Sickle cell disease (SCD) is an autosomal recessive genetic red cell disorder characterized by the production of a defective form of hemoglobin, hemoglobin-S, that is worldwide-distributed. The acute clinical manifestations of SCD are related to hemoglobin cyclic-polymerization and to the generation of rigid, dense red blood cells (RBCs). We studied RBCs membrane proteome from human sickle RBCs, fractioned according to density compared to normal RBCs. 2-DE followed by MS analysis was carried out. We identified 65 proteins differently expressed, divided into five major clusters according to their functions: (i) membrane-cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin-proteasome-system; (iv) flotillins; (v) chaperones. HSP27, HSP70 and peroxiredoxin-II (Prx-II) showed the most relevant changes. They were differently recruited to sickle RBCs membrane in response to in vitro hypoxia. Potential markers were then validated in a transgenic-mouse model for SCD, the SAD mice, exposed to hypoxia mimicking acute SCD vaso-occlusive-crisis (VOCs); we found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 h and 48 h of hypoxia, while Prx-II membrane binding was modulated during hypoxia. Our data indicate that HSP27 and HSP70 play a novel role as RBCs membrane protein protectors and as possibly new markers of severity of RBCs membrane damage during acute VOCs.
Primary melanoma of the lung is an extremely rare clinical entity. We found only 32 cases reported in literature, and in two of these multiple brain metastases were present. We describe a case of primary lung melanoma with brain and skin metastases that presented with an initial clinical diagnosis of pneumonia.A 55-year-old white man presented with cough productive of dark sputum and fever. A chest x-ray showed a right lung infiltration. After failure to respond to usual treatment for pneumonia, bronchoscopy examination and CT scan revealed a right pulmonary mass. The CT-guided biopsy confirmed a diagnosis of malignant melanoma. The primary lung origin of the tumor was demonstrated by the characteristic junctional pattern of melanoma cells. Further evaluation revealed metastases in the brain and in skin.Primary lung melanoma is an uncommon neoplasm that may be confused with more conventional types of lung cancer. Careful interpretation of histopathological information in correlation with all other clinical, laboratory and imaging studies may be needed to establish a diagnosis. Evaluation for metastases should include looking at the eyes, brain, skin. Due to the small number of cases reported in literature, there is no experience on the management and the prognosis of the disease.
Ischemic/reperfusion (I/R) injury plays a critical role in the pathogenesis of several clinical manifestations of sickle cell disease (SCD) and it results from complex interactions between cells and plasma factors. Hepatic dysfunction has been described in SCD and the pathophysiology of liver I/R damage in SCD is only partially known. The liver is highly vascular organ with high metabolic rate, susceptible to stress-induced by hypoxia. Here, we studied the effects of acute and chronic hypoxia on liver function and pathology in transgenic sickle mice (SAD mice). We evaluated the following parameters at baseline, at 4, 48 and 168 hours (hrs) of exposure to hypoxia (8% oxygen) followed by 2 hrs reoxygenation (21% oxygen): complete blood counts with retic, red cell density, serum AST and ALT levels, liver pathology and expression by RT-PCR of the following genes: TNF-a, IL-1b as inflammatory response markers; TgfB1 (involved in control cell growth and extracellular matrix formation), mmp9 (marker of hepatic matrix remodeling), NfKb (transcription effector involved in liver cell death and regeneration), Icam1 (endothelial damage and vascular remodeling marker), in addition to heme oxygenase-1 (Homx1), eNOS (Nos3) and iNOS (Nos2). SAD mice at baseline exhibited liver infiltrates of inflammatory cells, no thrombi and a mild hepatic injury (pathological score: 1.4±0.02; n=5); up-regulation of TgfB1, Icam1, Nos3 and down-regulation of Nfkb compared to normoxic WT mice. One SAD mice died at 48 hrs hypoxia and 2 SAD mice died at 96 hrs hypoxia. In SAD mice I/R induced: increased red cell density at 48 and 168 hrs of hypoxia; increased peripheral neutrophil count at 48 and 168 hrs; increased retic count at 168 hrs; increased serum levels of AST/ALT; increased liver cell inflammatory infiltrate at 48 hrs (n=5) and 168 hrs (n=6); increased pathological score at 48 hrs (2.3±0.02 n=5) and at 168 hrs (3.2±0.05 n=6; P<0.05). In SAD mice I/R induced changes in the expression of the following genes: 4hrs: up-regulation of Nfkb, Ilb1, Nos2 and down-regulation of Tgfb1, Icam1, homx1 and Nos3; 48 hrs: up-regulation of Icam1, Nos2, Tnfa and down-regulation of TgfB1, Nos3, homx1, Nfkb, Ilb1; 168 hrs: up-regulation of TgfB1, Icam1, Nos 3, homx1, Nfkb, Ilb1, Nos2 and down-regulation of Nos2. In WT mice I/R induced increased neutrophils and retic count at 168 hrs; increased serum levels of AST/ALT at 48 and 168 hrs; increased liver cell inflammatory infiltrate at 168 hrs (n=7) and pathological score at 168 hrs and modulation of the following genes at 4hrs: up-regulation of Nfkb, Icam1, Ilb1, Nos2, mmp9 and down-regulation of TgfB1 and homx1; at 48 hrs: up-regulation of TgfB1, Icam1, homx1, Nos2, Tnfa, mmp9 and down-regulation of Ilb1; at 168 hrs: up-regulation of Icam1, Nfkb, Nos 3, Nos2, Tnfa, mmp9 and down-regulation of Il1b, homox1, TnfB1. Then, we investigated the effects of a PDE-4 inhibitor (Rolipram) on hepatic I/R injury in sickle cell SAD mice, since cAMP cell content has been recently described to be crucial in I/R liver damage and PDE-4 is a key regulator of cAMP metabolism. In SAD mice, Rolipram reduced the hypoxia induced peripheral neutrophilia and serum AST/ALT increase, prevented the I/R liver injury as supported by reducing cell inflammatory infiltrate, pathological score (1.8±0.02, n= 6, P<0.05) and the modulation of the following genes: TgfB1, Icam1, Nos3, Ilb1, Tnfa and mmp9 These data suggest that the inhibition of PDE-4 protects from I/R related sickle cell liver injury, most likely by inducing over-expression of Nos3, reducing vascular activation, modulating matrix remodeling and immune-inflammatory response.
An otherwise healthy 32-year-old woman suffered from finger ischemia. An echocardiogram and CT scan revealed a mobile mass in the left ventricle that was attached to the anterior papillary muscle and did not involve the valve leaflets. The tumor was resected and histopathology confirmed it to be a papillary fibroelastoma. Our case emphasizes the significance of a comprehensive diagnostic workup for a peripheral ischemic lesion. This resulted in the discovery of an unusual intra-ventricular origin for a commonly benign tumor.
Sickle cell disease (SCD) is a worldwide-distributed hereditary red cell (RBCs) disorder characterized by the pathologic hemoglobin-S (HbS). The acute clinical manifestations of SCD are strictly related to HbS polymerization, to generation of dense RBCs, to their interaction with the abnormal activated vascular endothelial cells and to amplified inflammatory response. To identify new biomarkers associated with acute SCD events, we studied the RBCs membrane proteome from human SCD erythrocytes (n=20) fractioned according to density compared to normal RBCs (n=20), we divided RBCs into 2 fractions: fraction 1 (F1) corresponding to the least dense RBCs (density < 1.074) and fraction 2 (F2) corresponding to the densest RBCs (density > 1.095). None of the patients were under hydroxyurea treatment and they did not receive transfusion in the sixmonths before the study. Bi-dimensional electrophoresis (2-DE) followed by MS-analysis of RBCs F1 and F2 from both control and SCD subjects was carried out and 65 proteins differently expressed were identified. We focused on molecular chaperones. In SCD RBCs, we found that the amount of HSP27, HSP70 and Prx-2 recruited to the membrane was higher than in controls, Prx-2 was present as monomers and dimmers that were more abundant in SCD RBCs than in normal controls. Then, we exposed F1 and F2 fractions from normal and SCD patients to in vitro cellular stress (deoxygenation). In sickle RBCs, deoxygenation induced increase membrane recruitment of HSP27 in F1, modulation of HSP70 membrane binding in F2; reduction of Prx-2 monomers with increase Prx-2 dimerization in F1, but slightly changes in Prx-2 monomers in F2. These data suggest that in SCD RBCs HSP-27, -70 and Prx-2 are recruited to the membrane in response to cellular stress, acting as molecular chaperones to assist proteins in regaining their functional conformation. In order to evaluate their role in vivo and because increase serum levels of HSP70 has been recently reported in few SCD patients, we used transgenic sickle cell SAD mice as a model for SCD and exposed to hypoxia (8%-O2) respectively for 2–12–48 hours, mimicking acute human SCD vaso-occlusive-crisis (VOCs). We studies SAD mice and wild-type mice (C57/B6, WT) divided into groups of 6 animals each. We evaluated HSP70, HSP27 and Prx-2 expression on RBCs membrane, hematological parameters, RBCs density, and cation content. We found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 hours and 48 hours (hrs) of hypoxia, while Prx-2 membrane binding was modulated during hypoxia. At the different time schedule, we observed a marked reduction in red cell K+ at 12 and 48 hrs hypoxia, associated with significant increase of the dense RBCs at 48 hrs hypoxia. Our data indicate that in SCD, HSP70, HSP27 and Prx-2 membrane recruitment is modulated in vivo during acute VOCs, supporting their novel role as RBCs membrane protein protectors and as new markers of severity of RBCs membrane damage during acute sickle VOCs.
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