Bimodal Imaging of Inflammation with SPECT/CT and MRI Using Iodine-125 Labeled VCAM-1 Targeting Microparticle Conjugates.

Upregulation of cell adhesion molecules on endothelial cells is a hallmark of inflammation and an early feature of several neurological conditions. Here, we describe bimodal in vivo imaging of this inflammatory event in the brain using functionalized micron-sized particles of iron oxide. The particles were conjugated to anti-VCAM-1 antibodies and subsequently labeled with iodine-125. Radiolabeling of the antibody-coated particles was straightforward and proceeded in high radiochemical yields using commercially available iodination tubes. The corresponding contrast agent was evaluated in a rat model of cerebral inflammation based on intracerebral injection of tumor necrosis factor alpha and a rat model of status epilepticus. Biodistribution studies and phosphorimaging of cryosections were used to verify in vivo imaging data obtained with single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). The contrast agent showed rapid and highly localized binding to the vasculature of inflamed brain tissue, and was effectively cleared from the blood pool within 2 min postinjection. Overall, the pattern of hypointensities observed with MRI was in good agreement with the distribution of the contrast agent as determined with SPECT and phosphorimaging; however, conspicuous differences in the signal intensities were observed. The results demonstrate that radiolabeled micron-sized particles of iron oxide enable multimodal in vivo imaging with MRI and nuclear techniques, and highlight the value of validating different imaging methods against one another.

Noninvasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post-Cardiac Transplant.

Ischemia-reperfusion injury (IRI) is inevitable in solid organ transplantation, due to the transplanted organ being ischemic for prolonged periods prior to transplantation followed by reperfusion. The complement molecule C3 is present in the circulation and is also synthesized by tissue parenchyma in early response to IRI and the final stable fragment of activated C3, C3d, can be detected on injured tissue for several days post-IRI. Complement activation post-IRI was monitored noninvasively by single photon emission computed tomography (SPECT) and CT using (99m) Tc-recombinant complement receptor 2 ((99m) Tc-rCR2) in murine models of cardiac transplantation following the induction of IRI and compared to (99m) Tc-rCR2 in C3(-/-) mice or with the irrelevant protein (99m) Tc-prostate-specific membrane antigen antibody fragment (PSMA). Significant uptake with (99m) Tc-rCR2 was observed as compared to C3(-/-) or (99m) Tc-PSMA. In addition, the transplanted heart to muscle ratio of (99m) Tc-rCR2 was significantly higher than (99m) Tc-PSMA or C3(-/-) . The results were confirmed by histology and autoradiography. (99m) Tc-rCR2 can be used for noninvasive detection of activated complement and in future may be used to quantify the severity of transplant damage due to complement activation postreperfusion.

Significance of fractional exhaled nitric oxide measurements in detecting primary ciliary dyskinesia in Saudi children.

OBJECTIVE: To examine the usefulness of fractional exhaled nitric oxide (FENO) measurements in detecting primary ciliary dyskinesia (PCD) in children. METHODS: This observational study was conducted at the Department of Pediatrics and Physiology, College of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia from January 2011 to December 2011. The study population consisted of 22 children with symptoms suggestive of PCD and the diagnosis was confirmed by ciliary biopsy. Using the American Thoracic Society guidelines, measurements of FENO were performed in 22 subjects with proven PCD biopsies and in 11 healthy age-matched subjects. RESULTS: No significant differences were found on the basis of age or ventilatory function tests between the PCD patients and control groups. Fractional exhaled nitric oxide values were significantly lower in children with PCD (6.19+/-1.43) compared to control group (17.00+/-6.30) (CI: -14.854 to -5.927, p<0.0001). Rhinorrhea was seen in 7 (31.8%), recurrent acute otitis media in 16 (72.7%), chronic otitis media in 5 (22.7%), recurrent sinusitis in 5 (22.7%), chronic productive cough in 8 (36.4%), bronchospasm in 11 (50%), and dextrocardia in 3 (13.6%) subjects. There was no correlation between age, FENO, and ventilatory function parameters. CONCLUSION: The measurement of FENO appears to be a useful tool for screening children for PCD. It can complement other tests such as nasal biopsy and electron microscopy studies.

SPECT/CT lymphoscintigraphy of heterotopic cardiac grafts reveals novel sites of lymphatic drainage and T cell priming.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients' during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

Herpesvirus saimiri-based endothelin-converting enzyme-1 shRNA expression decreases prostate cancer cell invasion and migration.

The zinc metalloprotease, endothelin-converting enzyme-1 (ECE-1), which converts the mitogenic peptide endothelin-1 (ET-1) from its biologically inactive precursor big-ET-1, is commonly upregulated in prostate cancer (PC) cells. Consequently, we have sought to suppress ECE-1 expression by using RNAi as a potentially novel therapeutic approach. Therefore, a synthetic 64-nt short-hairpin RNA (shRNA), designed to target the ECE-1 gene, was expressed in an Herpesvirus saimiri (HVS)-based delivery vector. ECE-1 expression in cells transduced with the vector was examined by real-time PCR and Western blotting. The effects of ECE-1 knockdown on PC cell migration and invasion were studied using a scratch assay and Matrigel invasion. These studies, in vitro and ex vivo, demonstrated that the HVS-shRNA viruses could infect and silence ECE-1 expression effectively in human PC cells. Furthermore, it was observed that ECE-1 knockdown in either stromal cells or epithelial cells could significantly reduce invasion of PC-3 cells in coculture by 33 and 31%, respectively. In addition, suppressed migration was also observed in HVS-ECE-1 shRNA-infected PC-3 cells compared to uninfected and HVS-GFP-infected control cell cultures. These findings highlight the potential tumor-suppressing effect of ECE-1 knockdown in cancer cells and novel strategies for future therapeutic developments in advanced PC.

Expression and localization of endothelin-converting enzyme-1 in human prostate cancer.

Endothelin (ET)-1 can influence cancer invasion and metastasis by exerting an autocrine (epithelial) or paracrine (stromal) influence on growth. ET-1 is generated from big ET-1 by endothelin-converting enzyme (ECE)-1, which has four recognized isoforms, ECE-1a, ECE-1b, ECE-1c, and ECE-1d, differing only in their amino-terminal regions. This study investigated the expression and localization of the ECE-1 isoforms in prostate cancer (PC). The epithelial cell lines used were androgen-sensitive LNCaP, androgen-independent PC-3 and Du145, and nonmalignant transformed PNT1-a, PNT2-C2, and P4E6 prostate cells. Primary cells derived from malignant and benign tissue from radical prostatectomies were also exploited. Previously, we reported increased ECE-1 expression in androgen-independent PC cell lines, as compared with androgen-sensitive cells. Our present data show that transcripts for all ECE-1 isoforms were present in all epithelial cell lines analyzed. However, only the ECE-1c protein was detectable in PC-3, Du145, PNT2-C2, and PNT1-a cells. ECE-1c localized to both the cell surface and intracellular compartments in individual cell lines. In primary stromal cells, all individual ECE-1 isoforms were expressed at the mRNA level, with the exception of ECE-1a. ECE-1b and ECE-1c protein levels were higher in malignant stromal cells, as compared with benign cells. In stroma, ECE-1c protein was localized to the cell surface, with filamentous immunoreactivity throughout the cell, whereas ECE-1b immunoreactivity was punctate throughout the cytoplasm. The upregulation of the ECE-1c isoform in PC cell lines is being investigated further.

Endothelin system in oral squamous carcinoma cells: specific siRNA targeting of ECE-1 blocks cell proliferation.

The present study focused on the endothelin axis in human oral squamous cell carcinoma (SCC) cells. We investigated the expression and distribution of endothelin-1 (ET-1), its receptors (endothelin-A receptor (ET(A)R) and endothelin-B receptor (ET(B)R)) and isoforms of its specific converting enzyme (ECE-1a, 1b, 1c) and the report on their relative influences on cell proliferation. We also investigated the effect of an ECE-specific inhibitor (ECE-i) and siRNA targeting of the ECE-1 gene on SCC cell proliferation. We observed the expression of ET-1, ET(A)R, ET(B)R and all endothelin-converting enzyme-1 (ECE-1) isoforms in oral SCC cells, but only the expression of ET-1, ET(B)R and ECE-1 was increased when compared to normal human epidermal keratinocytes. ET-1 alone stimulated proliferation of oral SCC cells. Antagonists of either ET(A)R or ET(B)R inhibited ET-1-mediated proliferation. Decreased ECE-1 expression after ECE siRNA treatment reduced SCC cell proliferation. Antiproliferative effects were also observed by inhibiting ECE activity with ECE-i. In conclusion, the present study demonstrates that modulation of the endothelin system in oral SCC cells might provide a novel therapeutic protocol for oral cancer.

Differential expression of neutral endopeptidase-24.11 (neprilysin) and endothelin-converting enzyme in human prostate cancer cell lines.

Neutral endopeptidase-24.11 (neprilysin; NEP/CD10) is a cell surface metallopeptidase expressed by prostatic epithelial cells that degrades various bioactive peptides including endothelin. Endothelin-converting enzyme (ECE), the key enzyme of endothelin biosynthesis, catalyses the final processing step in the pathway. Neuropeptide substrates of NEP, including endothelin, have been implicated in the growth of androgen-independent prostate cancer. We have surveyed the expression of NEP and ECE in a range of prostate cancer cell lines. Western analysis reveals that ECE-1 is expressed abundantly in all the malignant cell lines tested, except for LNCaP. In contrast, LNCaP cells express high levels of NEP, while NEP was not detected in PC-3, DU145 and other metastatic cell lines that were tested. Of the normal immortalized prostate epithelial cell lines, PNT1a shows equivalent amounts of NEP and ECE. PNT2-C2 shows poor NEP expression but an abundance of ECE. P4E6, by comparison, has low levels of both ECE and NEP. These differences in expression may render these cell lines useful in experimental models for future study. Benign prostatic hyperplasia primary epithelial cells express much higher levels of NEP than malignant primary epithelial cells, but neither show ECE expression. On the other hand, surrounding stromal cell populations have detectable ECE levels. An absence of ECE in malignant and benign prostatic hyperplasia cells of primary epithelial origin suggests an important role for stromal interaction and paracrine production of ECE within the host. The upregulation of ECE expression in metastatic cells in culture may be indicative of its role in metastatic progression. A differential profile of ECE and NEP could contribute to an abundance of mitogenic peptides aiding the progression of androgen-independent prostate cancer.