Irf3 polymorphism alters induction of interferon beta in response to Listeria monocytogenes infection

Genetic makeup of the host plays a significant role in the course and outcome of infection. Inbred strains of mice display a wide range of sensitivities to Listeria monocytogenes infection and thus serve as a good model for analysis of the effect of genetic polymorphism. The outcome of L. monocytogenes infection in mice is influenced by the ability of this bacterium to induce expression of interferon beta mRNA, encoded in mouse by the Ifnb1 (interferon beta 1, fibroblast) gene. Mouse strains that lack components of the IFN beta signaling pathway are substantially more resistant to infection. We found that macrophages from the ByJ substrain of the common C57BL/6 inbred strain of mice are impaired in their ability to induce Ifnb1 expression in response to bacterial and viral infections. We mapped the locus that controls differential expression of Ifnb1 to a region on Chromosome 7 that includes interferon regulatory factor 3 (Irf3), which encodes a transcription factor responsible for early induction of Ifnb1 expression. In C57BL/6ByJ mice, Irf3 mRNA was inefficiently spliced, with a significant proportion of the transcripts retaining intron 5. Analysis of the Irf3 locus identified a single base-pair polymorphism and revealed that intron 5 of Irf3 is spliced by the atypical U12-type spliceosome. We found that the polymorphism disrupts a U12-type branchpoint and has a profound effect on the efficiency of splicing of Irf3. We demonstrate that a naturally occurring change in the splicing control element has a dramatic effect on the resistance to L. monocytogenes infection. Thus, the C57BL/6ByJ mouse strain serves as an example of how a mammalian host can counter bacterial virulence strategies by introducing subtle alteration of noncoding sequences

Application of serex-analysis for identification of human colon cancer antigens

Copyright © Experimental Oncology, 2015. Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II-IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREXtechnique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer

Spinal cord molecular and cellular changes induced by adenoviral vector- and cell-mediated triple gene therapy after severe contusion

© 2017 Izmailov, Povysheva, Bashirov, Sokolov, Fadeev, Garifulin, Naroditsky, Logunov, Salafutdinov, Chelyshev, Islamov and Lavrov. The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1)

Covalent Aurora A regulation by the metabolic integrator coenzyme A

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues

Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation

Application of serex-analysis for identification of human colon cancer antigens

Copyright © Experimental Oncology, 2015. Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II-IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREXtechnique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer

Application of serex-analysis for identification of human colon cancer antigens

Copyright © Experimental Oncology, 2015. Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II-IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREXtechnique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer

Application of serex-analysis for identification of human colon cancer antigens

Copyright © Experimental Oncology, 2015. Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II-IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREXtechnique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer

Combined supra-and sub-lesional epidural electrical stimulation for restoration of the motor functions after spinal cord injury in mini pigs

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This study evaluates the effect of combined epidural electrical stimulation (EES) applied above (C5) and below (L2) the spinal cord injury (SCI) at T8–9 combined with motor training on the restoration of sensorimotor function in mini pigs. The motor evoked potentials (MEP) induced by EES applied at C5 and L2 levels were recorded in soleus muscles before and two weeks after SCI. EES treatment started two weeks after SCI and continued for 6 weeks led to improvement in multiple metrics, including behavioral, electrophysiological, and joint kinematics outcomes. In control animals after SCI a multiphasic M-response was observed during M/H-response testing, while animals received EES-enable training demonstrated the restoration of the M-response and H-reflex, although at a lower amplitude. The joint kinematic and assessment with Porcine Thoracic Injury Behavior scale (PTIBS) motor recovery scale demonstrated improvement in animals that received EES-enable training compared to animals with no treatment. The positive effect of two-level (cervical and lumbar) epidural electrical stimulation on functional restoration in mini pigs following spinal cord contusion injury in mini pigs could be related with facilitation of spinal circuitry at both levels and activation of multisegmental coordination. This approach can be taken as a basis for the future development of neuromodulation and neurorehabilitation therapy for patients with spinal cord injury