Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages

Background and purpose:

Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.

Experimental approach:

Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.

Key results:

Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-κB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.

Conclusions and implications:

Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.     

Pharmacological effects of cannabinoids on the reference and working memory functions in mice

Rationale

Evidence indicates cannabinoid receptor agonists impair performance in procedures to assess memory that may also be confounded by motivational or motor effects, both of which occur with cannabinoids. Thus, convergence of evidence from a variety of procedures that differ in motivation, attention, arousal and response requirements, but share a common reliance on memory, is required. There are no current reports on cannabinoid effects on mice tested in the radial arm maze.

Objectives

The objective was to determine the effects of the cannabinoid agonist CP 55940 and the dependence of any such effects on the CB1 receptor using the CB1 receptor antagonist SR 141716A on two strains of mice in the eight-arm radial maze procedure.

Methods

Male C57BL/6J (N?=?36) and C3H/HEJ (N?=?12) mice were trained to a criterion and then were treated (IP) with vehicle?+?vehicle, SR 141716A?+?vehicle, vehicle?+?CP 55940 and SR 141716A?+?CP 55940 in a fully balanced mixed design prior to further tests in the maze. Reference (long-term) and working (short-term) memory were assessed.

Results

CP 55940 impaired performance of the reference memory task in the C57BL/6J strain but not the C3H/HEJ strain; SR 141716A reversed the effect of CP 55940 on these measures. CP 55940 also increased working memory errors in the C57BL/6J mice only, which was not affected by SR 141716A.

Conclusion

The present study provides evidence for a strain-specific effect of a dose of CP 55940 on reference memory. While the cannabinoid agonist also impaired working memory in one strain, this effect was apparently not mediated by CB1 receptors.     

The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling

Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies.     

Plasma osteoprotegerin levels increase with the severity of cerebral artery atherosclerosis

Objectives

Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily and suggested as a marker of atherosclerosis. We investigated whether plasma OPG levels were associated with the presence and severity of cerebral atherosclerosis.

Design and methods

We used an enzyme-linked immunosorbent assay to measure the plasma OPG levels of 107 patients with acute cerebral infarction. We compared the plasma OPG levels according to the presence and number of arteries with cerebral atherosclerosis (≥ 50% stenosis).

Results

Of 107 patients, 73 (68.2%) had cerebral atherosclerosis. OPG levels were increased in patients with cerebral atherosclerosis (374.69 ± 206.48 vs 261.17 ± 166.91 pg/mL, p = 0.006). OPG levels showed positive correlation with the number of cerebral arteries with atherosclerosis (Spearman's rho = 0.342, p < 0.001). After adjustment for vascular risk factors, OPG > 229.9 pg/mL was independently associated with the presence [OR 4.61, 95% CI 1.57–13.55, p = 0.005, binary logistic regression] of cerebral atherosclerosis and number [OR 3.20, 95% CI 1.26–8.12, p = 0.014, ordinal logistic regression] of arteries with cerebral atherosclerosis.

Conclusions

Plasma OPG levels were significantly associated with the presence and severity of cerebral atherosclerosis. This finding suggests that plasma OPG might have a role in cerebral atherosclerosis.     

The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases

We describe a revised and expanded database on human intermediate filament proteins, a major component of the eukaryotic cytoskeleton. The family of 70 intermediate filament genes (including those encoding keratins, desmins, and lamins) is now known to be associated with a wide range of diverse diseases, at least 72 distinct human pathologies, including skin blistering, muscular dystrophy, cardiomyopathy, premature aging syndromes, neurodegenerative disorders, and cataract. To date, the database catalogs 1,274 manually-curated pathogenic sequence variants and 170 allelic variants in intermediate filament genes from over 459 peer-reviewed research articles. Unrelated cases were collected from all of the six sequence homology groups and the sequence variations were described at cDNA and protein levels with links to the related diseases and reference articles. The mutations and polymorphisms are presented in parallel with data on protein structure, gene, and chromosomal location and basic information on associated diseases. Detailed statistics relating to the variants records in the database are displayed by homology group, mutation type, affected domain, associated diseases, and nucleic and amino acid substitutions. Multiple sequence alignment algorithms can be run from queries to determine DNA or protein sequence conservation. Literature sources can be interrogated within the database and external links are provided to public databases. The database is freely and publicly accessible online at www.interfil.org (last accessed 13 September 2007). Users can query the database by various keywords and the search results can be downloaded. It is anticipated that the Human Intermediate Filament Database (HIFD) will provide a useful resource to study human genome variations for basic scientists, clinicians, and students alike.     

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs.     

Imaging and quantification of metastatic melanoma cells in lymph nodes with a ferritin MR reporter in living mice

Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. This study demonstrates the feasibility of using the MR reporter ferritin for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice. A B16F10 murine melanoma cell line expressing human ferritin heavy chain (hFTH) and green fluorescent protein (GFP) was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4‐T MRI in vitro and in vivo, yielding significant changes in T₂* relative to control cells. In BALB/c nude mice, the presence of hFTH‐ and GFP‐expressing metastatic melanoma cells in deep‐seated axillary LNs was demonstrated as areas of low T₂* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T₂* MRI data, correlated well with tumor burden, which was determined by histology (r = ?0.8773, p = 0.0001). This study is the first to use MRI and an MR reporter gene for both the visualization and quantification of metastatic cancer cells in LNs. Copyright © 2011 John Wiley & Sons, Ltd.     

Current status of pediatric umbilical cord blood transplantation in Korea: a multicenter retrospective analysis of 236 cases

We report the outcome of 236 pediatric umbilical cord blood transplantations (UCBT) performed in Korea. Given that the sources of the grafts were mostly unrelated donors (n = 226; 95.8%), only the results of unrelated UCBT were included for all statistics. The most frequent primary disease was acute leukemia (n = 167). In total, 91.7% of recipients were seropositive for cytomegalovirus (CMV). The median doses of nucleated cells and CD34+ cells were 4.84 × 10(7)/kg and 2.00 × 10(5)/kg, respectively. The median times to neutrophil (>0.5 × 10(9)/L) and platelet recovery (>20 × 10(9)/L) were 18 and 45 days, respectively. Grade 2-4 acute graft-versus-host-disease (GVHD) and chronic GVHD developed in 41.1 and 36.1% of cases, respectively. Forty-five patients developed CMV disease. The 5-year overall and event-free survival were 47.5 and 36.9%, respectively. Multivariate analysis revealed that adverse factors for survival of the whole cohort were total body irradiation-based conditioning (P = 0.007), salvage transplant (P = 0.001), failure to achieve early complete chimerism (P < 0.0005), and CMV disease (P = 0.001). The outcomes of the single- and double-unit UCBT (n = 64) were similar, while double-unit recipients were heavier (P < 0.0005) and older (P < 0.0005). We conclude that double-unit UCBT is a reasonable option for older or heavier children and that the thorough surveillance of CMV infection and the development of an effective CMV therapeutic strategy may be especially important for Korean children, whose CMV seroprevalence exceeds 90%.