Secretome-based identification of Mac-2 binding protein as a potential oral cancer marker involved in cell growth and motility

Oral squamous cell carcinoma (OSCC) is the 11th most common cancer worldwide, and is associated with a high death rate. At present, there are no suitable markers for detecting and/or monitoring OSCC in body fluids/tissues. Here, we used 1D SDS-PAGE and MALDI-TOF MS to systematically analyze the secretomes of two OSCC cell lines (OEC-M1 and SCC4). The putative OSCC-related proteins identified in this analysis included the Mac-2 binding protein (Mac-2 BP), which was further found to be overexpressed in OSCC specimens and significantly elevated in the sera of OSCC patients compared to healthy controls. Finally, RNA interference-based knock-down of Mac-2 BP expression in OSCC cells revealed for the first time that Mac-2 BP is involved in regulating growth and motility of OSCC cells.     

Mac-2 binding protein is a novel e-selectin ligand expressed by breast cancer cells

Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.     

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) recognizes a novel ligand, Mac-2-binding protein, characteristically expressed on human colorectal carcinomas

Dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a type II transmembrane C-type lectin expressed on DCs such as myeloid DCs and monocyte-derived DCs (MoDCs). Recently, we have reported that DC-SIGN interacts with carcinoembryonic antigen (CEA) expressed on colorectal carcinoma cells. CEA is one of the most widely used tumor markers for gastrointestinal cancers such as colorectal cancer. On the other hand, other groups have reported that the level of Mac-2-binding protein (Mac-2BP) increases in patients with pancreatic, breast, and lung cancers, virus infections such as human immunodeficiency virus and hepatitis C virus, and autoimmune diseases. Here, we first identified Mac-2BP expressed on several colorectal carcinoma cell lines as a novel DC-SIGN ligand through affinity chromatography and mass spectrometry. Interestingly, we found that DC-SIGN selectively recognizes Mac-2BP derived from some colorectal carcinomas but not from the other ones. Furthermore, we found that the α1-3,4-fucose moieties of Le glycans expressed on DC-SIGN-binding Mac-2BP were important for recognition. DC-SIGN-dependent cellular interactions between immature MoDCs and colorectal carcinoma cells significantly inhibited MoDC functional maturation, suggesting that Mac-2BP may provide a tolerogenic microenvironment for colorectal carcinoma cells through DC-SIGN-dependent recognition. Importantly, Mac-2BP was detected as a predominant DC-SIGN ligand expressed on some primary colorectal cancer tissues from certain parts of patients in comparison with CEA from other parts, suggesting that DC-SIGN-binding Mac-2BP bearing tumor-associated Le glycans may become a novel potential colorectal cancer biomarker for some patients instead of CEA.     

Identification of potential serum markers for nasopharyngeal carcinoma from a xenografted mouse model using Cy-dye labeling combined with three-dimensional fractionation

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for improving NPC diagnosis, we established a proteomic platform for detecting aberrant serum proteins in nude mice bearing NPC xenografts. We first removed the three most abundant proteins from serum samples of tumor-bearing and control mice, and then labeled the samples with different fluorescent cyanine (Cy) dyes. The labeled serum proteins were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Differentially expressed proteins were identified by in-gel tryptic digestion and MALDI-TOF MS. We identified peroxiredoxin 2 (Prx-II) and carbonic anhydrase 2 (CA-II) as being elevated in the xenograft mouse model compared to controls. Western blot analysis confirmed up-regulation of Prx-II and CA-II in plasma from five NPC patients, and ELISA showed that plasma Prx-II levels were significantly higher in NPC patients (n = 84) versus healthy controls (n = 90) (3.03 +/- 4.47 versus 1.90 +/- 2.74 microg/mL, p = 0.047). In conclusion, Cy dye labeling combined with three-dimensional fractionation is a feasible strategy for identifying differentially expressed serum proteins in an NPC xenograft model, and Prx-II may represent a potential NPC biomarker.     

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin.

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultracentrifugation showed non-covalent association into oligomers with molar masses of 1000-1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30-40 nm. M2BP bound in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1 subunits but not to alpha2 and alpha6 subunits, RGD peptide or lactose. This distinguishes cell adhesion of M2BP from that of laminin and excludes involvement of lactose-binding galectin-3. Immunological assays demonstrated variable secretion by cultured human cells of M2BP, which was detected in the extracellular matrix of several mouse tissues.     

The differential roles of LFA-1 and Mac-1 in host defense against systemic infection with Streptococcus pneumoniae

Mice deficient in CD18, which lack all four CD11 integrins, have leukocytosis and increased susceptibility to bacterial infection. To determine the effect of deficiencies in LFA-1 (CD11a/CD18) or Mac-1 (CD11b/CD18) on host defense against systemic bacterial infection, knockout mice were inoculated i.p. with Streptococcus pneumoniae. Increased mortality occurred in both LFA-1(-/-) (15 of 17 vs 13 of 35 in wild type (WT), p < 0.01) and Mac-1(-/-) (17 of 34 vs 6 of 25, p < 0.01) mice. All deaths in LFA-1(-/-) mice occurred after 72 h, whereas most deaths in Mac-1(-/-) mice occurred within 24-48 h. At 24 h, 21 of 27 Mac-1(-/-) mice were bacteremic, vs 15 of 25 WT (p = 0.05); no difference was observed between LFA-1(-/-) and WT. Increased bacteria were recovered from Mac-1(-/-) spleens at 2 h (p = 0.03) and 6 h (p = 0.002) and from livers (p = 0.001) by 6 h. No difference was observed at 2 h in LFA-1(-/-) mice, but by 6 h increased bacteria were recovered from spleens (p = 0.008) and livers (p = 0.04). Baseline and peak leukocyte counts were similar between Mac-1(-/-) and WT, but elevated in LFA-1(-/-). At 8 h, peritoneal neutrophils were increased in Mac-1(-/-), but not significantly different in LFA-1(-/-). Histopathologically, at 24 h Mac-1(-/-) animals had bacteremia and lymphoid depletion, consistent with sepsis. LFA-1(-/-) mice had increased incidence of otitis media and meningitis/encephalitis vs WT at 72 and 96 h. Both Mac-1 and LFA-1 play important but distinct roles in host defense to S. pneumoniae.     

Identification Keratin 1 as a cDDP-resistant protein in nasopharyngeal carcinoma cell lines

Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy of tumors. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cis-diamminedichloroplatinum (CNE2/cDDP) were established from human nasopharyngeal carcinoma (NPC) cell lines CNE2. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and ESI-Q-TOF-MS were performed on protein extracted from CNE2 and CNE2/cDDP cell lines to screen drug resistance-related proteins. Keratin 1 (KRT1), cathepsin D (CTSD) and annexin a5 (ANXA5) were identified as three proteins showing higher expression in CNE2/cDDP compared to CNE2. Furthermore, suppression of KRT1 expression by siRNA resulted in decreased MDR in siRNA-CNE2/cDDP cells. And upregulation of KRT1 could result in increased of drug resistance in NPC cell lines. Taken together, KRT1 protein and its activity levels were higher in cDDP-resistant NPC cell lines compared to their parental cell lines. These data clearly linked KRT1 and cDDP resistance mechanisms. KRT1 could serve as a biomarker for chemotherapy sensitivity of NPC.     

鼻咽癌细胞系与永生化的鼻咽上皮细胞系蛋白质表达差异分析

鼻咽癌对我国南部居民的健康造成严重的威胁.为了研究鼻咽癌的发病机理,本研究采用了蛋白质组学技术分析和比较了鼻咽癌细胞系（HNE1和CNE1）与永生化的鼻咽上皮细胞系的蛋白质表达谱.采用双向凝胶电泳分离提取的全细胞蛋白质,通过PDQuest软件分析找出在肿瘤中表达变化的蛋白质点,用基质辅助激光解析电离飞行时间串联质谱(MALDI- TOF/TOF-MS)进行鉴定.共得到了15个在肿瘤细胞系中表达上调和18个在肿瘤细胞系中表达下调的蛋白质,并对其中一些蛋白质的表达进行免疫印迹的验证.这些表达差异的蛋白质与细胞的增殖和调亡、癌症的转移,细胞骨架,信号传导等有关.本研究鉴定了一批可能作为鼻咽癌治疗的药物靶标的蛋白质,并对研究鼻咽癌发病机理提供了相关的线索.     

Mac-2-binding glycoproteins. Putative ligands for a cytosolic beta-galactoside lectin

Mac-2, a galactose-binding lectin secretion by activated macrophages, is the major non-integrin laminin-binding protein in these cells. Mac-2 is also expressed by epithelial cells in the intestine and kidney. We wished to identify intestinal glycoproteins other than laminin that have a high affinity for Mac-2 and that could be considered as candidate ligands or partners for this lectin in intestinal epithelium. Certain lines of human colon adenocarcinoma cells produce two Mac-2-binding glycoproteins (M2BP-1 and M2BP-2) that were identified by their avid association with Mac-2 following detergent lysis and immunoprecipitation. These glycoproteins do not share a common epitope with Mac-2, and the interaction between Mac-2 and these proteins is mediated through the carbohydrate-binding domain of Mac-2 and sugar moieties on M2BP-1 and M2BP-2. M2BP-1 (98 kDa) and M2BP-2 (70 kDa) were purified by immunoaffinity chromatography and were specifically eluted with either galactose or lactose. Peptide maps revealed that M2BP-1 and M2BP-2 are structurally related. M2BP-1 is secreted and could conceivably associate with Mac-2 extracellularly. N-terminal sequence analysis of M2BP-2 suggests that these glycoproteins represent a unique subset of candidate ligands for this mammalian beta-galactoside lectin.     

心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的影响*

目的:探讨心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的作用。方法:雄性无特定病原体(SPF)级20只昆明小鼠随机分2组(每组10只),即慢性束缚应激(Stress)组和正常对照(Control)组。Stress组小鼠每天在自制式束缚器中限制活动2 h,其余时间两组小鼠在相同环境中自由饮水摄食,实验持续14 d,取血和白色脂肪组织(WAT);观察脂肪组织病理学改变,检测WAT中黄嘌呤氧化酶(XO)和烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(Nox-4)的蛋白水平,检测WAT组织中XO、Nox-4、超氧化物歧化酶(Mn SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、脂联素(ADPN)、单核细胞趋化蛋白1(MCP-1)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)、胰岛素受体底物1(IRS-1)、葡萄糖转运蛋白4(GLUT-4)、组织因子(TF)、纤溶酶原激活物抑制物1(PAI-1)的mRNA表达,检测血清和WAT组织中XO酶活性以及血清甘油三酯(TG)、总胆固醇(T-Cho)、游离脂肪酸(FFA)、尿酸(UA)的含量。结果:与control组比较,stress小鼠腹股沟WAT组织中XO免疫染色阳性着色细胞黄褐色沉淀深且丰富,WAT中出现大量的单核细胞、中性粒细胞、嗜酸性粒细胞及浆细胞浸润反应和炎症性的改变;血清XO浓度、WAT组织中XO mRNA水平和XO的酶活性显著升高(P＜0.01),血清游离脂肪酸(FFA)和尿酸(UA)的含量显著增高(P＜0.01),WAT组织中Nox-4蛋白、MCP-1、IL-6、TNF-α、TF、PAI-1mRNA的表达水平显著增高(P＜0.01),而Mn-SOD、GSH-Px、CAT、ADPN、IRS-1和GLUT-4的mRNA水平则显著降低(P＜0.01)。结论:心理应激可诱发脂肪XO过量表达及其活性增高,进而引起脂肪炎症、糖代谢及凝血酶原异常等反应。     

Synthesis and secretion of plasminogen activator inhibitor 1 by human endothelial cells in vitro. Effect of active site mutagenized tissue-type plasminogen activator

The effects of recombinant tissue-type plasminogen activator (rt-PA) and of an inactive mutant of rt-PA, obtained by mutagenesis of the active site Ser478 to Ala (rt-PA-Ala478), on the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture were studied. Under base-line conditions, PAI-1 antigen secretion was 4.3 +/- 1.0 micrograms (mean +/- S.D., n = 8) per 10(6) cells in 24 h. This PAI-1 had a low specific activity (6,000 +/- 1,600 units/mg) and Mr of 50,000, which was not altered by addition of rt-PA. In HUVEC cultured with 2 micrograms/ml rt-PA-Ala478, PAI-1 antigen secretion was 2.1 +/- 0.8 micrograms (n = 5) per 10(6) cells in 24 h with a specific activity of 120,000 +/- 42,000 units/mg and Mr of 50,000. Addition of rt-PA to this conditioned medium resulted in generation of three main components: 16% migrated as an Mr 106,000 rt-PA.PAI-1 complex, 16% as an Mr 81,000 degraded rt-PA.PAI-1 complex and the remainder as an Mr 45,000 degradation product of PAI-1. HUVEC cultured with 2 micrograms/ml rt-PA secreted 3.9 +/- 0.6 micrograms (n = 8) PAI-1 antigen per 10(6) cells within 24 h, of which 20-50% occurred as intact or degraded complexes with t-PA (Mr 106,000 and 81,000) and the rest as an inactive Mr 45,000 degradation product of PAI-1. PAI-1 mRNA levels, determined by Northern blot analysis and expressed relative to beta-actin mRNA levels, were very similar for HUVEC cultured in the absence or the presence of rt-PA or rt-PA-Ala478. It is concluded that PAI-1 is secreted by HUVEC in culture in fully active form which spontaneously inactivates. PAI-1 can be stabilized by addition of rt-PA-Ala478 to the culture medium, resulting in a 20-fold increase in specific activity. Interaction of rt-PA with active PAI-1 produces both t-PA.PAI-1 complex and an inactive degradation product of PAI-1.     

Quantitative proteomes and in vivo secretomes of progressive and regressive UV-induced fibrosarcoma tumor cells: mimicking tumor microenvironment using a dermis-based cell-trapped system linked to tissue chamber

The alterations of tumor proteome and/or in vivo secretome created by host-tumor cell interaction may be crucial factors for tumors to undergo progression or regression in a host system. Two UV-induced fibrosarcoma tumor cell lines (UV-2237 progressive cells and UV-2240 regressive cells) were used as models to address this issue. Hundreds of proteins including in vivo secretome have been identified and quantified via an isotope-coded protein label (ICPL) in conjunction with high-throughput NanoLC-LTQ MS analysis. A newly designed technology using a dermis-based cell-trapped system was employed to encapsulate and grow 3-D tumor cells. A tissue chamber inserted with a tumor cell-trapped dermis was implanted into mice to mimic the tumor microenvironment. The in vivo secretome created by host-tumor interaction was characterized from samples collected from tissue chamber fluids via ICPL labeling mass spectrometric analysis. Twenty-five proteins including 14-3-3 proteins, heat shock proteins, profilin-1, and a fragment of complement C3 with differential expression in proteomes of UV-2237 and UV-2240 cells were revealed. Three secreted proteins including myeloperoxidase, alpha-2-macroglobulin, and a vitamin D-binding protein have different abundances in the in vivo secretome in response to UV-2237 and UV-2240 cells. Differential tumor proteomes and in vivo secretome were thus accentuated as potential therapeutic targets to control tumor growth.     

The role of lipocortin I in macrophage-mediated immunosuppression in tumor-bearing mice

The soluble mediators and/or mechanisms involved in immunosuppression in tumor-bearing hosts are not well characterized, although macrophages have long been recognized as major participants. We have investigated the role of lipocortin I, a phospholipid-binding protein, in macrophage-mediated immunosuppression in tumor-bearing mice. Proliferation of splenic lymphocytes in response to the mitogens (PHA, Con A, LPS, and PWM) was severely suppressed in tumor (Sqc-NH-1 carcinoma)-bearing mice. This immunosuppression was associated with a decrease in T and B lymphocytes and an increase in macrophages in these spleens. Mac-2+ macrophages were found only in spleens from tumor-bearing mice. Splenic macrophages from tumor-bearing, but not normal, mice were responsible for this immunosuppression, as revealed by negative and positive selection experiments. The levels of lipocortin I mRNA expression were markedly increased in peripheral blood cells from tumor-bearing mice as compared with those from normal mice. Lipocortin I mRNA was strongly induced in splenic mononuclear cells from tumor-bearing mice. Furthermore, these cells displayed increased expression of lipocortin I protein, as judged by Western blot analysis with polyclonal anti-lipocortin I serum. Some nonimmune organs such as the heart, submaxillary gland, muscle, and bladder also displayed increased levels of lipocortin I mRNA expression in tumor-bearing mice. Mac-2+ macrophages among the splenic mononuclear cells in tumor-bearing mice expressed lipocortin I mRNA, as judged by negative and positive selection experiments. Most of these Mac-2+ macrophages also had Mac-1 and Mac-3 Ag. Lipocortin I protein was increased in the serum of tumor-bearing mice as compared with normal mice. The culture supernatants of splenic cells from tumor-bearing mice suppressed the mitogenic responses of splenic cells from normal mice, and addition of anti-lipocortin I antiserum inhibited this suppression. Furthermore, recombinant mouse lipocortin I suppressed mitogenic responses of splenic cells from normal mice. In summary, Mac-2+ macrophage-derived lipocortin I was largely involved in immunosuppression in tumor-bearing mice.     

Gender-dependent physiological implications of combined PAI-1 and TIMP-1 gene deficiency characterized in a mouse model

The endogenous proteinase inhibitors plasminogen activator inhibitor type 1 (PAI-1) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) are two distinct proteins with separate molecular pathways. However, a close relationship between PAI-1 and TIMP-1 has been proposed indicating some degree of functional overlap due to their involvement in ECM turnover, tissue remodeling, and cellular migration and signaling. To study the housekeeping physiological implications of PAI-1 and TIMP-1, we generated a combined PAI-1 and TIMP-1 gene-deficient mouse model. We present the results on generating this specific mouse model with particular emphasis on phenotypical characteristics, blood leukocyte counts, histology, and gene expression studies of PAI-1 and TIMP-1 in various organs. We observed a significant deviation in segregation of offspring only in male mice (P < 0.01) predominantly caused by PAI-1 deficiency. In addition, the body weight in 3- and 20-wk-old male and 20-wk-old female mice was significantly different between genotypes (P or= 0.13). Quantifying the relative expression of PAI-1 and TIMP-1 revealed upregulation of PAI-1 (P < 0.001) in male mice only. Our data highlight the complex roles of PAI-1 and TIMP-1 on physiological parameters such as segregation of offspring (embryonic development and survival), body weight (metabolism), blood leukocyte counts (immunity), and gene expression (regulatory redundancy). We conclude that PAI-1 and TIMP-1 seem to possess gender-dependent regulatory properties on various housekeeping physiological parameters and stress the potential implications in pathological conditions.     

Effects of a novel Mac-1 inhibitor, NPC 15669, on hemostatic parameters during preconditioned myocardial infarction.

NPC 15669, a member of the leumedins family, inhibits leukocyte adhesion to the endothelium by blockage of upregulation of a member of beta2 integrin family Mac-1 (CD11b/CD18). Inhibition of neutrophil-endothelial interactions may alter the course of myocardial reperfusion injury. However, the effects of NPC 15669 supplementation on the hemostatic profile during ischemia-reperfusion are unknown. The aim of the present study was to define changes in the certain hemostatic factors in the natural course of preconditioned myocardial infarction. Twelve consecutive Yorkshire swine underwent myocardial stunning (8 min. left anterior descending artery occlusion followed by 90 min. of reperfusion) and then preconditioned myocardial infarction (50 min. occlusion followed by 3 hours of reperfusion) experiments. NPC 15669 (10 mg/kg loading dose followed by constant infusion at 6 mg x kg(-1) x h(-1)) was administered in 6 animals; another 6 swine received saline and served as controls. Blood samples were obtained at baseline, twice during occlusion; and three times during reperfusion. The levels of antithrombin-III, Protein C, total Protein S, fibronectin, endothelin-1, as well as the stable metabolites of thromboxane (TxB2) and prostacyclin (6-keto-PGF1a), were determined. NPC 15669 treatment was associated with diminished endothelin-1, TxB2 levels and increased fibronectin, 6-keto-PGF1a, Protein C and total Protein S concentrations in the setting of preconditioned myocardial infarction. There were no changes in the plasma concentrations of antithrombin-III in NPC 15669 group when compared with controls. The increase in Protein C, total Protein S, and 6-keto-PGF1a (favoring antithrombosis), and decrease in endothelin-1 and TxB2 levels (favoring vasodilatation), following NPC 15669 may explain the reduction in infarct size previously reported with this agent.     

One year survival and significant reversal of motor deficits in parkinsonian rats transplanted with hESC derived dopaminergic neurons

We report the generation of functional dopaminergic neurons from human embryonic stem cells (hESCs) using a growth factor mediated multistep EB protocol and its therapeutic effects in vivo. Embryoid bodies (EBs) were cultured in insulin-transferrin-selenium fibronectin (ITSFn) media for the selection of neural precursor cells (NPC). The selected cells on exposure to N2 media supplemented with EGF, bFGF initially aggregated to generate spontaneous free floating neurospheres and on exposure to signaling molecules Shh and FGF-8 differentiated into dopaminergic neurons (40% TH+ cells/total neurons). The differentiated NPC expressed dopaminergic specific markers both at cellular and molecular levels. They secreted detectable levels of dopamine into the culture supernatant. The most unique feature of our protocol is the generation of free floating neurospheres which can be expanded for a longer period without losing their capability to differentiate into DA neurons. Further, transplantation of NPCs into the substantia nigra of 6-OHDA lesioned rat model of Parkinson’s disease elicited significant reversal of lesion induced motor deficits which was sustained upto the end of 1 year long study period. Immunohistochemical studies of the grafted area one year post transplantation revealed that transplanted hESC derived neural precursor cells survived, integrated in vivo and differentiated into dopaminergic neurons without teratoma formation.In summary, our results encourage the potential use of hESC derived dopaminergic neurons for future clinical application in Parkinson’s disease.     

Suppressor cell regulation of encephalitogenic T cell lines: generation of suppressor macrophages with cyclosporin A and myelin basic protein.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) can be adoptively transferred using myelin basic protein (BP)-specific helper T cell lines, and suppressor cells may be important in recovery from EAE. In order to generate suppressor cells, spleen cells obtained from BP-complete Freund's adjuvant (CFA) inoculated SJL/J mice and from normal mice were cultured for 7 days with medium, with cyclosporin A (CsA), or with CsA and antigen (BP or purified protein derivative of mycobacterium (PPD)). Cultured spleen cells were assayed for suppressor activity in vitro by coculture with BP-specific and PPD-specific helper T cell lines derived from SJL/J mice. Immunized donor spleen cells cultured with cyclosporin A (CsA) and BP were potent inhibitors of T cell line proliferation, and suppressor activity was increased 17-fold compared with control splenocytes. The number of suppressor cells required to suppress PPD-specific line proliferation by 50% (I50) was significantly higher than the number required to suppress BP-specific line proliferation, suggesting an antigen-specific component to the suppression. The major effector cell required for suppression was a large granular Mac-1+ cell with the functional characteristics of a macrophage. Suppressor activity persisted after depletion of Thy 1.2+ cells, but suppression was no longer antigen-specific, suggesting that culture of spleen cells with CsA and BP may generate suppressor macrophages which are antigen-nonspecific and Thy 1.2+ suppressor cells which are antigen-specific. These suppressor cells may be important in the regulation of CR-EAE and the techniques described for their generation may prove useful for treatment and prevention of disease.