Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

Systemic nanoparticle paclitaxel (nab-paclitaxel) for in-stent restenosis I (SNAPIST-I): a first-in-human safety and dose-finding study

BACKGROUND: Paclitaxel-eluting stents inhibit restenosis; however, this technology has drawbacks (e.g., stent thrombosis, requirement for long-term antiplatelet therapy, and cost--particularly for patients with multivessel disease). Systemic treatment with a novel 130-nm, albumin-bound particle form of paclitaxel (nab-paclitaxel) has been shown to reduce restenosis in animals. HYPOTHESIS: This study was designed to establish the safety and optimal dose of systemic nab-paclitaxel for reducing in-stent restenosis in humans. If well tolerated, systemic nab-paclitaxel may be used with any available bare-metal stent and at potentially lower cost than drug-eluting stents. METHODS: Patients received nab-paclitaxel 10, 30, 70, or 100 mg/m(2) intravenously after stenting of a single de novo lesion >or= 3 mm in diameter. Study endpoints included safety and major adverse cardiac events (MACE) at 2 and 6 months. RESULTS: Data were obtained for all 23 enrolled patients (mean age 66 +/- 10 years, 74% men, 26% with diabetes). No significant adverse events (AE) were attributable to nab-paclitaxel at 10 or 30 mg/m(2). Moderate neutropenia, moderate sensory neuropathy, and mild to moderate, reversible alopecia occurred only at doses of 70 and 100 mg/m(2); therefore, doses of 70 mg/m(2) or higher were considered unacceptable in this patient population. No MACE were reported at 2 months. At 6 months, 4 target lesion revascularizations (TLR) for restenoses were reported (2 each in the 10- and 100-mg/m(2)-dose groups). CONCLUSIONS: Systemic nab-paclitaxel was well tolerated at doses below 70 mg/m(2) in this group of patients; no unexpected AE were noted. Additional studies are under way to explore intravenous and intracoronary administration of nab-paclitaxel.     

STAT5b mediates the GH-induced expression of SOCS-2 and SOCS-3 mRNA in the liver

Suppressor of cytokine signalling (SOCS) proteins act as part of a classical negative feedback loop regulating cytokine signal transduction. Expression of SOCS proteins is induced in response to cytokines and down-regulates the cytokine signal by inhibiting the JAK/STAT pathway. Growth hormone (GH) was previously shown to induce strong transient expression of SOCS-3 and to a lesser extent CIS, SOCS-1 and SOCS-2 in mouse liver (Adams, T.E., Hansen, J.A., Starr, R., Nicola, N.A., Hilton, D.J., Billestrup, N., 1998. Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signalling. J. Biol. Chem. 273, 1285-1287.). In this work we have compared GH-induced SOCS gene expression in wild-type and STAT5b-deficient mice, and show that STAT5b is required for the induction of SOCS-2 and SOCS-3 in liver. In contrast, the absence of STAT5b has no effect on the GH-induced expression of CIS and SOCS-2 mRNA in the mammary gland. Suprisingly, there is no activation of SOCS-3 expression in mammary glands of wild-type and STAT5b mutant mice following GH administration. These results highlight both tissue- and factor-specific differences in the regulation of SOCS gene expression by STAT5a/b.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.     

HPV and precancerous lesions of anal canal in women: systematic review

Objective  

The infection caused by human papillomavirus (HPV) in the anogenital area is considered the most common sexually transmitted infection in the world. Although anal cancer is relatively uncommon in the general population, there has been a significant increase in incidence in recent years. In this review, we focused on research on anal lesions in women.     

Non-invasive methods for iron overload evaluation in dysmetabolic patients

IntroductionAlthough hyperferritinemia may reflect the inflammatory status of patients with non-alcoholic fatty liver disease (NAFLD), approximately 33% of hyperferritinemia cases reflect real hepatic iron overload.AimTo evaluate a non-invasive method for assessing mild iron overload in patients with NAFLD using 3T magnetic resonance imaging (MRI) relaxometry, serum hepcidin, and the expression of ferritin subunits.MethodsThis cross-sectional study assessed patients with biopsy-proven NAFLD. MRI relaxometry was performed using a 3T scanner in all patients, and the results were compared with iron content determined by liver biopsy. Ferritin, hepcidin, and ferritin subunits were assessed and classified according to ferritin levels and to siderosis identified by liver biopsy.ResultsA total of 67 patients with NAFLD were included in the study. MRI revealed mild iron overload in all patients (sensitivity, 73.5%; specificity, 70%). For mild (grade 1) siderosis, the transverse relaxation rate (R₂*) threshold was 58.9 s^?1 and the mean value was 72.5 s^?1 (SD, 33.9), while for grades 2/3 it was 88.2 s^?1 (SD, 31.9) (p < 0.001). The hepcidin threshold for siderosis was > 30.2 ng/mL (sensitivity, 87%; specificity, 82%). Ferritin H and ferritin L subunits were expressed similarly in patients with NAFLD, regardless of siderosis. There were no significant differences in laboratory test results between the groups, including glucose parameters and liver function tests.ConclusionsMRI relaxometry and serum hepcidin accurately assessed mild iron overload in patients with dysmetabolic iron overload syndrome.