Characterization of the Cardiac Overexpression of HSPB2 Reveals Mitochondrial and Myogenic Roles Supported by a Cardiac HspB2 Interactome.

Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.

Effects of redox state on the efficient uptake of cell permeable Peptide in Mammalian cells.

We investigated whether a cell-penetrating peptide linked via a disulfide bond to a fluorophore-labeled cargo peptide can be used to interrogate changes in cellular redox state. A fluorescence resonance energy transfer (FRET) pair was constructed so that the cargo peptide was labeled with fluorescein amidite (FAM) and the cell-penetrating peptide was attached to a quencher. Incubation of cells in culture with the FRET construct was visualized using live-cell, time-lapse imaging, which demonstrated earlier cellular uptake of the construct when cells were treated with the reducing agent n-acetylcysteine (NAC). The FRET peptide construct was easily detected in cells cultured in 96-well plates using a plate-reader. Treatment of cells with various classes of reducing or oxidizing agents resulted in an increase or decrease in FAM fluorescence, respectively. Changes in FAM fluorescence correlated significantly with redox-sensitive green fluorescent protein ratios in cells treated with hydrogen peroxide but not NAC. Detection of relative changes in cellular redox state was enhanced by the fact that uptake of the cell-penetrating peptide occurred more quickly in relatively reduced compared with oxidized cells. We conclude that cell-penetrating peptides coupled via disulfide bonds to detectable cargo is a novel and specific approach for assessment of relative changes in cellular thiol redox state.

A diagnostic dilemma of atypical gallbladder appearance on Tc-99m HIDA cholescintigraphy resolved with SPECT/CT.

Tc-99m HIDA cholescintigraphy is the diagnostic procedure of choice for acute cholecystitis. Acute cholecystitis is associated in vast majority of the cases with cystic duct obstruction. The demonstration of presence (cystic duct patency) or absence (cystic duct obstruction) of visualization of the gallbladder on cholescintigraphy is critical to the diagnosis of acute cholecystitis. The visualization of the gallbladder rules out acute cholecystitis in most of the cases. Although, in most cases, determination of visualization or nonvisualization of gallbladder is straight forward, occasionally it can be challenging. We describe a patient with suspected acute cholecystitis, in whom an unusual appearance of the gallbladder on hepatobiliary scintigraphy was clarified with SPECT/CT, an approach that is rarely used in Tc-99m HIDA cholescintigraphy.

Impact on reader performance for lesion-detection/ localization tasks of anatomical priors in SPECT reconstruction.

With increasing availability of multimodality imaging systems, high-resolution anatomical images can be used to guide the reconstruction of emission tomography studies. By measuring reader performance on a lesion detection task, this study investigates the improvement in image-quality due to use of prior anatomical knowledge, for example organ or lesion boundaries, during SPECT reconstruction. Simulated (67)Ga -citrate source and attenuation distributions were created from the mathematical cardiac-torso (MCAT) anthropomorphic digital phantom. The SIMIND Monte Carlo software was then used to generate SPECT projection data. The data were reconstructed using the De Pierro maximum a posteriori (MAP) algorithm and the rescaled-block-iterative (RBI) algorithm for comparison. We compared several degrees of prior knowledge about the anatomy: no knowledge about the anatomy; knowledge of organ boundaries; knowledge of organ and lesion boundaries; and knowledge of organ, lesion, and pseudo-lesion (non-emission uptake altering) boundaries. The MAP reconstructions used quadratic smoothing within anatomical regions, but not across any provided region boundaries. The reconstructed images were read by human observers searching for lesions in a localization receiver operating characteristic (LROC) study of the relative detection/localization accuracies of the reconstruction algorithms. Area under the LROC curve was computed for each algorithm as the comparison metric. We also had humans read images reconstructed using different prior strengths to determine the optimal trade-off between data consistency and the anatomical prior. Finally by mixing together images reconstructed with and without the prior, we tested to see if having an anatomical prior only some of the time changes the observer's detection/localization accuracy on lesions where no boundary prior is available. We found that anatomical priors including organ and lesion boundaries improve observer performance on the lesion detection/localization task. Use of just organ boundaries did not provide a statistically significant improvement in performance however. We also found that optimal prior strength depends on the level of anatomical knowledge, with a broad plateau in which observer performance is near optimal. We found no evidence that having anatomical priors use lesion boundaries only when available changes the observer's performance when they are not available. We conclude that use of anatomical priors with organ and lesion boundaries improves reader performance on a lesion-detection/localization task, and that pseudo-lesion boundaries do not hurt reader performance. However, we did not find evidence that a prior using only organ boundaries helps observer performance. Therefore we suggest prior strength should be tuned to the organ-only case, since a prior will likely not be available for all lesions.

A novel pretargeting method for measuring antibody internalization in tumor cells.

A novel pretargeting method has been developed to quantitate antibody cellular internalization. In this study, the antibody was conjugated with a phosphorodiamidate morpholino oligomer (MORF) specific for the complementary MORF (cMORF) as an effector. Half the tumor cells were incubated with the MORF-antibody (pretargeting group) and the other half with the same MORF-antibody at the same concentration but radiolabeled (direct targeting group). After incubation, the same dosage of radiolabeled cMORF was added to the wells of the pretargeting group. The radioactivity of the direct targeting cells represented the sum of both internalized and cell-surface-bound antibodies, whereas the radioactivity of the pretargeting cells resulted only from the surface-bound antibodies, as the radiolabeled cMORF does not penetrate the cell surface. Therefore, the difference in radioactivity accumulation between pretargeting and direct targeting provides the internalized fraction. In this example, the internalization of a MORF conjugated anti-prostate-specific membrane antigen antibody, 3C6, in LNCaP cells was examined, and the average cell-surface residence time was determined as 2 hours. This method of measuring antibody internalization is directly applicable to pretargeting applications but can be a universal alternative to the conventional acid-wash method, with the advantage of leaving the cell membrane undamaged.

Comparison of cardiac to hepatic uptake of 99mTc-tetrofosmin with and without adenosine infusion to predict the presence of haemodynamically significant coronary artery disease.

BACKGROUND: Coronary artery disease impairs cardiac vasodilatory reserve. A low ratio of cardiac to hepatic vasodilatory reserve may be diagnostic for coronary artery disease. AIM: To compare the ratio of cardiac to hepatic uptake of 99mTc-tetrofosmin during adenosine infusion and at rest in patients with and without coronary artery disease in order to determine whether the ratio was significantly different between the two groups. METHODS: Fifty-one patients who underwent coronary angiography and adenosine stress myocardial perfusion imaging using 99mTc-tetrofosmin were studied retrospectively. Anterior planar images from the single photon emission computed tomography (SPECT) raw data were used to draw regions of interest around the heart and liver. The counts per pixel in each region were used to calculate the stress ratio (SR) and the rest ratio (RR) as follows: SR = (cardiac counts per pixel)at stress/(hepatic counts per pixel)at stress; RR = (cardiac counts per pixel)at rest/(hepatic counts per pixel)at rest. The SR and SR/RR ratios were compared in patients with and without significant coronary artery disease. Receiver operating characteristic curves were drawn for SR and SR/RR. RESULTS: The SR and SR/RR ratios were significantly lower in patients with significant coronary artery disease than in patients without (P<0.001). A cut-off ratio of SR/RR = 1.00 yielded 87% sensitivity and 74% specificity for the detection of significant coronary artery disease. Combining SR/RR with standard SPECT image interpretation increased the sensitivity without substantially changing the specificity in comparison with standard SPECT image interpretation only. CONCLUSION: Comparison of cardiac to hepatic 99mTc-tetrofosmin concentration at rest and under adenosine stress provides useful diagnostic information for the assessment of the presence of significant coronary artery disease.

Effects of intravenous amino acid administration with Y-90 DOTA-Phe1-Tyr3-Octreotide (SMT487[OctreoTher) treatment.

Y-90-DOTA-Phe1-Tyr3-Octreotide (90Y-SMT 487, OctreoTher) has shown potential for effectively treating patients with neuroendocrine tumors. The dose-limiting organ for this agent is the kidney. The purpose of this work is to assess the effectiveness of a commercially available amino acid solution on reducing renal uptake of 90Y-SMT 487 and determine the safety profile of this solution. Subjects with In-111 pentetreotide positive tumors and normal creatinine levels were treated with 3 cycles of 90Y-SMT 487, 120 mCi/cycle, at 6-9 week intervals. During each treatment two liters of an amino acid solution containing arginine and lysine (Aminosyn II 7%, Abbott Laboratories, Abbott Park, IL) were infused IV over 4 hours. Adverse events were recorded. To assess the effect of Aminosyn II on renal uptake of 90Y-SMT 487, a subgroup of subjects underwent bremsstrahlung imaging 24 hours following infusion. Kidney to liver (K/L) count density ratios were generated from the baseline In-111 pentetreotide images (performed without amino acid infusion) and the 90Y bremsstrahlung images. Follow-up creatinine levels were obtained. Thirty-seven subjects received a total of 89 90Y-SMT 487 treatments. The number of amino-acid infusions associated with one or more episodes of emesis was 53 (62%). During 13 (15%) of these infusions, the Aminosyn II rate had to be reduced because of severe nausea and vomiting. Symptomatic flushing occurred during 16 (18%) of the infusions. One subject experienced a near syncopal event shortly after completing the infusion. Creatinine levels remained normal in 34 of 36 subjects during a mean follow-up period of 9.8 months. Fourteen subjects underwent bremsstrahlung imaging following infusion of 90Y-SMT 487. Kidney uptake appeared to decrease with administration of the amino acid solution in 13 of 14 subjects. For the 28 individual kidneys, the mean percent decrease in the Kidney/Liver uptake ratio with the amino acid solution was found to be 32%. We conclude that 2 L of Aminosyn II 7% infused over 4 hours appears to notably reduce renal uptake of 90Y-SMT 487. Aminosyn is generally well tolerated, particularly at lower infusion rates with occasional moderate to severe nausea and vomiting at higher rates.