A dual role for microglia in promoting tissue inhibitor of metalloproteinase (TIMP) expression in glial cells in response to neuroinflammatory stimuli

<p>Abstract</p> <p>Background</p> <p>By neutralizing the effect of the matrix metalloproteinases (MMPs), the tissue inhibitors of matrix metalloproteinases (TIMPs) play a critical role in maintaining tissue proteolysis in balance. As the major reactive glial cell types in the central nervous system (CNS), microglia and astrocytes play fundamental roles in mediating tissue breakdown and repair. As such, it is important to define the TIMP expression profile in these cells, as well as the mechanisms of regulation by neuroinflammatory stimuli.</p> <p>Methods</p> <p>Primary mixed glial cultures (MGC), pure microglia, and pure astrocytes were used in this study. To study astrocytes, we employed a recently described pure astrocyte culture system, which has the major advantage of totally lacking microglia. The three different types of culture were treated with lipopolysaccharide (LPS) or individual cytokines, and cell culture supernatants assayed for TIMP-1 or TIMP-2 protein expression by western blot.</p> <p>Results</p> <p>LPS induced TIMP-1 expression in MGC, but not in pure astrocyte or microglial cultures. When pure astrocytes were treated with the cytokines IL-1β, IFN-γ, TNF or TGF-β1, only IL-1β induced TIMP-1 expression. Significantly, astrocyte TIMP-1 expression was restored in LPS-treated astrocyte cultures after the addition of microglia, or conditioned medium taken from LPS-activated microglia (MG-CM). Furthermore, this effect was lost after depletion of IL-1β from MG-CM. By contrast, TIMP-2 was constitutively expressed by astrocytes, whereas microglia expressed TIMP-2 only after exposure to serum.</p> <p>Conclusions</p> <p>Taken together, these results demonstrate an important concept in glial interactions, by showing that microglia play a central role in regulating glial cell expression of TIMPs, and identify microglial IL-1β as playing a key role in mediating microglial-astrocyte communication.</p

Upregulation of Defensins in Burn Sheep Small Intestine

Objective: The aim of this study was to visualize and localize the sheep antimicrobials, β-defensins 1, 2, and 3, (SBD-1, SBD-2, SBD-3), sheep neutrophil defensin alpha (SNP-1), and the cathelicidin LL-37 in sheep small intestine after burn injury, our hypothesis being that these compounds would be upregulated in an effort to overcome a compromised endothelial lining. Response to burn injury includes the release of proinflammatory cytokines and systemic immune suppression that, if untreated, can progress to multiple organ failure and death, so protective mechanisms have to be initiated and implemented. Methods: Tissue sections were probed with antibodies to the antimicrobials and then visualized with fluorescently labeled secondary antibodies and subjected to fluorescence deconvolution microscopy and image reconstruction. Results: In both the sham and burn samples, all the aforementioned antimicrobials were seen in each of the layers of small intestine, the highest concentration being localized to the epithelium. SBD-2, SBD-3, and SNP-1 were upregulated in both enterocytes and Paneth cells, while SNP-1 and LL-37 showed increases in both the inner circular and outer longitudinal muscle layers of the muscularis externa following burn injury. Each of the defensins, except SBD-1, was also seen in between the muscle layers of the externa and while burn caused slight increases of SBD-2, SBD-3, and SNP-1 in this location, LL-37 content was significantly decreased. Conclusion: That while each of these human antimicrobials is present in multiple layers of sheep small intestine, SBD-2, SBD-3, SNP-1, and LL-37 are upregulated in the specific layers of the small intestine

Virtually Instantaneous, Room-Temperature [11C]-Cyanation Using Biaryl Phosphine Pd(0) Complexes

A new radiosynthetic protocol for the preparation of [[superscript 11]C]aryl nitriles has been developed. This process is based on the direct reaction of in situ prepared L·Pd(Ar)X complexes (L = biaryl phosphine) with [[superscript 11]C]HCN. The strategy is operationally simple, exhibits a remarkably wide substrate scope with short reaction times, and demonstrates superior reactivity compared to previously reported systems. With this procedure, a variety of [[superscript 11]C]nitrile-containing pharmaceuticals were prepared with high radiochemical efficiency.National Institutes of Health (U.S.) (NIH GM46059)National Science Foundation (U.S.) (NSF predoctoral fellowship (2010094243))Amgen Inc. (Educational donation)National Institutes of Health (U.S.) (NIH-NIDA postdoctoral fellowship (2T32DA015036)

Nuclear Localization of HBD-1 in Human Keratinocytes

Objective: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. Results: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. Conclusion: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions

Alterations in Content and Localization of Defensins in Rat Ileum and Jejunum Following Ischemia-Reperfusion. Specific Peptides, in Specific Places, for Specific Jobs?

Objective: To determine alterations in quantities and distributions of natural antimicrobials following ischemia-reperfusion injury. We hypothesized that these compounds would be upregulated in areas of small intestine where changes in permeability and cellular disruption were likely and where protective mechanisms would be initiated. Methods: Rats with ischemia-reperfusion underwent superior mesenteric artery clamping and reperfusion. Shams were subjected to laparotomy but no clamping. Ileum and jejunum were harvested and sectioned, and subjected to fluorescence deconvolution microscopy for determinations of content and localization of rat beta defensins, 1, 2, 3; rat neutrophil protein-1; and cathelicidin LL-37. Modeling was performed to determine cellular location of antimicrobials. Results: Ischemia-reperfusion increased neutrophil defensin alpha (RNP-1) in jejunum; rat beta defensin 1 was increased 2-fold in ileal mucosa and slightly reduced in jejunal mucosa; rat beta defensin 2 was reduced by ischemia-reperfusion in ileum, but slightly increased in jejunum; rat beta defensin 3 was concentrated in the muscularis externa and myenteric plexus of the jejunum; ischemia-reperfusion did not alter cathelicidin LL-37 content in the small intestine, although a greater concentration was seen in jejunum compared with ileum. Conclusion: Ischemia-reperfusion injury caused changes in antimicrobial content in defined areas, and these different regulations might reflect the specific roles of jejunum versus ileum

Arctic river temperature dynamics in a changing climate

Climate change in the Arctic is expected to have a major impact on stream ecosystems, affecting hydrological and thermal regimes. Although temperature is important to a range of in‐stream processes, previous Arctic stream temperature research is limited—focused on glacierised headwaters in summer—with limited attention to snowmelt streams and winter. This is the first high‐resolution study on stream temperature in north‐east Greenland (Zackenberg). Data were collected from five streams from September 2013 to September 2015 (24 months). During the winter, streams were largely frozen solid and water temperature variability low. Spring ice‐off date occurred simultaneously across all streams, but 11 days earlier in 2014 compared with 2015 due to thicker snow insulation. During summer, water temperature was highly variable and exhibited a strong relationship with meteorological variables, particularly incoming shortwave radiation and air temperature. Mean summer water temperature in these snowmelt streams was high compared with streams studied previously in Svalbard, yet was lower in Swedish Lapland, as was expected given latitude. With global warning, Arctic stream thermal variability may be less in summer and increased during the winter due to higher summer air temperature and elevated winter precipitation, and the spring and autumn ice‐on and ice‐off dates may extend the flowing water season—in turn affecting stream productivity and diversity