Genetic characteristics of archival noroviruses detected from the 1970s to the 1990s.

The genetic characterization of archival specimens is important for evaluating the evolutionary processes of noroviruses. Complete viral genome sequences, GVIII.1[GII.P28] and GIX.1[GII.P15], were determined from two archival specimens collected in Tokyo, Japan, in 1986 and 1995. In addition, complete VP1 and partial RdRp sequences of four samples collected between 1975 and 1983 were determined. Two viruses were classified as GI.5[P5] and GI.9[P9]; however, the viruses from the other two samples could not be assigned to any known genotypes using norovirus typing tools and phylogenetic analysis, suggesting that they might be untypable genotypes. Further evolutionary analysis of these viruses is warranted.

Profiling of Virus-Encoded MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma and Their Roles in Gastric Carcinogenesis.

UNLABELLED: Epstein-Barr virus (EBV) is one of the major oncogenic viruses and is found in nearly 10% of gastric carcinomas. EBV is known to encode its own microRNAs (miRNAs); however, their roles have not been fully investigated. The present report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in tissue samples from patients with EBV-associated gastric carcinoma. Several miRNAs were highly expressed in EBV-associated gastric carcinoma, and in silico analysis revealed that the target genes of these EBV miRNAs had functions associated with cancer-related pathways, especially the regulation of apoptosis. Apoptosis was reduced in EBV-associated gastric carcinoma tissue samples, and gastric carcinoma cell lines infected with EBV exhibited downregulation of the proapoptotic protein Bid (the BH3-interacting domain death agonist), a member of the Bcl-2 family. The luciferase activity of the reporter vector containing the 3' untranslated region of BID was inhibited by an ebv-miR-BART4-5p mimic in gastric cancer cell lines. Transfection of an ebv-miR-BART4-5p mimic reduced Bid expression in EBV-negative cell lines, leading to reduced apoptosis under serum deprivation. The inhibition of ebv-miR-BART4-5p expression was associated with partial recovery of Bid levels in EBV-positive cell lines. The results demonstrated the antiapoptotic role of EBV miRNA via regulation of Bid expression in EBV-associated gastric carcinoma. These findings provide novel insights in the roles of EBV miRNAs in gastric carcinogenesis, which would be a potential therapeutic target. IMPORTANCE: This report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in clinical samples from EBV-associated gastric carcinoma patients. Of the EBV miRNAs, ebv-miR-BART4-5p plays an important role in gastric carcinogenesis via regulation of apoptosis.

Study on the Umbilical Cord-Mesenchymal Stem Cell Manufacturing Using Clinical-Grade Culture Medium.

Mesenchymal stem/stromal cell (MSC)-based therapies have been gaining increasing attention owing to their application in various diseases and conditions. In this study, we aimed to identify the optimal condition for industrial-scale MSC manufacturing. MSCs were isolated from umbilical cord (UC) tissues by implementing the explant method (Exp) or a collagenase based-enzymatic digestion method (Col), using a good manufacturing practice-compatible serum-free medium developed in-house. Microarray analysis demonstrated that the gene expression profiles of Exp-MSCs and Col-MSCs did not significantly differ according to the method of isolation or the culture conditions used. The isolated UC-MSCs were then subjected to expansion using conventional static culture (ST) or microcarrier-based culture in stirred-tank bioreactors (MC). Metabolomic and cytokine array analyses were conducted to evaluate the biochemical status of the MSCs. However, no remarkable differences in the metabolic profile and cytokine secretome between ST-MSCs and MC-MSCs were observed. On the contrary, we observed for the first time that the hydrophobic components of ST-MSCs and MC-MSCs were different, which suggested that the cell membrane distribution of fatty acids and lipids was altered in the process of adaptation to shear stress in MC-MSCs. These results establish the flexibility of the isolation and expansion method for UC-MSCs during the manufacturing processes and provide new insights into the minor differences between expansion methods that may exert remarkable effects on MSCs. In conclusion, we demonstrated the feasibility of both Exp-MSCs and Col-MSCs and MC and ST culture methods for scale-up and scale-out of MSC production, as well as the equivalence of these cells. As for the industrialized mass production of MSCs, enzyme-based methods for isolation and cell expansion in a bioreactor were considered to be more suitable. The methods developed, which underwent comprehensive evaluation in this study, may contribute toward the provision of sufficient MSC sources and the establishment of cost-effective MSC therapies. Impact statement Our in-house-developed good manufacturing practice-grade serum-free medium could be used for both isolation (Exp and Col) and expansion (ST and MC) of umbilical cord (UC)-mesenchymal stem/stromal cells (MSCs). Characteristics of the obtained UC-MSCs were widely assessed with regard to gene expression, metabolome, and secretome. Cellular characteristics and efficacy were observed to be equivalently maintained among whichever technique was applied. In addition, our research presents the first evidence that bioreactor and microcarrier-based MSC cultures alter the fatty acid and phospholipid composition of MSCs. These results provide new insights into the differences between expansion methods that may exert remarkable effects on MSCs.

Umbilical cord derived mesenchymal stromal cells in microcarrier based industrial scale culture sustain the immune regulatory functions.

Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord-derived MSCs (UC-MSCs) are considered superior to other tissue-derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier-based cell culture in bioreactors, is required for the large-scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC-MSCs by using a good manufacturing practice (GMP)-compatible serum-free medium, developed in-house, and a small-scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC-MSCs cultured in microcarrier-based bioreactors (MC-UC-MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST-UC-MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC-UC-MSCs and ST-UC-MSCs in immunodeficient mice (graft-versus-host disease model). Lastly, we developed a semi-automated cell dispensing system, without bag-to-bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP-compatible serum-free and microcarrier-based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier-based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.

Inhibiting lentiviral replication by HEXIM1, a cellular negative regulator of the CDK9/cyclin T complex.

OBJECTIVE: Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. METHODS: HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. RESULTS: HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. CONCLUSION: These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.

High and inducible expression of human immunodeficiency virus type 1 (HIV-1) Nef by adenovirus vector does not disturb potent antigen presentation by monocyte-derived dendritic cells.

Numerous studies indicated that Nef is a pleiotropic factor. Although it has been shown that Nef impairs the antigen-presenting activity of dendritic cells, more recent studies have shown no such impairment. This issue is critical for designing a vaccine expressing Nef. To refine our knowledge regarding the effect of Nef on dendritic cells, we developed constitutive and inducible adenovirus vector systems that express high levels of Nef in monocyte-derived dendritic cells (MDDCs). We showed here that Nef expression clearly downregulated CD4 expression of MDDCs but had little or no effect on other surface molecules, including MHC class I. Nef also did not affect the functional maturation of MDDCs. Use of the inducible Nef-expression system clearly revealed that adenovirus infection per se modulates cytokine secretion and the expression of apoptosis-related molecules in MDDCs, whereas Nef had no effect on these functions. Moreover, the antigen-presenting activity of MDDCs was not disturbed by the presence of Nef. On the contrary, we found that Nef-expressing MDDCs generated from HIV-1-infected individuals efficiently activated Nef-reactive T cells. Therefore, although adenovirus vector may modulate some aspects of MDDC function, Nef-expressing adenovirus would be served as one of HIV vaccine candidates.

SOX9 expression and its methylation status in gastric cancer.

SOX9 is a member of the SOX [Sry-related high-mobility group (HMG) box] family and is required for the development and differentiation of multiple cell lineages. To clarify the significance of SOX9 in gastric carcinoma (GC), immunohistochemical expression of SOX9 and the CpG island methylation status of SOX9 were evaluated and compared with clinicopathological factors including overall survival. SOX9 expression was immunohistochemically evaluated in 382 GC tumors and the methylation status was examined in 121 GC tumors. SOX9 expression and its methylation status in six GC cell lines, their Epstein-Barr virus (EBV)-infected cell lines, and two EBV-associated GC cell lines was also examined. The SOX9 expression increased from non-neoplastic mucosa to early cancer. High expression of SOX9 was seen in 212 cases (56%). SOX9 expression was inversely related to advanced tumor stage, vessel infiltration, nodal metastasis, and EBV infection. Fifty-eight (48%) of 121 GC tumors had a methylated promoter in GC and the methylated status was related to low expression. The expression and methylation status were not related to prognosis. Three of six cell lines had increased methylation through EBV infection and decreased SOX9 expression. Upregulation of SOX9 is related to GC development. Downregulation by promoter methylation is related to GC progression and EBV infection. SOX9 is closely related to GC carcinogenesis and EBV-associated GC carcinogenesis.

ARID1A expression loss in gastric cancer: pathway-dependent roles with and without Epstein-Barr virus infection and microsatellite instability.

The AT-rich interactive domain 1A gene (ARID1A), which encodes one of the subunits in the Switch/Sucrose Nonfermentable chromatin remodeling complex, carries mutations and is responsible for loss of protein expression in gastric carcinoma, particularly with Epstein-Barr virus (EBV) infection and a microsatellite instability-high phenotype. We used immunohistochemistry to investigate the significance of ARID1A loss in 857 gastric carcinoma cases, including 67 EBV(+) and 136 MLH1-lost gastric carcinomas (corresponding to a microsatellite instability-high phenotype). Loss of ARID1A expression was significantly more frequent in EBV(+) (23/67; 34 %) and MLH1-lost (40/136; 29 %) gastric carcinomas than in EBV(-)MLH1-preserved (32/657; 5 %) gastric carcinomas (P < 0.01). Loss of ARID1A correlated with larger tumor size, advanced invasion depth, lymph node metastasis, and poor prognosis in EBV(-)MLH1-preserved gastric carcinoma. A correlation was found only with tumor size and diffuse-type histology in MLH1-lost gastric carcinoma, but no correlation was observed in EBV(+) gastric carcinoma. Loss of ARID1A expression in EBV(+) gastric carcinoma was highly frequent in the early stage of gastric carcinoma, although EBV infection did not cause downregulation of ARID1A: EBV-positive nasopharyngeal carcinomas (n = 8) and lymphomas (n = 15) failed to show loss of ARID1A, and EBV infection did not cause loss of ARID1A in gastric carcinoma cell lines. Taken together, loss of ARID1A may be an early change in carcinogenesis and may precede EBV infection in gastric epithelial cells, while loss of ARID1A promotes cancer progression in gastric cancer cells without EBV infection or loss of MLH1 expression. Loss of ARID1A has different and pathway-dependent roles in gastric carcinoma.

Downregulation of microRNA-200 in EBV-associated gastric carcinoma.

EBV-associated gastric carcinoma is a distinct gastric carcinoma subtype with characteristic morphologic features similar to those of cells that undergo epithelial-to-mesenchymal transition. The effect of microRNA abnormalities in carcinogenesis was investigated by measuring the expression of the epithelial-to-mesenchymal transition-related microRNAs, miR-200a and miR-200b, in 36 surgically resected gastric carcinomas using quantitative reverse transcription-PCR analysis. MiR-200 family expression was decreased in EBV-associated gastric carcinoma, as compared with that in EBV-negative carcinoma. Downregulation of the miR-200 family was found in gastric carcinoma cell lines infected with recombinant EBV (MKN74-EBV, MKN7-EBV, and NUGC3-EBV), accompanied by the loss of cell adhesion, reduction of E-cadherin expression, and upregulation of ZEB1 and ZEB2. E-cadherin expression was partially restored by transfection of EBV-infected cells with miR-200 family precursors. Reverse transcription-PCR analysis of primary precursors of miR-200 (pri-miR-200) revealed that the transcription of pri-miR-200 was decreased in EBV-infected cells. Transfection of MKN74 cells with BARF0, EBNA1, and LMP2A resulted in a decrease of pri-miR-200, whereas transfection with EBV-encoded small RNA (EBER) did not. These four latent genes contributed to the downregulation of the mature miR-200 family and the subsequent upregulation of ZEB1/ZEB2, resulting in the reduction of E-cadherin expression. These findings indicate that all the latency type I genes have a synergetic effect on the downregulation of the miR-200 family that leads to reduced E-cadherin expression, which is a crucial step in the carcinogenesis of EBV-associated gastric carcinoma.

Isolation and characterization of a replication-competent molecular clone of an HIV-1 circulating recombinant form (CRF33_01B).

A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs) have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1), a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5' LTR (long terminal repeat) region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone), showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4+, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.

Yeast-derived human immunodeficiency virus type 1 p55(gag) virus-like particles activate dendritic cells (DCs) and induce perforin expression in Gag-specific CD8(+) T cells by cross-presentation of DCs.

To evaluate the immunogenicity of human immunodeficiency virus (HIV) type 1 p55(gag) virus-like particles (VLPs) released by budding from yeast spheroplasts, we have analyzed the effects of yeast VLPs on monocyte-derived dendritic cells (DCs). Yeast VLPs were efficiently incorporated into DCs via both macropinocytosis and endocytosis mediated by mannose-recognizing receptors, but not the mannose receptor. The uptake of yeast VLPs induced DC maturation and enhanced cytokine production, notably, interleukin-12 p70. We showed that yeast membrane components may contribute to DC maturation partly through Toll-like receptor 2 signaling. Thus, Gag particles encapsulated by yeast membrane may have an advantage in stimulating Gag-specific immune responses. We found that yeast VLPs, but not the control yeast membrane fraction, were able to activate both CD4(+) and CD8(+) T cells of HIV-infected individuals. We tested the effect of cross-presentation of VLP by DCs in two subjects recruited into a long-term nonprogressor-slow progressor cohort. When yeast VLP-loaded DCs of these patients were cocultured with peripheral blood mononuclear cells for 7 days, approximately one-third of the Gag-specific CD8(+) T cells were activated and became perforin positive. However, some of the Gag-specific CD8(+) T cells appeared to be lost during in vitro culture, especially in a patient with a high virus load. Our results suggest that DCs loaded with yeast VLPs can activate Gag-specific memory CD8(+) T cells to become effector cells in chronically HIV-infected individuals, but there still remain unresponsive Gag-specific T-cell populations in these patients.

Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4+ T cells.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr appears to make a substantial contribution to the replication of HIV-1 in established T cell lines when HIV-1 is present at very low multiplicities of infection. However, the role of Vpr in viral replication in primary CD4+ T cells remains to be clarified. In this study, we generated a panel of viruses that encoded mutant forms of Vpr that lacked either the ability to accumulate in the nucleus and induce G2 arrest or the ability to induce apoptosis, which has been shown to occur independently of G2 arrest of the cell cycle. We demonstrate here that the nuclear localization of Vpr and consequent G2 arrest but not the induction of apoptosis by Vpr are important for viral replication in primary CD4+ T cells at both high and low multiplicities of infection. Viruses that encoded mutant forms of Vpr that failed to be imported into the nucleus in the presence of cytoplasmic extracts from primary CD4+ T cells in an in vitro nuclear import assay replicated at drastically reduced rates. Thus, Vpr might be a key regulator of the viral nuclear import process during infection in primary CD4+ T cells. By contrast, a mutant form of Vpr that exhibited diffuse cytosolic staining exclusively in an immunofluorescence assay of HeLa cells and was not imported into nucleus by the cytosol from HeLa cells was effectively imported into the nucleus by cytosol from primary CD4+ T cells. This Vpr mutant virus replicated well in primary CD4+ T cells, indicating that cellular factors in primary CD4+ T cells are indispensable for the accumulation of Vpr in the nucleus and, thus, for viral replication. Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of HIV-1.