cell cycle effects of IL-10 on malignant B-1 cells

IL-10 is overexpressed in human chronic lymphocytic leukemia (CLL), and is an autocrine growth factor involved in the development of malignant B1 clones in NZB mice, a murine model for CLL. Antisense IL-10 oligonucleotide treatment induces apoptosis and cell cycle disruption in these cells both in vitro and in vivo. In addition, NZB IL-10 knock-out mice fail to develop the B-1 clones. Dampening of IL-10 protein production via antisense IL-10 oligonucleotide treatment is correlated with decreased p27/Kip1 protein expression which results in increased cyclin D2, cyclin E and cyclin A associated kinase activity. The action of the antisense oligonucleotides is through alterations in cell cycle regulation, resulting in accelerated cell cycle progression, a G2/M block which culminates in apoptosis induction in the malignant cells. This implies that the role of IL-10 as an autocrine growth factor in malignant B-1 cells lies in its ability to inhibit apoptosis induction through the maintenance of sustainable cell cycle progression in malignant cells.     

Inhibition of alloreactivity by mAb MR6: differential effects on IL-2- and IL-4-producing human T cells

mAb MR6 has previously been shown to block both IL-4-lnducedT cell proliferation and IL-4-dependent IgE production, suggestinga functional association between the antigen detected by MR6(gp200-MR6) and the human IL-4 receptor. In this study the potentialmodulatory effects of mAb MR6 on IL-4 function have been furtheranalysed in alloantigen-specific assays for cytotoxlc and T_hcell maturation, mature cytotoxlc T cell killing, helper cellproliferation, and generation of IL-2- and IL-4-produclng T_hcells in a mixed lymphocyte reaction (MLR). Our data show thatmAb MR6 has an inhibitory effect on both clonal expansion andmaturation of cytotoxlc T lymphocyte precursors within the alloreactlveT cell population. mAb MR6 had no effect on the maturation ofT_h lymphocyte precursors (assayed by IL-2 production). However,this mAb had striking differential effects on cytoklne productionin MLR cultures, showing total ablation of IL-4 but no alterationof IL-2 levels in the supernatant medium. The absence of IL-4from culture supernatants could be due to the fact that mAbMR6 is blocking cytoMne production, that it is speeding up IL-4internalization and utilization or that it inhibits the expansionof the T cell subset(s) that secretes IL-4. The data demonstratethat the action of mAb MR6 is focused on the IL-4-producingpopulation and raise the possibility that gp200-MR6 may playan important role in this aspect of IL-4 function.     

Cytokine regulation of IL-12 receptor beta2 expression: differential effects on human T and NK cells

The biological activities of IL-12 are mediated through a specific, high-affinity receptor composed of IL-12 receptor(R)beta1 and IL-12Rbeta2 subunits that exist primarily on T and NK cells. Remarkably, the expression of IL-12Rbeta2 on CD4(+) T cells in mouse and humans appears to be differentially regulated by IFN-gamma and IFN-alpha, respectively. Using an antibody specific for the human IL-12Rbeta2 subunit, the effect of IFN-gamma, IFN-alpha, IL-12 and IL-2 on the regulation of IL-12R expression and IL-12 responsiveness of human T and NK cells was assessed. The presence of IFN-alpha or IFN-gamma in cultures enhanced IL-12Rbeta2 expression of CD4(+) and CD8(+) T cells. The enhancing effect of IFN-alpha and IFN-gamma was independent of endogenous IL-12. Furthermore, the clearest effects of IFN-alpha and IFN-gamma on IL-12Rbeta2 expression on T cells were seen by abrograting the inhibition induced by the presence of IL-4 in cultures. In contrast to T cells, IFN-alpha and IFN-gamma had little effect on regulating IL-12Rbeta2 expression on human NK cells. Taken together, these data show that there is differential regulation of IL-12Rbeta2 expression by IFN-alpha and IFN-gamma on human T and NK cells.     

IL-10 augments CD23 expression on U937 cells and down-regulates IL-4-driven CD23 expression on cultured human blood monocytes: effects of IL-10 and other cytokines on cell phenotype and phagocytosis.

The effects of human recombinant interleukin-10 (IL-14) on the expression of several markers on U937 and human peripheral blood monocytes was studied by immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. IL-10 augmented Fc IgE receptor (Fc epsilon RII/CD23) further enhanced by cotreatment with IL-4 or interferon-gamma (IFN-gamma). In contrast, the basal level of Fc epsilon RII expression on blood monocytes appeared to fall in response to IL-10, and this effect became more evident on IL-4-treated cells. Furthermore, the constitutive and IFN-gamma-triggered Fc gamma RI/CD64 expression was augmented on both monocytes and U937 cells. Thus the expression of Fc gamma RII/CD32, Fc gamma/RIII/CD16, Fc alpha R/CD89, the receptor for complement components (CR1/CD35, CD3/CD11b, CR4/CD11c) and the receptor for transferrin/CD71 was not significantly influenced on IL-10-treated cells. IL-10 modestly triggered CD14 antigen expression on monocytes but not U937. The expression of intercellular adhesion molecule-1 (ICAM-1)/CD54 on monocytes was significantly inhibited by IL-10. As expected, a marked reduction of the constitutive as well as of the IFN-gamma or IL-4-driven expression on HLA-DR, HLA-DP and HLA-DQ was observed on IL-10-cultured monocytes. On the other hand, the expression of major histocompatibility complex (MHC) class I molecules was slightly and dose-dependently induced on IL-10-treated monocytes. The ability of blood monocytes to phagocytose IgG-sensitized ox erythrocytes, and to bind and ingest opsonized Escherichia coli or latex particles, was amplified by IL-10. Our data demonstrate that IL-10 modulates the expression of a wide variety of structures on human mononuclear phagocytes, and augments their phagocytic capacity.     

子宫颈癌组织中HPV16癌基因及p53基因的检测

利用ＨＰＶ１６Ｅ６、Ｅ７基因特异性引物，ＰＣＲ技术及抗癌基因ｐ５３外显子７特异性引物，ＰＣＲ－ＳＳＣＰ技术对３５例进展期子宫颈癌组织进行研究，发现：（１）３５例标本中ＨＰＶ１６Ｅ６、Ｅ７ＤＮＡ的总检出率为７１．４２％（２５例），其中同时检出Ｅ６、Ｅ７为３１．４２％（１１例），另外８．５７％及３１．４２％（３例及１１例）仅分别检出Ｅ６、Ｅ７序列。（２）全组未见１例有ｐ５３基因外显子７的点突变及等位基因缺失。该结果说明ＨＰＶ１６与本地区妇女子宫颈癌发生有密切关系，并且癌组织中ＨＰＶ１６Ｅ６、Ｅ７亚基因的分布是不均一的，ｐ５３基因外显子７的改变并不常见。在实验中我们还建立了使用生物素标记的ｄＵＴＰ进行ＰＣＲ－ＳＳＣＰ的技术。     

AEG-1表达下调对人宫颈癌细胞细胞周期和侵袭能力的影响及其机制

 目的：研究星形细胞上调基因1（astrocyte elevated gene-1,AEG-1）在人宫颈鳞癌细胞和组织中的表达,探讨AEG-1表达下调对宫颈癌SiHa细胞细胞周期和侵袭能力的影响,并分析其可能的分子机制。方法：采用Western blotting检测正常宫颈组织、宫颈鳞癌组织、HeLa、SiHa和CaSki细胞中AEG-1蛋白的表达。将对照siRNA和AEG-1 siRNA分别转染SiHa细胞,利用Western blotting检测SiHa细胞中AEG-1蛋白的表达,采用流式细胞术检测细胞周期分布的变化,采用Boyden小室检测细胞侵袭能力的变化,最后采用Western blotting检测cyclin D1、细胞周期素依赖性激酶 2（CDK2）和基质金属蛋白酶9(MMP-9)蛋白表达的变化。结果：宫颈鳞癌组织中AEG-1蛋白表达显著高于正常宫颈组织（P<0.05）,同时3株宫颈癌细胞中AEG-1蛋白表达均显著高于正常宫颈组织,其中SiHa细胞中AEG-1蛋白表达最高（P<0.05）。此外,AEG-1 siRNA能显著下调SiHa细胞中AEG-1蛋白的表达（P<0.05）,其表达下调能明显促使SiHa细胞在G0/G1期的比例增加和降低其侵袭能力。Western blotting结果表明,AEG-1 siRNA组中cyclin D1、CDK2和MMP-9蛋白的表达均显著低于未处理组和对照siRNA组（P<0.05）。结论：AEG-1在宫颈癌中的高表达可能与宫颈癌的发生发展密切相关,其表达下调介导的细胞周期静止和侵袭能力降低可能与cyclin D1、CDK2和MMP-9蛋白表达下调密切相关。     

IL-2调节人外周血T淋巴细胞CTLA-4表达

目的 探讨人外周血T淋巴细胞CTLA-4的表达情况及IL-2对其表达的调节作用。方法 采用流式细胞仪定量测定人外周血T淋巴细胞内及细胞膜上CTLA-4的水平，半定量RT-PCR检测T淋巴细胞内CTLA-4mRNA的水平，并在体外用IL-2刺激T淋巴细胞后观察CTLA-4及CTLA-4mRNA水平的变化。结果 人外周血T淋巴细胞膜表面几乎不表达CTLA-4，7．6％-18．0％的T淋巴细胞有胞内表达，CD4^ T淋巴细胞表达CTLA-4的阳性比例略高于CD8^ T淋巴细胞；人T淋巴细胞可溶性形式的CTLA-4mRNA半衰期短于全长CTLA-4mRNA；IL-2可以通过诱导人T淋巴细胞CTLA-4mRNA的转录上调CTLA-4的表达，IL-2诱导的细胞多为CD25^ T淋巴细胞。结论 CTLA4多存在于人外周血T淋巴细胞内，参与T淋巴细胞活化过程的调节。IL-2的免疫抑制作用可能与其诱导T淋巴细胞内CTLA-4mRNA转录，从而上调CTLA-4的表达有关。     

Up-regulation of MSX2 enhances the malignant phenotype and is associated with twist 1 expression in human pancreatic cancer cells

MSX2 is thought to be a regulator of organ development and a downstream target of the ras signaling pathway; however, little is known about the role of MSX2 in the development of pancreatic cancers, most of which harbor a K-ras gene mutation. Therefore, we examined whether the presence of MSX2 correlates with the malignant behavior of pancreatic cancer cells. BxPC3 pancreatic cancer cells that stably overexpress MSX2 showed a flattened and scattered morphology accompanied by a change in localization of E-cadherin and beta-catenin from membrane to cytoplasm. Cell proliferation rate, cell migration, and anchorage-independent cell growth were enhanced in MSX2-expressing cells. Injection of MSX2-expressing cells into the pancreas of nude mice resulted in a significant increase in liver metastases and peritoneal disseminations compared with injection of control cells. Microarray analysis revealed a significant induction of Twist 1 expression in cells that express MSX2. When MSX2 was inactivated in pancreatic cancer cells following transfection with an MSX2-specific small interfering RNA, Twist 1 was down-regulated. Immunohistochemistry of human pancreatic carcinoma tissue revealed that MSX2 was frequently expressed in cancer cells, and that increased expression of MSX2 significantly correlated with higher tumor grade, vascular invasion, and Twist 1 expression. These data indicate that MSX2 plays a crucial role in pancreatic cancer development by inducing changes consistent with epithelial to mesenchymal transition through enhanced expression of Twist 1.     

Toll-like receptor 4 surface expression on human monocytes and B cells is modulated by IL-2 and IL-4

Human Toll-like receptor 4 (TLR4) has recently been identified, and it has been shown to be the main protein involved in recognizing gram-negative bacteria. We examined the regulation of TLR4 surface expression in human peripheral blood monocytes and B cells by interleukin-2 (IL-2) and IL-4. IL-2 up-regulated TLR4 surface expression on human peripheral blood monocytes, but did not change expression on human peripheral B cells. By contrast, IL-4 down-regulated TLR4 surface expression on human peripheral blood monocytes, but up-regulated TLR4 surface expression on human peripheral B cells. These results indicate that Th1 cytokine IL-2 enhances receptors involved in the response to gram-negative bacteria and that activation of cellular immunity may enhance defense against these pathogens through monocytes, but not B cells, whereas Th2 cytokine IL-4 modulates the receptor response to gram-negative bacteria and that activation of humoral immunity may enhance defense against these pathogens through B cells, but not monocytes.     

下调HMGA2基因表达对改善骨肉瘤U2OS细胞恶性表型的实验研究

目的:研究HMGA2表达在维持人骨肉瘤U2OS细胞恶性表型中的作用,为基因靶向治疗提供理论依据。方法:采用基于DNA的shRNA表达载体HMGA2-shRNA,稳定转染人骨肉瘤U2OS细胞,下调其HMGA2表达水平,并应用RT-PCR技术检测其对HMGA2基因的沉默效果;经Cell Counting Kit-8(CCK8)测定、Hoechst33342染色荧光显微镜观察及Boyden小室法分别检测细胞增殖、凋亡及迁移情况;实时定量RT-PCR检测mRNAs表达水平。结果:稳定转染靶向HMGA2的shRNA可特异性下调U2OS细胞HMGA2 mRNA表达水平;其作用结果使细胞的增殖和迁移能力受到明显抑制,自发凋亡率及Caspase 3和Caspas 9基因表达水平显著升高。结论:HMGA2基因异常表达在维持人骨肉瘤U2OS细胞恶性表型中起重要作用,靶向HM-GA2的基因治疗可能为骨肉瘤治疗带来新希望。     

Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells

Claudin proteins comprise a recently described family of tight junction proteins that differentially regulate paracellular permeability. Since other tight junction proteins show alterations in distribution or expression in inflammatory bowel disease (IBD) we assessed expression of claudins (CL) 2, 3 and 4 in IBD. CL 2 was strongly expressed along the inflamed crypt epithelium, whilst absent or barely detectable in normal colon. In contrast, CL 3 and 4 were present throughout normal colonic epithelium and were reduced or redistributed in the diseased surface epithelium. In a T84-cell culture model of the gut barrier, paracellular permeability decreased with time after plating and correlated with a marked decrease in the expression of CL 2. Addition of IFNgamma/TNFalpha led to further decreases in CL 2 and 3, the redistrbution of CL 4 and a marked increase in paracellular permeability. Conversely, IL-13 dramatically increased CL 2, with little effect on CL 3 or 4, but also resulted in increased paracellular permeability. Expression of CL 2 did not correlate with proliferation or junctional reorganisation after calcium ion depletion. Re-expression of CL 2 in response to IL-13 was inhibited by phophatidylinositol 3 kinase inhibitor, LY294002, which also restored the ion permeability to previous levels. CL 2 expression could be stimulated in the absence of IL-13 by activation of phospho-Akt in the phophatidylinositol 3 kinase pathway. These results suggest that INFgamma/TNFalpha and IL-13 have differential effects on CL 2, 3 and 4 in tight junctions, which may lead to increased permeability via different mechanisms.     

Increased expression of surface IgM but not IgD or IgG on human B cells in response to IL-4.

Surface IgM (sIgM) was increased up to 10 times on human tonsillar B cells activated with IL-4. No change was observed for surface IgD, IgG or IgE. Other activators of human B cells, such as TPA, EBV and anti-IgM resulted in increased expression of the low-affinity receptor for IgE (CD23), but had no effect on sIgM. IL-4 also increased sIgM expression on prolymphocytic leukaemic (PLL) B cells, whereas TPA significantly reduced the level of sIgM. The effect on sIgM thus seems specific for IL-4, and is consistent with the existence of a unique IL-4-dependent B-cell activation pathway. Preincubation with IL-4 did not 'prime' B cells to proliferate in response to subsequent exposure to anti-IgM, and slightly decreased the response to co-stimulation with IL-4 and anti-IgM. The increase in sIgM expression in response to IL-4, therefore, does not seem to be important for proliferation.