Genetic labeling reveals altered turnover and stability of innate lymphocytes in latent mouse cytomegalovirus infection.

Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B-eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H(+) and Ly49H(-) NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were "legacy" (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.

Five-year longitudinal bone evaluations in individuals with chronic complete spinal cord injury.

BACKGROUND/OBJECTIVES: Knowledge of spinal cord injury (SCI) bone changes has been derived primarily through cross-sectional studies, many of which are controvertible. Longitudinal studies are sparse, and long-term longitudinal chronic studies are unavailable. The objective of this study was to provide a clearer perception of chronic longitudinal bone variations in people with complete SCI. METHODS: Bone status of 31 individuals with chronic, complete SCI was assessed twice using dual-energy xray absorptiometry at an average interval of 5.06 +/- 0.9 years. Because the sample of women was small (4), the primary analyses of change and comparisons of those with paraplegia vs tetraplegia were confined to the male participants. RESULTS: Spine Z-scores showed a significant increase (P < 0.0001). The average Z-scores, initial and followup, were within the normal range. Hip Z-scores also showed a significant increase (P < 0.0001), and hip bone mineral density (BMD) increased in 48% of the participants. Knee BMD and lower extremity total bone mineral showed significant decreases (P < 0.003 and P < 0.02, respectively), but increases were seen in 33% and 26% at the respective sites. Individuals with tetraplegia had significantly lower values across all regions (P < 0.0001), and changes were significantly different compared with paraplegia (P < 0.0001). Bone values and changes in men vs women, despite the small sample of women, showed highly significant differences (P < 0.003-0.002). CONCLUSION: Chronic effects of complete SCI do not exclusively result in continued loss of BMD or a static state of lowered BMD; gain in BMD may occur. The nature and magnitude of the effects of complete SCI on BMD vary by site, with sex and level of injury, which has implications for treatment and its assessment.

Histamine stimulation of MMP-1(collagenase-1) secretion and gene expression in gastric epithelial cells: role of EGFR transactivation and the MAP kinase pathway.

BACKGROUND AND AIMS: GPCR stimulation by various ligands including histamine has been shown to transactivate the epidermal growth factor receptor (EGFR). This study examines the functional interactions between the H2 receptor and the EGFR in the regulation of matrix metalloproteinase-1 (MMP-1) secretion and gene expressions in cultured gastric epithelial cells. METHODS: AGS cells were incubated for up to 24 h with either histamine or heparin binding-epidermal growth factor (HB-EGF) and MMP-1 release was determined by immunoassay. MMP-1 responses to histamine and HB-EGF were further tested by the use of H2 receptor antagonist, EGFR inhibitor and mitogen activator protein kinase (MAPK) inhibitor. The role of EGFR in MMP-1 release was further tested in cells transfected with specific EGFR siRNA. EGFR and ERK1/2 phosphorylation was determined by Western blot analysis. MMP-1 gene expression was determined by RNase protection assay (RPA). RESULTS: Histamine and HB-EGF caused a dose-dependent release of MMP-1 with maximal responses that were 2.7- and 4.5-fold greater, respectively, than control, P<0.001. Famotidine prevented histamine-mediated MMP-1 release and AG1478 and EGFR siRNA completely inhibited MMP-1 secretion stimulated by both histamine and HB-EGF. Both histamine and HB-EGF stimulation of MMP-1 release was associated with activation of ERK1/2. MAPK inhibition also prevented histamine-and HB-EGF-induced MMP-1 secretion. Results of MMP-1 gene expression, either stimulatory or inhibitory, paralleled responses to MMP-1 secretion. CONCLUSION: Histamine stimulation of the H2 receptor on AGS cells evoked MMP-1 secretion and gene up regulation that was dependent on transactivation of the EGFR and downstream activation of MAPK.

Mitochondrial dysfunction in choline deficiency-induced apoptosis in cultured rat hepatocytes.

Our recent studies have demonstrated that generation of ROS is associated with choline deficiency (CD)-induced apoptosis in CWSV-1 cells, an immortalized rat hepatocyte that becomes tumorigenic by stepwise culturing in decreasing levels of choline. In the present study, we investigated the effect of CD on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by FASCAN assay. Our data demonstrate that MMP in CD-cultured cells was decreased in a time- and dose-dependent manner and that significant disruption occurred at 24 h, relative to high choline (HC, 70 microM) cultured cells. In order to investigate further the relationship among the CD-induced ROS, MMP collapse, and apoptosis, we examined the effects of different inhibitors on ROS production, MMP disruption, and apoptosis in CD or HC-cultured CWSV-1 cells. These data indicate that the disruption of MMP is an upstream event in CD-induced apoptosis, and mitochondrial dysfunction plays a key role in mediating CD-induced apoptosis in CWSV-1 cells.

Reactive oxygen species in choline deficiency-induced apoptosis in rat hepatocytes.

Choline deficiency (CD) is involved in hepatocellular carcinoma and CD-induced apoptosis may be implicated in cellular malignant transformation. In this report, we studied the effects of choline deficiency on generation of reactive oxygen species (ROS) using the fluorescent probe dichlorodihydrofluorescein diacetate and the possible role of ROS on CD-induced apoptosis in cultured CWSV-1 cells, an immortalized rat hepatocyte. This cell line is reported to become tumorigenic by step-wise culturing in lower levels of choline. Our data demonstrate that CD induces a time- and dose-dependent increase in ROS in CWSV-1 cells. The increase in ROS production may be related to dysfunction of the mitochondrial respiratory chain. Our data also demonstrated that ROS generation occurred before CD-induced apoptosis, suggesting ROS may play a key role in signaling CD-induced apoptosis in CWSV-1 cells.

Interleukin-10 overexpression mediates phenyl-N-tert-butyl nitrone protection from endotoxemia.

The free radical trapping compound phenyl N-tert-butylnitrone (PBN) provides potent protection against lethal endotoxemia in rodents, but the mechanism of this protection is not well understood. The objective of this study was to show that PBN administration in lipopolysaccharide- (LPS) induced endotoxemia promotes enhanced production of endogenous interleukin 10 (IL-10), and the expressed IL-10 is a causal factor in the protection from endotoxemia. We show the amplified expression of IL-10 in liver and plasma in PBN- (150 mg/kg) plus LPS- (4 mg/kg) treated rats using ribonuclease protection assay (RPA) and ELISA. In situ hybridization was utilized to visualize the overexpression of the IL-10 gene, and ELISA was used to determine plasma IL-10 and TNFalpha levels. Plasma IL-10 showed a 3-fold increase in PBN/LPS- treated rats compared to those treated with LPS alone, and in contrast, TNFalpha level decreased by more than 90%. However, the administration of PBN alone induced no IL-10 production. Immunoneutralization of IL-10 through anti-IL-10 antibody administration to PBN/LPS-treated rats abrogated PBN's suppression of systemic nitric oxide (NO) formation, a surrogate marker for the severity of endotoxemia, indicating that IL-10 is a causal factor for the protection. In these experiments, systemic NO level was quantified using an in vivo electron paramagnetic resonance (EPR) NO-trapping technique. Gel-shift and immunohistochemical analyses indicated that the transcription factor NF-kappaB was deactivated after PBN treatment, suggesting that NF-kappaB deactivation is closely involved in IL-10 overexpression.

Factors influencing body composition in persons with spinal cord injury: a cross-sectional study.

To determine the body composition differences across age, 133 men with chronic spinal cord injury (SCI) (66 with tetraplegia, 67 with paraplegia) were compared with an age-, height-, and ethnicity-matched able-bodied male reference population (n = 100) using two different dual-energy X-ray absorptiometry densitometers. The effects of duration of injury, level, and completeness of lesion were analyzed in the SCI population. Independent of age, total body and regional lean mass were lower and fat mass was higher in persons with SCI compared with controls. The SCI group was 13 +/- 1% (means +/- SE) fatter per unit of body mass index (kg/m2) compared with the control group (P < 0.0001). Advancing age was strongly associated with less lean mass and greater adiposity in those with SCI, whereas it was mildly related in the controls. Total body and regional arm and trunk, but not leg, lean tissues were lower in subjects with SCI, across all ages, than in the controls. In summary, persons with SCI were fatter for any body mass index and demonstrated significantly less lean and more adipose tissues for any given age compared with controls.

Germ-line and rearranged Tcrd transcription distinguish bona fide NK cells and NK-like gammadelta T cells.

NK cells and gammadelta T cells are distinct subsets of lymphocytes that contextually share multiple phenotypic and functional characteristics. However, the acquisition and the extent of these similarities remain poorly understood. Here, using T cell receptor delta locus-histone 2B-enhanced GFP (Tcrd-H2BEGFP) reporter mice, we show that germ-line transcription of Tcrd occurs in all maturing NK cells. We also describe a population of mouse NK-like cells that are indistinguishable from "bona fide" NK cells using standard protocols. Requirements for V(D)J recombination and a functional thymus, along with very low-level expression of surface TCRgammadelta but high intracellular CD3, define these cells as gammadelta T cells. "NK-like gammadelta T cells" are CD127+, have a memory-activated phenotype, express multiple NK cell receptors and readily produce interferon-gamma in response to IL-12/IL-18 stimulation. The close phenotypic resemblance between NK cells and NK-like gammadelta T cells is a source of experimental ambiguity in studies bridging NK and T cell biology, such as those on thymic NK cell development. Instead, it ascribes chronic TCRgammadelta engagement as a means of acquiring NK-like function.

Interleukin-10 protects against inflammation-mediated degeneration of dopaminergic neurons in substantia nigra.

Inflammation has been increasingly recognized to play an important role in the pathogenesis of Parkinson's disease (PD). Using immunocytochemistry and electron microscopy, we found that intranigral injection of lipopolysaccharide (LPS) caused marked microglial activation and a dose-dependent selective loss of dopaminergic neurons, which was mediated by apoptosis as evidenced by prominent TUNEL labeling. RNase protection assays revealed that mRNA for Bax, Fas and the pro-inflammatory cytokines interleukin (IL)-1α, IL-1ß, IL-6 and tumor necrosis factor (TNF)-α were significant increased ipsilaterally in LPS-injected side of SN, while expression of the anti-apoptotic gene Bcl-2 was decreased. Osmotic pump infusion of IL-10, a global inhibitor of cytokine synthesis, protected against LPS-induced cell death of dopaminergic neurons, with a corresponding decrease in the number of activated microglia, suggesting that the reduction in microglia-mediated release of inflammatory mediators may contribute to the anti-inflammatory effect of IL-10. Our results provide evidence that LPS induces apoptotic cell death in SNpc, which is likely through the expression of Fas, Bax, caspase-3, and the pro-inflammatory cytokines.

Antioxidant therapy with N-acetylcysteine plus mesalamine accelerates mucosal healing in a rodent model of colitis.

The aims of this study were to examine the ability of the antioxidant N-acetylcysteine (NAC) and mesalamine (5-ASA) alone and in combination to affect TNBS-induced colitis in rat. Three days following induction of TNBS colitis rats were randomized to receive daily intracolonic treatment with NAC, 5-ASA, and NAC plus 5-ASA for 5 or 8 days. At the end of the treatment period macroscopic and microscopic colonic injuries were scored. Myeloperoxidase (MPO) activity and cytokine gene expression were measured in colonic tissues. Results indicated that treatment with NAC plus 5-ASA caused a significantly greater reduction in colonic injury than either agent alone. Furthermore, combination therapy inhibited significantly MPO activity and inflammatory cytokine gene expression in the distal colon of TNBS-treated animals. The beneficial effects of NAC plus 5-ASA on reduction of colonic injury and promotion of healing were most evident after 8 days of treatment.

Toward in-cylinder absorption tomography in a production engine.

Design requirements for an 8000 frame/s dual-wavelength ratiometric chemical species tomography system, intended for hydrocarbon vapor imaging in one cylinder of a standard automobile engine, are examined. The design process is guided by spectroscopic measurements on iso-octane and by comprehensive results from laboratory phantoms and research engines, including results on temporal resolution performance. Novel image reconstruction techniques, necessary for this application, are presented. Recent progress toward implementation, including details of the optical access arrangement employed and signal-to-noise issues, is described. We present first cross-cylinder IR absorption measurements from a reduced channel-count (nontomographic) system and discuss the prospects for imaging.