ATF3 upregulation in glia during Wallerian degeneration: differential expression in peripheral nerves and CNS white matter

Background: Many changes in gene expression occur in distal stumps of injured nerves but the transcriptional control of these events is poorly understood. We have examined the expression of the transcription factors ATF3 and c-Jun by non-neuronal cells during Wallerian degeneration following injury to sciatic nerves, dorsal roots and optic nerves of rats and mice, using immunohistochemistry and in situ hybridization.Results: Following sciatic nerve injury-transection or transection and reanastomosis-ATF3 was strongly upregulated by endoneurial, but not perineurial cells, of the distal stumps of the nerves by 1 day post operation (dpo) and remained strongly expressed in the endoneurium at 30 dpo when axonal regeneration was prevented. Most ATF3+ cells were immunoreactive for the Schwann cell marker, S100. When the nerve was transected and reanastomosed, allowing regeneration of axons, most ATF3 expression had been downregulated by 30 dpo. ATF3 expression was weaker in the proximal stumps of the injured nerves than in the distal stumps and present in fewer cells at all times after injury. ATF3 was upregulated by endoneurial cells in the distal stumps of injured neonatal rat sciatic nerves, but more weakly than in adult animals. ATF3 expression in transected sciatic nerves of mice was similar to that in rats. Following dorsal root injury in adult rats, ATF3 was upregulated in the part of the root between the lesion and the spinal cord (containing Schwann cells), beginning at 1 dpo, but not in the dorsal root entry zone or in the degenerating dorsal column of the spinal cord. Following optic nerve crush in adult rats, ATF3 was found in some cells at the injury site and small numbers of cells within the optic nerve displayed weak immunoreactivity. The pattern of expression of c-Jun in all types of nerve injury was similar to that of ATF3.Conclusion: These findings raise the possibility that ATF3/c-Jun heterodimers may play a role in regulating changes in gene expression necessary for preparing the distal segments of injured peripheral nerves for axonal regeneration. The absence of the ATF3 and c-Jun from CNS glia during Wallerian degeneration may limit their ability to support regeneration

Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1.

BackgroundOncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response.MethodsTo sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R-). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R- mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor nude mouse xenograft models and systemic anti-tumor effects tested using dual flank syngeneic 4434 (melanoma), A20 (lymphoma) mouse tumor models alone and in combination with a murine anti-PD1 antibody, and 9 L (gliosarcoma) tumors in rats.ResultsThe twenty nine clinical strains of HSV-1 isolated and tested demonstrated a broad range of tumor cell killing abilities allowing the most potent strain to be identified which was then used for further development. Oncolytic ability was demonstrated to be further augmented by the expression of GALV-GP-R- in a range of tumor cell lines in vitro and in mouse xenograft models in nude mice. The expression of GALV-GP-R- was also demonstrated to lead to enhanced immunogenic cell death in vitro as confirmed by the increased release of HMGB1 and ATP and increased levels of calreticulin on the cell surface. Experiments using the rat 9 L syngeneic tumor model demonstrated that GALV-GP-R- expression increased abscopal uninjected (anenestic) tumor responses and data using mouse 4434 tumors demonstrated that virus treatment increased CD8+ T cell levels both in the injected and uninjected tumor, and also led to increased expression of PD-L1. A combination study using varying doses of a virus expressing GALV-GP-R- and mGM-CSF and an anti-murine PD1 antibody showed enhanced anti-tumor effects with the combination which was most evident at low virus doses, and also lead to immunological memory. Finally, treatment of mice with derivatives of this virus which additionally expressed anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L demonstrated enhanced activity, particularly in uninjected tumors.ConclusionThe new HSV-1 based platform described provides a potent and versatile approach to developing new oncolytic immunotherapies for clinical use. Each of the modifications employed was demonstrated to aid in optimizing the potential of the virus to both directly kill tumors and to lead to systemic therapeutic benefit. For clinical use, these viruses are expected to be most effective in combination with other anti-cancer agents, in particular PD1/L1-targeted immune checkpoint blockade. The first virus from this program (expressing GALV-GP-R- and hGM-CSF) has entered clinical development alone and in combination with anti-PD1 therapy in a number of tumor types (NCT03767348)

Severe propylthiouracil-induced hepatotoxicity in pregnancy managed successfully by liver transplantation: A case report

<p>Abstract</p> <p>Introduction</p> <p>Propylthiouracil-induced severe hepatotoxicity is a relatively rare occurrence, with very few cases reported in the literature. The management of this complication in pregnancy can be a challenge because of the effects of the various treatment options on the fetus.</p> <p>Case presentation</p> <p>We report a rare case of fulminant hepatic failure in a 36-year-old gravida 2 black woman of African descent that occurred at 17 weeks gestation following propylthiouracil treatment for Graves' disease. Her liver failure was managed by liver transplantation and thyroidectomy. Her pregnancy was continued to term, though with not so favorable early childhood sequelae.</p> <p>Conclusion</p> <p>This case illustrates a very rare complication of treatment with a presumed safe drug during pregnancy followed by adverse neonatal outcomes due to the extensive treatment.</p

Initial Solution Heuristic for Portfolio Optimization of Electricity Markets Participation

Meta-heuristic search methods are used to find near optimal global solutions for difficult optimization problems. These meta-heuristic processes usually require some kind of knowledge to overcome the local optimum locations. One way to achieve diversification is to start the search procedure from a solution already obtained through another method. Since this solution is already validated the algorithm will converge easily to a greater global solution. In this work, several well-known meta-heuristics are used to solve the problem of electricity markets participation portfolio optimization. Their search performance is compared to the performance of a proposed hybrid method (ad-hoc heuristic to generate the initial solution, which is combined with the search method). The addressed problem is the portfolio optimization for energy markets participation, where there are different markets where it is possible to negotiate. In this way the result will be the optimal allocation of electricity in the different markets in order to obtain the maximum return quantified through the objective function.This work has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641794 (project DREAM-GO) and from FEDER Funds through COMPETE program and from National Funds through FCT under the project UID/EEA/00760/2013.info:eu-repo/semantics/publishedVersio

Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Likely Role of APOBEC3G-Mediated G-to-A Mutations in HIV-1 Evolution and Drug Resistance

The role of APOBEC3 (A3) protein family members in inhibiting retrovirus infection and mobile element retrotransposition is well established. However, the evolutionary effects these restriction factors may have had on active retroviruses such as HIV-1 are less well understood. An HIV-1 variant that has been highly G-to-A mutated is unlikely to be transmitted due to accumulation of deleterious mutations. However, G-to-A mutated hA3G target sequences within which the mutations are the least deleterious are more likely to survive selection pressure. Thus, among hA3G targets in HIV-1, the ratio of nonsynonymous to synonymous changes will increase with virus generations, leaving a footprint of past activity. To study such footprints in HIV-1 evolution, we developed an in silico model based on calculated hA3G target probabilities derived from G-to-A mutation sequence contexts in the literature. We simulated G-to-A changes iteratively in independent sequential HIV-1 infections until a stop codon was introduced into any gene. In addition to our simulation results, we observed higher ratios of nonsynonymous to synonymous mutation at hA3G targets in extant HIV-1 genomes than in their putative ancestral genomes, compared to random controls, implying that moderate levels of A3G-mediated G-to-A mutation have been a factor in HIV-1 evolution. Results from in vitro passaging experiments of HIV-1 modified to be highly susceptible to hA3G mutagenesis verified our simulation accuracy. We also used our simulation to examine the possible role of A3G-induced mutations in the origin of drug resistance. We found that hA3G activity could have been responsible for only a small increase in mutations at known drug resistance sites and propose that concerns for increased resistance to other antiviral drugs should not prevent Vif from being considered a suitable target for development of new drugs

Studies on the Restriction of Murine Leukemia Viruses by Mouse APOBEC3

APOBEC3 proteins function to restrict the replication of retroviruses. One mechanism of this restriction is deamination of cytidines to uridines in (−) strand DNA, resulting in hypermutation of guanosines to adenosines in viral (+) strands. However, Moloney murine leukemia virus (MoMLV) is partially resistant to restriction by mouse APOBEC3 (mA3) and virtually completely resistant to mA3-induced hypermutation. In contrast, the sequences of MLV genomes that are in mouse DNA suggest that they were susceptible to mA3-induced deamination when they infected the mouse germline. We tested the possibility that sensitivity to mA3 restriction and to deamination resides in the viral gag gene. We generated a chimeric MLV in which the gag gene was from an endogenous MLV in the mouse germline, while the remainder of the viral genome was from MoMLV. This chimera was fully infectious but its response to mA3 was indistinguishable from that of MoMLV. Thus, the Gag protein does not seem to control the sensitivity of MLVs to mA3. We also found that MLVs inactivated by mA3 do not synthesize viral DNA upon infection; thus mA3 restriction of MLV occurs before or at reverse transcription. In contrast, HIV-1 restricted by mA3 and MLVs restricted by human APOBEC3G do synthesize DNA; these DNAs exhibit APOBEC3-induced hypermutation

An Integrative Approach to Understanding Counterproductive Work Behavior: The Roles of Stressors, Negative Emotions, and Moral Disengagement

Several scholars have highlighted the importance of examining moral disengagement (MD) in understanding aggression and deviant conduct across different contexts. The present study investigates the role of MD as a specific social-cognitive construct that, in the organizational context, may intervene in the process leading from stressors to counterproductive work behavior (CWB). Assuming the theoretical framework of the stressor-emotion model of CWB, we hypothesized that MD mediates, at least partially, the relation between negative emotions in reaction to perceived stressors and CWB by promoting or justifying aggressive responses to frustrating situations or events. In a sample of 1,147 Italian workers, we tested a structural equations model. The results support our hypothesis: the more workers experienced negative emotions in response to stressors, the more they morally disengaged and, in turn, enacted CW

Combining BRAF inhibition with oncolytic herpes simplex virus enhances the immune-mediated antitumor therapy of BRAF-mutant thyroid cancer.

BACKGROUND: The aggressive clinical behavior of poorly differentiated and anaplastic thyroid cancers (PDTC and ATC) has proven challenging to treat, and survival beyond a few months from diagnosis is rare. Although 30%-60% of these tumors contain mutations in the BRAF gene, inhibitors designed specifically to target oncogenic BRAF have shown limited and only short-lasting therapeutic benefits as single agents, thus highlighting the need for improved treatment strategies, including novel combinations. METHODS: Using a BRAFV600E-driven mouse model of ATC, we investigated the therapeutic efficacy of the combination of BRAF inhibition and oncolytic herpes simplex virus (oHSV). Analyses of samples from tumor-bearing mice were performed to immunologically characterize the effects of different treatments. These immune data were used to inform the incorporation of immune checkpoint inhibitors into triple combination therapies. RESULTS: We characterized the immune landscape in vivo following BRAF inhibitor treatment and detected only modest immune changes. We, therefore, hypothesized that the addition of oncolytic virotherapy to BRAF inhibition in thyroid cancer would create a more favorable tumor immune microenvironment, boost the inflammatory status of tumors and improve BRAF inhibitor therapy. First, we showed that thyroid cancer cells were susceptible to infection with oHSV and that this process was associated with activation of the immune tumor microenvironment in vivo. Next, we showed improved therapeutic responses when combining oHSV and BRAF inhibition in vivo, although no synergistic effects were seen in vitro, further confirming that the dominant effect of oHSV in this context was likely immune-mediated. Importantly, both gene and protein expression data revealed an increase in activation of T cells and natural killer (NK) cells in the tumor in combination-treated samples. The benefit of combination oHSV and BRAF inhibitor therapy was abrogated when T cells or NK cells were depleted in vivo. In addition, we showed upregulation of PD-L1 and CTLA-4 following combined treatment and demonstrated that blockade of the PD-1/PD-L1 axis or CTLA-4 further improved combination therapy. CONCLUSIONS: The combination of oHSV and BRAF inhibition significantly improved survival in a mouse model of ATC by enhancing immune-mediated antitumor effects, and triple combination therapies, including either PD-1 or CTLA-4 blockade, further improved therapy