Synthetic spatially graded Rac activation drives cell polarization and movement

Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.     

Giardia induces proliferation and interferon gamma production by intestinal lymphocytes

BACKGROUND: Murine intraepithelial lymphocytes kill Giardia lambia; responses of human intestinal lymphocytes to this parasite are unknown. AIMS: To examine giardia induced proliferation, interferon gamma production, migration, and cytotoxicity by lymphocytes from the human intestine and peripheral blood. METHODS: Giardia were added to intraepithelial lymphocytes, lamina propria lymphocytes, and peripheral blood lymphocytes, obtained from jejunal mucosa and blood of otherwise healthy patients undergoing gastric bypass surgery for morbid obesity. Proliferation was measured by 3H-thymidine incorporation; frequency of proliferation precursors, by limiting dilution analysis; interferon gamma production, by ELISA; cytotoxicity, by 51Cr release of radiolabelled giardia and by release of serine esterases by effector lymphocytes that mediate cytotoxicity. RESULTS: The CD4+ T lymphocytes from intestine and blood proliferated in response to giardia. The stimulus by the parasite was mitogenic rather than antigenic due to the fact that the peak response was on day 3 rather than day 6, and the large number of precursors was in the range of that for mitogens. CD4+ T lymphocytes from both sites produced interferon gamma in response to giardia. Lymphocytes did not migrate towards or kill the parasite. CONCLUSIONS: Giardia induced the same degree of proliferation and interferon gamma production by CD4+ T lymphocytes in intestine and blood, but did not trigger cytotoxicity or migration.     

Strategy and timing of peripheral nerve surgery

The authors review the latest theories of peripheral nerve regeneration and repair. They present their research on nerve regeneration including the alterations in the mother cell body, and in the distal part of the axon, and the time required to reach the best production of amino acids for cytoskeleton reconstruction. Other research of particular interest which is presented regards the chemotactic arrangement of motor and sensory axons inside a vein. This research has shown that the axons are able to find their way to the appropriate (sensory or motor) distal endoneural tubes.Adoption phenomena are also presented.The discussion of surgery includes the type (suture, glueing, grafts, tubulization) and the time of surgical repair. Timing and repair strategies are related to the site of the lesion (which can require that a greater or smaller amount of cytoskeleton be reconstructed), the type of the injury, the state of surrounding tissues, the age of the patients, injuries to muscles, tendons, bones, vessels and skin. A scheme of strategy is proposed.     

神经生长因子对雪旺氏细胞趋化移动的影响

目的：观察神经生长因子在雪旺氏细胞趋化移动中的作用,并探讨腺样囊性癌的侵袭机制。方法：将雪旺氏细胞接种于Transwell小室上室,下室中加入神经生长因子,空白对照组下室中无神经生长因子,于孵箱中孵育10h后计数移动到下室的雪旺氏细胞数。结果：与空白对照组相比,神经生长因子组移动到下室的雪旺氏细胞数显著增加。结论：神经生长因子可促进雪旺氏细胞趋化移动,可能是腺样囊性癌嗜神经性重要机理之一。     

Effect of Azelastine Hydrochloride on Macrophage Chemotaxis and Phagocytosis in Vitro

Azelastine, a newly synthesized anti-allergic agent, was tested for its effects on guinea pig macrophage chemotaxis and phagocytosis. As specific macrophage chemo-attractants, we used macrophage chemotactic factors a and c; separated and highly purified from inflamed skin sites. Macrophage chemotaxis induced by skin extract or chemotactic factors was significantly suppressed by a low concentration of the agent (1 microgram/ml); the effect was dose-dependent. The inhibition of chemotaxis was reversible, because chemotactic activity was restored when the agents was removed by washing cells before chemotactic assay. Inactivation of chemotactic factors was not detected by mixing azelastine and factors a and c. Azelastine may directly interact with macrophages to decrease their chemotactic responsiveness. beta-Glucuronidase activity in the medium and macrophages after phagocytosis of polystyrene latex particles was not affected by this agent at concentrations ranging from 1 to 10 micrograms/ml. The phagocytosis of latex particles or sheep red blood cells opsonized with IgG antibodies (EA) and anchoring of macrophages to substrate were not inhibited and azelastine did not damage the macrophages as determined by lactate dehydrogenase (LDH) release assay.     

Effect of dietary fish oil supplemented with different doses of vitamin e on neutrophil chemotaxis in healthy volunteers

The effect of fish oils supplemented with a high or low dose of vitamin E on neutrophil chemotaxis was investigated in a double-blind crossover study. Twelve healthy volunteers were given 30 mL of either oil (5.4 g eicosapentaenoic acid and 3.2 g docosahexaenoic acid) daily for three weeks The serum vitamin E (-tocopherol) concentration decreased and the plasma malondialdehyde concentration increased only after supplementation with the low vitamin E fish oil, the latter change indicating increased lipid peroxidation. Both oils decreased neutrophil chemotaxis significantly and there was no significant difference in this respect between the high and low vitamin E oils, indicating that the effect was due primarily to the fish oil and not to the vitamin E. Generation of leukotriene B₄ by neutrophils was unaltered. Dietary supplementation with n-3 fish oil could have beneficial effects in pathological conditions with activated neutrophils, such as ischemic heart disease.     

Targeted replacement of rodent CCR2 with the human orthologue CCR2B: A mouse model for in vivo analysis of human target‐selective small molecule MCP‐1 receptor antagonists

Rodent models for testing the efficacy of lead compounds are often invalidated by species selectivity of the compounds. The advent of mouse embryonic stem cell technology has allowed the development of genetically engineered mouse strains that incorporate a specific human gene in place of the orthologous mouse gene, a so‐called knock‐in mouse. This study describes the generation and validation of a mutant mouse line that expresses human CCR2B as a functional substitute for murine CCR2. The human CCR2B knock‐in mice are viable and appear normal. In vitro assays indicate that the CCR2B knock‐in is functionally expressed, giving a macrophage chemotactic profile in response to JE or MCP‐1 that is similar to human peripheral blood monocytes rather than that of a murine macrophage cell line. In addition, the human selective CCR2B antagonist, SB‐399721, was a more potent inhibitor of CCR2B knock‐in macrophages in response to hMCP‐1 than JE. The ability of the human CCR2B gene to functionally substitute for the mouse orthologue in vivo is demonstrated by a normal inflammatory response to intraperitoneal thioglycollate injection. Drug Dev. Res. 55:197–209, 2002. © 2002 Wiley‐Liss, Inc.     

Ex vivo studies of polymorphonuclear neutrophils from patients with early-onset forms of periodontitis (I).

Abstract The polymorphonuclear neutrophil (PMN) appears lo be an important cell in the protection of the host from pathogenic periodontal microorganisms and, despite some reports to the contrary, it is generally assumed that early-onset forms of periodontal disease including both juvenile and rapidly progressing periodontitis are associated with a defect in PMN chemotactic behaviour. The purpose of the present study was to examine the peripheral PMN chemotactic behaviour, using the under agarose method, in 4 groups, namely healthy periodontium group (n= 7), gingivitis group (n= 8), early-onset periodontitis group (n= 17) and adult periodontitis group (n= 8). PMN from early-onset periodontitis patients showed normal random and chemotactic locomotory behaviour when compared with those of PMN from subjects of the other groups. No statistically significant difference could be found among the 4 studied groups, with regard to spontaneous and oriented migration.     

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements.