Changes in DTI Diffusivity and fMRI Connectivity Cluster Coefficients for Students with and without Specific Learning Disabilities In Written Language: Brain's Response to Writing Instruction.

Before and after computerized writing instruction, participants completed assessment with normed measures and DTI and fMRI connectivity scanning. Evidence-based differential diagnosis was used at time 1 to assign them to diagnostic groups: typical oral and written language (n=6), dysgraphia (impaired handwriting, n=10), dyslexia (impaired word spelling and reading, n=20), and OWL LD (impaired syntax construction, n=6). The instruction was aimed at subword letter writing, word spelling, and syntax composing. With p <.001 to control for multiple comparisons, the following significant findings were observed in academic achievement, DTI (radial diffusivity RD, axial diffusivity AD, and mean diffusivity MD), and graph cluster coefficients for fMRI connectivity. A time effect (pre-post intervention increase) in handwriting and oral construction of sentence syntax was significant; but diagnostic group effects were significant for dictated spelling and creation of word-specific spellings, with the dyslexia and OWL LD groups scoring lower than the typical control or dysgraphia groups. For RD a time effect occurred in anterior corona radiata and superior frontal. For AD a time effect occurred in superior corona radiata, superior frontal region, middle frontal gyrus, and superior longitudinal fasciculus. For MD a time effect occurred in the same regions as AD and also anterior coronal radiata. A diagnostic group effect occurred for graph cluster coefficients in fMRI connectivity while writing the next letter in alphabet from memory; but the diagnostic group × time interaction was not significant. The only significant time × treatment interaction occurred in right inferior frontal gyrus associated with orthographic coding. Compared to time 1, cluster coefficients increased at time 2 in all groups except in the dysgraphia group in which they decreased. Implications of results are discussed for response to instruction (RTI) versus evidence-based differential diagnosis for identifying students with SLDs in writing which may be best understood at both the behavioral and brain levels of analysis.

A robust automated measure of average antibody staining in immunohistochemistry images.

Identifying and scoring cancer markers plays a key role in oncology, helping to characterize the tumor and predict the clinical course of the disease. The current method for scoring immunohistochemistry (IHC) slides is labor intensive and has inherent issues of quantitation. Although multiple attempts have been made to automate IHC scoring in the past decade, a major limitation in these efforts has been the setting of the threshold for positive staining. In this report, we propose the use of an averaged threshold measure (ATM) score that allows for automatic threshold setting. The ATM is a single multiplicative measure that includes both the proportion and intensity scores. It can be readily automated to allow for large-scale processing, and it is applicable in situations in which individual cells are hard to distinguish. The ATM scoring method was validated by applying it to simulated images, to a sequence of images from the same tumor, and to tumors from different patient biopsies that showed a broad range of staining patterns. Comparison between the ATM score and manual scoring by an expert pathologist showed that both methods resulted in essentially identical scores when applied to these patient biopsies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Site-specific labeling of annexin V with F-18 for apoptosis imaging.

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH 2 terminus, with the thiol-selective reagent (18)F-labeling agent N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide ([(18)F]FBABM). We also examined the cell binding affinity of the (18)F-labeled annexin V-128 ([(18)F]FAN-128). [(18)F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253-1259). The average yield of [(18)F]FBABM was 23 +/- 4% (n = 4, decay-corrected) and the specific activity was approximately 6000 Ci/mmol. The total synthesis time was approximately 92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [(18)F]FBABM with the thiol-containing annexin V-128 gave [(18)F]FAN-128 in 37 +/- 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [(18)F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [ (18)F]FAN-128 as an apoptosis imaging agent is warranted.

Regional hypoxia in glioblastoma multiforme quantified with [18F]fluoromisonidazole positron emission tomography before radiotherapy: correlation with time to progression and survival.

PURPOSE: Hypoxia is associated with resistance to radiotherapy and chemotherapy and activates transcription factors that support cell survival and migration. We measured the volume of hypoxic tumor and the maximum level of hypoxia in glioblastoma multiforme before radiotherapy with [(18)F]fluoromisonidazole positron emission tomography to assess their impact on time to progression (TTP) or survival. EXPERIMENTAL DESIGN: Twenty-two patients were studied before biopsy or between resection and starting radiotherapy. Each had a 20-minute emission scan 2 hours after i.v. injection of 7 mCi of [(18)F]fluoromisonidazole. Venous blood samples taken during imaging were used to create tissue to blood concentration (T/B) ratios. The volume of tumor with T/B values above 1.2 defined the hypoxic volume (HV). Maximum T/B values (T/B(max)) were determined from the pixel with the highest uptake. RESULTS: Kaplan-Meier plots showed shorter TTP and survival in patients whose tumors contained HVs or tumor T/B(max) ratios greater than the median (P < or = 0.001). In univariate analyses, greater HV or tumor T/B(max) were associated with shorter TTP or survival (P < 0.002). Multivariate analyses for survival and TTP against the covariates HV (or T/B(max)), magnetic resonance imaging (MRI) T1Gd volume, age, and Karnovsky performance score reached significance only for HV (or T/B(max); P < 0.03). CONCLUSIONS: The volume and intensity of hypoxia in glioblastoma multiforme before radiotherapy are strongly associated with poorer TTP and survival. This type of imaging could be integrated into new treatment strategies to target hypoxia more aggressively in glioblastoma multiforme and could be applied to assess the treatment outcomes.

Evaluation of 18F-annexin V as a PET imaging agent in an animal model of apoptosis.

UNLABELLED: Annexin V is a 36-kDa protein that binds with high affinity to phosphatidylserine lipids in the cell membrane. Because one of the earliest measurable events in apoptosis is the eversion of phosphatidylserine from the inner membrane leaflet to the outer cell surface, annexin V has proven useful for detecting the earliest stages of apoptosis. METHODS: Annexin V was radiolabeled with 18F using N-succinimidyl-4-18F-fluorobenzoic acid chemistry, to a specific activity of 555-925 kBq/mug of protein. 18F-Annexin V (14.8-51.8 MBq) was administered intravenously to rats after pretreatment with cycloheximide (5 mg/kg) to induce liver apoptosis, and the injected rats were imaged by PET over 2 h. After imaging, rats were dissected and individual organs were weighed and counted. RESULTS: Pretreatment of rats with cycloheximide resulted in a 3- to 9-fold increase in uptake of 18F-annexin V in the liver of treated animals at 2 h, compared with controls. By morphologic analysis, treated livers showed a 3- to 6-fold higher level of apoptosis than controls, with higher levels also seen with longer exposure to cycloheximide. Terminal deoxynucleotide end-labeling (TUNEL) assays performed on liver slices showed that cycloheximide induced a 5- to 8-fold increase in the number of TUNEL-positive nuclei. These TUNEL results correlated with the uptake of 18F-annexin V in dissected liver tissue, with an r2 value of 0.89. Biodistribution analysis of normal rats showed highest uptake of 18F-annexin V in the kidneys and urinary bladder, indicating rapid renal clearance of 18F-annexin V metabolites. CONCLUSION: The PET data, the organ-specific uptake data from dissection, and the morphologic and TUNEL measures of apoptosis together indicate that 18F-annexin V binds specifically to apoptotic tissues in this model of chemically induced apoptosis in rat liver. The short physical half-life of 18F-annexin V and the rapid clearance of its metabolites to the urinary system suggest that 18F-annexin V will be useful in early assessment of the clinical response to cancer therapy in individual patients.

Production of [F-18]fluoroannexin for imaging apoptosis with PET.

Recombinant human-annexin-V was conjugated with 4-[F-18]fluorobenzoic acid (FBA) via its reaction with the N-hydroxysuccinimidyl ester (FBA-OSu) at pH 8.5. A series of reactions using varying amounts of annexin-V, unlabeled FBA-OSu, and time produced products with different conjugation levels. Products were characterized by mass spectrometry and a cell-binding assay to assess the effect of conjugation. In each case, the conjugated protein was a mixture of proteins with a range of conjugation. Annexin-V could be conjugated with an average of two FBA mole equivalents without decreasing its affinity for red blood cells (K(d) 6-10 nM) with exposed phosphatidylserine. An average conjugation of 7.7 (range 3-13) diminished the binding 3-fold. Large-scale production and purification of [F-18]FBA-OSu from [F-18]fluoride was accomplished within 90 min and in 77% radiochemical yield (decay-corrected to the end of cyclotron bombardment). The conjugation reaction of annexin with [F-18]FBA-OSu was studied with respect to activity level, protein mass, and concentration. Under the most favorable conditions, >25 mCi [F-18]fluoroannexin (FAN) was isolated in 64% yield (decay-corrected for a 22 min conjugation process) from labeling 1.1 mg of annexin-V. A pilot PET imaging study of [F-18]fluoroannexin in normal rats showed high uptake in the renal excretory system and demonstrated sufficient clearance from most other internal organs within 1 h. [F-18]Fluoroannexin should prove useful in imaging targeted apoptosis.