Contrast-enhanced spectral mammography (CESM) versus MRI in the high-risk screening setting: patient preferences and attitudes.

PURPOSE: Our study evaluates patient preferences toward screening CESM versus MRI. MATERIALS AND METHODS: As part of a prospective study, high-risk patients had breast MRI and CESM. Patients completed an anonymous survey to evaluate preferences regarding the two modalities. RESULTS: 88% of participants completed the survey. 79% preferred CESM over MRI if the exams had equal sensitivity. 89% would be comfortable receiving contrast as part of an annual screening test. CONCLUSION: High-risk populations may accept CESM as a screening exam and may prefer it over screening MRI if ongoing trials demonstrate screening CESM to be clinically non-inferior MRI.

Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes.

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.

Vascular bed-specific endothelium-dependent vasomomotor relaxation in the hagfish, Myxine glutinosa.

The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates that lived some time more than 500 million years ago. Features that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. We recently reported that hagfish endothelium displays phenotypic heterogeneity in ultrastructure, lectin binding, and mechanisms of leukocyte adhesion. Thus, phenotypic cell heterogeneity evolved as an early feature of the endothelium. In the present study, we wanted to extend these observations by determining whether hagfish endothelium plays a role in mediating vasomotor tone. Response of mesenteric and skeletal muscle arteries to a variety of mediators was assayed by videomicroscopy. Phenylephrine and acetylcholine induced vasoconstriction of mesenteric and skeletal muscle arteries. Bradykinin (BK) and ADP promoted vasorelaxation in precontracted mesenteric arteries but not those from skeletal muscle. BK- and ADP-mediated vasorelaxation of the mesenteric artery was abrogated by mechanical denudation of the endothelium but was unaffected by N(G)-nitro-L-arginine methyl ester. Indomethacin significantly inhibited the vasodilatory response to ADP but not BK. The nitric oxide donor sodium nitroprusside resulted in endothelium-independent relaxation of both mesenteric and skeletal muscle arteries. Together, these data suggest that site-specific endothelium-dependent vasorelaxation is an evolutionarily conserved property of this cell lineage.

X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation.

We identified a family with gray platelet syndrome (GPS) segregating as a sex-linked trait. Affected males had a mild bleeding disorder, thrombocytopenia, and large agranular platelets characteristic of GPS, while obligate carrier females were asymptomatic but had dimorphic platelets on peripheral smear. Associated findings included mild erythrocyte abnormalities in affected males. Linkage analysis revealed a 63 cM region on the X chromosome between markers G10578 and DXS6797, which segregated with the platelet phenotype and included the GATA1 gene. Sequencing of GATA1 revealed a G-to-A mutation at position 759 corresponding to amino acid change Arg216Gln. This mutation was previously described as a cause of X-linked thrombocytopenia with thalassemia (XLTT) but not of gray platelet syndrome. Our findings suggest that XLTT is within a spectrum of disorders constituting the gray platelet syndrome, and we propose that GATA1 is an upstream regulator of the genes required for platelet alpha-granule biogenesis.

Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium.

Mammalian endothelial cells (ECs) display marked phenotypic heterogeneity. Little is known about the evolutionary mechanisms underlying EC heterogeneity. The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates, which lived some time more than 500 million years ago. Features of ECs that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. The goal of this study was to determine whether the hagfish endothelium displays phenotypic heterogeneity. Electron microscopy of the aorta, dermis, heart, and liver revealed ultrastructural heterogeneity of the endothelium. Immunofluorescent studies demonstrated marked differences in lectin binding between vascular beds. Intravital microscopy of the dermis revealed histamine-induced adhesion of leukocytes in capillaries and postcapillary venules, but no such adhesion in arterioles. Together, these data suggest that structural, molecular, and functional heterogeneity of the endothelium evolved as an early feature of this cell lineage.

Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.

The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion.

Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced alpha-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced alpha-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca(2+)- or guanosine triphosphate (GTP)-gamma-S-induced alpha-granule secretion. Latrunculin A-dependent alpha-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates alpha-granule and dense granule secretion.

Defective associations between blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking alphav integrins.

Mouse embryos genetically null for the alphav integrin subunit develop intracerebral hemorrhages at midgestation and die shortly after birth. A key question is whether the hemorrhage arises from primary defects in vascular endothelial cells or pericytes or from other causes. We have previously reported normal initiation of cerebral vessels comprising branched tubes of endothelial cells. Here we show that the onset of hemorrhage is not due to defects in pericyte recruitment. Additionally, most alphav-null vessels display ultrastructurally normal endothelium-pericyte associations and normal interendothelial cell junctions. Thus, endothelial cells and pericytes appear to establish their normal relationships in cerebral microvessels. However, by both light and electron microscopy, we detected defective associations between cerebral microvessels and the surrounding brain parenchyma, composed of neuroepithelial cells, glia, and neuronal precursors. These data suggest a novel role for alphav integrins in the association between cerebral microvessels and central nervous system parenchymal cells.