A novel 196Leu to Pro substitution in the beta3 subunit of the alphaIIbbeta3 integrin in a patient with a variant form of Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited disorder where an absence of platelet aggregation is associated with quantitative or qualitative abnormalities of the alphaIIbbeta3 integrin. In rare patients, amino acid substitutions have provided information on the functional significance of specific domains within alphaIIb or beta3. We now report an elderly male GT patient (R.M.) from the south west of France whose platelets possess a small residual expression of alphaIIbbeta3. Furthermore, the integrin failed to undergo the necessary conformational changes following platelet activation to permit the binding of fibrinogen or activation-dependent monoclonal antibodies despite the presence of an RGD-binding site. Screening of the alphaIIb and beta3 genes by PCR-SSCP revealed a heterozygous mutation at position 685 in exon 5 of the beta3 gene leading to a 196Leu to Pro substitution. 196Leu is a highly conserved amino acid of beta3. The other beta3 allele appeared to be silent. This mutation, inherited from his mother and present in other family members with intermediate levels of alphaIIbbeta3, was close to the MIDAS-like domain of beta3, a fact that appears to explain its effect on alphaIIbbeta3 activation and fibrinogen binding.     

Endogenous antioxidant defense induction by melon superoxide dismutase reduces cardiac hypertrophy in spontaneously hypertensive rats

We assessed the influence of SODB, a melon superoxide dismutase (SOD), on left ventricular (LV) hypertrophy in SHR. SODB (4 or 40U SOD) was given orally for 4 or 28 days to SHR. For each treatment period, LV weight index (LVWI) and cardiomyocytes size were measured. SOD, glutathione peroxidase (GPx) and catalase expressions, and LV production and presence of superoxide anion were determined. Pro-inflammatory markers were also measured. SODB reduced LVWI and cardiomyocytes size after 4 or 28 days. Cardiac SOD and GPx increased by 30–40% with SODB. The presence but not production of superoxide anion was significantly reduced by SODB. No effect of SODB was detected on inflammatory status in any group. The beneficial effect of SODB on cardiac hypertrophy seems to be related to the stimulation of endogenous antioxidant defense, suggesting that SODB may be of interest as a dietary supplementation during conventional antihypertensive therapy.     

Red wine phenolic compounds reduce plasma lipids and apolipoprotein B and prevent early aortic atherosclerosis in hypercholesterolemic golden Syrian hamsters (Mesocricetus auratus)

The effects of a red wine phenolic extract (PE) on plasma lipoproteins and early atherosclerosis were studied in hamsters. Hamsters (n = 32) were divided into 4 groups of 8 and fed an atherogenic diet for 8 wk. They received by force- feeding 7.14 mL/(kg. d) PE in 2.6 mol/L ethanol (E + PE) or PE in water (W + PE), mimicking a moderate consumption of red wine or alcohol-free red wine [30.4 mg/(kg. d)], or 2.6 mol/L ethanol (E-PE) or water (W-PE) as their respective controls. Plasma cholesterol and triglyceride concentrations were lower in groups that consumed PE. The decrease in plasma apolipoprotein (Apo) B concentration was due mainly to PE and was significantly lower in Group E + PE than in Group E-PE (-7.5%) and in Group W + PE than in Group W-PE (-40%). Apo-A1 was not affected. PE significantly increased plasma antioxidant capacity by 9% in Group E + PE and 18% in Group W + PE compared with their respective controls. Liver glutathione peroxidase activity was 67% greater in the group receiving PE in water compared with the group given water; there was no effect when PE was given in ethanol relative to its control. Aortic fatty streak area (AFSA) was significantly reduced in the groups receiving PE in ethanol (-32%) or PE in water (-29%) in comparison with their respective controls. Ethanol significantly reduced AFSA by 60% (Group E-PE vs. Group W-PE) or 62% (Group E + PE vs. Group W + PE). These data suggest that ethanol is a complementary component of phenolics in the benefits of red wine for hamsters and that chronic ingestion of PE in ethanol prevents the development of atherosclerosis through several mechanisms. With moderate consumption of red wine, ethanol can improve the effects of phenolic compounds. However, alcohol-free red wine appears to be a very good alternative to red wine.     

Evidence that the platelet integrin alphaIIb beta3 is regulated by the integrin-linked kinase,ILK, in a PI3-kinase dependent pathway

Platelet aggregation is mediated by the integrin alphaIIb beta3 which is activated by intracellular signals during platelet activation. We have attempted to determine if ILK ("Integrin-Linked Kinase") is involved in the regulation of alphaIIb beta3 function. ILK co-immunoprecipitated with beta3 in stimulated platelets. Using confocal microscopy, ILK was detected in the cytoplasm of resting platelets. ADP or PMA stimulation led to its translocation to the plasma membrane. In parallel, there was a transient increase in ILK kinase activity, association with and phosphorylation of beta3. Inhibition of PI3-kinase by two unrelated inhibitors (wortmannin and LY294002) prevented ILK-related functions. However, it did not prevent the conformational change in alphaIIb beta3 (shown by PAC-1 binding), although integrin affinity for fibrinogen was decreased as measured using FITC-fibrinogen. Furthermore, aggregate formation was reduced. Thus ILK transiently associates with and phosphorylates beta3 in a PI3-kinase dependent manner suggesting that it participates at an intermediate stage in a critical mechanism for assuring large stable aggregates.     

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome

We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking alpha-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor gamma-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C gamma 2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the literature.     

Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells

In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.     

Respiratory physiotherapy to prevent pulmonary complications after abdominal surgery: a systematic review

OBJECTIVES: To examine the efficacy of respiratory physiotherapy for prevention of pulmonary complications after abdominal surgery. METHODS: We searched in databases and bibliographies for articles in all languages through November 2005. Randomized trials were included if they investigated prophylactic respiratory physiotherapy and pulmonary outcomes, and if the follow-up was at least 2 days. Efficacy data were expressed as risk differences (RDs) and number needed to treat (NNT), with 95% confidence intervals (CIs). RESULTS: Thirty-five trials tested respiratory physiotherapy treatments. Of 13 trials with a "no intervention" control group, 9 studies (n = 883) did not report on significant differences, and 4 studies (n = 528) did: in 1 study, the incidence of pneumonia was decreased from 37.3 to 13.7% with deep breathing, directed cough, and postural drainage (RD, 23.6%; 95% CI, 7 to 40%; NNT, 4.3; 95% CI, 2.5 to 14); in 1 study, the incidence of atelectasis was decreased from 39 to 15% with deep breathing and directed cough (RD, 24%; 95% CI, 5 to 43%; NNT, 4.2; 95% CI, 2.4 to 18); in 1 study, the incidence of atelectasis was decreased from 77 to 59% with deep breathing, directed cough, and postural drainage (RD, 18%; 95% CI, 5 to 31%; NNT, 5.6; 95% CI, 3.3 to 19); in 1 study, the incidence of unspecified pulmonary complications was decreased from 47.7% to 21.4 to 22.2% with intermittent positive pressure breathing, or incentive spirometry, or deep breathing with directed cough (RD, 25.5 to 26.3%; NNT, 3.8 to 3.9). Twenty-two trials (n = 2,734) compared physiotherapy treatments without no intervention control subjects; no conclusions could be drawn. CONCLUSIONS: There are only a few trials that support the usefulness of prophylactic respiratory physiotherapy. The routine use of respiratory physiotherapy after abdominal surgery does not seem to be justified.     

Persistent activation of the Fyn/ERK kinase signaling axis mediates imatinib resistance in chronic myelogenous leukemia cells through upregulation of intracellular SPARC

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization, growth factor availability, cell adhesion, differentiation, and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study, we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity, whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably, we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly, SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings, increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together, our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC.