Novel properties of statins: suppression of the systemic and bone marrow responses induced by exposure to ambient particulate matter (PM10) air pollution

Exposure to ambient particulate matter (PM(10)) elicits systemic inflammatory responses that include the stimulation of bone marrow and progression of atherosclerosis. The present study was designed to assess the effect of repeated exposure of PM(10) on the turnover and release of polymorphonuclear leukocytes (PMNs) from the bone marrow into the circulation and the effect of lovastatin on the PM(10)-induced bone marrow stimulation. Rabbits exposed to PM(10) three times a week for 3 wk, were given a bolus of 5'-bromo-2'-deoxyuridine to label dividing cells in the marrow to calculate the transit time of PMNs in the mitotic or postmitotic pool. PM(10) exposure accelerated the turnover of PMNs by shortening their transit time through the marrow (64.8 ± 1.9 h vs. 34.3 ± 7.4 h, P < 0.001, control vs. PM(10)). This was predominantly due to a rapid transit of PMNs through the postmitotic pool (47.9 ± 0.7 h vs. 21.3 ± 4.3 h, P < 0.001, control vs. PM(10)) but not through the mitotic pool. Lovastatin delayed the transit time of postmitotic PMNs (38.2 ± 0.5 h, P < 0.001 vs. PM(10)) and shifted the postmitotic PMN release peak from 30 h to 48 h. PM(10) exposure induced the prolonged retention of newly released PMNs in the lung, which was reduced by lovastatin (P < 0.01). PM(10) exposure increased plasma interleukin-6 levels with significant reduction by lovastatin (P < 0.01). We conclude that lovastatin downregulates the PM(10)-induced overactive bone marrow by attenuating PM(10)-induced systemic inflammatory responses.     

Synchronous fluorescence, UV-visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

Constant wavelength synchronous fluorescence spectroscopy (CW-SFS), UV-visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)(2)](2+)) and a fluorescence probe, neutral red dye (NR), in a tris-hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)(2)](2+)and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)(2)](2+) decreased on addition of the DNA and the intercalated [Cu(phen)(2)](2+)-DNA complex formed (beta = (4.14 +/- 0.24) x 10(3)). CW-SFS measurements were facilitated by the use of the three-way resolution of the CW-SFS for NR, [Cu(phen)(2)](2+), and NR-DNA. The important constant wavelength (CW) interval, Deltalambda, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR-DNA, and [Cu(phen)(2)](2+), respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Deltalambda is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW-SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)(2)](2+), and NR-DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)(2)](2+) molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)(2)](2+), approximately 50% of NR was replaced by [Cu(phen)(2)](2+) at a concentration of 0.45 x 10(-5) mol L(-1), and nearly all of the NR was replaced at a [Cu(phen)(2)](2+) concentration of 2.50 x 10(-5) mol L(-1). This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.     

The immunosuppressant cyclosporin A antagonizes human formyl peptide receptor through inhibition of cognate ligand binding

Cyclosporin A (CsA) is a fungus-derived cyclic undecapeptide with potent immunosuppressive activity. Its analog, cyclosporin H (CsH), lacks immunosuppressive function but can act as an antagonist for the human formyl peptide receptor (FPR). More recent studies have shown that CsA also inhibits fMLF-induced degranulation in differentiated HL-60 promyelocytic leukemia cells. However, it is unclear whether CsA interferes with ligand-receptor interaction, G protein activation, or other downstream signaling events. In this study we used human neutrophils, differentiated HL-60 cells, and rat basophilic leukemia (RBL)-2H3 cells expressing human FPR (RBL-FPR) to identify the action site of CsA. In functional assays, CsA inhibited fMLF-stimulated degranulation, chemotaxis, calcium mobilization, and phosphorylation of the MAPKs ERK 1/2 and the serine/threonine protein kinase Akt. CsA also blocked Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm)-induced functions in RBL-FPR cells. Concentrations for half-maximal inhibition with CsA are generally 6- to 50-fold higher than that of CsH. CsA was compared with another immunosuppressant, ascomycin, relative to the inhibitory effects on FPR-mediated chemotaxis, calcium mobilization, and degranulation. In these experiments, ascomycin produced no inhibitory effects at low micromolar concentrations (1-4 microM), whereas the inhibitory effects of CsA were prominent at comparable concentrations. Finally, CsA dose-dependently inhibited the uptake of fNle-Leu-Phe-Nle-Tyr-Lys-fluoresceine and [3H]fMLF or [125I]WKYMVm binding to FPR. However, CsA and CsH did not show any obvious inhibitory effect on FPR-like 1-mediated cellular functions. These results demonstrate that CsA is a selective antagonist of FPR and that its inhibition of fMLF-stimulated leukocyte activation is at the level of cognate ligand binding.     

Enhanced regulatory T cell activity is an element of the host response to injury

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-beta1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.     

Genetic variation in Lumnitzera racemosa, a mangrove species from the Indo-West Pacific

The genetic structure of 18 populations of Lumnitzera racemosa from the Indo-West Pacific, including South China, Malay Peninsula, Sri Lanka, and North Australia, was assessed by inter simple sequence repeat (ISSR) markers. Our results showed a relatively high level of genetic variation at the species level (P = 87.04%, H_e = 0.260). The value of G_st was 0.642, suggesting significant genetic differentiation among populations. At the population level, however, genetic diversity was low (P = 32.17%, H_e = 0.097). When populations were grouped according to geographic regions, i.e., South China Sea, the East Indian Ocean, and North Australia, it was inferred from AMOVA that more than half the total variation (55.37%) was accounted for by differentiation between regions. A UPGMA dendrogram based on genetic distance also revealed a deep split between populations from these regions, indicating that Malay Peninsula and the Indonesia archipelago may play an important part on the genetic differentiation in L. racemosa. The high degree of population differentiation between regions and low genetic variation within populations recorded here highlights the need for appropriate conservation measures for this species, both in terms of incorporating further populations into protected areas, and the restoration strategies for separate regions.     

Identification and modification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from Ran antigen

Ran is considered to be a promising target for tumor-specific immunotherapy because its protein is exclusively expressed in tumor tissues, though its mRNA can be expressed in most normal tissues. In our study, we obtained four candidate wild-type epitopes designated Ran1, Ran2, Ran3, and Ran4, derived from the Ran antigen with the highest predicted affinity with MHC-I, indicated by affinity prediction plots and molecular dynamics simulation. However, in vitro affinity assays of these epitopes showed only a moderate affinity with MHC-I. Thus, we designed altered peptide ligands (APLs) derived from Ran wild-type epitopes with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. Of the eight tested peptides, the 1Y analog had the strongest binding-affinity and lowest-dissociation rate to HLA-A*0201. Additionally, we investigated the CTLs activities induced by Ran wild-type peptides and the APLs in human PBMCs and in HLA-A*0201/K^b transgenic mice. Ran1 1Y was superior to other APLs and wild-type peptides in eliciting epitope-specific CTL immune responses both in vitro and in vivo. In summary, a wild-type epitope of the tumor-specific antigen Ran, expressed broadly in many tumors, was identified and designated Ran1. An APL of Ran1, Ran1 1Y, was further designed and verified in vitro and in vivo and found to elicit a stronger Ran-specific CTL response, indicating a potential anti-tumor application in the future.     

A non‐redundant role for MKP5 in limiting ROS production and preventing LPS‐induced vascular injury

There are at least 11 mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and only 3 major groups of MAPKs, raising the question of whether these phosphatases have non-redundant functions in vivo. Using a modified mouse model of local Shwartzman reaction, we found that deletion of the MKP5 gene, but not the MKP1 gene, led to robust and accelerated vascular inflammatory responses to a single dose of LPS injection. Depletion of neutrophils significantly reduced the vascular injury in Mkp5^−/− mice, whereas adoptive transfer of Mkp5^−/− neutrophils replicated the LPS-induced skin lesions in wild-type recipients. Neutrophils isolated from Mkp5^−/− mice exhibited augmented p38 MAPK activation and increased superoxide generation on activation. The p38 MAPK inhibitor, SB203580, significantly reduced p47^phox phosphorylation and diminished superoxide production in neutrophils. p38 MAPK phosphorylated mouse p47^phox, and deletion of the p47^phox gene ablated the LPS-induced vascular injury in Mkp5^−/− mice. Collectively, these results show an earlier unrecognized and non-redundant function of MKP5 in restraining p38 MAPK-mediated neutrophil oxidant production, thereby preventing LPS-induced vascular injury.     

Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells

Wnt/β‐catenin pathway plays an important role in regulating embryonic development. Hepatocytes differentiate from endoderm during development. Hepatic progenitor cells (HPCs) have been isolated from fetal liver and extrahepatic tissues. Most current studies in liver development and hepatic differentiation have been focused on Wnts, β‐catenin, and their receptors. Here, we sought to determine the role of Wnt antagonists in regulating hepatic differentiation of fetal liver‐derived HPCs. Using mouse liver tissues derived from embryonic day E12.5 to postnatal day (PD) 28, we found that 13 of the 19 Wnt genes and almost all of Wnt receptors/co‐receptors were expressed in most stages. However, Wnt antagonists SFRP2, SFRP3, and Dkk2 were only detected in the early stages. We established and characterized the reversible stable HPCs derived from E14.5 mouse fetal liver (HP14.5). HP14.5 cells were shown to express high levels of early liver progenitor cell markers, but low levels or none of late liver markers. HP14.5 cells were shown to differentiate into mature hepatocytes upon dexamethasone (Dex) stimulation. Dex‐induced late marker expression and albumin promoter activity in HP14.5 cells were inhibited by exogenous expression of SFRP3. Furthermore, Dex‐induced glycogen synthesis of PAS‐positive HP14.5 cells was significantly inhibited by SFRP3. Therefore, our results have demonstrated that the expression of Wnt antagonists decreases as hepatic differentiation progresses, suggesting that a balanced Wnt signaling may be critical during mouse liver development and hepatic differentiation. J. Cell. Biochem. 108: 295–303, 2009. © 2009 Wiley‐Liss, Inc.