Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.     

Stress-Induced Visceral Hypersensitivity in Maternally Separated Rats Can Be Reversed by Peripherally Restricted Histamine-1-Receptor Antagonists

Background

The histamine-1 receptor (H1R) antagonist ketotifen increased the threshold of discomfort in hypersensitive IBS patients. The use of peripherally restricted and more selective H1R antagonists may further improve treatment possibilities. We examined the use of fexofenadine and ebastine to reverse post-stress visceral hypersensitivity in maternally separated rats.

Methods

The visceromotor response to colonic distension was assessed in adult maternally separated and nonhandled rats pre- and 24 hours post water avoidance. Subsequently rats were treated with vehicle alone or different dosages of fexofenadine (1.8 and 18 mg/kg) or ebastine (0.1 and 1.0 mg/kg) and re-evaluated. Colonic tissue was collected to assess relative RMCP-2 and occludin expression levels by Western blot and histamine-1 receptor by RT-qPCR. β-hexosaminidase release by RBL-2H3 cells was used to establish possible mast cell stabilizing properties of the antagonists.

Key results

Water avoidance only induced enhanced response to distension in maternally separated rats. This response was reversed by 1.8 and 18 mg/kg fexofenadine. Reversal was also obtained by 1.0 but not 0.1 mg/kg ebastine. RMCP-2 expression levels were comparable in these two ebastine treatment groups but occludin was significantly higher in 1.0 mg/kg treated rats. There were no differences in histamine-1 receptor expression between nonhandled and maternally separated rats. Fexofenadine but not ebastine showed mast cell stabilizing quality.

Conclusions

Our results indicate that the peripherally restricted 2^nd generation H1-receptor antagonists fexofenadine and ebastine are capable of reversing post stress visceral hypersensitivity in rat. These data justify future IBS patient trials with these well tolerated compounds.     

Breeding biology of Frances's Sparrowhawk Accipiter francesii in a lowland rainforest of northeastern Madagascar

Lily-Arison, R.de R. 2000. Breeding biology of Frances's Sparrowhawk Accipiter francesii in a lowland rainforest of northeastem Madagascar. Ostrich 71 (1 & 2): 332–335.

Frances's Sparrowhawks Accipiter fmncesii were studied during the breeding seasons 1994 and 1995 on Masoala Peninsula of northeastern Madagascar. Breeding coincides with the dry season, nest building in October, egg laying in November, hatching in December and fledging in January (beginning of the wet season). All but one of 10 pairs built new nests in 1995 and the mean distance of nest site from 1994 and 1995 nests was 105± 100 m (n = 6, ranging from 0 to 250m). Nests in primary forest were incubated for 79% of the observation time by females, 3% by males and eggs were unattended for 18% of the observation time. On a numerical basis the diet was composed of lizards (56%) and birds (23%), making up more than 79% of the identified prey items. A total of 33 eggs was laid in 13 nests (average clutch size 2.5). In 13 breeding attempts 29 (88%) hatched and all of those hatchlings fledged. Of the 14 My-documented breeding attempts 2.1 young fledged per breeding attempt and overall nesting success was 93%.

L'Épervier de France Accipiter francesii a été étudié pendant la saison de reproduction en 1994 et 1995 dans la Presqu'Ile Masoala, au partie Nord-Est de Madagascar. La saison de reproduction de cette espkce coincide avec la saison sèche: la construction du nid en Octobre, la ponte en Novembre, I'élosion en Déembre et l'envol du poussin en Janvier (début de la saison pluviale). Tous, sauf une des 10 paires ont construit des nouveaw nids en 1995 et la distance moyenne de I'endroit du nid de I'anné 1994 et 1995 est de 105 + 100 m (n = 6, variant de 0 à 250m). Dans la for[ebar]t primaire, les oeufs ont éeté incubés par la femelle pendant 79% du temps d'observation, 3% par le mile et non incubés pendant 18% du temps. Les proies sont principalement composées de lkzards (56%) et oiseaux (23%), atteignant plus de 79% des proies identifiés. 33 oeufs ont été pondus dans 13 nids (en moyenne 2.5 oeufs par nid). Pour ces 13 nids, 29 oeufs (88%) ont été éclos et tous ces poussins (100%) sont capables de voler. Sur 14 nids observés, 2.1 poussins par nid s'envolent et le tam de succés est de 93%.