Distinct Pattern of Human Vδ1 γδ T Cells Recognizing MICA

γδT cells represent one unique recognition pattern,the limited recognition,which distinguishes from the specificrecognition for αβT cells and pattern recognition for macrophages.Vδ1 γδT cell is the major subset of human γδT cells,which predominates in mucosal tissue including the intestinal epithelia.Presently,a few antigens thathuman Vδ1TCR can recognize have been identified.Among them,MHC class I chain-related molecules A (MICA)have been studied most intensively.Besides Vδ1TCR,MICA is also the ligand of NKG2D,a C-type lectin-likeactivating immunoreceptor.In human,only Vδ1 cells can simultaneously express both types of receptors of MICAwhile NK cells,αβT cells and other subsets of γδT cells likewise express NKG2D.Although the precisemechanisms are still enigmatic,this distinct pattern of Vδ1 cells recognizing MICA predicts unique biologicalsignificance of Vδ1 cells in immune defense.Recent years,some progresses have been made in this issue.In thisreview we summarize the related reports and put forward some novel views based on our group's studies.Cellular& Molecular Immunology.2005;2(4):253-258.     

Suitability of Human Tenon’s Fibroblasts as Feeder Cells for Culturing Human Limbal Epithelial Stem Cells

Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon’s fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α⁺LSCs co-cultured onto TFs was significantly higher than those on DFs (p?=?0.032) and 3T3-J2 (p?=?0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p?<?0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.     

Identification and Isolation of Human Dental Pulp Stem Cells

1 IntroductionDentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. Adult pulp stem cells was fo…     

Phagocytosis of IgA Immune Complexes by Human U937 Cells

Human promonocytic cell line U937 can express both IgAFc receptors ( FcαRⅠ, CD89 ) and IgG Fc receptors(FcαγⅠ, FcγRⅡ and FcγRⅢ)[1]. These receptors canmediate a variety of cell reactions including phagocytosis ofimmune complexes ( ICs ), degranulation, respiratorybursts, release of cytokines and enhancement of antibody dependent cell mediated cytotoxicity (ADCC) [2]. AfterIgA and IgG form ICs with their corresponding antigens,the ICs are bound by FcR on phagocyt…     

Expression of Nerve Growth Factor in Human Pancreatic β Cells

Nerve growth factor (NGF) is an important modulator of rat pancreatic β-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy.

We found that in normal human pancreas, insulin and NGF are co-expressed in β cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in β cells.

Pancreatic β cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor.

In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate.

We demonstrate that human β cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat β cells.     

Overexpression of the �� Subunit of Human Chorionic Gonadotropin Promotes the Transformation of Human Ovarian Epithelial Cells and Ovarian Tumorigenesis

Ovarian carcinoma is the most lethal gynecologic malignancy, however underlying molecular events remain elusive. Expression of human chorionic gonadotropin β subunit (β-hCG) is clinically significant for both trophoblastic and nontrophoblastic cancers; however, whether β-hCG facilitates ovarian epithelial cell tumorigenic potential remains uncharacterized. Immortalized nontumorigenic ovarian epithelial T29 and T80 cells stably overexpressing β-hCG were examined for alterations in cell cycle and apoptotic status by flow cytometry, expression of proteins regulating cell cycle and apoptosis by Western blot, proliferation status by MTT assay, anchorage-independent colony formation, and mouse tumor formation. Immunoreactivity for β-hCG was evaluated using mouse xenografts and on human normal ovarian, fallopian tube, endometrium, and ovarian carcinoma tissues. T29 and T80 cells overexpressing β-hCG demonstrated significantly increased proliferation, anchorage-independent colony formation, prosurvival Bcl-X_L protein expression, G2-checkpoint progression, elevated cyclins E/D1 and Cdk 2/4/6, and decreased apoptosis. Collectively, these transformational alterations in phenotype facilitated increased xenograft tumorigenesis (P < 0.05). Furthermore, β-hCG immunoreactivity was elevated in malignant ovarian tumors, compared with normal epithelial expression in ovaries, fallopian tube, and endometrium (P < 0.001). Our data indicate that elevated β-hCG transforms ovarian surface epithelial cells, facilitating proliferation, cell cycle progression, and attenuated apoptosis to promote tumorigenesis. Our results further decipher the functional role and molecular mechanism of β-hCG in ovarian carcinoma. β-hCG may contribute to ovarian cancer etiology, which introduces a new therapeutic intervention target for ovarian cancer.Ovarian cancer is the most lethal form of gynecologic cancer in the United States, accounting for an estimated 21,550 new cases and 14,600 deaths in 2009.¹ Survival rates can approach 90% when ovarian cancer is diagnosed at an early stage; however, early detection is challenging, because the relatively nonspecific symptoms of ovarian lesions may be overlooked until abdominal distension by ascites fluid or by large tumor masses becomes unmistakable. Even with extensive surgical debulking and aggressive chemotherapy, the prognosis for women with ovarian cancer currently is not hopeful. Several studies have indicated that different histological subtypes of ovarian carcinoma are associated with different causes and underlying mechanisms, including gene amplification, genetic predisposition, and various carcinogens.^2–5 Nonetheless, the origin and causes of ovarian carcinoma remain to be elucidated.Human chorionic gonadotropin (hCG) has a physiologically significant role during pregnancy. It is produced as a heterodimeric glycoprotein complex by the placenta over the course of the first 3 months of gestation. The heterocomplex consists of an α subunit and a hormone-specific β subunit, which collectively act as a ligand in activating the luteinizing hormone/hCG receptor (LH/hCGR) in gonadal cells to regulate sex hormone synthesis and reproductive processes.⁶ The β subunit of the hCG complex (β-hCG) is an accurate marker for diagnosis and monitoring of trophoblastic tumors and ovarian germ cell tumors.^7,8 Recently, it was shown that elevation in levels of β-hCG in serum, urine, or tumor tissue correlates with patient outcome in a variety of nontrophoblastic tumors of diverse primary tissue origin.^9–15 Moreover, elevated β-hCG was associated with aggressive disease, poor prognosis, and predicted resistance to therapy in bladder cancer patients.¹⁶ Additionally, increased β-hCG expression simultaneously stimulates proliferation and inhibits apoptosis of cancer cells derived from the bladder and the cervix in vitro.^17,18 Furthermore, ovarian carcinoma tissue displays an overexpression not only of the hormone-specific β-hCG subunit, but also the cognate receptor hCGR.^9,19 The functional role and molecular mechanism of β-hCG within ovarian cancer tumorigenesis have yet to be characterized. Recent evidence has strongly implicated epithelial cells derived from the fallopian tube, especially the fimbriated ends, as the likely origin for high-grade serous carcinoma.²⁰ To test whether this molecule plays a direct role in facilitating ovarian epithelial cell activation and tumorigenic potential, we designed a panel of experiments using both in vitro and in vivo methods, including evaluation of β-hCG expression in the normal fallopian tube.     

FcγRI-Mediated Activation of Human Mast Cells Promotes Survival and Induction of the Pro-survival Gene Bfl-1

Activation of mast cells through either FcɛRI or FcγRI leads to release of mediators contributing to the inflammatory response. One of the biologic characteristics of mast cells in allergic pathology is that these cells have the capacity to recover and regranulate after aggregation of FcɛRI. We have previously demonstrated that the pro-survival protein A1/Bfl-1 is required for mast cells to survive IgE-mediated activation. In the present study, we have investigated whether human mast cells show similar induction of bfl-1 and activation-induced survival after aggregation of FcγRI. Human cord blood-derived mast cells were activated by aggregation of either FcɛRI or FcγRI, and activation-induced survival and induction of bfl-1 was measured. We found that aggregation of FcγRI-induced expression of bf-1 and caused a comparable activation-induced mast cell survival as FcɛRI does. These data suggests that activation through Fc-receptors contribute to mast cell survival during antibody-dependent mast cell mediated inflammatory responses.     

Malignant Transformation of Human Embryonic Liver Cells Induced by Hepatitis B Virus and Aflatoxin B_1

Hepatocellular carcinoma (HCC) is a worldwide distribut ed disease accounting for 473 000 newly developed cases ofHCC annually in the world and 235 000 cases in China[1]. Epidemiological and laboratory investigations havedemonstrated that hepatitis…     

Effect of α1-Acidic Glycoprotein in the Ascitic Fluid of Cancer Patients on Human NK Cells: Selective Suppression of Interferon-Induced NK Activation

The in vitro effect of C-AGP (pure 1-acid glycoprotein from the ascitic fluid of cancer patients) on NK cell cytotoxicity was tested using normal healthy human PBMC. C-AGP had no inhibitory effect on basal NK cell activity. C-AGP selectively suppressed the augmentation of NK cell activity by rIFNA and rIFN, but C-AGP did not prevent the NK activation by rIL-2. NK cells in PBMC treated with C-AGP for 12 h and then washed just once, to remove the C-AGP, fully recovered the ability to respond to rIFNA. However, after the treatment of PBMC with C-AGP for 5 or 6 days, NK cells failed to respond to rIFNA, in spite of washing to remove C-AGP from the cultures. Monocytes were necessary for the suppressive effect of C-AGP on rIFNA activation of NK cells. Indomethacin restored the ability of NK cells to respond to rIFNA in C-AGP-treated PBMC. These results suggest that monocytes are able to selectively suppress the response of NK cells to IFNs in the presence of, or following treatment with C-AGP.     

Intracellular Distributing and Interferon-γ Secretion of Human Interleukin-18 in BxPC-3 Cells

Objective: To investigate the characteristics of interleukin-18 (IL-18) in vitro, explore IL-18, interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretive activity in BxPC-3 line cells with interleukin-18 mutants.Methods: Human IL-18 full-length gene (hIL-18-F) and the hIL-18 presumed mature protein gene (hIL-18-M) were inserted into the expression vector pEGFP-N1, to construct recombinant plasmids as Mu0, Mu1, Mu2, Mu3, and Mu4, and the recombinant plasmids were then transferred into BxPC-3 line cells. There are significant differences between Mu1, Mu2 and the pEGFP-C1 control group (P<0.05) by 3-(4,5-dimethiazol- 2-yl)- 2,5-diphenyltetrazolium bromide (MTT) for a proliferation assay, and the fluorescence of the Mu1 and Mu 2 appeared targeted to the membranous region in the BxPC-3 cells after transfected 24h by confocal laser scanning microscope (OLSM).To characterize the intracellular distribution of hIL-18, recombinant IL-18 were each fused to the enhanced green fluorescent protein gene, and expressed in BxPC-3 cells.Results: Results showed that the Mu1 tended to the membranous region in BxPC-3 cells, this indicates that the N-terminal former amino acid peptide helped ChIL-18 target to BxPC-3 cellS membranes. ELISA results demonstrated that IFN-γ and IL-18 secreted levels of BxPC-3 cells transfecting with recombinant plasmid showed an significant difference (P<0.01); refers to IL-2 expression, the two BxPC-3 cells groups transfecting with recombinant plasmid have no significant function (P>0.05).Conclusions: The results showed that hIL-18 and hIL-18 presumed mature protein can induce the secretion of IFN-γ in BxPC-3 cells, and increase the expression of IL-18, but they have no effects on IL-2.     

Adenosine A2A Receptor and TNF-α Regulate the Circadian Machinery of the Human Monocytic THP-1 Cells

Morning stiffness and increased symptoms of inflammatory arthritis are among the most common manifestations of rheumatoid arthritis (RA). Tumor necrosis alpha (TNF-α), an important mediator of inflammation in RA, regulates the circadian expression of clock proteins, and adenosine A_2A receptors (A_2AR) mediate many of the anti-inflammatory and antirheumatic actions of methotrexate, the cornerstone drug in the treatment of RA. We found that A_2AR activation and TNF-α activated the clock core loop of the human monocytic THP-1 cell line. We further observed that interleukin (IL)-10, but not IL-12, mRNA expression fluctuates in a circadian fashion and that TNF-α and A_2AR stimulation combined increased IL-10 expression. Interestingly, TNF-α, but not CGS21680, dramatically inhibited IL-12 mRNA expression. The demonstration that A_2AR and TNF-α regulate the intrinsic circadian clock in immune cells provides an explanation for both the pathologic changes in circadian rhythms in RA and for the adverse circadian effects of methotrexate, such as fatigue.     

Effects of Transforming Growth Factor-βl and Phorbol Ester on PALI-1 and PA Genes in Human Lung Cells

Abstract

Transforming growth factor-β (TGF-β) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-β and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-β in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-β involves PKC. TGF-β1 and PMA increased expression of TGF-βl mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-β2 and TGF-β3 were more complex; while TGF-β2 and TGF-p3β mRNAs increased transiently in response to TGF-p1 in NHBE cells and TGF-β3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCLH727 where TGF-pZ mRNA increased dramatically. TGF-β1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contmst, while TGF-βl also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-β1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-βl, PAI-1 and PA by TGF-β1 and PMA in NSCLC, but differential regulation of the genes for PAL1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-β1, PAI-1 and PA to TGF-βl and PMA were inhibited by the serind threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-β1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-p responses by inhibiting a protein serindthmnine kinase(s). Because the effects of TGF-p and PMA on the different TGF-p isoforms, PA, PA1 and fibronectin in NHBE and NSCLC cells are complex, our data suggest that there are distinct mechanisms for controlling the different TGF-p isoforms, PA, PA1 and extracellular matrix proteins in normal lung and lung cancer cells.     

Distinct Pattern of Human Vδ/51 γδ/5 T Cells Recognizing MICA

γδ T cells represent one unique recognition pattern, the limited recognition, which distinguishes from the specific recognition for αβ T cells and pattern recognition for macrophages. Vδ1 γδT cell is the major subset of human γδT cells, which predominates in mucosal tissue including the intestinal epithelia. Presently, a few antigens that human Vδ1TCR can recognize have been identified. Among them, MHC class I chain-related molecules A （MICA） have been studied most intensively. Besides Vδ1TCR, MICA is also the ligand of NKG2D, a C-type lectin-like activating immunoreceptor. In human, only Vδ1 cells can simultaneously express both types of receptors of MICA while NK cells, αβ T cells and other subsets of γδT cells likewise express NKG2D. Although the precise mechanisms are still enigmatic, this distinct pattern of Vδ1 cells recognizing MICA predicts unique biological significance of Vδ1 cells in immune defense. Recent years, some progresses have been made in this issue. In this review we summarize the related reports and put forward some novel views based on our group＇s studies.     

Quantitative Peripheral Blood Perturbations of γδ T Cells in Human Disease and Their Clinical Implications

Human γδ T cells, which play innate and adaptive, protective as well as destructive, roles in the immune response, were discovered in 1986, but the clinical significance of alterations of the levels of these cells in the peripheral blood in human diseases has not been comprehensively reviewed. Here, we review patterns of easily measurable changes of this subset of T cells in peripheral blood from relevant publications in PubMed and their correlations with specific disease categories, specific diagnoses within disease categories, and prognostic outcomes. These collective data suggest that enumeration of γδ T cells and their subsets in the peripheral blood of patients could be a useful tool to evaluate diagnosis and prognosis in the clinical setting.