Expression Levels and Clinical Significance of Serum miR-497, CEA, CA24-2, and HBsAg in Patients with Colorectal Cancer.

The objective of the current study was to look at the levels of blood micro ribonucleic acid- (miR-) 497, carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 24-2, and hepatitis B surface antigen (HBsAg) in patients with colorectal cancer (CRC), as well as the clinical importance of these markers in CRC patients. The serum levels of miR-497, CEA, CA24-2, and HBsAg were compared between 60 patients with CRC (observation group) and another 60 patients with colorectal polyps (control group). The 4 indicators in patients with lymph node metastasis and liver metastasis were compared. The diagnostic effects of 4 detection methods and the combined detection were analyzed, and the influence of 4 indicators on the 5-year cumulative survival rate of patients was discussed. The results showed that the serum levels of miR-497 and HBsAg were lower, and the levels of CEA and CA24-2 were higher in the observation group (P < 0.05). The combined detection had the best diagnostic effect, and CEA alone had the best prediction effect. The serum level of miR-497 was significantly lower in patients with lymphatic metastasis, with the significantly higher levels of CEA and CA24-2 (P < 0.05). The HBsAg level of patients with liver metastases was greatly lower than that of patients without liver metastases (P < 0.05). The 5-year cumulative survival rate of patients with high levels of CEA and CA24-2 was significantly lower than that of patients with low level of CEA. The 5-year cumulative survival rate was lower in patients with low level of HBsAg, but the difference was small. The 5-year cumulative survival rate of patients with elevated serum miR-497 was observably lower. In conclusion, combined detection could diagnose CRC more accurately. Serum miR-497, CEA, and CA24-2 were important in the diagnosis of lymph node metastasis of CRC. HBsAg did a better job of predicting liver metastases in CRC patients. High level of CEA significantly reduced the cumulative survival rate of CRC patients and could predict the long-term survival rate of patients. Serum levels of miR-497, CEA, CA24-2, and HBsAg played a positive role in the diagnosis and evaluation of CRC and could identify lymph node and liver metastases, having a high clinical guidance value.

Diagnosis value of miR-181, miR-652, and CA72-4 for gastric cancer.

PURPOSE: To find a useful disease marker for early diagnosis of gastric cancer, we tried to explore the expression of serum miR-181, miR-652, and carbohydrate antigen 72-4 (CA72-4). PATIENTS AND METHODS: According to clinical pathologic stages, 112 patients with gastric cancer were divided into early gastric cancer group (n = 60) and advanced gastric cancer group (n = 52), stage I-II (n = 65), and stage III-IV (n = 47). Another 50 cases of gastric benign lesions and 40 healthy controls were also selected. Real-time quantitative PCR together with chemiluminescence were applied to detect expression levels. ROC curve was applied to judge their diagnostic efficiency. Pearson's correlation analysis was put into use to investigate the relevance of three indicators. RESULTS: Compared with benign lesions group and control group, significantly higher expression levels were found in patients of gastric cancer (all p < 0.001). Similarly, compared with early gastric cancer group, significantly higher expression levels were found in advanced gastric cancer group (all p < 0.001). The same result was also found in stage III-IV (all p < 0.001). The best cutoff values were 0.93, 2.38, and 16.94 U/ml, respectively. The area under the curve (0.917, 95%CI: 0.856-0.975) of the three combined diagnosis of early gastric cancer was the largest, and its sensitivity and specificity were 92.5% and 86.8%. And miR-181 and miR-652 were positively correlated with CA72-4 (r = 0.772, p < 0.001, r = 0.853, p < 0.001). CONCLUSION: Serum miR-181, miR-652, and CA72-4 are closely linked to the occurrence and development of gastric cancer. Combination of three indicators has diagnostic value for early gastric cancer.

LncRNA NEAT1 promotes cell proliferation, migration, and invasion via the miR-186-5p/PTP4A1 axis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive and malignant tumor. In this study, the effect and molecular mechanism of nuclear enriched abundant transcript 1 (NEAT1) in CCA were elucidated. The expressions of NEAT1, microRNA-186-5p (miR-186-5p), and PTP4A1 were measured by quantitative real-time PCR. The protein levels were measured by Western blotting. Kaplan-Meier analysis was performed to create survival curves. The interactions between NEAT1, miR-186-5p, and PTP4A1 were assessed through the dual luciferase reporter assay. Additionally, the cell proliferation, apoptosis, migration, and invasion were measured by colony formation, flow cytometry, the Transwell assay, and the wound healing assay, respectively. NEAT1 and PTP4A1 were significantly upregulated in CCA tissues and cells, but miR-186-5p was downregulated. NEAT1 expression was negatively correlated with the survival of CCA patients and has remarkable correlation with serum CA199 levels and lymph node metastasis. Besides, NEAT1 could act as a molecular sponge for miR-186-5p to upregulate PTP4A1 expression. More importantly, the knockdown of NEAT1 or overexpression of miR-186-5p inhibited the proliferation, migration and invasion of CCA cells, and the inhibition of miR-186-5p reversed the effects of the knockdown of NEAT1. In addition, NEAT1 could also activate the PI3K/AKT signaling pathway and regulate the epithelial-mesenchymal transition (EMT) through the miR-186-5p/PTP4A1 axis. In conclusion, NEAT1 was involved in cell proliferation, migration and invasion in CCA, and the NEAT1/miR-186-5p/PTP4A1/PI3K/AKT axis indicated novel regulatory mechanisms and therapeutics for the treatment of CCA.

Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria.

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

Extended half-life target module for sustainable UniCAR T-cell treatment of STn-expressing cancers.

BACKGROUND: Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn). METHODS: The extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM. RESULTS: The novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM. CONCLUSION: In a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.

A novel nonsense and inactivating variant of ST3GAL3 in two infant siblings suffering severe epilepsy and expressing circulating CA19.9.

Three missense variants of ST3GAL3 are known to be responsible for a congenital disorder of glycosylation determining a neurodevelopmental disorder (intellectual disability/epileptic encephalopathy). Here we report a novel nonsense variant, p.Y220*, in two dichorionic infant twins presenting a picture of epileptic encephalopathy with impaired neuromotor development. Upon expression in HEK-293T cells, the variant appears totally devoid of enzymatic activity in vitro, apparently accumulated with respect to the wild-type or the missense variants, as detected by western blot, and in large part properly localized in the Golgi apparatus, as assessed by confocal microscopy. Both patients were found to efficiently express the CA19.9 antigen in the serum despite the total loss of ST3GAL3 activity, which thus appears replaceable from other ST3GALs in the synthesis of the sialyl-Lewis a epitope. Kinetic studies of ST3GAL3 revealed a strong preference for lactotetraosylceramide as acceptor and gangliotetraosylceramide was also efficiently utilized in vitro. Moreover, the p.A13D missense variant, the one maintaining residual sialyltransferase activity, was found to have much lower affinity for all suitable substrates than the wild-type enzyme with an overall catalytic efficiency almost negligible. Altogether the present data suggest that the apparent redundancy of ST3GALs deduced from knock-out mouse models only partially exists in humans. In fact, our patients lacking ST3GAL3 activity synthesize the CA19.9 epitope sialyl-Lewis a, but not all glycans necessary for fine brain functions, where the role of minor gangliosides deserves further attention.

Globo H expression is associated with driver mutations and PD-L1 expressions in stage I non-small cell lung cancer.

BACKGROUND: Globo H is a tumor-associated carbohydrate antigen exclusively expressed in cancer cells rather than normal tissue. Globo H has been found on many cancers of epithelial origins, and become an attractive target for cancer vaccine. OBJECTIVES: We aimed to study the expression of Globo H in non-small cell lung cancer (NSCLC) patients, and correlated its expression with common driver mutations, clinical outcomes, and status of immune checkpoint, programmed death-ligand 1 (PD-L1). METHODS: The study enrolled 228 patients with surgically resected stage I NSCLC, including 139 patients with adenocarcinoma (ADC) and 89 patients with squamous cell carcinoma (SqCC). Using immunohistochemistry, tumors with moderate to strong membranous staining in ⩾ 1% tumor cells per section were scored as positive Globo H expression. Driver mutations including EGFR, KRAS, BRAF were detected by direct sequencing, while ALK, PI3KCA, FGFR1 and PD-L1 expression was detected by immunohistochemical (IHC) staining. RESULTS: Positive Globo H expression was detected in 88 of the 228 (38.6%) patients. These included 51 of 139 (36.7%) patients with ADC and 37 of 89 (41.6%) patients with SqCC. Positive Globo H expression was significantly associated with EGFR mutation and PD-L1 expression in the ADC group, and PI3KCA overexpression in the SqCC group. The survival analysis showed that Globo H expression was not an independent prognostic factor in stage I NSCLC. CONCLUSIONS: Globo H expression was correlated with specific driver mutations in ADC and SqCC NSCLC tumors, as well as PD-L1 status. Immunotherapy targeting Globo H may have potential application in lung cancer treatment.

Sialyl-Tn identifies muscle-invasive bladder cancer basal and luminal subtypes facing decreased survival, being expressed by circulating tumor cells and metastases.

OBJECTIVES: To evaluate the potential of sialyl-Tn (STn), a cancer-associated glycan antigen present in membrane glycoproteins, to improve a recent molecular model for stratification and prognostication of advanced stage bladder tumors based on keratins (KRT14, 5, and 20) expression. In addition, determine the association between STn and disease dissemination based on the evaluation of circulating tumor cells (CTCs) and the metastasis, which is a critical matter to improve patient management. PATIENTS AND METHODS: A retrospective series of 80 muscle-invasive primary bladder tumors and associated metastasis were screened for KRT14, 5, and 20 and STn by real-time polymerase chain reaction and immunohistochemistry. Peripheral blood was collected in a patients' subset, CTCs were isolated through a size-based microfluidic chip and screened for KRTs and STn. RESULTS: Basal-like lesions presented worse cancer-specific and disease-free survival compared to luminal tumors. STn antigen inclusion discriminated patients with worst survival in each subgroup (P = 0.047 for luminal; P = 0.027 for basal-like tumors). STn expression in CTCs and distant metastasis was also demonstrated. CONCLUSION: This work reinforces the potential of the KRT-based model for bladder cancer management and the association of STn with aggressiveness, supporting its inclusion in predictive molecular models toward patient-tailored precision medicine. Moreover, we describe for the first time that CTCs and the metastasis present a basal phenotype and express the STn antigen, highlighting its link with disease dissemination. Future studies should focus on determining the biological and clinical significance of these observations in the context of liquid biopsies. Given the membrane nature of STn, highly specific targeted therapeutics may also be envisaged.

CD3/CD28 dynabeads induce expression of tn antigen in human t cells accompanied by hypermethylation of the cosmc promoter.

Glycosylation is an important protein post-translational modification. In this process, the intermediate product, Tn antigen, arises from somatic mutations in core1ß3-galactosyltransferase-specific molecular chaperone (Cosmc), which is required for the formation of active core1ß3-galactosyltransferase (T-synthase). As a type of tumor-associated carbohydrate antigen, Tn antigen is mainly expressed in many human tumor cells and is absent in normal cells. Surprisingly, it is also expressed in normal activated T cells after in vitro stimulation, but the mechanism underlying its expression remains unclear. This study demonstrated that Tn antigen was expressed in activated T cells and that the percentage of positive (Tn+) cells increased and subsequently decreased within 72h after stimulation with CD3/CD28 Dynabeads, with peak expression occurring at 48h. During activation, interleukin-4 (IL-4) expression in the T-cell supernatant consistently increased with Tn+ cells, and was inversely correlated with serum interferon gamma (IFN-γ) levels. Compared with unactivated (without CD3/CD28 Dynabead stimulation) T cells, the level of T-synthase transcription in activated T cells did not significantly change, whereas T-synthase activity and Cosmc transcription significantly decreased, accompanied by a further increase in methylation of the Cosmc promoter. The results also showed that Cosmc transcription and translation decreased and then increased, and that Cosmc promoter methylation was a dynamic process during T cell activation. These data suggest that hypermethylation of the Cosmc promoter may induce the expression of Tn antigen in activated T cells.

Apical membrane expression of distinct sulfated glycans represents a novel marker of cholangiolocellular carcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver neoplasm, followed by hepatocellular carcinoma. ICC can be further subclassified as (i) perihilar and (ii) peripheral types, the latter histologically resembling small-sized intrahepatic bile ducts, such as interlobular bile ducts, cholangioles/ductules and the canals of Hering. Cholangiolocellular carcinoma (CoCC), now classified by the World Health Organization as a subtype of combined hepatocellular-cholangiocarcinoma, is currently regarded as a subtype of peripheral-type ICC. The present study was undertaken to determine whether sulfated glycans recognized by the MECA-79 monoclonal antibody could serve as a CoCC marker. Using immunohistochemistry, we show that MECA-79 sulfated glycans are preferentially expressed at the apical membrane of cholangiocytes found in small-sized intrahepatic bile ducts in normal liver and in canalicular structures formed in CoCC. We also report that apical membrane MECA-79 sulfated glycan expression colocalizes with that of mucin 1 (MUC1) core proteins. We also present immunoblotting of Chinese hamster ovary cells overexpressing FLAG-tagged MUC1 to show that MUC1 serves as a MECA-79 scaffold. Furthermore, we report that SSP-25 human ICC cells overexpressing N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), but not GlcNAc6ST-1, exhibit membrane expression of MECA-79 sulfated glycans, suggesting that GlcNAc6ST-2 catalyzes MECA-79 epitope biosynthesis in cholangiocytes. Moreover, both wild-type and GlcNAc6ST-1 knockout mice exhibit apical membrane MECA-79 expression in small-sized intrahepatic bile ducts, namely interlobular bile ducts, whereas MECA-79 expression was completely absent in comparable tissues from GlcNAc6ST-1 and GlcNAc6ST-2 double knockout mice. These data collectively indicate that apical membrane localization of MUC1 proteins decorated with GlcNAc6ST-2-dependent MECA-79 sulfated glycans may mark cholangiocytes with cholangiolar/ductular differentiation and could serve as a useful CoCC marker.

A Novel Chemoenzymatic Synthesis of Sulfated Type†2 Tumor-Associated Carbohydrate Antigens by Transglycosylation of Sulfated Lewis X Oxazoline Catalyzed by Keratanase†II.

Sulfated type 2 carbohydrate chains are known tumor-associated carbohydrate antigens (TACAs). Many reports on cancer vaccines employing TACAs as specific antigens have been published, but structurally specified sulfated TACAs have not been used because of the low natural abundance and difficulties in chemical synthesis. We demonstrate for the first time the synthesis of the sulfated type 2 TACAs with an l-fucose branch by keratanase-II-catalyzed transglycosylation of the sulfated Lewis X (Galß(1→4)[Fucα(1→3)]GlcNAc(6-OSO3- ); su-Lex ) oxazoline derivative. Two keratanase IIs (from Bacillus sp. Ks36 and Bacillus circulans KsT202) efficiently catalyzed the transglycosylation reaction of the su-Lex oxazoline derivative, thereby giving the su-Lex dimer as the main product in good yields. Structural analysis of the oligomers confirmed exclusive formation of the ß(1→3) glycosidic bond.

Muse Cells, a New Type of Pluripotent Stem Cell Derived from Human Fibroblasts.

A new type of mesenchymal stem cells (MSCs) that expresses stage-specific embryonic antigen 3 (SSEA-3) and the mesenchymal cell marker CD105 are known as multilineage-differentiating stress-enduring (Muse) cells. Studies have shown that stem cells in suspension cultures are more likely to generate embryoid body-like stem cell spheres and maintain an undifferentiated phenotype and pluripotency. We separated Muse cells derived from human dermal fibroblasts by long-term trypsin incubation (LTT) through suspension cultures in methylcellulose. The Muse cells obtained expressed several pluripotency markers, including Nanog, Oct4, Sox2, and SSEA-3, and could differentiate in vitro into cells of the three germ layers, such as hepatocytes (endodermal), neural cells (ectodermal) and adipocytes, and osteocytes (mesodermal cells). These cells showed a low level of DNA methylation and a high nucleo-cytoplasmic ratio. Our study provides an innovative and exciting platform for exploring the potential cell-based therapy of various human diseases using Muse cells as well as their great possibility for regenerative medicine.

Vitamin C stimulates human gingival stem cell proliferation and expression of pluripotent markers.

Gingival stem cells (GSCs) are a novel source of mesenchymal stem cells (MSCs) that are easily accessed from the oral cavity. GSCs were considered valuable autograft MSCs with particular characteristics. However, the limitation in the number of available GSCs remains an obstacle. Therefore, this study aimed to stimulate GSC proliferation by ascorbic acid (AA) and determined the effects of AA on GSC pluripotent potential-related gene expression. GSCs were isolated from gum tissue by explant culture and continuously subcultured before analysis of stemness and effects of AA on pluripotent-related gene expression. GSCs cultured with various concentrations of AA showed increased proliferation in a dose-dependent manner. AA-treated GSCs showed significantly higher expression of SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 compared with control cells. More importantly, GSCs also maintained their stemness with MSC phenotypes and failed to cause tumors in nude athymic mice. Our results show that AA is a suitable factor to stimulate GSC proliferation.

What controls the expression of the core-1 (Thomsen-Friedenreich) glycotope on tumor cells?

Malignant transformation is tightly connected with changes in the glycosylation of proteins and lipids, which in turn are contributing to the invasive and metastatic behavior of tumor cells. One example of such changes is demasking of the otherwise hidden core-1 structure, also known as Thomsen-Friedenreich antigen, which is a highly tumor-specific glycotope and potentially a cancer stem cell marker. This review summarizes what is known about the mechanism(s) of its expression on tumor cells. New data reveal a close connection between tumor metabolism and Golgi function. Based on these data, we suggest that the expression of this antigen is also a marker of aerobic glycolysis.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer.

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29.

The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn+ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn+ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn+ as well as in HT-29-Tn- and Tn- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn+ and HT-29-Tn- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn+ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function.

Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers.

Stage-specific embryonic antigen-3 (SSEA-3)-positive multipotent mesenchymal cells (multilineage differentiating stress-enduring [Muse] cells) were isolated from cultured human adipose tissue-derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic-activated cell sorting into positive and negative fractions, a SSEA-3+ cell-enriched fraction (Muse-rich) and the remaining fraction (Muse-poor). Muse-rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse-poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse-poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse-rich cells significantly accelerated wound healing compared with treatment with Muse-poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell-depleted or ischemic conditions of various organs and tissues.

Expression of MUC1 and CD176 (Thomsen-Friedenreich antigen) in papillary thyroid carcinomas.

The incidence of thyroid cancer has appeared as an increasing trend globally, especially in Asian countries. In this study, the expression of mucin-1 (MUC1) and Thomsen-Friedenreich antigen, Galß1-3GalNAcα1-R (CD176) was investigated by immunohistochemistry in papillary thyroid carcinomas (PTCs), which accounts for approximately 80 % of all thyroid cancer. We found that 78 % of PTC overexpressed MUC1. Importantly, we observed firstly that CD176 was expressed in 63 % of PTC, but was faintly or not expressed in normal thyroid tissues and benign thyroid disease tissues, indicating that CD176 is also a tumour-associated antigen for PTCs. Moreover, expression of CD176 was strongly correlated with MUC1 by immunohistochemical staining in PTCs. Furthermore, we used the immunochemical method to confirm that MUC1 is a common and main carrier of CD176 in PTCs. Our data demonstrated that MUC1 and CD176 might be promising biomarkers for thyroid cancer.

A Photobacterium sp. α2-6-sialyltransferase (Psp2,6ST) mutant with an increased expression level and improved activities in sialylating Tn antigens.

In order to improve the catalytic efficiency of recombinant Photobacterium sp. JT-ISH-224 α2-6-sialyltransferase Psp2,6ST(15-501)-His6 in sialylating α-GalNAc-containing acceptors for the synthesis of tumor-associated carbohydrate antigens sialyl Tn (STn), protein crystal structure-based mutagenesis studies were carried out. Among several mutants obtained by altering the residues close to the acceptor substrate binding pocket, mutant A366G was shown to improve the sialyltransferase activity of Psp2,6ST(15-501)-His6 toward α-GalNAc-containing acceptors by 21-115% without significantly affecting its sialylation activity to ß-galactosides. Furthermore, the expression level was improved from 18-40 mg L(-1) for the wild-type enzyme to 72-110 mg L(-1) for the A366G mutant. In situ generation of CMP-sialic acid in a one-pot two-enzyme system was shown effective in overcoming the high donor hydrolysis of the enzyme. Mutant A366G performed better than the wild-type Psp2,6ST(15-501)-His6 for synthesizing Neu5Acα2-6GalNAcαOSer/Thr STn antigens.

Medullary thymic epithelial stem cells maintain a functional thymus to ensure lifelong central T cell tolerance.

Medullary thymic epithelial cells (mTECs) are crucial for central T cell self-tolerance. Although progenitors of mTECs have been demonstrated in thymic organogenesis, the mechanism for postnatal mTEC maintenance remains elusive. We demonstrate that implantation of embryonic TECs expressing claudin-3 and claudin-4 (Cld3,4) in a medulla-defective thymic microenvironment restores medulla formation and suppresses multiorgan autoimmunity throughout life. A minor SSEA-1(+) fraction within the embryonic Cld3,4(hi) TECs contained self-renewable clonogenic TECs, capable of preferentially generating mature mTECs in vivo. Adult SSEA-1(+)Cld3,4(hi) TECs retained mTEC reconstitution potential, although the activity decreased. The clonogenicity of TECs also declined rapidly after birth in wild-type mice, whereas it persisted in Rag2(?/?) adult mice with defective thymopoiesis. The results suggest that unipotent mTEC-restricted stem cells that develop in the embryo have the capacity to functionally reconstitute the thymic medulla long-term, thus ensuring lifelong central T cell self-tolerance.