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In this paper we present a brief review of studies that have reported therapeutic benefits of elevated cAMP on plasticity and regeneration after injury to the central nervous system (CNS). We also provide new data on the cellular mechanisms by which elevation of cyclic adenosine monophosphate (cAMP) promotes cytokine driven regeneration of adult CNS axons, using the visual system as the experimental model. cAMP is a second messenger for many intracellular signalling pathways. Elevation of cAMP in the eye by intravitreal injection of the cell permeant analogue (8-(4-chlorophenylthio)-adenosine-3′,5′-cyclic monophosphate; CPT-cAMP), when added to recombinant ciliary neurotrophic factor (rCNTF), significantly enhances rCNTF-induced regeneration of adult rat retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafted onto transected optic nerve. This effect is mediated to some extent by protein kinase A (PKA) signalling, but CPT-cAMP also acts via PI3K/Akt signalling to reduce suppressor of cytokine signalling protein 3 (SOCS3) activity in RGCs. Another target for cAMP is the exchange protein activated by cAMP (Epac), which can also mediate cAMP-induced axonal growth. Here we describe some novel results and discuss to what extent the pro-regenerative effects of CPT-cAMP on adult RGCs are mediated via Epac as well as via PKA-dependent pathways. We used the established PN–optic nerve graft model and quantified the survival and regenerative growth of adult rat RGCs after intravitreal injection of rCNTF in combination with a selective activator of PKA and/or a specific activator of Epac. Viable RGCs were identified by βIII-tubulin immunohistochemistry and regenerating RGCs retrogradely labelled and quantified after an injection of fluorogold into the distal end of the PN grafts, 4 weeks post-transplantation. The specific agonists of either PKA or Epac were both effective in enhancing the effects of rCNTF on RGC axonal regeneration, but interestingly, injections that combined rCNTF with both agonists were significantly less effective. The results are discussed in relation to previous CPT-cAMP studies on RGCs, and we also consider the need to modulate cAMP levels in order to obtain the most functionally effective regenerative response after CNS trauma. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation. [ABSTRACT FROM AUTHOR]

Abstract: The mature CNS has limited intrinsic capacity for repair after injury; therefore, strategies are needed to enhance the viability and regrowth of damaged neurons. Here we review gene therapy studies in the eye, aimed at improving the survival and regeneration of injured retinal ganglion cells (RGCs). To target RGCs most current methods use recombinant adeno-associated viral vectors (AAV), usually serotype-2 (AAV2), that are injected into the vitreal chamber of the eye. This vector provides long-term transduction of adult RGCs. Strong, constitutive promoters such as CMV and/or β-actin are commonly used but cell-specific promoters have also been tested. Transgenes encoded by AAV have been selected to limit cell death, enhance growth factor expression, or promote growth cone responsiveness. We have assessed the effects of AAV vectors in adult rodent models (i) after optic nerve (ON) crush and (ii) after transplantation of peripheral nerve (PN) onto the cut ON, a procedure that induces injured RGCs to regenerate axons over longer distances. AAV–CNTF–GFP promotes RGC survival and axonal regrowth in mice after ON crush, and in rats after ON crush or PN transplantation. In rats, intravitreal injection of AAV–BDNF–GFP also increases RGC viability but does not promote regeneration. RGC viability and axonal regrowth is further enhanced when AAV–CNTF–GFP is injected into transgenic mice that over-express bcl-2. Reconstituted PN grafts containing Schwann cells that were transduced ex vivo with lentiviral (LV) vectors encoding a secretable form of CNTF support RGC axonal regrowth, however grafts containing Schwann cells transduced with LV–BDNF or LV–GDNF are less successful. We have also quantified the transduction efficiency and tropism of different AAV vectors injected intravitreally. AAV 2/2 and AAV 2/6 showed highest levels of transduction, AAV 2/8 the lowest, and each serotype displayed different transduction profiles for retinal cells. We are also studying the long-term impact of AAV2-mediated CNTF or BDNF expression on the dendritic morphology of RGCs in normal and PN grafted retinas. Analysis of regenerating RGCs intracellularly injected with lucifer yellow indicates gene-specific changes in dendritic structure that likely impact upon visual function. [Copyright &y& Elsevier]

Objective: To create a bioengineered uterine patch for uterine repair of a partially defect uterus.Design: Three different decellularized uterine scaffolds were recellularized in vitro with primary uterine cells and green fluorescent protein- (GPF-) labeled bone marrow-derived mesenchymal stem cells (GFP-MSCs). The patches were transplanted in vivo to investigate their tissue adaptation and supporting capacity during pregnancy.Setting: Research laboratory.Animal(s): Female Lewis rats (n = 9) as donors to generate whole-uterus scaffolds using three different protocols (n = 3 per protocol); Sprague Dawley rats (n = 40) for primary uterus cell isolation procedures (n = 10) and for transplantation/pregnancy studies (n = 30); and male Sprague Dawley rats (n = 12) for mating.Intervention(s): Decellularization was achieved by whole-uterus perfusion with buffered or nonbuffered Triton-X100 and dimethyl sulfoxide (DMSO; group P1/P2) or with sodium deoxycholate (group P3). Primary uterine cells and GFP-MSCs were used to develop uterine tissue constructs, which were grafted to uteri with partial tissue defects.Main Outcome Measure(s): Recellularization efficiency and graft quality were analyzed morphologically, immunohistochemically, and by real-time quantitative polymerase chain reaction (PCR). The location and number of fetuses were documented during pregnancy days 16-20.Result(s): Pregnancy and fetal development were normal in groups P1 and P2, with fetal development over patched areas. Group P3 showed significant reduction of fetal numbers, and embryos were not seen in the grafted area. Quantitative PCR and immunohistochemistry revealed uterus-like tissue in the patches, which had been further reconstructed by infiltrating host cells after transplantation.Conclusion(s): Primary uterine cells and MSCs can be used to reconstruct decellularized uterine tissue. The bioengineered patches made from triton-X100+DMSO-generated scaffolds were supportive during pregnancy. These protocols should be explored further to develop suitable grafting material to repair partially defect uteri and possibly to create a complete bioengineered uterus. [ABSTRACT FROM AUTHOR]

The present study (VINGEM) is the first randomised trial comparing vinflunine/gemcitabine (VG) to standard carboplatin/gemcitabine (CG) in patients with advanced urothelial carcinoma (aUC) ineligible for treatment with cisplatin. Patients with aUC, creatinine clearance 30–60 ml/min, performance status ≤1 and no prior chemotherapy for metastatic disease were randomised to the experimental arm (vinflunine 280 or 250 mg/m2 day 1, gemcitabine 1000 mg/m2 days 1 and 8, q21 days) or the control arm (carboplatin AUC 4.5 day 1, gemcitabine 1000 mg/m2 days 1 and 8, q21 days). Primary end-point was progression-free survival (PFS). Sixty-two patients were randomised; a total of 59 patients were treated (29 VG, 30 CG). There was no significant difference in PFS between the treatment arms: median 6.2 months for VG versus 6.3 months for CG (hazard ratio [HR]: 0.75, 95% confidence interval [CI]: 0.44–1.28; P = 0.293). Median overall survival was 12.5 months for VG versus 10.6 months for CG. The overall response rate (ORR) was higher in the VG arm than in the CG arm (63% versus 40%) but was not statistically significant in the intention-to-treat analysis. Furthermore, VG showed a high complete response (CR) rate, 22% versus 3% in CG. In the per-protocol group, both ORR and CR were significantly higher for VG than for CG. The most common adverse events (AEs) were fatigue, haematological toxicities, gastrointestinal disorders and nausea/vomiting. Common grade III/IV AEs were neutropenia (VG 62%, CG 43%), thrombocytopenia (VG 7%, CG 37%) and febrile neutropenia (VG 31%, CG 7%). The combination of VG did not improve PFS compared with standard treatment with CG in patients unfit for cisplatin due to renal impairment. The response rate of VG indicates, however, an active regimen and warrants further studies. NCT02665039. • This was the first trial comparing vinflunine/gemcitabine (VG) vs carboplatin/gemcitabine (CG) in advanced urothelial cancer. • VG showed clinically meaningful efficacy with high overall and complete response rates. • VG did not improve median progression-free survival but was comparable with CG. • VG showed high incidence of neutropenia but with overall feasible side-effects. [ABSTRACT FROM AUTHOR]

Neuroinflammation is significant in the pathogenesis and development of Alzheimer's disease (AD). Previously, we showed lipopolysaccharide (LPS)-induced neuroinflammation caused memory impairment. We investigated the possible preventive effects of punicalagin (PUN), a component of pomegranate, on memory deficiency caused by LPS, along with the fundamental mechanisms. LPS-treated cultured astrocytes and microglial BV-2 cells were investigated for anti -neuroinflammatory effects of PUN. PUN (1.5 mg/kg) ameliorates LPS (250 μg/kg daily 7 times)-induced memory impairment as well as prevents the LPS-induced expression of inflammatory proteins. In in vitro study, we also found that PUN (1 μg/ml) inhibited the LPS-(10, 20 and 50 μM) induced expression of iNOS and Cox-2 as well as the production of ROS, NO, TNF-α and IL-1β. PUN also suppress activation of NF-κB via inhibition of IκB degradation as well as p50 and p65 translocation into the nucleus in LPS treated mouse brain and cultured astrocytes and microglial BV-2 cells. Consistent with the inhibitory effect on neuro inflammation, PUN inhibited LPS-induced Aβ 1-42 generation through down-regulation of APP and BACE1 expression in in vivo and in vitro study. Moreover, PUN directly binds to NF-κB subunit p50 evidenced by a docking model and pull down assay. These results suggest that PUN inhibits LPS-induced memory impairment via anti-inflammatory and anti -amylogenic mechanisms through inhibition of NF-κB activation. [ABSTRACT FROM AUTHOR]

Summary: Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses. [Copyright &y& Elsevier]