OBJECTIVETo determine whether the transfusion-transmitted virus (TTV) is hepatotropic.

METHODSTotal DNA was extracted from various tissues of 5 experimentally infected Rhesus monkeys during the viremic period. A dot hybridization was done with viral double stranded DNA probes or single antisense probes.

RESULTSThe double-stranded probe was hybridized with DNA from the liver, bone marrow, spleen,stomach, small intestine and colon. The single-stranded antisense probe was hybridized with DNA from the liver, small intestine and bone marrow of all 5 monkeys, but not with that from other tissues.

CONCLUSIONSAs the viral genome was of negative polarity, the plus-stranded fragment identified in our study might be a replicative intermediate, and was only demonstrated in the liver, small intestine, and bone marrow by dot blot hybridization with single-stranded antisense probes. It is suggested that the TTV replicates in the liver, bone marrow and small intestine, and TTV might be hepatotropic.

Animals ; Bone Marrow ; virology ; DNA Virus Infections ; virology ; DNA, Viral ; analysis ; Intestine, Small ; virology ; Liver ; virology ; Macaca mulatta ; Torque teno virus ; genetics ; isolation & purification ; physiology ; Virus Replication

This study was aimed to explore the infection characteristics of murine mononuclear cell subpopulations in bone marrow with murine cytomegalovirus (MCMV). Subpopulations of mononuclear cells, including lin(+), lin(-), lin(-)CD117(+) and lin(-)CD117(-) cells, were infected with MCMV after being separated by MACS, and induced to differentiation by adding cytokines or inducer, then nucleic acid and proteins were detected. The results indicated that the MCMV DNA, IE transcripts and IE protein could be detected in the lin(+) cells infected with MCMV; no virus products were detected in infected lin(-) cells without adding any stimulating factors, while IE and E transcripts and proteins were detected after adding GM-CSF, rhEPO or phorbol ester in the lin(-) cells infected with MCMV. Furthermore, no IE or E gene transcripts were detected in the lin(-)CD117(+) and lin(-)CD117(-) cells, but the cell colony formation of lin(-)CD117(+) hematopoietic stem and progenitor cells was inhibited after MCMV infection and expression of CD117 antigen on cell surface of the lin(-) cells was downregulated. It is concluded that MCMV can latently infect subpopulations of mononuclear cells in the murine bone marrow. Cells which are of characteristics of primitive stem and progenitor cells are not susceptible to MCMV, but infection of these cells with MCMV can inhibit functions of cells and downregulate the expression of antigen on cells surface.
Animals ; Bone Marrow ; virology ; Cytomegalovirus Infections ; Mice ; Mice, Inbred BALB C ; Monocytes ; virology ; Muromegalovirus ; physiology ; Proto-Oncogene Proteins c-kit ; Stem Cells ; virology

BACKGROUND/AIMS: Epstein-Barr virus (EBV) is involved in the pathogenesis of angioimmunoblastic T-cell lymphoma (AILT), but its precise role and prognostic impact are not clear. This study aimed to evaluate the incidence of EBV-postitivity in the tumor and bone marrow (BM) samples from AILT patients, and their correlations with the clinical variables and patient survival. METHODS: Seventy AILT cases were identified over a period of 8 years. Twenty seven cases were investigated for their EBV tumor status, and 10 BM samples of these patients were investigated for their EBV status with using in situ hybridization (ISH). EBV PCR was performed for the BM mononuclear cells in 8 cases. RESULTS: Among the 27 tumor specimens, ten (37%) were EBV-positive. Only CD20-negativity in tumor correlated with the EBV-positivity (p=0.035). In 13 (48%) patients, gross tumor involvement was recognized by hematoxylin-eosin staining at the time of diagnosis. Among the 10 patients who had additional BM slides available, there were 3 with BM involvement, and none showed EBV positive results on ISH. EBV PCR of the BM mononuclear cells revealed one-positive case among 8 patients. This patient was negative for both BM involvement and EBV ISH. The median overall survival of the 25 treated patients was 48.9 months (95% CI: 18.6~79.2 months). Neither overall survival nor progression-free survival was related with EBV-positivity of the tumor. CONCLUSIONS: EBV-positivity of tumor had no impact on the prognosis of AILT patients.
Adolescent ; Adult ; Aged ; Bone Marrow/virology ; DNA, Viral/isolation & purification ; Female ; Herpesvirus 4, Human/*isolation & purification ; Humans ; Immunoblastic Lymphadenopathy/mortality/*virology ; In Situ Hybridization ; Lymphoma, T-Cell/mortality/*virology ; Male ; Middle Aged ; Polymerase Chain Reaction ; Prognosis ; Survival Analysis

OBJECTIVE: To investigate the transduction efficiency of recombinant adeno-associated virus 2 ( rAAV2) in human bone marrow CD34+ hematopoietic stem/progenitor cells and mesenchyme stem cells. METHODS: The rAAV2 containing green fluorescent protein genes (rAAV2/GFP) were constructed, packaged and purified. CD34+ hematopoietic stem/progenitor cells and mesenchyme stem cells were infected with the rAAV2/GFP. After transduction for 48 hours, the expression of GFP was detected under fluorescence microscope. Furthermore, the transduction efficiency of AAV transduced CD34+ with hydroxyurea (HU) pretreatment and that of untreated were compared. RESULTS GFP genes were expressed in 5.3% +/- 1.7% CD34+ cells. After pretreatment with HU, the expression of the GFP gene in CD34+ cells increased to 13.2% +/- 2.8%, and 23% +/- 3.6% mesenchyme stem cells expressed the GFP gene. Conclusion The transduction efficiency of mesenchyme stem cells is higher than that of CD34+ hematopoietic stem/progenitor cells. HU pretreatment can obviously increase the transduction efficiency of CD34+ hematopoietic stem/progenitor cells.
Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; virology ; Dependovirus ; genetics ; Genetic Therapy ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hematopoietic Stem Cells ; metabolism ; virology ; Humans ; Mesenchymal Stem Cells ; metabolism ; virology ; Recombination, Genetic ; Transduction, Genetic

Human cytomegalovirus (CMV) infection may cause life-threatening complications and lead to failure in transplantation. So, it is very important to explore the laboratory methods which can detect the CMV sufficiently early and accurately. This review discusses methodological aspects of quantitative CMV assays with emphasis on recently developed antigen detection assays and molecular biological methods.
Antigens, Viral ; blood ; Bone Marrow Transplantation ; adverse effects ; methods ; Cytomegalovirus ; immunology ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; etiology ; virology ; Humans ; Transplantation, Homologous

OBJECTIVETo evaluate the therapeutic effect of in vitro induced autologous bone marrow-derived liver stem cell transplantation for posthepatitic cirrhosis.

METHODSBetween Jun 2008 and Mar 2009, 12 patients with posthepatitic cirrhosis and portal hypertensive underwent azygousportal disconnection and splenectomy in our department. The patients were then divided into two groups to receive autologous bone marrow-deprived liver stem cell infusion via the hepatic artery after in vitro induction for 7 days (n=6) or saline (n=6). The therapeutic effects of the operations on the liver functions and liver fibrosis index were evaluated.

RESULTSAll the patients recovered uneventfully and no side effect of the operation was found. After the operation, the patients receiving bone marrow-deprived liver stem cell infusion showed better hepatic function improvement than those receiving saline infusion (P<0.05).

CONCLUSIONTransplantation of in vitro induced autologous bone marrow-derived liver stem cell via the hepatic artery is safe and effective for treatment of posthepatitic cirrhosis.

Adult ; Bone Marrow Cells ; cytology ; Female ; Hepatitis, Viral, Human ; complications ; Humans ; Liver Cirrhosis ; etiology ; therapy ; virology ; Male ; Middle Aged ; Stem Cell Transplantation ; Transplantation, Autologous

Adenoviridae ; genetics ; Animals ; Bone Marrow Purging ; methods ; Genes, p53 ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; virology ; Humans ; Receptors, Virus ; genetics ; Transplantation, Autologous ; methods

OBJECTIVETo investigate the efficient transfection of green fluorescent protein gene (GFP) mediated by recombinant adenovirus vector(Ad-GFP) to rat bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODSWistar rat bone marrow-derived MSCs were separated and purified in vitro by Percoll density gradient centrifugation combined with adherent cell culture followed by identification with flow cytometry. MSCs infected by Ad-GFP were observed and the transfection efficiency was assessed by fluorescence microscope. The proliferative ability of these cells was tested by CCK-8.

RESULTSThe transfection efficiency was as high as 90.0%. Expression of GFP gene of infected MSCs was stable for 1 month after infection. There was no statistically difference in proliferative ability between the infected MSCs and non-infected ones (P>0.05).

CONCLUSIONThe infected MSCs with Ad-GFP expressed GFP with high efficiency and retain the ability of proliferation as non-infected MSCs. Transgection with Ad-GFP is a highly effective method for labeling MSCs.

Adenoviridae ; genetics ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; metabolism ; virology ; Cell Proliferation ; Cells, Cultured ; Female ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; virology ; Mice ; Rats ; Rats, Wistar ; Transduction, Genetic ; methods

To culture bone marrow mesenchymal stem cells (BMSCs) of rat in vitro and observe HSV-1 infection on BMSCs, BMSCs were separated from the bone marrow and identified by alizarin red staining and detection of ALP. The morphology of HSV-1 infected BMSCs and the CPE were observed. The total DNA was extracted from HSV-1 infected BMSCs and the desired specific gene fragment of 477bp of HSV-1 was amplified by PCR. Results showed that after BMSCs were induced by mineral-fluid for 14 days, the ALP level was increased and the nodule calcification was formed. The induced BMSCs were manifested to have the characteristics of osteoblasts. CPE couldn't be found in HSV-1 latently infected BMSCs but the 477bp gene fragment was still detectable. HSV-1 could establish latent infection in BMSCs after 7 passages. This study indicated that rat BMSCs could be induced to differentiate into osteoblasts in vitro, therefore they can be used as the seed cells for the tissue engineering. HSV-1 can infect rat BMSCs and develop the latent infection in vitro.
Alkaline Phosphatase ; analysis ; Animals ; Bone Marrow Cells ; virology ; Cell Differentiation ; Female ; Herpesvirus 1, Human ; physiology ; Male ; Mesenchymal Stromal Cells ; cytology ; virology ; Polymerase Chain Reaction ; Rats ; Rats, Wistar ; Tissue Engineering ; Virus Latency

OBJECTIVETo compare the transduction efficiencies of adenoviral vector, adeno-associated viral vector, baculoviral vector, and plasmid vector in human bone-marrow-derived mesenchymal stem cells (hBMSCs).

METHODSThe hBMSCs were cultured in vitro and transducted with the adenoviral vector, adeno-associated viral vector, baculoviral vector, and plasmid vector. The expression of target protein was observed by inverted fluorescent microscopy and flow cytometry.

RESULTSInverted fluorescent microscopy showed that some of the hBMSCs after transduction expressed the green fluorescent protein (GFP) and the hBMSCs transducted with baculoviral vector expressed more GFP than those of other three vectors. Flow cytometry showed that the transduction efficiencies and mean fluorescence intensities of the adenoviral vector, adeno-associated viral vector, and plasmid vector were 42%, 37%, and 22% and 158, 115, and 77, respectively, which were significantly lower than those of baculoviral vector (70%, P < 0.01; 212, P < 0.05; respectively).

CONCLUSIONCompared with the adenoviral vector, adeno-associated viral vector, and plasmid vector, the baculoviral vector has higher transduction efficiency in hBMSCs and therefore may be a more suitable gene vector for research in human gene therapy.

Adenoviridae ; genetics ; metabolism ; Baculoviridae ; genetics ; metabolism ; Bone Marrow Cells ; metabolism ; virology ; Cells, Cultured ; Dependovirus ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Hematopoietic Stem Cells ; metabolism ; virology ; Humans ; Plasmids ; genetics ; metabolism ; Transduction, Genetic ; methods