Effect of age on susceptibility to post-traumatic infection in the elderly

Previous work has demonstrated an age-related decline in neutrophil function, including a decline in phagocytic capacity, with age in healthy individuals. This decline in function may contribute to increased susceptibility to bacterial infections in the elderly population. The present study has investigated the effects of age on susceptibility to infection and neutrophil function in elderly humans following mild trauma. Specifically, we have measured neutrophil function in 44 patients, all of whom had no significant co-morbidity, were over 65 years old (mean age 82.5 years) and had sustained a fractured neck of femur. We obtained neutrophils and examined the process of microbial engulfment by phagocytosis and the bactericidal mechanism of superoxide production. In the 5-week period after trauma, almost half of the elderly trauma patients succumbed to bacterial or fungal infection, with a predominance of chest and urinary tract infections. When examining neutrophil function, a decline in superoxide production was observed in neutrophils from the elderly trauma group at the time of hip fracture when compared with those from healthy elderly controls, and this was maintained 5 weeks after trauma. This was accompanied by an age-related reduction in phagocytic function during this period. We propose that trauma and an age-related decline in neutrophil function combine to decrease the immune response to bacteria in the elderly.     

New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (betac) and the IL-2 receptor beta chain (IL-2Rbeta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y-->F and "add-back" mutants of betac shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rbeta that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.     

Cell cycle programs of gene expression control morphogenetic protein localization

Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms.     

Glycoprotein degradation: do sugars hold the key?

Secretory glycoproteins that fail to fold or assemble correctly are retained in the endoplasmic reticulum and eventually degraded. Recent evidence shows that trimming of their N-linked oligosaccharide chains plays a key role in targeting glycoproteins for destruction.     

Enhancement of tumor perfusion and oxygenation by carbogen and nicotinamide during single- and multifraction irradiation

Numerous experimental and clinical studies have been completed regarding the effects of carbogen and nicotinamide on tumor oxygenation and radiosensitivity. The current study incorporates three physiological measurement techniques to further define spatial variations in oxygen availability and development of hypoxia after single- and multifraction irradiation in KHT murine fibrosarcomas. Distances to anatomical and perfused blood vessels were measured using immunohistochemical and fluorescent staining, intravascular oxygen levels were determined cryospectrophotometrically, and tumor hypoxia was quantified using uptake of EF5, a marker of hypoxia. Carbogen, nicotinamide, and the combination of both all increased intravascular oxygen availability compared to controls. While nicotinamide had no effect on the number of perfused blood vessels in nonirradiated tumors, carbogen produced a substantial closing of vessels. After a single dose of 4 Gy, only the combination of nicotinamide and carbogen produced significant improvements in oxygen availability, while numbers of perfused vessels were significantly increased for nicotinamide, unchanged for the combination of nicotinamide and carbogen, and significantly decreased for carbogen. After 4 x 4-Gy fractions, oxygen availability was increased substantially with the combination of nicotinamide and carbogen, somewhat with carbogen, and not at all with nicotinamide. Tumor oxygenation changes were estimated by EF5/Cy3 intensity distributions, which demonstrated that manipulative agents could produce disparate effects on tumor hypoxia when combined with either single- or multifraction irradiation.     

Decreased lateral aggregation of a variant recombinant fibrinogen provides insight into the polymerization mechanism

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.     

Long-term response of the biotic community to fluctuating water levels and changes in water quality in Cootes Paradise Marsh,a degraded coastal wetland of Lake Ontario

During the early 1900s, more than 90% of the surface area of Cootes Paradise Marsh was covered with emergent vegetation; currently, less than 15% of the surface is covered with aquatic vegetation and the remainder is wind-swept, turbid, open water. The loss of emergent cover is significantly correlated with mean annual water levels that increased more than 1.5 m over the past 60 years. Species diversity and the percent cover of the submerged macrophtye community also declined dramatically after the 1940s, coincident with decreased water clarity and increased nutrients from pollution by sewage and stormwater effluent. Phosphorus levels in the marsh dropped ten-fold after the sewage plant was upgraded to a tertiary-treatment facility in 1978; however, there was no measurable improvement in water clarity, in spite of a decrease in chlorophyll concentrations. Long-term changes in the composition of the planktonic, benthic and fish communities accompanied changes in water clarity, nutrient status and macrophyte cover. Phytoplankton changed from a community dominated by diverse taxa of green algae and diatoms during the 1940s, to a less diverse community dominated by a few taxa of green and blue-green algae in the 1970s, then to a much more diverse community recently, including many taxa of green algae, diatoms and chrysophytes; however, because water turbidity continues to be high, and algae tolerant of low light levels are now very abundant. Daphnia, which were prominent during the 1940s (especially in the vegetated sites) were replaced in the 1970s by smaller zooplankton such as the cladoceran, Bosmina, and several rotifer species including Brachionus, Asplanchna and Keratella. In the recent survey conducted in 1993 and 1994, small-bodied forms still dominate the turbid open-water areas, while medium-sized cladocerans such as Moina were common near macrophyte beds. Generally, total herbivorous zooplankton biomass tended to be highest next to Typha beds and declined with increasing distance from the plants. Conversely, biomass of edible algae at these sites increased with distance from the macrophytes. Species diversity of aquatic insects declined dramatically over the past 40 years, from 57 genera (23 families and 6 orders) in 1948, to 9 genera (6 families and 3 orders) in 1978, to only 5 genera (3 families and 2 orders) in 1995. The diverse benthic community present 5 decades ago has now been replaced by a community consisting primarily of chironomid larvae, oligochaetes and other worms associated with low-oxygen environments. These successional changes illustrate the impact of natural (fluctuating water levels) and anthropogenic (deterioration in water quality) stressors on the character of the biotic communities, and reveal the complex interactions among the various trophic levels and the abiotic environment as degradation and remediation proceeded.     

Spatial Organization of EphA2 at the Cell-Cell Interface Modulates Trans-Endocytosis of EphrinA1

EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell’s microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2’s cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell’s microenvironment and may have implications in physical aspects of tumor biology.     

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.