Demonstration of stable, long-term operation of a nanosecond pulsed DPSSL at 10 J, 100 Hz.

We report on stable, long-term operation of a diode-pumped solid-state laser (DPSSL) amplifying 15 ns pulses at 1029.5 nm wavelength to 10 J energy at 100 Hz pulse rate, corresponding to 1 kW average power, with 25.4% optical-to-optical efficiency. The laser was operated at this level for over 45 minutes (∼3 · 105 shots) in two separate runs with a rms energy stability of 1%. The laser was also operated at 7 J, 100 Hz for 4 hours (1.44 · 106 shots) with a rms long-term energy stability of 1% and no need for user intervention. To the best of our knowledge, this is the first time that long-term reliable amplification of a kW-class high energy nanosecond pulsed DPSSL at 100 Hz has been demonstrated.

High energy, high pulse rate laser operation using crystalline adhesive-free bonded Yb:YAG slabs.

We report on the successful amplification of 10 ns pulses to 10 J energy at 10 Hz in a DiPOLE laser amplifier using crystalline Yb:YAG/Cr:YAG composite slabs manufactured using adhesive-free bonding (AFB) technology. We demonstrate that bonded slabs are suitable for operation in high energy cryogenic laser amplifiers. We also report on frequency doubling of the beam amplified in the bonded slabs. When the pulse energy of the output infrared beam is set to 5 J, a pulse energy of 3.9 J is achieved in the green (corresponding to 78% conversion efficiency). Results demonstrate that AFB technology is suitable for producing large-sized gain material slabs and can overcome current limitations in the manufacture of large-aperture gain material pieces. We believe this work will facilitate energy scaling of high energy lasers where aperture scaling of optical elements is not achievable via conventional manufacturing techniques.

Modelling and measurement of thermal stress-induced depolarisation in high energy, high repetition rate diode-pumped Yb:YAG lasers.

In this paper, we present a model to predict thermal stress-induced birefringence in high energy, high repetition rate diode-pumped Yb:YAG lasers. The model calculates thermal depolarisation as a function of gain medium geometry, pump power, cooling parameters, and input polarisation state. We show that model predictions are in good agreement with experimental observations carried out on a DiPOLE 100 J, 10 Hz laser amplifier. We show that single-pass depolarisation strongly depends on input polarisation state and pumping parameters. In the absence of any depolarisation compensation scheme, depolarisation varies over a range between 5% and 40%. The strong dependence of thermal stress-induced depolarisation on input polarisation indicates that, in the case of multipass amplifiers, the use of waveplates after every pass can reduce depolarisation losses significantly. We expect that this study will assist in the design and optimisation of Yb:YAG lasers.

Second and third harmonic conversion of a kilowatt average power, 100-J-level diode pumped Yb:YAG laser in large aperture LBO.

We report on the successful demonstration of second and third harmonic conversion of a high pulse energy, high average power 1030 nm diode pumped Yb-doped yttrium aluminum garnet (Yb:YAG) nanosecond pulsed laser in a large aperture lithium triborate (LBO) crystal. We demonstrated generation of 59.7 J at 10 Hz (597 W) at 515 nm (second harmonic) and of 65.0 J at 1 Hz (65 W) at 343 nm (third harmonic), with efficiencies of 66% and 68%, respectively. These results, to the best of our knowledge, represent the highest energy and power reported for frequency conversion to green and UV-A wavelengths.

Interleukin (IL)-4 promotes T helper type 2-biased natural killer T (NKT) cell expansion, which is regulated by NKT cell-derived interferon-gamma and IL-4.

CD1d-restricted natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and Th2 cytokines and also play regulatory or pathological roles in immune responses. NKT cells are able to expand when cultured with alpha-galactosylceramide (alpha-GalCer) and interleukin (IL)-2 in a CD1d-restricted manner. However, the expansion ratio of human NKT cells is variable from sample to sample. In this study, we sought to determine what factor or factors are responsible for efficient in vitro expansion of NKT cells from various inbred mouse strains. Although the proportion of NKT cells in the spleen was nearly identical in each mouse strain, the growth rates of NKT cells cultured in vitro with alpha-GalCer and IL-2 were highly variable. NKT cells from the B6C3F1 and BDF1 mouse strains expanded more than 20-fold after 4 days in culture. In contrast, NKT cells from the strain C3H/HeN did not proliferate at all. We found that cell expansion efficiency correlated with the level of IL-4 detectable in the supernatant after culture. Furthermore, we found that exogenous IL-4 augmented NKT cell proliferation early in the culture period, whereas interferon (IFN)-gamma tended to inhibit NKT cell proliferation. Thus, the ratio of production of IL-4 and IFN-gamma was important for NKT cell expansion but the absolute levels of these cytokines did not affect expansion. This finding suggests that effective expansion of NKT cells requires Th2-biased culture conditions.

Pancreatic endocrine and exocrine cell ontogeny from renal capsule transplanted embryonic stem cells in streptozocin-injured mice.

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.

Modulation of acute graft-versus-host disease and chimerism after adoptive transfer of in vitro-expanded invariant Valpha14 natural killer T cells.

Mouse natural killer T cells with an invariant Valpha14-Jalpha18 TCR rearrangement (Valpha14i NKT cells) are able to regulate immune responses through rapid and large amounts of Th1 and Th2 cytokine production. It has been reported that in vivo administration of the Valpha14i NKT cell ligand, alpha-galactosylceramide (alpha-GalCer) significantly reduced morbidity and mortality of acute graft-versus-host disease (GVHD) in mice. In this study, we examined whether adoptive transfer of in vitro-expanded Valpha14i NKT cells using alpha-GalCer and IL-2 could modulate acute GVHD in the transplantation of spleen cells of C57BL/6 mice into (B6xDBA/2) F(1) mice. We found that the adoptive transfer of cultured spleen cells with a combination of alpha-GalCer and IL-2, which contained many Valpha14i NKT cells, modulated acute GVHD by exhibiting long-term mixed chimerism and reducing liver damage. Subsequently, the transfer of Valpha14i NKT cells purified from spleen cells cultured with alpha-GalCer and IL-2 also inhibited acute GVHD. This inhibition of acute GVHD by Valpha14i NKT cells was blocked by anti-IL-4 but not by anti-IFN-gamma monoclonal antibody. Therefore, the inhibition was dependent on IL-4 production by Valpha14i NKT cells. Our findings highlight the therapeutic potential of in vitro-expanded Valpha14i NKT cells for the prevention of acute GVHD after allogeneic hematopoietic stem cell transplantation.

Long-term maintenance of liver-specific functions in cultured ES cell-derived hepatocytes with hyaluronan sponge.

This study investigated the three-dimensional culture of hepatocytes differentiated from mouse embryonic stem (ES) cells with a porous hyaluronan (HA) sponge support. Hepatocytes were immobilized within the pores of the support. Spheroids could be observed within the support, each containing between 20 and 50 hepatocytes. To examine the liver-specific functions of the hepatocytes in the culture, the levels of albumin secreted into the medium were analyzed. The secretion of albumin was stable over the course of 32 days, longer than that in both conventional monolayer and collagen sponge cultures. To elucidate further the liver-specific functions of hepatocytes embedded in the HA sponge, metabolic activities of the hepatocytes were examined for their ability to eliminate ammonia from culture media and the synthesis of urea nitrogen. While rates of ammonia removal and urea nitrogen synthesis were similar to those in both conventional monolayer and in collagen sponge cultures, these functions were maintained for longer duration in cells embedded in the HA sponge. These results demonstrate that the porous HA sponge is an effective support for the in vitro culture of ES-derived hepatocytes used for both basic and applied studies for cell transplantation.

Recombinant adenovirus vector activates and protects human monocyte-derived dendritic cells from apoptosis.

The aim of this study was to examine the effect of two of the most commonly used viral vectors, that is, retrovirus and adenovirus, on the antigen presentation of dendritic cells (DCs). DCs were generated from CD34(+) hematopoietic precursors and CD14(+) monocytes of the same prostate cancer patients. Adenoviral transduction of monocyte-derived DCs (MO-DCs) resulted in upregulation of CD80, CD86, and CD83 expression. Adenovirus-transduced MO-DCs were also more potent stimulators of allogeneic lymphocytes, produced increased amounts of the cytokines tumor necrosis factor alpha and interleukin 12 p70, and exhibited increased expression of NF-kappaB and antiapoptotic molecules Bcl-X(L) and Bcl-2. Enhanced expression of the antiapoptotic molecules correlated with increased resistance of adenovirus-transduced MO-DCs to spontaneous as well as Fas-mediated cell death. In contrast to the adenoviral construct, no significant transduction of MO-DCs with the retrovirus could be obtained. Transduction of CD34(+) cell-derived DCs with the retrovirus or the adenovirus did not significantly alter expression of the costimulatory molecules or cytokines studied. At lower stimulation ratios, CD34(+) cell-derived DCs transduced with retrovirus were less potent in their ability to stimulate allogeneic lymphocytes in comparison with nontransduced DCs. Our results indicate that adenoviral vectors may be more suitable for gene delivery to DCs for immunotherapy.

Interferon-induced SCYL2 limits release of HIV-1 by triggering PP2A-mediated dephosphorylation of the viral protein Vpu.

Human cells respond to infection by retroviruses through the actions of proteins that inhibit the spread of viruses to other cells. One example is bone marrow stromal cell antigen 2 (BST2; also known as tetherin), which is an interferon (IFN)-inducible protein that restricts the release of progeny virions from infected cells. The HIV-1 accessory protein Vpu (viral protein U) causes degradation of BST2, and phosphorylation of Vpu at residues Ser(52) and Ser(56) is required for this function. We report that the host protein SCY1-like protein 2 (SCYL2) mediates the dephosphorylation of Vpu, antagonizing Vpu function and facilitating BST2-dependent restriction of HIV-1 release. SCYL2 reduced the number of virus particles released from cells infected with wild-type HIV-1, but not a strain lacking vpu, in a BST2-dependent manner. SCYL2 stimulated the dephosphorylation of Vpu on Ser(52) and Ser(56) by recruiting protein phosphatase 2A (PP2A) to Vpu. Conversely, depletion of SCYL2 resulted in enhanced phosphorylation of Vpu and increased viral particle release. Moreover, SCYL2 was produced in response to type I IFN and contributed to IFN-mediated viral restriction. Together, these results suggest that SCYL2 serves as a regulatory factor for Vpu, reducing the extent of Vpu phosphorylation and consequently enhancing BST2-mediated viral restriction.

Embryonic stem cell transplantation correlates with endogenous neurogenin 3 expression and pancreas regeneration in streptozotocin-injured mice.

Pancreatic beta cell regeneration remains poorly understood, yet stimulation of adult beta cell neogenesis could lead to therapies for type 1 and type 2 diabetes. We studied the effect of embryonic stem (ES) cell transplantation on pancreas regeneration following beta cell injury. Female Balb/c nude mice were treated with streptozotocin to induce hyperglycemia and received an ES cell transplant 24 hr later beneath the renal capsule. Transplantation of ES cells prevented hyperglycemia in a subset of mice, maintaining euglycemia and mild glucose tolerance up to 5 weeks. Pancreata of euglycemic mice showed histological evidence of beta cell regeneration and expression of pancreas and duodenum transcription factor-1 (PDX-1) and neurogenin 3 (Ngn3) in ductal epithelium. Cell tracing analysis indicated that significant beta cell neogenesis from progenitor cells occurred between 2 to 3 weeks following injury in ES cell-transplanted mice but not in sham-transplanted animals. Significantly, whereas pancreas-localized ES cells or their derivatives were adjacent to sites of regeneration, neogenic pancreatic epithelia, including Ngn3+ cells, were endogenous. In conclusion, transplanted ES cells can migrate to the injured pancreas. Transplantation is associated with enhanced endogenous regeneration characterized by expression of Ngn3 and increased beta cell differentiation from endogenous progenitor cells.

Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes.

UNLABELLED: Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver. CONCLUSION: Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.

Direct hepatic fate specification from mouse embryonic stem cells.

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fate-specification from ES cells by using simple adherent monolayer culture conditions. ES cell-derived hepatocytes showed liver-specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell-derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. Supplementary material for this article can be found on the HEPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells.

Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3beta/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se.

Packaging of human endogenous retrovirus sequences is undetectable in porcine endogenous retrovirus particles produced from human cells.

The chronic shortage of human donor organs and tissues for allotransplantation could be relieved if clinical xenotransplantation were to become a viable clinical therapy. Balanced against the benefits of xenotransplantation are the possible consequences of zoonotic infections, and in particular, infection by porcine endogenous retrovirus (PERV). An often-proclaimed risk of PERV infection is the possible recombination of PERV with human endogenous retroviruses (HERV). To address this issue, we examined the potential for HERV sequences to be cross-packaged into PERV particles produced from infected human 293 cells. Although HERV-K, W, E, R, and ERV-9 RNA transcripts are expressed in 293 cells, we did not detect cross-packaging of any of these HERV groups. Quantitative analysis indicated that less than approximately 1 in 10(4)-10(7) PERV particles might contain HERV sequences. In comparison, we found that murine leukemia virus (MLV)-based vector transcripts were cross-packaged at a rate of approximately one copy in 10(4) PERV particles. Our results indicate that the potential for recombination of PERV and HERV sequences is low and that novel viruses generated by this mechanism are unlikely to represent a significant risk for xenotransplantation.

Control of Fine Particulate (PM2.5) Emissions from Restaurant Operations.

This paper describes efforts to reduce particulate matter (PM) emissions from restaurant operations, including application of an existing control method to a new equipment type. Commercial charbroiling in the South Coast Air Basin results in emissions of approximately 10 tons/day of fine particulate matter (PM2.5) and 1.3 tons/day of volatile organic compounds (VOCs). Over a seven-year period, the South Coast Air Quality Management District worked with industry to develop test methods for measuring emissions from various cooking operations, evaluate control technologies, and develop a rule to reduce these emissions. Of the two basic types of charbroilers-chain-driven and underfired-underfired produce four times the emissions when equivalent amounts of product are cooked. Cost-effective control technology is currently available only for chain-driven charbroilers. The application of flameless catalytic oxidizers to chain-driven charbroilers was found to effectively reduce emissions by at least 83% and is cost-effective. The catalysts have been used worldwide at restaurants for several years. Research efforts are underway to identify control options for underfired charbroilers. Implementation of Rule 1138, Control of Emissions from Restaurant Operations, adopted November 14, 1997, will result in reductions of 0.5 tons/day of PM2.5 and 0.2 tons/day of VOCs. Future rules will result in reductions from underfired charbroilers and possibly other restaurant equipment when cost-effective solutions are available.

Differentiation of embryonic stem cells into hepatocytes: biological functions and therapeutic application.

Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies.

Genotyping of porcine endogenous retroviruses from a family of miniature swine.

The identification of animals in an inbred miniature swine herd that consistently fail to produce replication- competent humantropic porcine endogenous retrovirus (PERV) has prompted studies on the biology of PERV in transmitter and nontransmitter animals. We analyzed PERV RNA transcript profiles in a family of inbred miniature swine (SLA(d/d) haplotype) in which individual members differed in their capacity to generate humantropic and ecotropic (i.e., pigtropic) virus. We identified unique HaeIII and HpaII gag restriction fragment length polymorphism (RFLP) profiles resulting from single nucleotide polymorphisms in blood cells; these were found only in animals that produced humantropic PERV. These HaeIII and HpaII gag RFLP profiles proved to be components of humantropic PERV as they were transmitted to 293 human target cells in vitro. The humantropic HaeIII and HpaII gag RFLP genotypes in the family of study were not present in other miniature swine in the herd that produced humantropic PERV, indicating that these RFLP profiles relate specifically to this family's lineage.

Characterization of germline porcine endogenous retroviruses from Large White pig.

Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.