No abstract

Photoacoustic imaging allows absorption-based high-resolution spectroscopic in vivo imaging at a depth beyond that of optical microscopy. Until recently, photoacoustic imaging has largely been restricted to visualizing the vasculature through endogenous haemoglobin contrast, with most non-vascularized tissues remaining invisible unless exogenous contrast agents are administered. Genetically encodable photoacoustic contrast is attractive as it allows selective labelling of cells, permitting studies of, for example, specific genetic expression, cell growth or more complex biological behaviours in vivo. In this study we report a novel photoacoustic imaging scanner and a tyrosinase-based reporter system that causes human cell lines to synthesize the absorbing pigment eumelanin, thus providing strong photoacoustic contrast. Detailed threedimensional images of xenografts formed of tyrosinase-expressing cells implanted in mice are obtained in vivo to depths approaching 10 mm with a spatial resolution below 100 μm. This scheme is a powerful tool for studying cellular and genetic processes in deep mammalian tissues.O ptical techniques such as fluorescence or bioluminescence imaging are widely used to visualize biological tissues in vivo 1-4 . However, strong optical scattering fundamentally limits the penetration depth or spatial resolution. Microscopy and other techniques that utilize ballistic photons 4 can provide cellular resolution, but only to sub-millimetre penetration depths, while diffuse optical methods such as fluorescence optical tomography 1 can provide greater penetration depths (on the scale of centimetres) but with only limited spatial resolution (on the scale of millimetres). Photoacoustic imaging (PAI) offers the prospect of overcoming these limitations 5-8 . Here, ultrasound waves generated by the absorption of laser light by tissue chromophores are used to produce images of biological tissues based on optical absorption. Because acoustic waves are scattered much less than photons in soft tissues, PAI avoids the depth and spatial resolution limitations of purely optical imaging techniques: depths of a few centimetres with scalable spatial resolution ranging from tens to hundreds of micrometres (depending on depth) are readily achievable.Although strong absorption by haemoglobin enables the acquisition of exquisite three-dimensional photoacoustic (PA) images of the vasculature 9-14 , most cells and tissues are relatively weakly absorbing at visible and near-infrared wavelengths and are thus indistinguishable in the absence of exogenous contrast. The latter can be provided by nanoparticle-or dye-based targeted contrast agents 15-17 , but these can present challenges in achieving effective specific targeting and clearance. The use of reporter genes to provide genetically encoded exogenous PA contrast would avoid these limitations and has the further advantage of providing opportunities to study more complex biological behaviours such as cell growth dynamics and intracellular processes such as gene expressi...

SummaryLevels of circulating red blood cell (RBC)-derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6-fold greater in SCA and 4-fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40-50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)-treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD؉D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 ؋ 10 9 /L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P ‫؍‬ .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P ‫؍‬ .04, but no significant difference in relapse risk (28% vs 23%; P ‫؍‬ .5) or overall survival (59% vs 67%; P ‫؍‬ .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n ‫؍‬ 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD ؉ cells, but this inhibition was reduced in the presence of ATRA. IntroductionMost cases of acute promyelocytic leukemia (APL) are characterized by t(15;17)(q22;q21) leading to formation of the promyelocytic leukemia-retinoic acid receptor ␣ (PML-RARA) fusion protein. 1 PML-RARA plays a critical role in determining disease phenotype, mediating the characteristic differentiation block through the repression of genes implicated in myelopoiesis, which is overcome by pharmacologic levels of retinoic acid. 1 However, evidence derived largely from transgenic mouse models has suggested that PML-RARA is insufficient for leukemogenesis, 2,3 although the precise nature of the cooperating events implicated in generating the full disease phenotype remains uncertain. A number of potential candidates have been proposed to play a role in this process. These include the reciprocal fusion gene product RARA-PML, which is expressed in approximately 75% of patients [4][5][6] and has been postulated to contribute to leukemogenesis by promoting genomic instability, thereby predisposing to the acquisition of additional oncogenic lesions. 7 There has also been considerable interest in the potential role of activating mutations of genes encoding receptor tyrosine kinases (RTKs), which commonly accompany acute myelocytic leukemia (AML)-associated translocations including t(15;17), giving rise to the proposition that they could provide a common class of cooperating mutation in the development of the disease. 8 Fms-like tyrosine kinase 3 (FLT3) is an RTK expressed on hematopoietic progenitors. Mutation of the FLT3 gene is common in AML. [9][10][11][12] Numerous mutations have been identified. The majority, present in approximately 25% of patients, are internal tandem duplications (ITDs) that lead to in-frame insertions within the juxtamembrane region of the receptor. Le...

As a dendritic cell (DC) matures, it becomes more potent as an antigen-presenting cell. This functional change is accompanied by a change in DC immunophenotype. The signal transduction events underlying this process are poorly characterized. In this study, we have investigated the signal transduction pathways involved in the lipopolysaccharide (LPS)-induced maturation of human monocyte–derived DCs (MoDCs) in vitro. We show that exposure of immature MoDCs to LPS activates the p38 stress-activated protein kinase (p38SAPK), extracellular signal–regulated protein kinase (ERK), phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and nuclear factor (NF)-κB pathways. Studies using inhibitors demonstrate that PI3 kinase/Akt but not the other pathways are important in maintaining survival of LPS-stimulated MoDCs. Inhibiting p38SAPK prevented activation of the transcription factors ATF-2 and CREB and significantly reduced the LPS-induced up-regulation of CD80, CD83, and CD86, but did not have any significant effect on the LPS-induced changes in macropinocytosis or HLA-DR, CD40, and CD1a expression. Inhibiting the NF-κB pathway significantly reduced the LPS-induced up-regulation of HLA-DR as well as CD80, CD83, and CD86. Inhibiting the p38SAPK and NF-κB pathways simultaneously had variable effects depending on the cell surface marker studied. It thus appears that different aspects of LPS-induced MoDC maturation are regulated by different and sometimes overlapping pathways.