Sepsis induced by S. aureus was used to investigate whether neutrophil migration failure to infectious focus correlates with lethality in Gram‐positive bacteria‐induced sepsis in mice. By contrast with the sub‐lethal (SL‐group), the lethal (L‐group) intraperitoneal inoculum of S. aureus caused failure of neutrophil migration (92% reduction), high CFU in the exudate, bacteremia and impairment of in vitro neutrophil chemotactic activity. Pre‐treatments of L‐group with adequate doses of aminoguanidine prevented the neutrophil migration failure and improved the survival of the animals (pre‐treated: 43%; untreated: 0% survival). Thus, the impairment of neutrophil migration in the L‐group appears to be mediated by nitric oxide (NO). The injection of S. aureus SL‐inoculum in iNOS deficient (−/−) or aminoguanidine‐treated wild‐type mice (pre‐ and post‐treatment), which did not present neutrophil migration failure, paradoxically caused severe peritonitis and high mortality. This fact is explainable by the lack of NO dependent microbicidal activity in migrated neutrophils. In conclusion, although the NO microbicidal mechanism is active in neutrophils, the failure of their migration to the infectious focus may be responsible for the severity and outcome of sepsis. British Journal of Pharmacology (2002) 136, 645–658; doi:10.1038/sj.bjp.0704734

Abstract-Focal adhesion kinase (Fak) has been implicated as a signaling molecule involved in the early response of cardiac myocytes to mechanical stress. The mechanism of Fak activation by mechanical stimuli is not clear. In this study, we report the load-induced Fak activation and its association with myosin heavy chain in cardiac myocytes. Pressure overload lasting from 3 to 60 minutes was shown to induce Fak phosphorylation at Tyr-397, -576/7, -861, and -925 as detected by phosphospecific antibodies. This was paralleled by increases of Fak/Src association and Src activity (Tyr-418 phosphorylation). Yeast two-hybrid screening of an adult rat cDNA library revealed an interaction between Fak and C-terminal coiled-coil region of ␣-myosin heavy chain. This was confirmed by pulldown assay with GST-C-terminal myosin fragment and native Fak from rat left ventricle. Such interaction was confirmed by coimmunoprecipitation assay with anti-Fak and anti-heavy chain cardiac myosin antibodies, confocal microscopy of double-labeled isolated cardiac myocytes and immunoelectron microscopy with anti-Fak antibody. Fak activation by mechanical stress was accompanied by a reduction of Fak/myosin heavy chain association and its relocation at subcellular sites such as costameres, Z-discs, and nuclei. Thus, our present data identify Fak interaction with C-terminal region of myosin heavy chain adding comprehensive data on Fak activation by mechanical stress and mechanotransduction in cardiac myocytes. Key Words: focal adhesion kinase Ⅲ mechanotransduction Ⅲ cell signaling Ⅲ hypertrophy Ⅲ myosin M echanical stress is a major factor involved in the development of myocardial adaptive and maladaptive changes in heart diseases. Local mechanical forces activate signaling mechanisms in cardiac myocytes inducing the expression of specific genetic programs linked to myocardial structural and functional remodeling. 1,2 Although mechanical forces might directly trigger signaling mechanisms in cardiac myocytes, the mechanism by which they are sensed and converted to biochemical signals remains elusive.Structures such as sarcomeric lattice, cytoskeleton, and the extracellular matrix operate in the transmission of either passive or active forces in cardiac myocytes. 3,4 Studies have confirmed the critical importance of the molecular integrity of Z-disc and cytoskeleton to the expression of genetic program induced by mechanical stress in cardiac myocytes. Z-disc structure is organized by N-terminal titin Z repeats linked to ␣-actinin and associated proteins such as MLP, ALP, telethonin (T-cap), cypher/Zasp, and myotilin. 4,5 Notably, MLP null mice were shown to fail to upregulate brain and atrial natriuretic factors mRNA in response to stretch. 6 Human titin mutations as well as deletion of the ␣-actinin binding proteins such as ALP or MLP in the mouse causes dilated cardiomyopathy. 6 -10 However, the mechanisms by which these structures and proteins detect physical forces and initiate biochemical signals are yet unclear.The link of Z-discs to sarco...

Umbilical cord blood (UCB), an ideal source for transplantable hematopoietic stem cells (HSC), is readily available and is rich in progenitor cells. Identification of conditions favoring UCB-HSC ex vivo expansion and of repopulating potential remains a major challenge in hematology. CD133+ cells constitute an earlier, less-differentiated HSC group with a potentially higher engraftment capacity. The presence of SCF, Flt3-L, and TPO are essential for CD133+ and/or CD34+ cells ex vivo expansion; however, IL-3 and IL-6 influence has not yet been clearly established. We investigated this influence on CD133+ cells from UCB ex vivo expansion and the effect of these cytokines upon cell phenotype. Immediately after isolation an 85% of CD133+ cell purity was obtained, diminishing after 4 and 8 days of ex vivo expansion. CD133+ fold-increase was higher using IMDM with SCF, Flt3-L, and TPO (BM)+IL-3 or BM+IL-3+IL-6 on day 8 (13.83- and 17.47-fold increase, respectively). BM+IL-6 presented no significant difference from BM alone. We demonstrated that 5.1% of the CD133+ cells expressed IL-6 receptor (IL-6R) after isolation. After 4 and 8 days in culture, the percentage of CD133+ cells that expressed IL-6R was as follows: BM alone (9.8% and 22.02%, respectively); BM+IL-3 (8.33% and 16.74%); BM+IL-6 (9.2% and 17.67%); and BM+IL-3+IL-6 (12.5% and 61.20%). Cell cycle analysis revealed quiescent cells after isolation, 95.5% CD133+ cells in the G0/G1 phase. Regardless of culture period or cytokine incubation, CD133+ cell cycle altered to 70% of CD133+ in the G0/G1 phase. Colony-forming unit (CFU) doubled in BM+IL-3+IL-6 after 8 days of incubation compared with BM group. SOX-2 and NANOG-relative gene expression was detected on day 0 after isolation. BM+IL-6 prevented the decrease in NANOG and SOX-2 gene expression level compared to BM+IL-3 or BM+IL-3+IL-6 incubated cells. Our results indicated that UCB-isolated CD133+ cells were better ex vivo expanded in the presence of SCF, Flt3-L, TPO, IL-3+IL-6. IL-3 probably promotes higher CD133+ cell expansion and IL-6 maintains immature phenotype.