Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage.

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb's oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb's destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.

MAGE-C1/CT-7 expression in plasma cell myeloma: sub-cellular localization impacts on clinical outcome.

Plasma cell myelomas (PMs) have a poor prognosis. Cancer-testis (CT) antigens are immunogenic proteins, representing potential targets for tumor vaccination strategies. The expression of the CT antigens GAGE, MAGE-A4, MAGE-C1/CT-7, and NY-ESO-1 was investigated on paraffin-embedded bone marrow biopsies from 219 PM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients. The frequency and prognostic impact of these CT antigens were compared with known morphological prognostic markers (i.e. Mib1 labeling index) and the presence of the translocations t(4;14)(p16.3; q32) and t(11;14)(q13;q32). We show that MAGE-C1/CT-7 is the most prevalent CT antigen, expressed in 57% of PMs in a high percentage of tumor cells. While MAGE-C1/CT-7 was absent in non-malignant plasma cells, plasma cells of patients with MGUS did express MAGE-C1/CT-7, but no other CT antigens. MAGE-C1/CT-7 was more frequently expressed in PMs with an elevated proliferation rate (Mib1 >10%) compared to PMs with a low proliferation rate (Mib1

Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts.

Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.