A microPET/CT system for in vivo small animal imaging

A microCT scanner was designed, fabricated and integrated with a previously reported microPET II scanner (Tai et al 2003 Phys. Med. Biol. 48 1519, Yang et al 2004 Phys. Med. Biol. 49 2527), forming a dual modality system for in vivo anatomic and molecular imaging of the mouse. The system was designed to achieve high-spatial-resolution and high-sensitivity PET images with adequate CT image quality for anatomic localization and attenuation correction with low x-ray dose. The system also has relatively high throughput for screening, and a flexible gantry and user interface. X-rays were produced by a 50 kVp, 1.5 mA fixed tungsten anode tube, with a focal spot size of 70 microm. The detector was a 5 x 5 cm(2) photodiode detector incorporating 48 microm pixels on a CMOS array and a fast gadolinium oxysulfide (GOS) intensifying screen. The microCT system has a flexible C-arm gantry design with adjustable detector positioning, which acquires CT projection images around the common microPET/CT bed. The design and the initial characterization of the microCT system is described, and images of the first mouse scans with microPET/CT scanning protocols are shown.     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.     

Branding and a child’s brain: an fMRI study of neural responses to logos

Branding and advertising have a powerful effect on both familiarity and preference for products, yet no neuroimaging studies have examined neural response to logos in children. Food advertising is particularly pervasive and effective in manipulating choices in children. The purpose of this study was to examine how healthy children’s brains respond to common food and other logos. A pilot validation study was first conducted with 32 children to select the most culturally familiar logos, and to match food and non-food logos on valence and intensity. A new sample of 17 healthy weight children were then scanned using functional magnetic resonance imaging. Food logos compared to baseline were associated with increased activation in orbitofrontal cortex and inferior prefrontal cortex. Compared to non-food logos, food logos elicited increased activation in posterior cingulate cortex. Results confirmed that food logos activate some brain regions in children known to be associated with motivation. This marks the first study in children to examine brain responses to culturally familiar logos. Considering the pervasiveness of advertising, research should further investigate how children respond at the neural level to marketing.     

Evolution of neuronal and glial tau isoforms in chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy characterized by accumulation of hyperphosphorylated tau (p‐tau) in perivascular aggregates in neurons and glia at the depths of neocortical sulci and progresses to diffuse neocortical, allocortical and brainstem structures. The strongest risk factor is exposure to repetitive head impacts acquired most commonly through contact sports and military service. Given that CTE can only be definitively diagnosed after death, a better understanding of the cellular and molecular changes in CTE brains may lead to identification of mechanisms that could be used for novel biomarkers, monitoring progression or therapeutic development. Disruption of alternative pre‐mRNA splicing of tau mRNA plays a pathogenic role in tauopathy, with multiple characteristic patterns of isoform accumulation varying among tauopathies. Limited data are available on CTE, particularly at early stages. Using biochemical and histological approaches, we performed a detailed characterization of tau isoform signatures in post‐mortem human brain tissue from individuals with a range of CTE stages (n = 99). In immunoblot analyses, severity was associated with decreased total monomeric tau and increased total oligomeric tau. Immunoblot with isoform‐specific antisera revealed that oligomeric tau with three and four microtubule binding domain repeats (3R and 4R) also increased with CTE severity. Similarly, immunohistochemical studies revealed p‐tau accumulation consisting of both 3R and 4R in perivascular lesions. When the ratio of 4R:3R was analyzed, there was mixed expression throughout CTE stages, although 4R predominated in early CTE stages (I‐II), a 3R shift was observed in later stages (III‐IV). While neurons were found to contain both 3R and 4R, astrocytes only contained 4R. These 4R‐positive cells were exclusively neuronal at early stages. Overall, these findings demonstrate that CTE is a mixed 4R/3R tauopathy. Furthermore, histologic analysis reveals a progressive shift in tau isoforms that correlates with CTE stage and extent of neuronal pathology.     

Neuropathological profile of long‐duration amyotrophic lateral sclerosis in military Veterans

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting both the upper and lower motor neurons. Although ALS typically leads to death within 3 to 5 years after initial symptom onset, approximately 10% of patients with ALS live more than 10 years after symptom onset. We set out to determine similarities and differences in clinical presentation and neuropathology in persons with ALS with long vs. those with standard duration. Participants were United States military Veterans with a pathologically confirmed diagnosis of ALS (n = 179), dichotomized into standard duration (<10 years) and long‐duration (≥10 years). The ALS Functional Rating Scale‐Revised (ALSFRS‐R) was administered at study entry and semi‐annually thereafter until death. Microglial density was determined in a subset of participants. long‐duration ALS occurred in 76 participants (42%) with a mean disease duration of 16.3 years (min/max = 10.1/42.2). Participants with long‐duration ALS were younger at disease onset (P = 0.002), had a slower initial ALS symptom progression on the ALSFRS‐R (P < 0.001) and took longer to diagnose (P < 0.002) than standard duration ALS. Pathologically, long‐duration ALS was associated with less frequent TDP‐43 pathology (P < 0.001). Upper motor neuron degeneration was similar; however, long‐duration ALS participants had less severe lower motor neuron degeneration at death (P < 0.001). In addition, the density of microglia was decreased in the corticospinal tract (P = 0.017) and spinal cord anterior horn (P = 0.009) in long‐duration ALS. Notably, many neuropathological markers of ALS were similar between the standard and long‐duration groups and there was no difference in the frequency of known ALS genetic mutations. These findings suggest that the lower motor neuron system is relatively spared in long‐duration ALS and that pathological progression is likely slowed by as yet unknown genetic and environmental modifiers.