Person:

Carter, Edward

Sepsis remains the major cause of death in patients with major burn injuries. In the present investigation we evaluated the interaction between burn injuries of varying severity and preexisting distant infection. We used Gram-negative bacteria (Pseudomonas aeruginosa and Proteus mirabilis) that were genetically engineered to be bioluminescent, which allowed for noninvasive, sequential optical imaging of the extent and severity of the infection. The bioluminescent bacteria migrated from subcutaneous abscesses in the leg to distant burn wounds on the back depending on the severity of the burn injury, and this migration led to increased mortality of the mice. Treatment with ciprofloxacin, injected either in the leg with the bacterial infection or into the burn eschar, prevented this colonization of the wound and decreased mortality. The present data suggest that burn wounds can readily become colonized by infections distant from the wound itself.

Washed rat colonic rings were incubated in closed flasks under N2 at physiologic pH and temperature levels. In the absence of an exogenous substrate, negligible H2 but some CH4 concentrations were detected in vitro after one hour of incubation, but high concentrations (H2 > 100 ppm, CH4 > 10 ppm) of both gases were found after 24 hours of culture. Production of H2 and CH4 by the washed colonic rings was stimulated by lactose addition. Maximum H2 production occurred at about pH 7.0, while maximum CH4 production occurred between pH 4.0 and 6.0. The increased production of both gases at 24 hours was associated with dramatic increases (104-fold) in anaerobic bacteria colony counts on the colonic rings and in the incubation media, as well as dramatic increases (100-fold) in acetate concentrations in the media, while lactate concentrations first rose and then fell significantly. These results suggest that gas production in colonic ring preparations is subject to quantitative changes in microbiota, pH, and metabolite formation analogous to in vivo conditions. In addition, microbiota firmly attached to colonic tissue appears to utilize colonic tissue to support its growth in the absence of an exogenous substrate.

Background—Interventions to increase brown adipose tissue (BAT) volume and activation are being extensively investigated as therapies to decrease the body weight in obese subjects. Noninvasive methods to monitor these therapies in animal models and humans are rare. We investigated whether contrast ultrasound (CU) performed in mice could detect BAT and measure its activation by monitoring BAT blood flow. After validation, CU was used to study the role of uncoupling protein 1 and nitric oxide synthases in the acute regulation of BAT blood flow. Methods and Results—Blood flow of interscapular BAT was assessed in mice (n=64) with CU by measuring the signal intensity of continuously infused contrast microbubbles. Blood flow of BAT estimated by CU was 0.5±0.1 (mean±SEM) dB/s at baseline and increased 15-fold during BAT stimulation by norepinephrine (1 µg·kg−1·min−1). Assessment of BAT blood flow using CU was correlated to that performed with fluorescent microspheres (R2=0.86, P<0.001). To evaluate whether intact BAT activation is required to increase BAT blood flow, CU was performed in uncoupling protein 1–deficient mice with impaired BAT activation. Norepinephrine infusion induced a smaller increase in BAT blood flow in uncoupling protein 1–deficient mice than in wild-type mice. Finally, we investigated whether nitric oxide synthases played a role in acute norepinephrine-induced changes of BAT blood flow. Genetic and pharmacologic inhibition of nitric oxide synthase 3 attenuated the norepinephrine-induced increase in BAT blood flow. Conclusions—These results indicate that CU can detect BAT in mice and estimate BAT blood flow in mice with functional differences in BAT.

Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.