The extracellular concentrations of adenosine are elevated during sepsis and adenosine receptors regulate the host’s response to sepsis. Here, we investigated the role of the adenosine generating ectoenzyme, ecto-5′-nucleotidase (CD73) in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased activation of caspase-3, poly(ADP-ribose) polymerase and NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and elevated cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using AMPCP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.

Purpose: Breast cancer micrometastases in the bone marrow are resistant to chemotherapy. They can remain dormant for years before some begin to proliferate. We seek to understand survival mechanisms and develop targeted approaches to eliminating these cells.Experimental Design:In an in vitro model of dormancy, basic fibroblast growth factor 2 (FGF-2), abundant in the bone marrow, inhibits the growth of well-differentiated cells in the 2-to 10-cell stage and up-regulates integrin A 5 B 1 . Through this integrin, cells bind fibronectin, spread out, and acquire a survival advantage, partly through activation of the phosphatidylinositol 3-kinase/Akt pathway. We investigated the effects of Taxotere, flavopiridol, and mitogenactivated protein/extracellular signal-regulated kinase (ERK) kinase and p38 inhibitors on survival of dormant clones and that of flavopiridol on expression of integrins, adhesion strength, and phosphorylation of Akt, ERK 1/2, and p38.Results: Dormant MCF-7 and T-47D cell clones were resistant to Taxotere concentrations 10-fold higher than needed to eliminate growing clones but were almost completely eradicated by 200 nmol/L flavopiridol. Flavopiridol caused a decrease in FGF-2 -induced expression of integrins, including A 5 and B 1 , and decreased FGF-2 -induced specific adhesion to fibronectin. It diminished Akt phosphorylation, but reexpression of active Akt was not sufficient to reverse dormant clone inhibition. Flavopiridol did not affect phosphorylation of ERK 1/2 and p38 but diminished total protein levels. Chemical inhibition of these pathways partially abrogated dormant clone survival.Conclusions: Flavopiridol has pleiotropic effects on key targets involved with survival of dormant breast cancer cells and may represent a useful approach to eliminating cells dependent on multiple signal pathways for survival.

Adenylyl cyclase (AC) types 5 and 6 (AC5 and AC6) are the two major AC isoforms expressed in the mammalian heart that mediate signals from ␤-adrenergic receptor stimulation. Because of the unavailability of isoform-specific antibodies, it is difficult to ascertain the expression levels of AC5 protein in the heart. Here we demonstrated the successful generation of an AC5 isoform-specific mouse monoclonal antibody and studied the expression of AC5 protein during cardiac development in different mammalian species. The specificity of the antibody was confirmed using heart and brain tissues from AC5 knockout mice and from transgenic mice overexpressing AC5. In mice, the AC5 protein was highest in the brain but was also detectable in all organs studied, including the heart, brain, lung, liver, stomach, kidney, skeletal muscle, and vascular tissues. Western blot analysis showed that AC5 was most abundant in the neonatal heart and declined to basal levels in the adult heart. AC5 protein increased in the heart with pressure-overload left ventricular hypertrophy. Thus this new AC5 antibody demonstrated that this AC isoform behaves similarly to fetal type genes, such as atrial natriuretic peptide; i.e., it declines with development and increases with pressureoverload hypertrophy. adenylyl cyclase isoforms; monoclonal antibody; pressure overload; hypertrophy ADENYLYL CYCLASE (AC) is an enzyme that catalyzes the conversion of ATP to cAMP. The complexity in understanding the signaling pathway of cAMP can be attributed, in part, to 10 isoforms of AC: nine membrane bound and one soluble, which have been cloned and characterized in mammals (4,6,9,10,29). Each of these membrane-bound isoforms consists of two hydrophobic domains (with 6 transmembrane spans) and two cytoplasmic domains. The cytoplasmic domains constitute the catalytic site, which is subject to intracellular regulation (6). The AC isoforms have high amino acid homology in their cytoplasmic domains but differ in the sequence of the transmembrane region. The specificity of tissue distribution, the functional properties, and the chromosomal location of the corresponding genes also differentiate these isoforms (4, 10, 12). The major AC isoforms expressed in the heart are type 5 (AC5) and type 6 (AC6) (7,11,30). Tissue distribution and developmental expression of AC5 and AC6 mRNA have been previously studied in rats, chicks, and humans (7,30,31). However, in the absence of an AC5-specific antibody, it has not been possible to determine the levels of protein expression. It is very important to study the differential regulation of AC5 and AC6 in the heart since AC5 and AC6 behave differently in the pathogenesis of heart failure. We demonstrated that the AC5 knockout (KO) mouse model lives longer and is resistant to stress (32), including chronic pressure overload (21) and chronic catecholamine (22) stress. However, studies by others have suggested that the overexpression of AC6 is beneficial and might be considered for heart failure therapy (23). In addition, based on mRNA...

IRAK1 is a key regulatory protein in TLR/IL1R-mediated cell activation during the inflammatory response. Studies indicated that pending on the nature of the used inflammatory model, down-regulation of IRAK1 may be beneficial or detrimental. However the role of IRAK1 in affecting outcome in polymicrobial sepsis is unknown. We tested this question using an IRAK1 deficient mouse strain and the cecal ligation and puncture (CLP) procedure, which is a clinically relevant rodent septic model. Sepsis-induced mortality was markedly lower in IRAK1-deficient mice (35%) compared to WT (85%). Sepsis-induced increases in blood IL-6 and IL-10 levels were blunted at 6h post-CLP in IRAK1 deficiency compared to WT but cytokine levels were similar at 20h post-CLP. Sepsis induced blood granulocytosis and depletion of splenic B cells were also blunted in IRAK1 deficient mice as compared to WT. Analysis of TLR-mediated cytokine responses by IRAK1 deficient and WT macrophages ex vivo indicated a TLR4-dependent down-regulation of IL-6 and IL1β in IRAK1 deficiency, whereas TLR2 dependent responses were unaffected. TLR7/8-mediated IL-6, IL1β and IL-10 production was also blunted in IRAK1 macrophages as compared to WT. The study shows that IRAK1 deficiency impacts multiple TLR-dependent pathways and decreases early cytokine responses following polymicrobial sepsis. The delayed inflammatory response caused by the lack of IRAK1 expression is beneficial, as it manifests a markedly increased chance of survival after polymicrobial sepsis.

Bone marrow (BM) dysfunction is an important component of immunomodulation. This study investigated alterations in cell content, apoptotic responses, and cell proliferation in BM, blood, and spleen in endotoxemic mice (LPS from Escherichia coli). As the decreased antioxidant status associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency has been shown to modulate the innate immune response, we also tested whether a G6PD mutation (80% decrease in cellular enzyme activity) alters BM responses during endotoxemia. LPS decreased BM myeloid (CD45(+)CD11b(+)) and B lymphoid (CD45(+)CD19(+)CD11b(-)) cell content compared with controls. In contrast, LPS increased CD11b(+) myeloid but decreased T and B cell counts in the circulation. Endotoxemia inhibited spontaneous, heat shock, and H(2)O(2)-induced apoptosis as well as proliferative activity in BM lymphoid cells. In contrast, BM myeloid cell apoptosis was not altered, and their proliferative activity was increased during endotoxemia. Following LPS, splenic myeloid cell content was increased, and T and B cell content was unchanged; furthermore, splenocytes showed increased apoptosis compared with controls. BM cell content, including lymphoid and myeloid cells, was greater in G6PD mutant than wild-type (WT) mice, and LPS decreased BM cell counts to a greater degree in mutant than WT mice. Endotoxemia caused widespread inhibition of BM cytokine and chemokine production; however, IL-6 production was increased compared with controls. LPS-induced IL-6 production was decreased in G6PD mutant animals compared with WT. This study indicates that endotoxin inversely affects BM myeloid and lymphoid cell production. LPS-induced down-regulation of B cell production contributes to the generalized lymphopenia and lymphocyte dysfunction observed following nonspecific immune challenges.

Cellular X chromosome mosaicism, which is unique to females may be advantageous during pathophysiological challenges as compared to the single X chromosome machinery of males and may also contribute to gender dimorphism in the inflammatory response. We tested the hypothesis whether cellular mosaicism for the X-linked gp91phox (NOX2) deficiency, the catalytic component of the superoxide anion generating NADPH oxidase complex, is advantageous during polymicrobial sepsis. Deficient, WT and heterozygous/mosaic mice were compared following polymicrobial sepsis initiated by cecal ligation and puncture. As compared to WT littermates, sepsis-induced mortality was improved in deficient mice, as well as, in mosaics animals carrying deficient and WT phagocyte subpopulations simultaneously. In contrast, blood bacterial counts were the highest in deficient mice as compared to mosaic or WT animals. Consistent with poor survival, WT mice also showed the worst organ damage following sepsis. In mosaic animals, the deficient neutrophil subpopulations displayed increased organ recruitment and elevated CD11b membrane expression as compared to WT neutrophil subpopulations within the same animal. The dynamics of sepsis-induced blood and organ cytokine content and white blood cell composition changes including lymphocyte subsets in blood and BM showed differences among WT, deficient and mosaic subjects indicating that mosaic mice are not simply the average of the deficient and WT responses. Upon oxidative burst, interchange of oxidants between WT and deficient neutrophil subpopulations occurred in mosaic mice. The study suggests that mice mosaic for gp91phox expression have multiple advantages in comparison to WT as well as deficient mice during the septic course.

We measured the background level and spatial variation of carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) and associated basic soil parameters along the depth profile for 3 years, in 3 habitats (woodland, wetland, and grassland), in Keoladeo National Park (KNP), an important water fowl reserve and a world heritage site in India. The study examined soils at 5 depths (0, 0.25, 0.50, 0.75 and 1.0 m) for 3 years, i.e. from 2003 to 2005. Total organic C (TOC), total N (TN), total available P (TAP), and total available S (TAS), irrespective of the habitat type and year, were found to be highest in the litter layer, gradually declining with depth. Elemental ratios (C : N, C : P, and C : S) followed the same declining trend, whereas the N : P ratio increased down the soil profile. The high C : N and C : S ratios in the litter layer suggest the relatively low mobilisation of N and S from the decaying detritus layer. All the variables studied varied significantly among the soil layers (GLM-ANOVA, P < 0.05). Principal component analysis (PCA) showed 4 components based on examination of the scree plot. The first component accounted for 27.1% of the total variance in soil characteristics among samples, reflecting the influence of soil variables such as P, TOC, and pH. The second component accounted for 23.5% of the total variance, reflecting the influence of total dissolved solids (TDS) and TAS. The influence of C on elemental ratio (C : S) was pointed by the third component, accounted for 14.2% of the total variance. The fourth component accounted for 13.6% of the variance, indicating the influence of soil TN. Thus, the 4 PCA components that accounted for 78.4% of the total variance in the data can be qualified as N : P/soil P/C, TDS/TAS, C : S ratio, and soil TN, respectively.

Objective-To test the hypothesis, using an animal model, whether female X-chromosome mosaicism for inflammatory gene expression could contribute to the gender dimorphic response during the host response. X-chromosome-linked genetic polymorphisms present a unique biological condition because females display heterozygous cellular mosaicism, due to the fact that either the maternal or the paternal X chromosomes are inactivated in each individual cell in females. This is in contrast with the conditions in males who carry exclusively the maternal X chromosome. Design-Prospective, randomized, laboratory investigation. Settings-University research laboratory.Subjects-Female mice deficient, heterozygous (mosaic) or WT for the X-linked gp91phox.Interventions-We compared selected inflammatory markers among heterozygous (mosaics), WT and homozygous deficient animals in response to in vivo lipopolysaccharide (Escherichia coli, 20 mg/kg body weight). To test individual mosaic subpopulations of polymorphonuclear neutrophil responses, we also developed a flow cytometry assay that identifies the active parental X chromosomes in individual cells, using gp91phox expression as a marker. Measurements and MainResults-Heterozygous mosaic mice presented white blood cell trafficking patterns similar to that observed in WT mice, despite the fact that the deficient subpopulation in mosaic animals displayed increased cell activation as reflected in elevated neutrophil CD11b expression and splenic infiltration. Mosaic animals also displayed splenic neutrophil infiltration, which was skewed toward the deficient subpopulation. Observations on splenic T-cell depletion and post lipopolysaccharide interleukin-10 responses indicated that the inflammatory response in mosaic animals does not simply display an average of the deficient and WT responses, but the mosaic subjects display a uniquely characteristic response.Conclusions-The study supports the notion that female X chromosome mosaicism for polymorphic gene expression represents a unique condition, which may contribute to the gender dimorphic character of the inflammatory response. Mosaicism for X-linked polymorphisms may have clinical significance and needs consideration in genetic association or gender-related clinical studies.