Devkumar Mustafi

Quantitative Analysis of Dynamic Contrast Enhanced MRI for Assessment of Bowel Inflammation in Crohn's Disease: Pilot Study

RATIONALE AND OBJECTIVES The aim of this study was to evaluate the feasibility of quantitative analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data in the detection of bowel inflammation in patients with Crohns disease. MATERIALS AND METHODS Eleven patients who underwent magnetic resonance enterography for known or suspected Crohns disease and had colonoscopy or surgery within 4 weeks of imaging were included in this study. DCE-MRI data were acquired using a 1.5-T scanner with temporal resolution of 5 to 12 seconds for approximately 5 to 7 minutes. A two-compartment pharmacokinetic model was used to analyze the data to obtain the volume transfer constant (K(trans)) and the extravascular extracellular space (v(e)). Additionally, semiquantitative parameters were derived using an empirical mathematical model to fit the contrast concentration curves. Endoscopic, surgical, and pathologic results were compared to the MRI data. Receiver-operating characteristic analysis was performed to compare the diagnostic accuracy of the parameters in the task of distinguishing normal tissue from inflammation. RESULTS A total of 51 bowel segments (19 with inflammation, 32 normal) were included in the analyses. Inflamed bowel segments had faster K(trans) values, larger v(e) values, increased contrast uptake, larger initial areas under the contrast concentration curve, and steeper initial enhancement slopes than normal bowel segments (P < .05). The areas under the receiver-operating characteristic curve for these parameters ranged from 0.70 to 0.86. CONCLUSION These preliminary results demonstrate that the quantitative analysis of DCE-MRI data is possible for the assessment of bowel inflammation in patients with Crohns disease. Future studies need be performed on larger numbers of patients to correlate the severity and type of inflammation with kinetic parameters.

Intranasal Delivery of Mesenchymal Stem Cells Significantly Extends Survival of Irradiated Mice with Experimental Brain Tumors

Treatment options of glioblastoma multiforme are limited due to the blood-brain barrier (BBB). In this study, we investigated the utility of intranasal (IN) delivery as a means of transporting stem cell-based antiglioma therapeutics. We hypothesized that mesenchymal stem cells (MSCs) delivered via nasal application could impart therapeutic efficacy when expressing TNF-related apoptosis-inducing ligand (TRAIL) in a model of human glioma. ¹¹¹In-oxine, histology and magnetic resonance imaging (MRI) were utilized to track MSCs within the brain and associated tumor. We demonstrate that MSCs can penetrate the brain from nasal cavity and infiltrate intracranial glioma xenografts in a mouse model. Furthermore, irradiation of tumor-bearing mice tripled the penetration of (¹¹¹In)-oxine-labeled MSCs in the brain with a fivefold increase in cerebellum. Significant increase in CXCL12 expression was observed in irradiated xenograft tissue, implicating a CXCL12-dependent mechanism of MSCs migration towards irradiated glioma xenografts. Finally, MSCs expressing TRAIL improved the median survival of irradiated mice bearing intracranial U87 glioma xenografts in comparison with nonirradiated and irradiated control mice. Cumulatively, our data suggest that IN delivery of stem cell-based therapeutics is a feasible and highly efficacious treatment modality, allowing for repeated application of modified stem cells to target malignant glioma.

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of kcat and kcat/Km were 4.5 ± 0.1 s−1 and 0.49 ± 0.1 × 106 M−1⋅s−1 in the absence of divalent metal ions, 14.7 ± 2.2 s−1 and 25.1 ± 7.4 × 106 M−1⋅s−1 in the presence of Mg2+ or Mn2+, and 24.2 ± 3.6 s−1 and 2.4 ± 0.7 × 106 M−1⋅s−1 when supported by VO2+ or bis(acetylacetonato)oxovanadium(IV). Binding of VO2+ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg2+. Electron paramagnetic resonance spectra of the ternary enzyme–VO2+-chelate–dUDP complex revealed a pattern of 31P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO2+ ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO2+. We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO2+ and the VO2+-chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.

High-resolution magnetic resonance colonography and dynamic contrast-enhanced magnetic resonance imaging in a murine model of colitis

Inflammatory bowel disease, including ulcerative colitis, is characterized by persistent or recurrent inflammation and can progress to colon cancer. Colitis is difficult to detect and monitor noninvasively. The goal of this work was to develop a preclinical imaging method for evaluating colitis. Herein, we report improved MRI methods for detecting and characterizing colitis noninvasively in mice, using high‐resolution in vivo MR images and dynamic contrast‐enhanced MRI studies, which were confirmed by histologic studies in a murine model of colitis. C57Bl6/J male mice were treated with 2.5% dextran sulfate sodium in their drinking water for 5 days to induce colitis. MR images were acquired using a 9.4‐T Bruker scanner from 5–25 days following dextran sulfate sodium treatment. In dynamic contrast‐enhanced MRI studies, Gd uptake (Ktrans) and its distribution (ve) were measured in muscle and normal and inflamed colons after administering Gd‐diethyltriaminepentaacetic acid (Gd‐DTPA). T2‐weighted MR images distinguished normal colon from diffusely thickened colonic wall occurring in colitis (P <0.0005) and correlated with histologic features. Values of Ktrans and ve obtained from dynamic contrast‐enhanced MRI were also significantly different in inflamed colons compared to normal colon (P < 0.0005). The results demonstrate that both T2‐weighted anatomic imaging and quantitative analysis of dynamic contrast‐enhanced MRI data can successfully distinguish colitis from normal colon in mice. Magn Reson Med 63:922–929, 2010.

Sensitivity to tumor microvasculature without contrast agents in high spectral and spatial resolution MR images

Contrast‐enhanced (CE)‐MRI is sensitive to cancers but can produce adverse reactions and suffers from insufficient specificity and morphological detail. This research investigated whether high spectral and spatial resolution (HiSS) MRI detects tumor vasculature without contrast agents, based on the sensitivity of the water resonance line shape to tumor blood vessels. HiSS data from AT6.1 tumors inoculated in the hind legs of rats (N = 8) were collected pre‐ and post–blood pool contrast agent (iron‐oxide particles) injection. The waterline in small voxels was significantly more asymmetric at the tumor rim compared to the tumor center and normal muscle (P < 0.003). Composite images were synthesized, with the intensity in each voxel determined by the Fourier component (FC) of the water resonance having the greatest relative image contrast at that position. We tested whether regions with high contrast in FC images (FCIs) contain vasculature by comparing FCIs with CE‐MRI as the “gold standard” of vascular density. The FCIs had 75% ± 13% sensitivity, 74% ± 10% specificity, and 91% ± 4% positive predictive value (PPV) for vasculature detection at the tumor rim. These results suggest that tumor microvasculature can be detected using HiSS imaging without the use of contrast agents. Magn Reson Med 61:291–298, 2009.

Use of a reference tissue and blood vessel to measure the arterial input function in DCEMRI

Accurate measurement of the arterial input function is critical for quantitative evaluation of dynamic contrast enhanced magnetic resonance imaging data. Use of the reference tissue method to derive a local arterial input function avoided large errors associated with direct arterial measurements, but relied on literature values for Ktrans and ve. We demonstrate that accurate values of Ktrans and ve in a reference tissue can be measured by comparing contrast media concentration in a reference tissue to plasma concentrations measured directly in a local artery after the 1–2 passes of the contrast media bolus—when plasma concentration is low and can be measured accurately. The values of Ktrans and ve calculated for the reference tissue can then be used to derive a more complete arterial input function including the first pass of the contrast bolus. This new approach was demonstrated using dynamic contrast enhanced magnetic resonance imaging data from rodent hind limb. Values obtained for Ktrans and ve in muscle, and the shape and amplitude of the derived arterial input function are consistent with published results. Magn Reson Med, 2010.

The Renin–Angiotensin System Mediates EGF Receptor–Vitamin D Receptor Cross-Talk in Colitis-Associated Colon Cancer

Purpose: We previously showed that EGF receptor (EGFR) promotes tumorigenesis in the azoxymethane/dextran sulfate sodium (AOM/DSS) model, whereas vitamin D suppresses tumorigenesis. EGFR–vitamin D receptor (VDR) interactions, however, are incompletely understood. Vitamin D inhibits the renin–angiotensin system (RAS), whereas RAS can activate EGFR. We aimed to elucidate EGFR–VDR cross-talk in colorectal carcinogenesis. Experimental Design: To examine VDR–RAS interactions, we treated Vdr+/+ and Vdr−/− mice with AOM/DSS. Effects of VDR on RAS and EGFR were examined by Western blotting, immunostaining, and real-time PCR. We also examined the effect of vitamin D3 on colonic RAS in Vdr+/+ mice. EGFR regulation of VDR was examined in hypomorphic EgfrWaved2 (Wa2) and Egfrwild-type mice. Angiotensin II (Ang II)–induced EGFR activation was studied in cell culture. Results: Vdr deletion significantly increased tumorigenesis, activated EGFR and β-catenin signaling, and increased colonic RAS components, including renin and angiotensin II. Dietary VD3 supplementation suppressed colonic renin. Renin was increased in human colon cancers. In studies in vitro, Ang II activated EGFR and stimulated colon cancer cell proliferation by an EGFR-mediated mechanism. Ang II also activated macrophages and colonic fibroblasts. Compared with tumors from EgfrWaved2 mice, tumors from Egfrwild-type mice showed upregulated Snail1, a suppressor of VDR, and downregulated VDR. Conclusions: VDR suppresses the colonic RAS cascade, limits EGFR signals, and inhibits colitis-associated tumorigenesis, whereas EGFR increases Snail1 and downregulates VDR in colonic tumors. Taken together, these results uncover a RAS-dependent mechanism mediating EGFR and VDR cross-talk in colon cancer. Clin Cancer Res; 20(22); 5848–59. ©2014 AACR.

Characterization of calcium binding properties of lithostathine

Abstract. The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichiacoli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with 45Ca2+ showed that LIT binds Ca2+ with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO2+) ion as a paramagnetic substitute for Ca2+ also showed that VO2+ binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO2+ competes with Ca2+ in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95–100%) of binding of Ca2+ and VO2+, showing that these residues are critical for calcium binding by LIT.

The effective position of the electronic point dipole of the nitroxyl group of spin labels determined by ENDOR spectroscopy

Abstract The effective position of the electronic point dipole of the nitroxyl group of the spin label 2.2,5,5-tetra methyl-l-oxypyrroline-3-carboxamide has been assigned by electron-nuclear double resonance and molecular modeling. From proton ENDOR spectra of the spin label in frozen solutions of perdeuterated dimethyl sulfoxide:chloroform: toluene (50:25: 25 v/v), we have identified the principal hyperfine coupling (hfc) components of the vinylic proton of the pyrrolinyl ring and of the two protons of the carboxamide side chain. The ENDOR-based dipolar He components are in excellent agreement with calculation when they are evaluated for each of the vinyl and carboxamide protons as the sum of two separate interactions with the nitroxyl nitrogen and oxygen atoms, weighted according to their respective EPR-assigned spin densities. Within experimental error, the corresponding electron-proton vectors belonging to the vinylic and the two carboxamide protons intersect on the NO bond. This intersection point is shown to be identical to that calculated as the effective position of the unpaired electron as a point dipole along the NO bond according to the EPR-determined fractional spin density associated with the nitrogen atom of the nitroxyl group.

Magnetic Resonance Imaging of Changes in Muscle Tissues after Membrane Trauma

Abstract: A pure electroporation injury leads to cell membrane disruption and subsequent osmotic swelling of the tissue. The state of water in the injured area of a tissue is changed and differs from a healthy tissue. Magnetic resonance imaging (MRI), which is very sensitive to the quality of the interaction between mobile (water) protons and a restricted (protein) proton pool, is therefore a useful tool to characterize this injury. Here, we present a protocol designed to measure the difference between the values of the transverse magnetic relaxation time (T2) in MRIs of healthy and electrically injured tissue. In addition, we present a method to evaluate the two main contributions to the MRI contrast, the degree of structural alteration of the cellular components (including a major contribution from membrane pores), and edema. The approach is useful in assessing the level of damage that electric shocks produce in muscle tissues, in that edema will resolve in time whereas structural changes require active repair mechanisms.