Pitavastatin prevents NMDA-induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.     

Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD(50)), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD(50) to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD(50) for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD(50) required for CTL to achieve 50% efficiency of infected cell killing [KE(50)]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE(50) despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE(50), demonstrating epitope dependence of KE(50). Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE(50). Thus, defining KE(50) values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.     

Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome

It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-alpha, IFN-gamma, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.     

Characterization of the metabolic activation of hepatitis C virus nucleoside inhibitor beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) and identification of a novel active 5'-triphosphate species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.     

Phorbol ester-induced shedding of the prostate cancer marker transmembrane protein with epidermal growth factor and two follistatin motifs 2 is mediated by the disintegrin and metalloproteinase-17

The transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed in prostate and brain and shed from the cell surface in a metalloproteinase-dependent fashion. Neither the sheddase(s) responsible for TMEFF2 shedding nor the physiological significance or activity of the soluble TMEFF2 ectodomain (TMEFF2-ECD) has been identified. In the present study we present new evidence that a disintegrin and metalloproteinase-17 (ADAM17) is responsible for phorbol 12-myristate 13-acetate-induced release of TMEFF2-ECD using small interfering RNA to ablate ADAM17 expression or by inhibiting enzymatic activity. A single well shedding assay monitoring the release of alkaline phosphatase-tagged TMEFF2-ECD into medium and the generation of 22- and 14-kDa C-terminal fragments in lysates were dependent on ADAM17 activity. A gamma-secretase inhibitor prevented the formation of a 10-kDa fragment in cell lysates, thus establishing TMEFF2 as a novel substrate for regulated intramembrane proteolysis. We assigned proliferation-inducing activity to TMEFF2. Inhibition of TMEFF2 shedding using synthetic metalloproteinase inhibitors or small interfering RNA targeting TMEFF2 expression yielded a statistically significant reduction of cell proliferation in the lymph node-derived prostate cancer cells (LNCaPs) and a human embryonic kidney (HEK293) cell line overexpressing TMEFF2. The TMEFF2-ECD was able to induce ERK1/2 phosphorylation in an epidermal growth factor receptor (or ErbB1)-dependent manner in HEK293 cells. Our data suggest that TMEFF2 contributes to cell proliferation in an ADAM17-dependent autocrine fashion in cells expressing this protein.     

A simplified approach to quasi-linear viscoelastic modeling

The fitting of quasi-linear viscoelastic (QLV) constitutive models to material data often involves somewhat cumbersome numerical convolution. A new approach to treating quasi-linearity in 1-D is described and applied to characterize the behavior of reconstituted collagen. This approach is based on a new principle for including nonlinearity and requires considerably less computation than other comparable models for both model calibration and response prediction, especially for smoothly applied stretching. Additionally, the approach allows relaxation to adapt with the strain history. The modeling approach is demonstrated through tests on pure reconstituted collagen. Sequences of "ramp-and-hold" stretching tests were applied to rectangular collagen specimens. The relaxation force data from the "hold" was used to calibrate a new "adaptive QLV model" and several models from literature, and the force data from the "ramp" was used to check the accuracy of model predictions. Additionally, the ability of the models to predict the force response on a reloading of the specimen was assessed. The "adaptive QLV model" based on this new approach predicts collagen behavior comparably to or better than existing models, with much less computation.     

A model-based parametric study of impact force during running

This paper deals with the impact force during foot-ground impact activities such as the running. A previously developed model is used for this study. The model is a lumped-parameter one consisting of four masses connected to each other via linear springs and viscous dampers. A shoe-specific nonlinear function is used for representation of the ground reaction force. The authors have previously showed that the previous version of the model as well as its simulation is incorrect. This paper slightly modifies the previous model so as it is able to produce results in agreement with the experiments. Then, the modified model is simulated for two typical shoe types. A parametric study is also conducted. The parametric study concerns with the effects of masses, mass ratios, stiffness constants, and damping coefficients on the dynamics of the impact. It is shown that the impact forces increase as the rigid and wobbling masses increase. However, the increase in the impact forces is not the same for all the masses. It is found that the impact force increases as the touchdown velocities increase. Simulations imply that the variations of the damping coefficients result in larger variations of the impact force compared to the stiffness. The effect of the variation of gravity on the simulated impact force is also explored. It is concluded that both the first and the second peaks of the impact force are increased with gravity. An in-depth discussion is included to compare results of the current paper with results of other investigators.     

CHIP chaperones wild type p53 tumor suppressor protein

Wild type p53 exists in a constant state of equilibrium between wild type and mutant conformation and undergoes conformational changes at elevated temperature. We have demonstrated that the co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein), which suppressed aggregation of several misfolded substrates and induced the proteasomal degradation of both wild type and mutant p53, physically interacts with the amino terminus of WT53 and prevented it from irreversible thermal inactivation. CHIP preferentially binds to the p53 mutant phenotype and restored the DNA binding activity of heat-denatured p53 in an ATP-independent manner. In cells under elevated temperatures that contained a higher level of p53 mutant phenotype, CHIP restored the native-like conformation of p53 in the presence of geldanamycin, whereas CHIP-small interfering RNA considerably increased the mutant form. Further, under elevated temperatures, the levels of CHIP and p53 were higher in nucleus, and chromatin immunoprecipitation shows the presence of p53 and CHIP together upon the DNA binding site in the p21 and p53 promoters. We propose that CHIP might be a direct chaperone of wild type p53 that helps p53 in maintaining wild type conformation under physiological condition as well as help resurrect p53 mutant phenotype into a folded native state under stress condition.     

Use of periodic abstinence in Bangladesh: do they really understand?

Data from a nationally representative study in Bangladesh (BDHS 1996-97) were analysed to identify significant predictors of use of periodic abstinence, in comparison with other modern contraceptive methods. The study found that women in Bangladesh mostly use modern methods during their peak reproductive years, after which some of them switch to periodic abstinence. These women tend to be more from educated and from higher socioeconomic backgrounds and with at least one living son. Another set of data from the Matlab DSS was analysed and the results were in the same direction. Focus group discussions found that women were using the periodic abstinence method incorrectly, abstaining for more days than is necessary. For Bangladeshi contraceptive users to reach a higher degree of use-effectiveness for period abstinence, more IEC materials need to be developed.     

Susceptibility of bollworm and tobacco budworm (Lepidoptera: Noctuidae) to Cry2Ab2 insecticidal protein

Susceptibilities of 82 bollworm, Helicoverpa zea (Boddie), and 44 tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), populations to Cry2Ab2 protein were measured in diet incorporated assays at the University of Arkansas from 2002 to 2005. Resulting data were used to calculate overall (pooled data) estimates of species susceptibility for future benchmarks of resistance. Variabilities among populations also were studied by comparing regressions for individual populations and calculating mean susceptibilities for different subgroups of the colonies studied. Individual lethal concentration (LC50) estimates for nine laboratory, seven laboratory-cross, and 28 field populations of H. virescens varied up to 48-fold when adjusted for the response of the most susceptible laboratory colony studied. Mean susceptibilities of all laboratory, laboratory-cross, or field colonies varied only two-fold. When grouped by host plants, populations collected on tobacco, Nicotiana tabacum (L.), seemed to be less susceptible than those collected on other host plants. Individual LC50 values for 82 laboratory, laboratory-cross and field populations of H. zea varied up to 37-fold. Mean LC50 values of all laboratory, laboratory-cross, or field populations varied only three-fold. Susceptibilities of populations from Bollgard cotton were up to four-fold less than those from Bacillus thuringiensis corn, Zea mays L. Field populations collected during late season were generally less susceptible than those collected early in the season. Across the two species, H. zea was less sensitive to Cry2Ab2 than H. virescens. Both species seem to be less sensitive to Cry2Ab2 than to CrylAc.