Mesenteric ischemia-reperfusion injury up-regulates certain CC, CXC, and XC chemokines and results in multi-organ injury in a time-dependent manner.

INTRODUCTION: Trauma patients who develop multi-organ dysfunction have increased systemic levels of chemotactic cytokines. Ischemia-reperfusion (IR) injury to the gut may play a role. The purpose of this study was to examine chemokine production in a mouse model of mesenteric IR injury. Given the pre-eminent role of the neutrophil, there has been much investigation of the CXC chemokines, but very limited research on the CC and XC chemokines. We hypothesized that intestinal IR injury would induce remote organ injury and enhance serum CC and XC chemokine levels. METHODS: Fasted female C57BL6 mice were anesthetized prior to laparotomy. In IR animals, the superior mesenteric artery (SMA) was occluded for 30, 45, or 75 min, while controls underwent sham laparotomy, n = 5-7 per group. After the indicated time point, the incision was closed and the mouse was allowed to recover for six hours. Following euthanasia, serum levels of 15 chemokines (10 CC, 4 CXC, and 1 XC) were assessed and histopathologic analyses performed. RESULTS: Seventy-five minutes of SMA occlusion was the key time frame for significant serum cytokine level up-regulation, intestinal and remote organ injury, and neutrophil influx into tissues. With 75 min of intestinal ischemia, significantly elevated serum levels, as compared to shams, were noted for seven CC chemokines: MCP-1, MCP-3, MIP-1ß, MIP-3ß, eotaxin, MDC, and RANTES. Levels of the XC chemokine lymphotactin also increased. Levels of MIP-2, IP-10, and KC/GRO (CXC chemokines) rose significantly. MIP-1α levels were only significantly increased at 45 min IR. We did not find any significant IR injury-induced changes in levels of MCP-5, MIP-1γ, or GCP-2, at any ischemia time frame. Serum levels of IL-6 correspondingly increased significantly with longer ischemia times. CONCLUSIONS: The novel finding of this study is the demonstration of significant systemic increases in the CC chemokines eotaxin, MCP-3, MDC, MIP-3ß in a time-dependent manner, along with tissue injury. The data suggest a complex response to IR injury whereby chemokines that are active on a variety of leukocytes may play a role in inducing local and remote tissue injury.

Methamphetamine administration targets multiple immune subsets and induces phenotypic alterations suggestive of immunosuppression.

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear to show signs of perturbation. CD4 and CD8 T cell subsets were affected by methamphetamine, both showing a reduction in antigen-experienced subsets. CD4 T cells also exhibited signs of activation, with increased expression of CD150 on CD226-expressing cells and an expansion of KLRG1(+), FoxP3(-) cells. These results exhibit that meth has the ability to disrupt immune homeostasis and impact key subsets of leukocytes which may leave users more vulnerable to pathogens.

Spatial resolution of a hard x-ray CCD detector.

The spatial resolution of an x-ray CCD detector was determined from the widths of the tungsten x-ray lines in the spectrum formed by a crystal spectrometer in the 58 to 70 keV energy range. The detector had 20 microm pixel, 1700 by 1200 pixel format, and a CsI x-ray conversion scintillator. The spectral lines from a megavolt x-ray generator were focused on the spectrometer's Rowland circle by a curved transmission crystal. The line shapes were Lorentzian with an average width after removal of the natural and instrumental line widths of 95 microm (4.75 pixels). A high spatial frequency background, primarily resulting from scattered gamma rays, was removed from the spectral image by Fourier analysis. The spectral lines, having low spatial frequency in the direction perpendicular to the dispersion, were enhanced by partially removing the Lorentzian line shape and by fitting Lorentzian curves to broad unresolved spectral features. This demonstrates the ability to improve the spectral resolution of hard x-ray spectra that are recorded by a CCD detector with well-characterized intrinsic spatial resolution.

Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function.

Exposure to cigarette smoke is associated with airway epithelial mucus cell hyperplasia and a decrease in cilia and ciliated cells. Few models have addressed the long-term effects of chronic cigarette smoke exposure on ciliated epithelial cells. Our previous in vitro studies showed that cigarette smoke decreases ciliary beat frequency (CBF) via the activation of protein kinase C (PKC). We hypothesized that chronic cigarette smoke exposure in an in vivo model would decrease airway epithelial cell ciliary beating in a PKC-dependent manner. We exposed C57BL/6 mice to whole-body cigarette smoke 2 hours/day, 5 days/week for up to 1 year. Tracheal epithelial cell CBF and the number of motile cells were measured after necropsy in cut tracheal rings, using high-speed digital video microscopy. Tracheal epithelial PKC was assayed according to direct kinase activity. At 6 weeks and 3 months of smoke exposure, the baseline CBF was slightly elevated (~1 Hz) versus control mice, with no change in ß-agonist-stimulated CBF between control mice and cigarette smoke-exposed mice. By 6 months of smoke exposure, the baseline CBF was significantly decreased (2-3 Hz) versus control mice, and a ß-agonist failed to stimulate increased CBF. The loss of ß-agonist-increased CBF continued at 9 months and 12 months of smoke exposure, and the baseline CBF was significantly decreased to less than one third of the control rate. In addition to CBF, ciliated cell numbers significantly decreased in response to smoke over time, with a significant loss of tracheal ciliated cells occurring between 6 and 12 months. In parallel with the slowing of CBF, significant PKC activation from cytosol to the membrane of tracheal epithelial cells was detected in mice exposed to smoke for 6-12 months.

Inhalation of tobacco smoke induces increased proliferation of urinary bladder epithelium and endothelium in female C57BL/6 mice.

Cigarette smoking is the major environmental risk factor for bladder cancer in humans. Aromatic amines, potent DNA-reactive bladder carcinogens present in cigarette smoke, contribute significantly. However, increased cell proliferation, caused by direct mitogenesis or in response to cytotoxicity, may also play a role since urothelial hyperplasia has been observed in human cigarette smokers. We examined the urothelial effects of cigarette smoke (whole body inhalation exposure (Teague) system) in female C57BL/6 mice at various times in two studies, including reversibility evaluations. In both studies, no urothelial hyperplasia was observed by light microscopy in any group. However, in study 1, the Ki-67 labeling index (LI) of the urothelium was significantly increased in the smoke exposed group compared to controls through 3 months, but was not present at 6, 9 or 12 months even with continued exposures. In the groups that discontinued smoke exposure, it returned to the same levels as controls or lower. In study 2, the bromodeoxyuridine LI was similar to controls on day 1 but significantly increased at 5 days in the smoke exposed group. In the group that discontinued smoke exposure for 2 days, the LI was increased compared to controls but not significantly. Superficial urothelial cell cytotoxicity and necrosis were detectable by scanning electron microscopy at 5 days. Changes in LI of submucosal endothelial cells generally followed those of the urothelium and effects were reversible upon cessation of exposure. The increased urothelial proliferation appeared to be due to superficial cell cytotoxicity with consequent regeneration.

Cells derived from the circulation contribute to the repair of lung injury.

Bone marrow (stem/progenitor) cells have been shown to "differentiate" into cells in multiple tissues, including lung. A low number of hematopoietic stem/progenitor cells also circulate in peripheral blood. The physiologic roles of these cells are still uncertain. This study was designed to test, using parabiotic mice that were joined surgically, whether stem/progenitor cells in blood contributed to the regeneration of lung after injury. Parabiotic mice were generated surgically by joining green fluorescent protein transgenic mice and wild-type littermates. These mice developed a common circulation (approximately 50% green cells in blood) by 2 weeks after surgery. The wild-type mouse was either uninjured or lethally irradiated or received intratracheal elastase or the combination of radiation with intratracheal elastase injection. Radiation or the combination of radiation with elastase significantly increased the proportion of bright green cells in the lungs of the wild-type mice. Morphologically, interstitial monocytes/macrophages, subepithelial fibroblast-like interstitial cells, and additionally type I alveolar epithelial cells immunostained for green fluorescent protein in wild-type mice. Approximately 5 to 20% of lung fibroblasts primary cultured from injured wild-type mice were green fluorescent protein expressing cells, indicating their blood derivation. This study demonstrates that stem/progenitor cells in blood contribute to the repair of lung injury in irradiated mice.

Lung cells transplanted to irradiated recipients generate lymphohematopoietic progeny.

Bone marrow (stem) cells can differentiate into cells in multiple tissues, including lung. Conversely, there are reports that cells of nonhematopoietic tissues (brain, muscle) can give rise to lymphohematopoietic cells. Here we show that the lung contains cells capable of giving rise to lymphohematopoietic cells when transplanted to irradiated recipients. Whole lung cell suspensions, lung side population (SP) cells, and CD45(+/-) lung cells obtained from male transgenic enhanced green fluorescent protein-expressing mice were transplanted intravenously to total body irradiated female mice. Green fluorescent cells were recovered from the circulation and phenotyped for their expression of lymphohematopoietic markers (CD3, CD4, CD8, B220, Gr-1, and Mac-1). Lung SP cells were composed of heterogeneous populations and had less ability to give rise to lymphohematopoietic cells than did bone marrow SP cells. Furthermore, the ability of cells from the lung of aged mice to generate lymphohematopoietic progeny was equivalent to that of cells from young mice. Cells from lung with radioprotective and lymphohematopoietic reconstituting abilities were CD45(+). CD45(+) cells in the lung cells have lymphohematopoietic stem/progenitor cell characteristics, and this has implications for cell or gene therapy applications.

X-ray absolute calibration of the time response of a silicon photodiode.

The time-dependent response of a 1-mm2 silicon photodiode was characterized by use of pulsed synchrotron radiation in the 4- to 16-nm-wavelength range. Modeling the input radiation pulse and the electrical response of the photodiode allowed the photodiode's capacitance as a function of wavelength and applied bias voltage to be determined. The capacitance was in the 7- to 19-pF range and resulted in response fall times as small as 0.4 ns. The capacitance determined by pulsed x-ray illumination was in good agreement with the capacitance determined by pulsed optical laser illumination. The absolute responsivity was measured by comparison with the responsivity of a calibrated photodiode.