New insight into the microbiome, resistome, and mobilome on the dental waste water in the context of heavy metal environment.

Object: Hospital sewage have been associated with incorporation of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) into microbes, which is considered as a key indicator for the spread of antimicrobial resistance (AMR). The compositions of dental waste water (DWW) contain heavy metals, the evolution of AMR and its effects on the water environment in the context of heavy metal environment have not been seriously investigated. Thus, our major aims were to elucidate the evolution of AMR in DWW. Methods: DWW samples were collected from a major dental department. The presence of microbial communities, ARGs, and MGEs in untreated and treated (by filter membrane and ozone) samples were analyzed using metagenomics and bioinformatic methods. Results: DWW-associated resistomes included 1,208 types of ARGs, belonging to 29 antibiotic types/subtypes. The most abundant types/subtypes were ARGs of multidrug resistance and of antibiotics that were frequently used in the clinical practice. Pseudomonas putida, Pseudomonas aeruginosa, Chryseobacterium indologenes, Sphingomonas laterariae were the main bacteria which hosted these ARGs. Mobilomes in DWW consisted of 93 MGE subtypes which belonged to 8 MGE types. Transposases were the most frequently detected MGEs which formed networks of communications. For example, ISCrsp1 and tnpA.5/4/11 were the main transposases located in the central hubs of a network. These significant associations between ARGs and MGEs revealed the strong potential of ARGs transmission towards development of antimicrobial-resistant (AMR) bacteria. On the other hand, treatment of DWW using membranes and ozone was only effective in removing minor species of bacteria and types of ARGs and MGEs. Conclusion: DWW contained abundant ARGs, and MGEs, which contributed to the occurrence and spread of AMR bacteria. Consequently, DWW would seriously increase environmental health concerns which may be different but have been well-documented from hospital waste waters.

Hepatitis B virus surface protein induces sperm dysfunction through the activation of a Bcl2/Bax signaling cascade triggering AIF/Endo G-mediated apoptosis.

BACKGROUND: Hepatitis B virus (HBV) was found to exist in semen and male germ cells of patients with chronic HBV infection. Our previous studies demonstrated that HBV surface protein (HBs) could induce sperm dysfunction by activating a calcium signaling cascade and triggering caspase-dependent apoptosis. However, the relationship between sperm dysfunction caused by HBs and caspase-independent apoptosis has not been investigated. OBJECTIVES: To evaluate the effects of HBs exposure on sperm dysfunction by activating caspase-independent apoptosis. MATERIALS AND METHODS: Spermatozoa were exposed to HBs at concentrations of 0, 25, 50, and 100 µg/mL for 3 h. Flow cytometry, qRT-PCR, immunofluorescence assay, ELISA, and zona-free hamster oocyte penetration assays were performed. RESULTS: With increasing concentrations of HBs, various parameters of the spermatozoa changed. The number of Bcl2-positive cells declined and that of both Bax-positive cells and Apaf-1-positive cells increased. The transcription level of Bcl2 increased and that of both Bax and Apaf-1 declined. The average levels of AIF and Endo G declined in mitochondria and increased in the cytoplasm and nucleus. The sperm DNA fragmentation index increased. The mean percentages of live spermatozoa declined and that of both injured and dead spermatozoa increased; and the sperm penetration rate declined. For the aforementioned parameters, the differences between the test and the control groups were statistically significant. CONCLUSION: HBs exposure can activate the Bax/Bcl2 signaling cascade that triggers AIF/Endo G-mediated apoptosis, resulting in sperm DNA fragmentation, sperm injury, and death, and a decrease in the sperm fertilizing capacity. This new knowledge will help to evaluate the negative impact of HBV on male fertility in HBV-infected patients.

Transcription and regulation of hepatitis B virus genes in host sperm cells.

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo.

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.

Circulating tight junction proteins mirror blood-brain barrier integrity in leukaemia central nervous system metastasis.

The aim of this study was to evaluate the clinical significance of circulating tight junction (TJ) proteins as biomarkers reflecting of leukaemia central nervous system (CNS) metastasis. TJs [claudin5 (CLDN5), occludin (OCLN) and ZO-1] concentrations were measured in serum and cerebrospinal fluid (CSF) samples obtained from 45 leukaemia patients. Serum ZO-1 was significantly higher (p < 0.05), but CSF ZO-1 levels were not significantly higher in the CNS leukaemia (CNSL) compared to the non-CNSL. The CNSL patients also had a lower CLDN5/ZO1 ratio in both serum and CSF than in non-CNSL patients (p < 0.05). The TJ index was negatively associated with WBCCSF , ALBCSF and BBB values in leukaemia patients. Among all of the parameters studied, CLDN5CSF had the highest specificity in discriminating between CNSL and non-CNSL patients. Therefore, analysing serum and CSF levels of CLDN5, OCLN and the CLDN5/ZO1 ratio is valuable in evaluating the potential of leukaemia CNS metastasis. Copyright © 2016 John Wiley & Sons, Ltd.

Diagnostic value of platelet indices and bone marrow megakaryocytic parameters in immune thrombocytopenic purpura.

Platelet indices could mirror megakaryopoietic activity in immune thrombocytopenic purpura (ITP), but its specificity and sensitivity need to be studied. The diagnostic performance of platelet indices was analyzed by receiver-operating characteristic curves, and the probability of true positive (sensitivity) and true negative (specificity) in predicting ITP, myelodysplasia, or controls was determined. Mean platelet volume (MPV) was higher, whereas plateletcrit (PCT) was significantly lower in ITP than in myelodysplasia and controls. The platelet distribution width in ITP patients was lower than in myelodysplasia, but higher than in controls. Increased megakaryocytes were only observed in ITP. A strong positive correlation was found between MPV and quantities of granular megakaryocytes, whereas a negative relationship existed between MPV and platelet-form megakaryocytes. In receiver-operating characteristic analysis, MPV and PCT gave a sensitivity of 70.3% (89.8%) and specificity of 74.8% (84.7%) at a cutoff of 9.35 (0.085) in diagnosis of ITP. Combined parallel test of MPV and PCT increased the sensitivity to 97.5 with 64.1% specificity, whereas series test increased the specificity to 94.7 with 62.7% sensitivity. Our results suggest that MPV, PCT, and platelet distribution width represent megakaryopoietic activity in bone marrow and may be reliable markers in ITP diagnosis.

The prognostic significance of serum and cerebrospinal fluid MMP-9, CCL2 and sVCAM-1 in leukemia CNS metastasis.

Metastasis to the central nervous system (CNS) is the primary obstacle in leukemia treatment. Matrix metalloproteinase-9 (MMP-9), chemokine ligand-2 (CCL2) and soluble vascular adhesion molecule-1 (sVCAM-1) play crucial roles in tumor cell adhesion, motivation and survival, but their roles in leukemia CNS metastasis remain to be elucidated. We investigated the prognostic significance of serum and cerebrospinal fluid (CSF) MMP-9, CCL2 and sVCAM-1 in leukemia patients to explore their potential as predictive biomarkers of the development of CNS leukemia (CNSL). MMP-9, CCL2 and sVCAM-1 were measured in paired CSF and serum samples collecting from 33 leukemia patients with or without CNS metastasis. Other risk factors related to CNSL prognosis were also analyzed. sVCAM-1Serum and CCL2Serum/CSF were significantly higher in the CNSL group than in the non-CNSL group and the controls (p < 0.05). MMP-9Serum was insignificantly lower in the CNSL group than in the non-CNSL group and the controls (p > 0.05). No differences were found for the sVCAM-1Serum, CCL2Serum, and MMP-9Serum levels between non-CNSL patients and controls (p > 0.05). MMP-9CSF was significantly higher in the CNSL group than both the non-CNSL and the control groups (p < 0.05). The indexes of sVCAM-1, CCL2, and MMP-9 in the CNSL group were lower than in the controls (p < 0.05). Positive correlations were determined between the MMP-9CSF and the ALBCSF/BBB value/WBCCSF, between sVCAM-1Serum and the WBCCSF/BBB value. Negative correlations existed between MMP-9Serum and the ALBCSF/BBB value/WBCCSF, and between the CCL2 index and ALBCSF. sVCAM-1Serum was positively associated with event-free survival (EFS), and patients with higher levels of ALBCSF, MMP-9CSF/Serum, CCL2CSF/Serum, and sVCAM-1CSF/Serum had shorter EFS. MMP-9CSF, CCL2CSF and sVCAM-1CSF are the first three principal components analyzed by cluster and principal component analysis. Our data suggest that MMP-9, CCL2 and sVCAM-1 in the CSF may be more potent than serum in predicting the possibility of leukemia metastatic CNS and the outcome of CNSL patients.

The integrated HIV-1 provirus in patient sperm chromosome and its transfer into the early embryo by fertilization.

Complete understanding of the route of HIV-1 transmission is an important prerequisite for curbing the HIV/AIDS pandemic. So far, the known routes of HIV-1 transmission include sexual contact, needle sharing, puncture, transfusion and mother-to-child transmission. Whether HIV can be vertically transmitted from human sperm to embryo by fertilization is largely undetermined. Direct research on embryo derived from infected human sperm and healthy human ova have been difficult because of ethical issues and problems in the collection of ova. However, the use of inter-specific in vitro fertilization (IVF) between human sperm and hamster ova can avoid both of these problems. Combined with molecular, cytogenetical and immunological techniques such as the preparation of human sperm chromosomes, fluorescent in situ hybridization (FISH), and immunofluorescence assay (IFA), this study mainly explored whether any integrated HIV provirus were present in the chromosomes of infected patients' sperm, and whether that provirus could be transferred into early embryos by fertilization and maintain its function of replication and expression. Evidence showed that HIV-1 nucleic acid was present in the spermatozoa of HIV/AIDS patients, that HIV-1 provirus is present on the patient sperm chromosome, that the integrated provirus could be transferred into early embryo chromosomally integrated by fertilization, and that it could replicate alongside the embryonic genome and subsequently express its protein in the embryo. These findings indicate the possibility of vertical transmission of HIV-1 from the sperm genome to the embryonic genome by fertilization. This study also offers a platform for the research into this new mode of transmission for other viruses, especially sexually transmitted viruses.

In vitro study on vertical transmission of the HIV-1 gag gene by human sperm.

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.

[Effect of liquorice decoction on rat intestinal P-glycoprotein].

OBJECTIVE: To investigate the effect of liquorice in functional modulation of intestinal P-glycoprotein (P-gp) in rats. METHODS: An in vitro diffusion chamber system (Ussing chamber) was used to examine the direct effect of liquorice decoction on rhodamine 123 (a subtrate of P-gp) transport and evaluate the permeability of rhodamine 123 or fluorescein sodium through rat jejunum membranes after oral administration of liquorice decoction. RESULTS: Direct application of liquorice decoction did not obviously affect rhodamine 123 transport across the intestinal mucosa. Oral administration of liquorice decoction (10 g/kg, twice daily for a week) significantly increased the absorption of rhodamine 123 and also enhanced rhodamine 123 secretion across the jejunum mucosa. Liquorice had no obvious effect on the transport of CF across the jejunum mucosa. CONCLUSION: Liquorice may slightly inhibit P-gp function in the intestinal mucosa to increase the intestinal absorption of rhodamine 123.

In vivo study on vertical transmission of the HIV-1 gag gene via mouse oocytes.

Acquired immunodeficiency syndrome (AIDS) is a major public health problem worldwide. This study was performed to explore the feasibility of vertical transmission of human immunodeficiency virus-1 (HIV-1) gag gene via oocyte. The recombinant plasmid (pIRES2-EGFP-gag) was injected into mouse ovaries to transfect germ cells. Induction of superovulation and then animal mating were performed to collect oocytes and two-cell embryos. Positive FISH signals for HIV-1 gag DNA were detected in the nuclei of oocytes and embryos, and in chromosomes of mature oocytes, indicated integration of the gene into the oocyte genome and gene replication in the embryo. HIV-1 gag cDNA positive bands detected by RT-PCR in oocytes and embryos indicated successful gene transcription, while positive immunofluorescence signals for HIV-1 gag protein indicated successful translation in both oocytes and embryos. The HIV-1 gag gene was transmitted vertically to the next generation via oocytes and it retained its function in replication, transcription and translation following at least one mitotic division in embryos.

Effects of hepatitis B virus S protein on human sperm function.

BACKGROUND: Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function. METHODS: Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed. RESULTS: HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs-HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 microg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential. CONCLUSIONS: HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.

In vitro and in vivo studies evaluating recombinant plasmid pCXN2-mIzumo as a potential immunocontraceptive antigen.

PROBLEMS: Study on feasibility of pCXN2-mIzumo as a potential immunocontraceptive antigen. METHOD OF STUDY: Two groups of mice received 100 microg/mouse plasmids of pCXN2-mIzumo and pCXN2 respectively. RT-PCR Immunofluorescence assay and ELISA were performed to observe pCXN2-mIzumo expression and antibody response in the inoculated mice. Sperm penetration assay and animal mating were employed to detect differences of in vitro fertilization (IVF) rate and mean litter size between the experimental and control groups. RESULTS: Izumo cDNA positive bands were detected in sample from mice immunized with pCXN2-mIzumo. IgG response started to rise at 2 weeks after first boost and reached the highest antibody titers at 2 weeks after third boost of immunization with pCXN2-mIzumo in the experimental mice. In vitro fertilization rate in the experimental group (11.57%) was significantly lower than that in control (36.60%). Significant difference of mean litter size between female experimental and control groups was observed, and there was significant negative correlation between individual anti-serum titers and litter size (r = -0.308, P < 0.05). CONCLUSION: pCXN2-mIzumo plasmid possesses appreciable anti-fertility potential.

In vitro and in vivo studies evaluating antisemen antibodies as a potential spermicidal agent in hamsters.

OBJECTIVE: To determine the spermicidal activity of antisemen antibodies in the hamster model. DESIGN: Prospective, controlled study. SETTING: Advanced preclinical sciences center. ANIMAL(S): Subgroups of 10 and 14 golden hamsters. INTERVENTION(S): Ex vitro and in vivo treatment of sperm with antisemen antibodies or normal rabbit serum. MAIN OUTCOME MEASURE(S): The EC(50) value of antisemen antibodies, the time required for 50% motility loss of progressively motile spermatozoa exposed to antisemen antibodies, the average sperm mitochondrion fluorescence intensity, the rate of fertilization, and the scoring of histologic changes in the hamster vaginal tissue. RESULT(S): The EC(50) value of antisemen antibodies was found 70 microg/mL, and the time required for 50% motility loss of progressively motile spermatozoa exposed to antisemen antibodies (at 70 microg/mL) was 5 minutes; for the experimental and control groups, the average fluorescence intensities of sperm mitochondria were respectively 180.28 +/- 82.24 and 309.74 +/- 148.37, the fertilization rates in vitro were 0.09% and 45%, the rates of fertilization with intrauterine sperm injection were 0 and 15.0%. There was a significant difference between two groups. None of the four hamsters that received antisemen antibodies in gel-polyoxyl-40-stearate had epithelial disruption characteristic of inflammation. CONCLUSION(S): Antisemen antibodies possess appreciable spermicidal potential, which may be explored as an effective constituent of spermicide.

A sensitive and rapid assay for investigating vertical transmission of hepatitis B virus via male germ line using EGFP Vector as reporter.

Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed.

An improved experimental model for studying vertical transmission of hepatitis B virus via human spermatozoa.

Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line.

Investigation of recombinant mouse sperm protein izumo as a potential immunocontraceptive antigen.

PROBLEM: To determine if the recombinant mouse Izumo (mIzumo) could be used as a potential immunocontraceptive antigen. METHOD OF STUDY: The recombinant mIzumo fused with 6His tag (6His-mIzumo) was purified by immobilized Ni2+ affinity chromatography. Enzyme-linked immunosorbent assay and Western blot were used to detect anti-6His-mIzumo activities of serum from the mice immunized with 6His-mIzumo. Inhibition of the anti-6His-mIzumo antibody on mouse sperm-egg fusion in vitro was performed using the zona free oocytes and acrosome reacted sperm. Fertility of the 6His-mIzumo immunized male and female mice was compared with control mice. RESULTS: The recombinant mIzumo was successfully produced. Female and male mice inoculated with 6His-mIzumo developed a specific serum antibody and the highest antibody titer lasted at least 6 weeks. The serum anti-6His-mIzumo antibody almost completely blocked mouse sperm-egg fusion in vitro. However, there was no significant reduction in fertility for both male and female mice immunized with 6His-mIzumo compared with control mice. CONCLUSION: The circulated anti-mIzumo antibody can block mouse sperm-egg fusion in vitro but has no effect on fertility in vivo. It seems that application of Izumo as a candidate antigen in development of contraceptive vaccine needs further investigation.

[Biological characteristics of human umbilical cord-derived mesenchymal stem cells and their differentiation into neurocyte-like cells].

OBJECTIVE: To investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells. METHODS: The growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR. RESULTS: A population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed. CONCLUSION: The human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome.

AIM: To detect the expression of hepatitis B virus (HBV) genes (HB S and C genes) in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization (IVF) technique. METHODS: Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction (PCR) was used to detect HB S and pre-Core/Core (pre-C/C) coding genes both in one- and two-cell embryos. Reverse transcription-PCR (RT-PCR) analysis was used to study the expression of the two genes. Fluorescence in situ hybridization (FISH) analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. RESULTS: Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. CONCLUSION: Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells.

BACKGROUND: The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells. METHODS: MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and beta-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM). The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and beta-mercaptoethanol also induced MSCs to express nestin, beta-tubulinIII, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed. CONCLUSIONS: MSCs could be isolated from human umbilical cord Wharton's Jelly. They were capable of differentiating into nerve-like cells using Salvia miltiorrhiza or beta-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.