Fabrication and Characterization of Electrospun Thermoplastic Polyurethane/Fibroin Small-Diameter Vascular Grafts for Vascular Tissue Engineering.

The demand for small-diameter blood vessel substitutes has been increasing due to a shortage of autograft vessels and problems with thrombosis and intimal hyperplasia with synthetic grafts. In this study, hybrid small-diameter vascular grafts made of thermoplastic polyurethane (TPU) and silk fibroin, which possessed a hybrid fibrous structure of an aligned inner layer and a random outer layer, were fabricated by the electrospinning technique using a customized striated collector that generated both aligned and random fibers simultaneously. A methanol post-treatment process induced the transition of fibroin protein conformation from the water-soluble, amorphous, and less ordered structures to the water-insoluble ß-sheet structures that possessed robust mechanical properties and relatively slow proteolytic degradation. The methanol post-treatment also created crimped fibers that mimicked the wavy structure of collagen fibers in natural blood vessels. Ultrafine nanofibers and nanowebs were found on the electrospun TPU/fibroin samples, which effectively increased the surface area for cell adhesion and migration. Cyclic circumferential tensile test results showed compatible mechanical properties for grafts made of a soft TPU/fibroin blend compared to human coronary arteries. In addition, cell culture tests with endothelial cells after 6 and 60 days of culture exhibited high cell viability and good biocompatibility of TPU/fibroin grafts, suggesting the potential of applying electrospun TPU/fibroin grafts in vascular tissue engineering.

A randomised phase II study of pegylated arginine deiminase (ADI-PEG 20) in Asian advanced hepatocellular carcinoma patients.

BACKGROUND: Human hepatocellular carcinoma (HCC) cells are largely deficient of argininosuccinate synthetase and thus auxotrophic for arginine. This study aims to investigate the efficacy and pharmacodynamics of pegylated arginine deiminase (ADI-PEG 20), a systemic arginine deprivation agent, in Asian HCC patients. METHODS: Patients with advanced HCC who were not candidates for local therapy were eligible and randomly assigned to receive weekly intramuscular injections of ADI-PEG 20 at doses of 160 or 320 IU m(-2). The primary end point was disease-control rate (DCR). RESULTS: Of the 71 accruals, 43.6% had failed previous systemic treatment. There were no objective responders. The DCR and the median overall survival (OS) of the intent-to-treat population were 31.0% (95% confidence interval (CI): 20.5-43.1) and 7.3 (95% CI: 4.7-9.9) months respectively. Both efficacy parameters were comparable between the two study arms. The median OS of patients with undetectable circulating arginine for more than or equal to and <4 weeks was 10.0 (95% CI: 2.1-17.9) and 5.8 (95% CI: 1.4-10.1) months respectively (P=0.251, log-rank test). The major treatment-related adverse events were grades 1-2 local and/or allergic reactions. CONCLUSIONS: ADI-PEG 20 is safe and efficacious in stabilising the progression of heavily pretreated advanced HCC in an Asian population, and deserves further exploration.

Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone.

To reduce the number of animals required for controlled studies of marmoset oocytes and early embryos, a superovulation protocol was developed for the common marmoset. Females were given up to 50 i.u./day recombinant human follicle stimulating hormone (FSH)--(r-hFSH) for 6 days. Ovaries were visualized by a modified laparoscopic technique and follicular aspiration was performed using a needle and suction apparatus inserted directly through an otoscope speculum. The number of follicles + ovulation points (+/- S.E.) was 2.9 (+/- 0.2) in controls and 14.1 (+/- 1.6; P < or = 0.001) in the 50 i.u. r-hFSH per day animals. Oocytes, typically at the germinal vesicle stage at collection, extruded a first polar body within 26 hours. In vitro fertilization was performed and embryos developed to the hatched blastocyst stage (34%). With many high quality oocytes and the ability to synchronize cycles, the marmoset is a valuable primate model for examining nuclear reprograming and early embryonic events.

The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.

Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens.

Cell-surface antigens provide invaluable tools for the identification of cells and for the analysis of cell differentiation. In particular, stage-specific embryonic antigens that are developmentally regulated during early embryogenesis are widely used as markers to monitor the differentiation of both mouse and human embryonic stem (ES) cells and their malignant counterparts, embryonic carcinoma (EC) cells. However, there are notable differences in the expression patterns of some such markers between human and mouse ES/EC cells, and hitherto it has been unclear whether this indicates significant differences between human and mouse embryos, or whether ES/EC cells correspond to distinct cell types within the early embryos of each species. We now show that human ES cells are characterized by the expression of the cell-surface antigens, SSEA3, SSEA4, TRA-1-60, and TRA-1-81, and by the lack of SSEA1, and that inner cell mass cells of the human blastocyst express a similar antigen profile, in contrast to the corresponding cells of the mouse embryo.

Human ES cells--haematopoiesis and transplantation strategies.

Human embryonic stem (ES) cells provide a novel opportunity to study early developmental events in a human system. We have used human ES cell lines, including clonally derived lines, to evaluate haematopoiesis. Co-culture of the human ES cells with irradiated bone marrow stromal cell lines in the presence of fetal bovine serum (FBS), but without other exogenous cytokines, leads to differentiation of the human ES cells within a matter of days. A portion of these differentiated cells express CD34, the best-defined marker for early haematopoietic cells. Haematopoietic colony-forming cells (CFCs) are demonstrated by methylcellulose assay. Myeloid, erythroid, megakaryocyte and multipotential CFCs can all be derived under these conditions. Enrichment of CD34+ cells derived from the human ES cells markedly increases the yield of CFCs, as would be expected for cells derived from adult bone marrow or umbilical cord blood. Transcription factors are also expressed in a manner consistent with haematopoietic differentiation. This system now presents the potential to evaluate specific conditions needed to induce or support events in early human blood development. Human ES cells are also a novel source of cells for transplantation therapies. The immunogenicity of ES cell-derived cells is unknown. The unique properties of ES cells afford the opportunity to explore novel mechanisms to prevent immune-mediated rejection. Potential strategies to overcome rejection will be presented, including creation of haematopoietic chimerism as a means to successfully transplant cells and tissues derived from human ES cells.

In vitro differentiation of transplantable neural precursors from human embryonic stem cells.

The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube-like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell-derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.

Hodgkin's disease complicated by the nephrotic syndrome in a man with Kugelberg-Welander disease.

A case of nephrotic syndrome due to minimal change glomerulonephritis complicating Hodgkin's disease in a man with a longstanding neurological disorder is presented. Treatment with combination chemotherapy resulted in a rapid improvement in the nephrotic syndrome, and complete remission of the Hodgkin's disease. Disease relapse occurred less than 12 months later without recurrence of the nephrotic syndrome and was refractory to further treatment. The association of minimal change glomerulonephritis with Hodgkin's disease and the possible pathogenesis of this association are discussed.

Hematopoietic colony-forming cells derived from human embryonic stem cells.

Human embryonic stem (ES) cells are undifferentiated, pluripotent cells that can be maintained indefinitely in culture. Here we demonstrate that human ES cells differentiate to hematopoietic precursor cells when cocultured with the murine bone marrow cell line S17 or the yolk sac endothelial cell line C166. This hematopoietic differentiation requires fetal bovine serum, but no other exogenous cytokines. ES cell-derived hematopoietic precursor cells express the cell surface antigen CD34 and the hematopoietic transcription factors TAL-1, LMO-2, and GATA-2. When cultured on semisolid media with hematopoietic growth factors, these hematopoietic precursor cells form characteristic myeloid, erythroid, and megakaryocyte colonies. Selection for CD34(+) cells derived from human ES cells enriches for hematopoietic colony-forming cells, similar to CD34 selection of primary hematopoietic tissue (bone marrow, umbilical cord blood). More terminally differentiated hematopoietic cells derived from human ES cells under these conditions also express normal surface antigens: glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. The in vitro differentiation of human ES cells provides an opportunity to better understand human hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies.

Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos.

Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal-fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture of in vitro fertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription-PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.

Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro.

A cell culture system consisting of mouse S17 stromal cells supplemented with cytokines was developed for hematopoietic differentiation of rhesus monkey embryonic stem (ES) cells. The differentiated colonies that formed contained clusters of hematopoietic-like cells, as well as structures similar in appearance to embryonic blood islands. When this culture system was supplemented with bone morphogenetic protein 4 (BMP-4), the numbers of primary hematopoietic clusters increased by an average of 15 fold. The primary hematopoietic clusters containing clonogenic precursors (expandable hematopoietic clusters) increased by 18 fold. Immunofluorescence analysis showed that a substantial percentage of the hematopoietic-like cells were CD34(+), with morphologic features of undifferentiated blast cells. Enrichment of the CD34(+) cells was associated with enhanced stromal-dependent, cytokine-driven formation of cobblestone colonies on secondary plating. The hematopoietic identity of the precursors was further indicated by their expression of genes associated with hematopoietic differentiation, as well as morphologic assessments that showed erythroid and myeloid lineages among the progeny cells. In addition, reverse transcriptase-polymerase chain reaction analysis of BMP-4-treated rhesus monkey ES cells demonstrated an up-regulation of early-expressed genes responsible for embryonic hematopoiesis and angiogenesis during the first 7 days of culture. These observations suggest that embryonic mesoderm regulatory protein may mimic physiologic signals that are required for the onset of embryonic hematopoiesis and stem cell formation in rhesus monkey ES cells.

Short tandem repeat profiling provides an international reference standard for human cell lines.

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Multilineage differentiation from human embryonic stem cell lines.

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation. A wide variety of adult mammalian tissues harbors stem cells, yet "adult" stem cells may be capable of developing into only a limited number of cell types. In contrast, embryonic stem (ES) cells, derived from blastocyst-stage early mammalian embryos, have the ability to form any fully differentiated cell of the body. Human ES cells have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture. Furthermore, they can differentiate into derivatives of all three embryonic germ layers when transferred to an in vivo environment. Data are now emerging that demonstrate human ES cells can initiate lineage-specific differentiation programs of many tissue and cell types in vitro. Based on this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms underlying many facets of human development. Because they have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, human ES cells could potentially provide an unlimited supply of tissue for human transplantation. Though human ES cell-based transplantation therapy holds great promise to successfully treat a variety of diseases (e.g., Parkinson's disease, diabetes, and heart failure) many barriers remain in the way of successful clinical trials.

CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing.

It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce a strong CD8(+) response. Additional desirable elements are multispecificity and a focus on conserved epitopes. The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means to achieve these goals. To test this concept in a relevant disease model we sought to identify multiple simian immunodeficiency virus (SIV)-derived CD8(+) epitopes bound by a single nonhuman primate major histocompatibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu-A*01(2), a common rhesus macaque MHC class I molecule that presents the immunodominant SIV gag-derived cytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for the presence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined using purified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent K(d) values of 500 nM or lower, with 21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infected Mamu-A*01(+) macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This study reveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents an important step toward the design of a multiepitope vaccine for SIV and HIV.

Analysis of disputed single-parent/child and sibling relationships using 16 STR loci.

This study describes the validation of short tandem repeat (STR) systems for the resolution of cases of disputed parentage where only a single parent is available for testing or where the claimed relationship of both parents is in doubt and also cases where sibship must be tested. Three separate multiplex systems the Second Generation Multiplex, Powerplex 1.2 and FFFL have been employed, giving a total of 16 STR loci. Both empirical and theoretical approaches to the validation have been adopted. Appropriate equations have been derived to calculate likelihood ratios for different relationships, incorporating a correction for subpopulation effects. An F(ST) point estimate of 1% has been applied throughout. Empirically, 101 cases of alleged father, alleged mother and child where analysed using six SLP systems and also using the three multiplex STR systems. Of the 202 relationships tested, 197 were independently resolved by both systems, providing either clear evidence of non-parentage or strong support for the relationship.

An aldose reductase homolog from the resurrection plant Xerophyta viscosa Baker.

An aldose reductase homologue (ALDRXV4) was cloned from the resurrection plant Xerophyta viscosa Baker using complementation by functional sufficiency in Escherichia coli. A cDNA library constructed from X. viscosa leaves dehydrated to 85%, 37% and 5% relative water contents (RWC) was converted into an infective phagemid library. Escherichia coli (sr1::Tn10) cells transformed with ds-pBluescript phagemids were selected on minimal medium plates supplemented with 1 mM isopropyl beta-D-thiogalactopyranoside and 1.25 M sorbitol. Nine cDNA clones that conferred tolerance to the osmotically stressed E. coli cells were selected. The phagemid from one clone contained the ALDRXV4 insert. The E. coli cells expressing ALDRXV4 were capable of tolerating the osmotic stress, whereas control cultures were not. The ALDRXV4 insert contained an open reading frame that can code for 319 amino acids, and the predicted protein had a calculated Mr of 35,667. Amino acid sequence comparisons revealed significant similarity to several aldose reductases, with the highest similarity to aldose reductase proteins from Hordeum vulgare, Bromus inermis and Avena fatua, in the order of 66%, 65% and 65% respectively. Northern blot analysis revealed that ALDRXV4 was expressed only under dehydration conditions in X. viscosa leaves. Western blot analysis detected a protein of 36 kDa under dehydration conditions only. Aldose reductase activity levels in X. viscosa leaves increased as the leaf RWC decreased, whereas there was no significant change in aldose reductase activity in Sporobolus stafianus as the leaf RWC decreased.

Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.

Embryonic stem (ES) cell lines derived from human blastocysts have the developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture. Here we describe the clonal derivation of two human ES cell lines, H9.1 and H9.2. At the time of the clonal derivation of the H9.1 and H9.2 ES cell lines, the parental ES cell line, H9, had already been continuously cultured for 6 months. After an additional 8 months of culture, H9.1 and H9.2 ES cell lines continued to: (1) actively proliferate, (2) express high levels of telomerase, and (3) retain normal karyotypes. Telomere lengths, while somewhat variable, were maintained between 8 and 12 kb in high-passage H9.1 and H9.2 cells. High-passage H9.1 and H9.2 cells both formed teratomas in SCID-beige mice that included differentiated derivatives of all three embryonic germ layers. These results demonstrate the pluripotency of single human ES cells, the maintenance of pluripotency during an extended period of culture, and the long-term self-renewing properties of cultured human ES cells. The remarkable developmental potential, proliferative capacity, and karyotypic stability of human ES cells distinguish them from adult cells.