In vivo osteopontin-induced macrophage accumulation is dependent on CD44 expression

In order to assess the role of osteopontin (OPN) in leukocyte accumulation in inflammatory conditions, native OPN and its thrombin cleaved form (OPN + Thr) were studied in vivo using a rodent subcutaneous air pouch model (AP). Both forms of OPN-induced macrophage infiltration into the AP in wild-type mice. In animals lacking CD44, macrophage numbers were significantly reduced within the cavity, but cells still accumulated along the subcutaneous lining. In animals lacking endogenous OPN, no differences were found in exogenous OPN-induced macrophage accumulation, although macrophage exhibited increased α4 integrin expression. These studies reveal that both OPN and OPN + Thr attract macrophages in vivo through CD44.     

Haemonchus contortus: evaluation of two signal sequence trapping systems for detection of secreted molecules

Given that signal sequences between secreted proteins of different species can be interchanged, it is reasonable to expect that both mammalian and yeast signal sequence trapping (SST) systems would secrete Haemonchus contortus proteins with similar efficiency and quality. To determine if H. contortus cDNAs that contain a signal sequence could re-establish secretion of a reporter protein, mammalian and yeast SST vectors were designed, 10 H. contortus genes selected, and their respective cDNAs cloned into these two SST vectors. The selected molecules included genes known to code for excretory/secretory or membrane-bound proteins as potential test 'positives', and genes known to code for non-secreted proteins as test 'negatives'. While differentiation between secretion and non-secretion was evident in both systems, the results indicated greater efficiency was achieved when the mammalian system was used. Therefore, mammalian SST using COS cells would be a more useful tool to screen H. contortus cDNA libraries for potential secreted and type-1 integral membrane proteins than yeast SST.     

Metal complexation chemistry used for phosphate and nucleotide determination: an investigation of the Yb3+–pyrocatechol violet sensor

Metal complexation reactions can be used effectively as sensors to measure concentrations of phosphate and phosphate analogs. Recently, a method was described for the detection of phosphate or ATP in aqueous solution based on the displacement by these ligands of pyrocatechol violet (PV) from a putative 2:1 (Yb³⁺)₂PV complex. We have not been able to reproduce this stoichiometry and report this work in order to correct the coordination chemistry upon which sensor applications are based. In our work, colorimetric and spectrophotometric detection of phosphate was confirmed qualitatively (blue PV + Yb³⁺; yellow + P_i); however, the sequence of visual changes on the titration of PV with 2 equiv. of Yb³⁺ and back titration with ATP as described previously could not be reproduced. In contrast to the linear response to P_i that was reported previously, the absorbance response at 443 or 623 nm was found to be sigmoidal using the recommended 2:1 Yb³⁺:PV solution (100 μM:50 μM, pH 7, HEPES). Furthermore, both continuous variation titration and molar ratio analysis (Job plot) experiments are consistent with 1:1, not 2:1, YbPV complex stoichiometry at pH 7 in HEPES buffer, indicating that the deviation from linearity is produced by excess Yb³⁺. Indeed, using a 1:1 Yb³⁺:PV ratio produces a linear response in ΔAbs at 443 or 623 nm on back titration with analyte (phosphate or ATP). In addition, speciation analysis of the Yb–ATP system demonstrates that a 1:1 complex containing Yb³⁺ and ATP predominates in solution at μM metal ion and ATP concentrations. Paramagnetic ¹H NMR spectroscopy directly establishes the formation of Yb³⁺–solute complexes in dilute aqueous solution. The 1:1 YbPV complex can be used for the colorimetric measurement of phosphate and ATP concentrations from ~2 μM. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Randomised controlled trial assessing the impact of a nurse delivered,flow monitored protocol for optimisation of circulatory status after cardiac surgery

Objective To assess whether a nurse led, flow monitored protocol for optimising circulatory status in patients after cardiac surgery reduces complications and shortens stay in intensive care and hospital.Design Randomised controlled trial.Setting Intensive care unit and cardiothoracic unit of a university teaching hospital.Participants 174 patients who underwent cardiac surgery between April 2000 and January 2003.Interventions Patients were allocated to conventional haemodynamic management or to an algorithm guided by oesophageal Doppler flowmetry to maintain a stroke index above 35 ml/m².Results 26 control patients had postoperative complications (two deaths) compared with 17 (four deaths) protocol patients (P = 0.08). Duration of hospital stay in the protocol group was significantly reduced from a median of nine (interquartile range 7-12) days to seven (7-10) days (P = 0.02). The mean duration of hospital stay was reduced from 13.9 to 11.4 days, a saving in hospital bed days of 18% (95% confidence interval -12% to 47%). Usage of intensive care beds was reduced by 23% (-8% to 59%).Conclusion A nurse delivered protocol for optimising circulatory status in the early postoperative period after cardiac surgery may significantly shorten hospital stay.     

Mitochondrial DNA deletion mutations: a causal role in sarcopenia.

Mitochondrial DNA (mtDNA) deletion mutations accumulate with age in tissues of a variety of species. Although the relatively low calculated abundance of these deletion mutations in whole tissue homogenates led some investigators to suggest that these mutations do not have any physiological impact, their focal and segmental accumulation suggests that they can, and do, accumulate to levels sufficient to affect the metabolism of a tissue. This phenomenon is most clearly demonstrated in skeletal muscle, where the accumulation of mtDNA deletion mutations remove critical subunits that encode for the electron transport system (ETS). In this review, we detail and provide evidence for a molecular basis of muscle fiber loss with age. Our data suggest that the mtDNA deletion mutations, which are generated in tissues with age, cause muscle fiber loss. Within a fiber, the process begins with a mtDNA replication error, an error that results in a loss of 25-80% of the mitochondrial genome. This smaller genome is replicated and, through a process not well understood, eventually comprises the majority of mtDNA within the small affected region of the muscle fiber. The preponderance of the smaller genomes results in a dysfunctional ETS in the affected area. As a consequence of both the decline in energy production and the increase in oxidative damage in the region, the fiber is no longer capable of self-maintenance, resulting in the observed intrafiber atrophy and fiber breakage. We are therefore proposing that a process contained within a very small region of a muscle fiber can result in breakage and loss of muscle fiber from the tissue.     

Distinct region of the granulocyte colony-stimulating factor receptor mediates proliferative signaling through activation of Janus kinase 2 and p44/42 mitogen-activated protein kinase.

The granulocyte colony-stimulating factor receptor (G-CSFR) regulates the proliferation, differentiation and survival of neutrophilic progenitor cells. In these studies, we introduced mutant G-CSFRs with cytoplasmic domains truncated approximately every 30 amino acids from the C-terminus into interleukin-3 (IL-3)-dependent myeloid LGM-1 cells. The G-CSFR membrane proximal region containing the Box 2 homology sequence was determined to be critical for proliferative signaling, as well as for activation of Janus kinase (JAK2) and p44/42 mitogen-activated protein kinase (MAPK) following G-CSF stimulation. In the presence of increasing concentrations of JAK2 or p44/42 MAPK inhibitors, LGM-1 cells expressing the full-length G-CSFR exhibited a decreased capacity to proliferate in response to G-CSF. These results demonstrate that JAK2 and p44/42 MAPK activation is involved in proliferative signaling through the G-CSFR membrane proximal region containing the Box 2 homology sequence.     

Birth weight of offspring and insulin resistance in late adulthood: cross sectional survey

ObjectiveTo investigate the association between birth weight of offspring and mothers'' insulin resistance in late adulthood.DesignCross sectional survey.SettingGeneral practitioner''s surgeries in 23 towns in Great Britain.Participants4286 women aged 60-79 years.ResultsBirth weight of offspring was inversely related to maternal insulin resistance in late adulthood. For each 1 kg higher birth weight of offspring, women had a 15% reduction in the odds of being in the fourth with highest insulin resistance, compared to other fourths (odds ratio 0.85; 95% confidence interval 0.71 to 1.00). This increased to 27% (0.73; 0.60 to 0.90) after adjusting data for potential confounders. A U shaped relation between birth weight of offspring and diabetes in older age was found; women with the lightest and heaviest offspring had the highest prevalence of diabetes.ConclusionsBirth weight of offspring is inversely related to the mother''s insulin resistance in late adulthood, despite the association of glucose intolerance during pregnancy with heavier offspring at birth. Common genetic factors contribute to the relation between birth weight and risk of cardiovascular disease and diabetes in adults.

What is already known on this topic

Small birth weight is related to increased risk of cardiovascular disease and diabetes in adulthood; the underlying mechanisms are unclearSmall birth weight of offspring is related to parental cardiovascular disease, suggesting that common genetic factors affect birth weight and the risk of disease in adulthoodGenetic factors associated with the metabolism of insulin are plausible in linking birth weight and cardiovascular disease (the fetal insulin hypothesis)

What this study adds

Birth weight of offspring is inversely related to maternal insulin resistance in older ageGenetic factors related to both insulin resistance and birth weight explain at least part of the association between birth weight and risk of cardiovascular disease and diabetes in adulthood     

Wild Type Mesenchymal Cells Contribute to the Lung Pathology of Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.