Regulation of RhoA by STAT3 coordinates glial scar formation.

Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3+/- mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.

Stabbing simulations and DNA transfer.

Technical developments have made it possible to analyze very low amounts of DNA. This has many advantages, but the drawback of this technological progress is that interpretation of the results becomes increasingly complex: the number of mixed DNA profiles increased relatively to single source DNA profiles and stochastic effects in the DNA profile, such as drop-in and drop-out, are more frequently observed. Moreover, the relevance of low template DNA material regarding the activities alleged is not as straightforward as it was a few years ago, when for example large quantities of blood were recovered. The possibility of secondary and tertiary transfer is now becoming an issue. The purpose of this research is twofold: first, to study the transfer of DNA from the handler and secondly, to observe if handlers would transfer DNA from persons closely connected to them. We chose to mimic cases where the offender would attack a person with a knife. As a first approach, we envisaged that the defense would not give an alternative explanation for the origin of the DNA. In our transfer experiments (4 donors, 16 experiments each, 64 traces), 3% of the traces were single DNA profiles. Most of the time, the DNA profile of the person handling the knife was present as the major profile: in 83% of the traces the major contributor profile corresponded to the stabber's DNA profile (in single stains and mixtures). Mixture with no clear major/minor fraction (12%) were observed. 5% of the traces were considered of insufficient quality (more than 3 contributors, presence of a few minor peaks). In that case, we considered that the stabber's DNA was absent. In our experiments, no traces allowed excluding the stabber, however it must be noted that precautions were taken to minimize background DNA as knives were cleaned before the experiments. DNA profiles of the stabber's colleagues were not observed. We hope that this study will allow for a better understanding of the transfer mechanism and of how to assess and describe results given activity level propositions. In this preliminary research, we have focused on the transfer of DNA on the hand of the person. Besides, more research is needed to assign the probability of the results given an alternative activity proposed by the defense, for instance when the source of the DNA is not contested, but that the activities are.

Deletion of N-myc downstream-regulated gene 2 attenuates reactive astrogliosis and inflammatory response in a mouse model of cortical stab injury.

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the CNS. In this study, we examined the expression and the role of Ndrg2 after cortical stab injury. We observed that Ndrg2 expression was elevated in astrocytes surrounding the wounded area as early as day 1 after injury in wild-type mice. Deletion of Ndrg2 resulted in lower induction of reactive astroglial and microglial markers in the injured cortex. Histological analysis showed reduced levels of hypertrophic changes in astrocytes, accumulation of microglia, and neuronal death in Ndrg2(-/-) mice after injury. Furthermore, activation of the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway, including the expression of IL-6 family cytokines and phosphorylation of STAT3, was markedly reduced in Ndrg2(-/-) mice after injury. In a culture system, both of Il6 and Gfap were up-regulated in wild-type astrocytes treated with forskolin. Deletion of Ndrg2 attenuated induction of these genes, but did not alter proliferation or migration of astrocytes. Adenovirus-mediated reexpression of Ndrg2 rescued the reduction of IL-6 expression after forskolin stimulation. These findings suggest that Ndrg2 plays a key role in reactive astrogliosis after cortical stab injury through a mechanism involving the positive regulation of IL-6/STAT3 signaling.

Profiling circulating microRNA expression in experimental sepsis using cecal ligation and puncture.

The levels of circulating microRNAs (miRNAs) in mice with experimental sepsis induced by cecal ligation and puncture (CLP) were determined using whole blood samples obtained from C57BL/6 mice at 4, 8, and 24 h after CLP; miRNA expression analysis was performed in these samples using an miRNA array. Microarray analysis revealed upregulation of 10 miRNA targets (miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451). The expression of these miRNA targets in the whole blood, serum, and white blood cells (WBCs) of CLP mice was quantified using quantitative real-time PCR; these values were compared to those in sham-operated C57BL/6 mice, and the results indicated that these miRNA targets were significantly up-regulated in the whole blood and serum but not in the WBCs. In addition, the levels of these 10 miRNA targets in the serum of Tlr2-/-, Tlr4-/-, and NF-κB-/- mice at 8 h after CLP did not decrease significantly., which indicated that the transcription of these miRNAs was not directly mediated by the TLR2/NF-κB or TLR4/NF-κB pathway, and pathways induced by exposure to the gram-positive or gram-negative bacteria. Immunoprecipitation with the Argonaute 2 ribonucleoprotein complex revealed significantly increased expression of the 10 miRNA targets in the serum of mice after CLP, and the levels of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of these 10 miRNA targets increased significantly in exosomes isolated using ExoQuick precipitation solution. In this study, we identified circulating miRNAs that were up-regulated after CLP and determined the increase in the levels of these miRNAs, and our results suggest that circulating Ago2 complexes and exosomes may be responsible for the stability of miRNAs in the serum.

Long-term gene expression profile dynamics following cecal ligation and puncture in the rat.

BACKGROUND: Despite the fact that the treatment options for septic patients have been significantly improved, the pathophysiologic changes caused by various septic cases have not been well understood. One commonly observed clinical phenomenon is the onset of a polymicrobial infection caused by bacteria that originate in the intestine but enter the peritoneum via translocation from the gut. This triggers a systemic inflammatory response via the innate immune system, which needs to be well characterized. Cecal ligation and puncture (CLP) is considered to be the gold-standard animal model by establishing infection with mixed bacterial flora and necrotic tissue to induce an inflammatory response. The aim of this study is to analyze the long-term gene expression dynamics in the rats subject to CLP in order to characterize the impact of sepsis upon liver function over an 8-d time period. METHODS: Rats received CLP or its control, sham CLP (SCLP), and then they were sacrificed at 9 am on days 0 (no treatment), 1, 2, 5, and 8 post injury to collect liver samples for microarray analysis. Differentially expressed probe sets in CLP versus SCLP (q value <0.001 and P value <0.001) were combined to form one single matrix, which was then clustered using the approach of "consensus clustering" to identify subsets of transcripts with coherent expression patterns. Finally, the gene expression patterns of the clusters were further transformed into principal components, which account for 65% of the total data. RESULTS: Three major clusters were obtained. The first cluster, which is mainly related to genes of anti-inflammatory response and antioxidative properties, is suppressed early in the CLP condition and later upregulated compared to the SCLP condition. Cluster 2 represents pro-inflammatory responses and signaling, along with amino acid metabolism. Cluster 3 is also associated with pro-inflammatory response. The genes of toll-like receptor signaling and hypermetabolism were identified in this cluster as well. Clusters 2 and 3 are both suppressed in the long-term response following CLP. Clusters 1 and 2 acting in concert return to the time 0 baseline in both groups, indicating resolution of both the anti-inflammatory and pro-inflammatory response; however, the SCLP response in cluster 3 shows persistent downregulation. CONCLUSIONS: Characterization of long-term hepatic responses to injury is critical to understanding the dynamics of transcriptional changes following the induction of the inflammatory response, and to monitoring its effective resolution. These results showed that each condition has unique dynamics that indicate fundamental differences in the response. Furthermore, the gene ontologies suggest a link to oxidative stress over the long term that may be able to be explored for clinical treatments.

Dynamics of hepatic gene expression profile in a rat cecal ligation and puncture model.

BACKGROUND: Sepsis remains a major clinical challenge in intensive care units. The difficulty in developing new and more effective treatments for sepsis exemplifies our incomplete understanding of the underlying pathophysiology of it. One of the more widely used rodent models for studying polymicrobial sepsis is cecal ligation and puncture (CLP). While a number of CLP studies investigated the ensuing systemic inflammatory response, they usually focus on a single time point post-CLP and therefore fail to describe the dynamics of the response. Furthermore, previous studies mostly use surgery without infection (herein referred to as sham CLP, SCLP) as a control for the CLP model, however, SCLP represents an aseptic injurious event that also stimulates a systemic inflammatory response. Thus, there is a need to better understand the dynamics and expression patterns of both injury- and sepsis-induced gene expression alterations to identify potential regulatory targets. In this direction, we characterized the response of the liver within the first 24 h in a rat model of SCLP and CLP using a time series of microarray gene expression data. METHODS: Rats were randomly divided into three groups: sham, SCLP, and CLP. Rats in SCLP group are subjected to laparotomy, cecal ligation, and puncture while those in CLP group are subjected to the similar procedures without cecal ligation and puncture. Animals were saline resuscitated and sacrificed at defined time points (0, 2, 4, 8, 16, and 24 h). Liver tissues were explanted and analyzed for their gene expression profiles using microarray technology. Unoperated animals (Sham) serve as negative controls. After identifying differentially expressed probesets between sham and SCLP or CLP conditions over time, the concatenated data sets corresponding to these differentially expressed probesets in sham and SCLP or CLP groups were combined and analyzed using a "consensus clustering" approach. Promoters of genes that share common characteristics were extracted and compared with gene batteries comprised of co-expressed genes to identify putatative transcription factors, which could be responsible for the co-regulation of those genes. RESULTS: The SCLP/CLP genes whose expression patterns significantly changed compared with sham over time were identified, clustered, and finally analyzed for pathway enrichment. Our results indicate that both CLP and SCLP triggered the activation of a proinflammatory response, enhanced synthesis of acute-phase proteins, increased metabolism, and tissue damage markers. Genes triggered by CLP, which can be directly linked to bacteria removal functions, were absent in SCLP injury. In addition, genes relevant to oxidative stress induced damage were unique to CLP injury, which may be due to the increased severity of CLP injury versus SCLP injury. Pathway enrichment identified pathways with similar functionality but different dynamics in the two injury models, indicating that the functions controlled by those pathways are under the influence of different transcription factors and regulatory mechanisms. Putatively identified transcription factors, notably including cAMP response element-binding (CREB), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and signal transducer and activator of transcription (STAT), were obtained through analysis of the promoter regions in the SCLP/CLP genes. Our results show that while transcription factors such as NF-κB, homeodomain transcription factor (HOMF), and GATA transcription factor (GATA) were common in both injuries for the IL-6 signaling pathway, there were many other transcription factors associated with that pathway which were unique to CLP, including forkhead (FKHD), hairy/enhancer of split family (HESF), and interferon regulatory factor family (IRFF). There were 17 transcription factors that were identified as important in at least two pathways in the CLP injury, but only seven transcription factors with that property in the SCLP injury. This also supports the hypothesis of unique regulatory modules that govern the pathways present in both the CLP and SCLP response. CONCLUSIONS: By using microarrays to assess multiple genes in a high throughput manner, we demonstrate that an inflammatory response involving different dynamics and different genes is triggered by SCLP and CLP. From our analysis of the CLP data, the key characteristics of sepsis are a proinflammatory response, which drives hypermetabolism, immune cell activation, and damage from oxidative stress. This contrasts with SCLP, which triggers a modified inflammatory response leading to no immune cell activation, decreased detoxification potential, and hyper metabolism. Many of the identified transcription factors that drive the CLP-induced response are not found in the SCLP group, suggesting that SCLP and CLP induce different types of inflammatory responses via different regulatory pathways.

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation.

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP(+) astrocytes (21% of FABP7(+) cells) and NG2(+) oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7(+)/NG2(+) cells, while there was a significant increase in FABP7(+)/GFAP(+) cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU(+) astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.

Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase.

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin.

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.

[Human genotyposcopy: the identification of the species, sex and individual by DNA genetic imprints in a case connected with a murder attempt]. / Genotiposkopiia cheloveka: identifikatsiia vida, pola i lichnosti po geneticheskim otpechatkam DNK v sluchae, sviazannom s pokusheniem na ubiistvo.

Blood stains on a knife were identified by DNA genotyposcopy. The statistical validation method has confirmed that the blood stains on material evidence belonged to the victim, the probability of random coincidence being less than 10(-11). The efficacy of using hypervariable locus-specific DNA probes and the possibility of detecting DNA impressions in blood stains stored for more than 3 months have been demonstrated.