PURPOSE: Preimplantation Genetic Diagnosis (PGD) has proven to be a useful reproductive option for carriers of some chromosome rearrangements. The data presented in this study compares the impact of one versus two blastomere biopsy on the likelihood of achieving a PGD result, as well as the effect on subsequent embryo development and clinical outcomes. METHODS: IVF-PGD couples had either one or two blastomeres biopsied from all embryos with ≥7 blastomeres on day 3 post oocyte collection. These blastomeres were assessed for the specific chromosome rearrangement using Fluorescent In-situ Hybridisation (FISH). Further embryo development was monitored on days 4 and 5. Clinical outcomes were assessed retrospectively. RESULTS: The data shows that statistically more embryos achieved a PGD result following two blastomere biopsy, compared with one blastomere biopsy (92 % versus 88 %, respectively). Furthermore it was found that embryo development and clinical outcomes were similar between the two biopsy groups. CONCLUSIONS: Based on this analysis it appears that the biopsy of two blastomeres from embryos with ≥7 blastomeres on day 3 is a valid and successful approach for couples presenting for IVF-PGD for a chromosome rearrangement.

Transverse dispersion represents an important mixing process for transport of contaminants in groundwater and constitutes an essential prerequisite for geochemical and biodegradation reactions. Within this context, this work describes the detailed numerical simulation of highly controlled laboratory experiments using uranine, bromide and oxygen depleted water as conservative tracers for the quantification of transverse mixing in porous media. Synthetic numerical experiments reproducing an existing laboratory experimental set-up of quasi two-dimensional flow through tank were performed to assess the applicability of an analytical solution of the 2D advection-dispersion equation for the estimation of transverse dispersivity as fitting parameter. The fitted dispersivities were compared to the "true" values introduced in the numerical simulations and the associated error could be precisely estimated. A sensitivity analysis was performed on the experimental set-up in order to evaluate the sensitivities of the measurements taken at the tank experiment on the individual hydraulic and transport parameters. From the results, an improved experimental set-up as well as a numerical evaluation procedure could be developed, which allow for a precise and reliable determination of dispersivities. The improved tank set-up was used for new laboratory experiments, performed at advective velocities of 4.9m d(-1) and 10.5m d(-1). Numerical evaluation of these experiments yielded a unique and reliable parameter set, which closely fits the measured tracer concentration data. For the porous medium with a grain size of 0.25-0.30mm, the fitted longitudinal and transverse dispersivities were 3.49×10(-4)m and 1.48×10(-5)m, respectively. The procedures developed in this paper for the synthetic and rigorous design and evaluation of the experiments can be generalized and transferred to comparable applications.

OBJECTIVE.: Tissue fibrosis caused by a pathological activation of SSc fibroblasts is a major hallmark of systemic sclerosis (SSc). The aims of the present study were to investigate whether JAK2 contributes to the pathologic activation of fibroblasts in SSc and to evaluate the anti-fibrotic potential of JAK2 inhibition for the treatment of systemic sclerosis. METHODS.: Activation of JAK2 in human skin and in experimental fibrosis was determined by immunohistochemistry. JAK2 signaling was inhibited with the selective JAK2 inhibitor TG 101209 or by siRNA. Bleomycin-induced dermal fibrosis and tight-skin 1 (Tsk-1) mice were used to evaluate the anti-fibrotic potential of a specific JAK2 inhibition in vivo. RESULTS.: Increased activation of JAK2 was detected in the skin of SSc patients, particularly in fibroblasts. The activation of JAK2 was TGFβ dependent and persisted in cultured SSc fibroblasts. Inhibition of JAK2 reduced the basal collagen synthesis selectively in SSc fibroblasts but not in resting healthy dermal fibroblasts. Moreover, inhibition of JAK2 prevented the stimulatory effects of TGFβ on fibroblasts. Treatment with TG 101209 did not only prevent bleomycin-induced fibrosis, but also effectively reduced skin fibrosis in Tsk-1 mice. CONCLUSION.: We demonstrated that JAK2 is activated in a TGFβ dependent manner in SSc. Considering the potent anti-fibrotic effects of JAK2 inhibition, our study might have direct translational implications, because inhibitors of JAK2 are currently evaluated in clinical trials for myeloproliferative disorders and would be also available for evaluation in SSc patients.

Brain inflammation plays a central role in multiple sclerosis (MS). Besides lymphocytes, the astroglia and microglia mainly contribute to the cellular composition of the inflammatory infiltrate in MS lesions. Several studies were able to demonstrate that cortical lesions are characterized by lower levels of inflammatory cells among activated microglia/macrophages. The underlying mechanisms for this difference, however, remain to be clarified. In the current study, we compared the kinetics and extent of microglia and astrocyte activation during early and late cuprizone-induced demyelination in the white matter tract corpus callosum and the telencephalic gray matter. Cellular parameters were related to the expression profiles of the chemokines Ccl2 and Ccl3. We are clearly able to demonstrate that both regions are characterized by early oligodendrocyte stress/apoptosis with concomitant microglia activation and delayed astrocytosis. The extent of microgliosis/astrocytosis appeared to be greater in the subcortical white matter tract corpus callosum compared to the gray matter cortex region. The same holds true for the expression of the key chemokines Ccl2 and Ccl3. The current study defines a model to study early microglia activation and to investigate differences in the neuroinflammatory response of white vs. gray matter.

PURPOSE OF REVIEW: Fibrosis is a key feature of systemic sclerosis (SSc) and arises from excessive release of collagens by pathologically activated fibroblasts. Affecting the skin and many internal organs, fibrosis represents a major cause for the high morbidity and mortality in SSc. So far, effective therapies to treat fibrosis in SSc and other fibrotic diseases are not available in clinical routine. Nevertheless, promising antifibrotic agents are emerging from translational studies with some having already entered clinical trials. RECENT FINDINGS: In this review, we focus on recent advances in the development of antifibrotic treatment strategies in SSc. We have selected for targeted therapeutic approaches that have proven high efficacy and tolerability in preclinical fibrosis models of SSc and/or are already in clinical evaluation. Applying these criteria, we discuss a large repertory of candidate antifibrotic therapies that block inflammatory pathways, inhibit profibrotic growth factors, modulate epigenetic signaling, and interfere with morphogenic pathways. SUMMARY: Many antifibrotic candidate therapies have proven efficacy and tolerability in preclinical models of SSc. So far, early clinical studies have tested only few of these agents. Besides discovering novel molecular treatment strategies, SSc research will now have to translate its findings into clinical practice.

The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

OBJECTIVES: Tissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach.METHODS: The activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice.RESULTS: Hedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis.CONCLUSIONS: Inhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.

The p7 protein of Hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells and infectious chimeric virions were only produced when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in NS2, T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of the above mutations per se restored the infectivity of the parental chimeric genome suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B co-localized in cellular compartments, and the NS5B mutation did not affect the co-localization pattern. The NS5B K151R mutation did not increase viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.

In innate immunity, dead and dying cells release internal constituents that can serve as damage-associated molecular patterns (DAMPs) or alarmins. This release occurs more abundantly during necrosis than apoptosis and may account for the differences in the immunologic properties of these death forms. To elucidate DAMP release in necrosis, we compared the levels of two nuclear molecules (DNA and HMGB1, a non-histone protein with alarmin activity) in media following necrosis of Jurkat T cells by freeze-thawing, ethanol, heat or hydrogen peroxide treatment. In our experiments, DNA release was measured by fluorimetry with the dye PicoGreen, while HMGB1 was measured by Western blotting. As the results of our study show, each form of necrosis is associated with a distinct pattern of DNA and HMGB1 release with respect to kinetics and amounts. Of these, freeze-thawing produced the highest and most rapid increase in HMGB1 and DNA levels, although the released DNA was subject to nuclease digestion; in addition, freeze-thawing led to the production of particles measured by flow cytometry. Together, these results indicate that experimental necrosis leads to diverse patterns of nuclear molecule release which could affect their immunologic activity.