Establishing Simultaneous T Cell Receptor Excision Circles (TREC) and K-Deleting Recombination Excision Circles (KREC) Quantification Assays and Laboratory Reference Intervals in Healthy Individuals of Different Age Groups in Hong Kong.

The clinical experience gathered throughout the years has raised awareness of primary immunodeficiency diseases (PIDD). T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) assays for thymic and bone marrow outputs measurement have been widely implemented in newborn screening (NBS) programs for Severe Combined Immunodeficiency. The potential applications of combined TREC and KREC assay in PIDD diagnosis and immune reconstitution monitoring in non-neonatal patients have been suggested. Given that ethnicity, gender, and age can contribute to variations in immunity, defining the reference intervals of TREC and KREC levels in the local population is crucial for setting up cut-offs for PIDD diagnosis. In this retrospective study, 479 healthy Chinese sibling donors (240 males and 239 females; age range: 1 month-74 years) from Hong Kong were tested for TREC and KREC levels using a simultaneous quantitative real-time PCR assay. Age-specific 5th-95th percentile reference intervals of TREC and KREC levels (expressed in copies per µL blood and copies per 106 cells) were established in both pediatric and adult age groups. Significant inverse correlations between age and both TREC and KREC levels were observed in the pediatric age group. A significant higher KREC level was observed in females than males after 9-12 years of age but not for TREC. Low TREC or KREC levels were detected in patients diagnosed with mild or severe PIDD. This assay with the established local reference intervals would allow accurate diagnosis of PIDD, and potentially monitoring immune reconstitution following haematopoietic stem cell transplantation or highly active anti-retroviral therapy in the future.

Attenuation of hind-limb ischemia in mice with endothelial-like cells derived from different sources of human stem cells.

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as "off-the-shelf" format for the treatment of tissue ischemia.

Reversal of endothelial progenitor cell dysfunction in patients with type 2 diabetes using a conditioned medium of human embryonic stem cell-derived endothelial cells.

BACKGROUND: The potential clinical application of bone marrow or peripheral blood-derived progenitor cells for cardiovascular regeneration in patients with diabetes mellitus (DM) is limited by their functional impairment. We sought to determine the mechanisms of impaired therapeutic efficacy of peripheral blood-derived progenitor cells in type 2 DM patients and evaluated the use of cell-free conditioned medium obtained from human embryonic stem cell-derived endothelial-like cells (ESC-ECs) to reverse their functional impairment. METHODS: The angiogenic potential of late outgrowth endothelial cells (OECs) and cytokine profile of the conditional medium of proangiogenic cells (PACs) derived from peripheral blood-mononuclear cells of healthy control and DM patients and ESC-ECs was compared by in vitro tube formation assay and a multiplex bead-based immunoassay kit, respectively. The in vivo angiogenic potential of ESC-ECs derived conditioned medium in rescuing the functional impairment of PB-PACs in DM patients was investigated using a hindlimb ischemia model. RESULTS: Human ESC-ECs had similar functional and phenotypic characteristics as OECs in healthy controls. Cytokine profiling showed that vascular endothelial growth factor, stromal cell-derived factor 1 and placental growth factor were down-regulated in PACs from DM patients. Tube formation assay that revealed functional impairment of OECs from DM patients could be rescued by ESC-ECs conditioned medium. Administration of ESC-ECs conditioned medium restored the therapeutic efficacy of PB-PACs from DM patients in a mouse model of hindlimb ischemia. CONCLUSIONS: Our results showed that peripheral blood-derived progenitor cells from DM patients have impaired function because of defective secretion of angiogenic cytokines, which could be restored by supplementation of ESC-ECs conditioned medium.

Granulin-epithelin precursor as a therapeutic target for hepatocellular carcinoma.

UNLABELLED: Primary liver cancer, hepatocellular carcinoma (HCC), is the fifth most common cancer and the third leading cancer killer in the world. There is no effective therapeutic option for most HCC patients. A new therapeutic strategy is essential. Granulin-epithelin precursor (GEP, also called progranulin, acrogranin, or PC-derived growth factor) was identified as a potential therapeutic target for HCC from our earlier genome-wide expression profiles. We aimed to conduct a detailed investigation with in vitro and animal experiments. We developed the anti-GEP monoclonal antibody (mAb), and examined its effect on hepatoma cells and normal liver cells in vitro. A nude mice model transplanted with human HCC was used to investigate if anti-GEP mAb can inhibit tumor growth in vivo. We demonstrated that anti-GEP mAb inhibited the growth of hepatoma cells but revealed no significant effect on normal liver cells. In the nude mice model transplanted with human HCC, anti-GEP mAb decreased the serum GEP level and inhibited the growth of established tumors in a dose-dependent manner. The anti-GEP mAb reduced tumor cell proliferation via the p44/42 MAPK and Akt pathways, and reduced tumor angiogenesis to deprive the nutrient supply with reduced microvessel density and tumor vascular endothelial growth factor level. CONCLUSION: We have shown that anti-GEP antibody can inhibit HCC growth, providing evidence that GEP is a therapeutic target for HCC treatment.

Inhibition of hepatocellular carcinoma invasion by suppression of claudin-10 in HLE cells.

Previously, we showed that down-regulation of claudin-10 (CLDN-10) in hepatocellular carcinoma is associated with prolonged disease-free survival after curative surgery. Claudins are important tight junction components. Increasing evidence shows that claudins are involved in cancer progression but each member of claudins is specifically expressed in a variety of malignancies. The biological role of CLDN-10 in hepatocellular carcinoma is unexplored. In the current study, we investigated the CLDN-10 function in two different hepatocellular carcinoma cell lines by in vitro assays with the CLDN-10 overexpression and small interfering RNA-mediated knockdown transfectants. We observed that overexpression of CLDN-10 conferred malignant phenotypes to hepatocellular carcinoma cells, Hep3B, which lack CLDN-10 expression, by promoting cancer cell survival, motility, and invasiveness. More importantly, matrix metalloproteinase 2 (MMP2) was up-regulated. Increase in mRNA transcription and protein expression of membrane type 1-MMP (MT1-MMP) was also observed in the CLDN-10 transfectants, where MT1-MMP was a protease shown to promote intrahepatic metastasis in hepatocellular carcinoma in our earlier study. In addition, CLDN-1, CLDN-2, and CLDN-4 was up-regulated in CLDN-10 overexpression transfectants, indicating that the expression of CLDN-10 in cancer cells might affect the expression levels of its family members. On the contrary, small interfering RNA-based knockdown of CLDN-10 in HLE, an invasive cell line with high level of CLDN-10 expression, abolished invasion and strongly decreased activation of MMPs and claudin members expression. These findings showed that CLDN-10 is functionally involved in hepatocellular carcinoma invasion and is a potential target for hepatocellular carcinoma therapy.

Down-regulation of retinol binding protein 5 is associated with aggressive tumor features in hepatocellular carcinoma.

PURPOSE: Acyclic retinoid (ACR) has been shown to be a promising chemopreventive agent for hepatocellular carcinoma (HCC) after curative resection. The effects of retinoid are mediated by retinol-binding proteins (RBPs) through regulating cell proliferation and differentiation. PATIENTS AND METHODS: This study investigated the clinical significance of RBP5 in HCC. RBP5 mRNA level was examined by real-time quantitative PCR on 52 matched tumor and adjacent non-tumor liver tissues, and on ten normal livers. Expression of RBP5 protein was examined using Western blotting analysis and immunohistochemistry. RESULTS: Down-regulation of RBP5 was found in HCC tissues at both mRNA and protein levels. Decreased RBP5 level was closely related to poor differentiation (P=0.02) and large tumor size (P=0.01). Low level of RPB5 was associated with poor overall survival (P=0.02), and was an independent prognostic factor for HCC. CONCLUSIONS: Our study revealed that RBP5 down-regulation in HCC was associated with aggressive tumor features, suggesting an important role of RPB5 in HCC progression.

Preoperative plasma transcript AA454543 level is an independent prognostic factor for hepatocellular carcinoma after partial hepatectomy.

BACKGROUND: We have previously reported that tissue expression levels of transcript AA454543 in hepatocellular carcinoma (HCC) are significantly higher than those of normal livers, livers with cirrhosis, and livers with hepatitis. In addition, a higher level of transcript AA454543 in tumor tissues is associated with poor prognosis. We aim to examine whether quantitative measurement of preoperative plasma transcript AA454543 can provide similar prognostic information. PATIENTS AND METHODS: Blood samples were obtained from 84 HCC patients before surgery. Real-time quantitative reverse transcription-polymerase chain reaction, using TaqMan system, was employed to measure plasma transcript AA454543 and alpha-fetoprotein (AFP) RNA levels. We assessed their prediction power in prognosis using univariate and multivariate analyses. RESULTS: High plasma transcript AA454543 RNA levels were associated with poor overall survival (log-rank test, P < .01). Patients with different plasma AFP RNA levels revealed no difference in overall survival (log-rank test, P = .88). By multivariate Cox regression analysis, plasma transcript AA454543 RNA level (hazard ratio = 4.8, P < .01) and tumor stage (hazard ratio = 1.7, P < .01) were determined to be independent risk factors for the prediction of overall survival. CONCLUSION: Preoperative plasma transcript AA454543 RNA level can provide prognostic information for HCC patients receiving curative partial hepatectomy.

Increased expression of glycosyl-phosphatidylinositol anchor attachment protein 1 (GPAA1) is associated with gene amplification in hepatocellular carcinoma.

Glycosyl-phosphatidylinositol (GPI) anchor attachment protein 1 (GPAA1) transcript level was frequently up-regulated in our earlier study on gene expression profile. We therefore analyzed the potential involvement of GPAA1 in hepatocellular carcinoma (HCC) as GPAA1 gene locates at chromosome 8q24.3 which chromosome region is frequently amplified in HCCs. In this study, we observed that GPAA1 transcript in the HCCs (n = 93) showed a significantly higher expression level compared with their paralleled adjacent nontumor liver tissues, cirrhosis (n = 15) and normal (n = 16) liver tissues using real-time quantitative RT-PCR (p < 0.005). We also demonstrated that GPAA1 protein up-regulation was common in HCCs (90%, 9/10), and GPAA1 gene was frequently amplified (73%, 11/15) using quantitative microsatellite analysis. Increased GPAA1 expression was significantly associated with HCCs poor cellular differentiation (p = 0.011) and poor prognosis (p = 0.010). We then modulated the GPAA1 expression level in HCC cells (Hep3B) by transfection experiments, which was shown to positively regulate cell adhesion ability (p = 0.004) and proliferation rate (p = 0.037). Our data revealed GPAA1 gene amplification with overexpression of RNA and protein in HCC. GPAA1 is a potential amplification target of chromosome 8q and responsible to regulate tumor cells behavior.

Transcript AA454543 is a novel prognostic marker for hepatocellular carcinoma after curative partial hepatectomy.

BACKGROUND: We have previously reported on the cDNA microarray gene expression profiles of hepatocellular carcinomas (HCCs). Among the genes that show prognostic significance and are overexpressed in tumor compared with adjacent nontumorous liver, transcript AA454543 may have potential for practical use. Our aim is to validate the prognostic significance of transcript AA454543 by alternative research methods and in a separate group of HCC patients. METHODS AND RESULTS: The data of transcript AA454543 derived from microarray analysis of 48 patients having curative partial hepatectomy (group 1) were verified by quantitative reverse transcription polymerase chain reaction (r = 0.618, P < .001). A separate sample set of HCCs obtained from 53 patients (group 2) was examined and the association of AA454543 expression level with overall survival was again validated (P = .027). By Cox regression analysis, transcript AA454543 [hazard ratio (HR) = 3.0, P = .017] and pathologic tumor node metastasis (pTNM) stage (HR = 3.3, P = .010) were independent prognostic factors for overall survival. The accuracy of prediction for 3-year overall survival for transcript AA454543 (74.2%, P = .001) and pTNM stage (76.4%, P = .001) was comparable as measured by the area under the receiver operating characteristic curve. CONCLUSION: Transcript AA454543 is potentially useful molecular prognostic marker for overall survival after curative partial hepatectomy for HCC.

Decreased expression of cytochrome P450 2E1 is associated with poor prognosis of hepatocellular carcinoma.

Cytochrome P450-2E1 (CYP2E1) is one of the major hepatic enzymes involved in the metabolism of procarcinogen. Our study aimed to investigate the differential expression level of CYP2E1 and its clinicopathological significance in hepatocellular carcinoma (HCC). CYP2E1 revealed low level of expression in 70% of the tumor tissues, when compared to the adjacent nontumor tissues, at both mRNA and protein levels. The low expression of CYP2E1 was significantly correlated with the aggressive tumor phenotype, including poor differentiation status (by the Edmondson grading system) (p=0.038), absence of tumor capsule (p=0.030) and younger age of the patients (p=0.002). Multivariate analysis indicated that CYP2E1 expression level and pTNM stage were independent prognostic factors for disease-free survival. CYP2E1 was also shown to have a differential expression level in different liver tissues. The level of CYP2E1 was significantly higher in nontumor tissues from HCC patients compared to the intermediate level in cirrhosis livers from noncancer patients and normal livers from healthy persons. Tumor tissues were shown to have the lowest expression level. In conclusion, our results have shown that CYP2E1 is upregulated in the nontumor tissue and downregulated in tumor tissue, which is associated with aggressive tumor type and poor prognosis of the patients. It suggested that the differential expression of CYP2E1 may play an important role in HCC tumorigenesis.

Cell-cycle-dependent localisation of Ulp1, a Schizosaccharomyces pombe Pmt3 (SUMO)-specific protease.

We report here on the characterisation of Ulp1, a component of the SUMO modification process in S. pombe. Recombinant S. pombe Ulp1 has de-sumoylating activity; it is involved in the processing of Pmt3 (S. pombe SUMO) and can, to a limited extent, remove Pmt3 from modified targets in S. pombe cell extracts. ulp1 is not essential for cell viability, but cells lacking the gene display severe cell and nuclear abnormalities. ulp1-null (ulp1.d) cells are sensitive to ultraviolet radiation in a manner similar to rad31.d and hus5.62, which have mutations in one subunit of the activator and the conjugator for the ubiquitin-like protein SUMO respectively. However ulp1.d cells are less sensitive to ionising radiation and hydroxyurea (HU) than are rad31.d and hus5.62. ulp1-null cells are defective in processing precursor Pmt3 and display reduced levels of Pmt3 conjugates compared with wild-type cells. The slow growth phenotype of ulp1 null cells is not substantially rescued by over-expression of the mature form of Pmt3 (Pmt3-GG), suggesting that the de-conjugating activity of Ulp1 is required for normal cell cycle progression. During the S and G2 phases of the cell cycle the Ulp1 protein is localised to the nuclear periphery. However, during mitosis the pattern of staining alters, and during anaphase, Ulp1 is observed within the nucleus. Ulp1 localisation at the nuclear periphery is generally re-established by the time of septation (S phase).