Carina Mari

Adipose-derived adult stromal cells heal critical-size mouse calvarial defects

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84–99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

Small-Animal SPECT and SPECT/CT: Important Tools for Preclinical Investigation

The need to study dynamic biologic processes in intact small-animal models of disease has stimulated the development of high-resolution nuclear imaging methods. These methods are capable of clarifying molecular interactions important in the onset and progression of disease, assessing the biologic relevance of drug candidates and potential imaging agents, and monitoring therapeutic effectiveness of pharmaceuticals serially within a single-model system. Single-photon–emitting radionuclides have many advantages in these applications, and SPECT can provide 3-dimensional spatial distributions of γ- (and x-) ray–emitting radionuclide imaging agents or therapeutics. Furthermore, combining SPECT with CT in a SPECT/CT system can assist in defining the anatomic context of biochemical processes and improve the quantitative accuracy of the SPECT data. Over the past decade, dedicated small-animal SPECT and SPECT/CT systems have been developed in academia and industry. Although significant progress in this arena has been realized through system development and biologic application, further innovation continues to address challenges in camera sensitivity, spatial resolution, and image reconstruction and quantification. The innumerable applications of small-animal SPECT and SPECT/CT in drug development, cardiology, neurology, and oncology are stimulating further investment in education, research, and development of these dedicated small-animal imaging modalities.

Multi-modality Imaging Identifies Key Times for Annexin V Imaging as an Early Predictor of Therapeutic Outcome

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.

Use of somatostatin analogue scintigraphy in the localization of recurrent medullary thyroid carcinoma

Abstract.Detection of recurrence of medullary thyroid carcinoma (MTC) remains a diagnostic problem. Increased serum tumour marker levels frequently indicate recurrence while conventional imaging techniques (CIT) are non-diagnostic. In this study, we performed indium-111 octreotide scintigraphy and CIT in a series of 20 patients with MTC presenting with elevated serum tumour markers after surgery. 111In-octreotide whole-body studies detected 15 pathological uptake foci in 11 of the 20 patients studied and CIT detected 17 lesions in 11 of the 20 patients. Ten patients underwent reoperation, five of them with positive 111In-octreotide scintigraphy and CIT and two with positive isotopic exploration and negative CIT. Surgical findings demonstrated that the results of isotopic study and CIT had been false-positive for MTC in one case (sarcoidosis). The six patients with true-positive 111In-octreotide studies had significantly higher basal calcitonin (CT) and carcinoembryonic antigen (CEA) levels than the patients with negative isotopic studies. The expression of somatostatin receptor (SSTR) subtypes by PC-PCR could be investigated in four cases with a positive isotopic study. Among the three cases with a true-positive study, SSTR2, the SSTR subtype that preferentially binds to the somatostatin analogue octreotide, was detected in two, SSTR5 was demonstrated in the three, and SSTR3 was detected in one. No subtype of SSTR was detected in the case with a final diagnosis of sarcoidosis. We conclude that 111In-octreotide has limited sensitivity in detecting recurrence in patients with MTC, although its sensitivity may improve with high serum CT levels. This radionuclide imaging technique should be employed when conventional imaging techniques are negative or inconclusive or when the presence of somatostatin receptors may provide the basis for treatment with somatostatin analogues.

Nitrate administration to enhance the detection of myocardial viability by technetium-99m tetrofosmin single-photon emission tomography

A comparison was performed between technetium-99m tetrofosmin myocardial perfusion tomography at baseline and after nitrate administration, using a 2-day protocol, and rest-reinjection thallium-201 single-photon emission tomography (SPET) studies in order to assess whether nitrates enhance the detection of viable myocardium with99mTc-tetrofosmin. Fifteen patients with coronary artery disease, previous myocardial infarction and a left ventricular ejection fraction <40% underwent201T1 rest-injection and99mTc-tetrofosmin. baseline-postnitroglycerin (0.4 mg sublingually) SPET studies, within 48 h. Tomograms based on the three spatial planes were divided into 15 segments and regional tracer uptake was quantitatively analysed. Viability was defined as presence of tracer uptake >50% of peak activity on baseline studies or after reversibility. The percentage of peak activity of99mTc-tetrofosmin at baseline correlated with that of 201T1 (r=0.82,P <0.001). On baseline99mTc-tetrofosmin studies, 73 of the 225 segments that were analysed had <50% of peal. activity. Fifteen percent of these segments showed reversibility after nitrate administration, with an increase in99mTc-tetrofosmin uptake from 40%±9% to 57%±9% of peak activity (P=0.003). All reversible segments after nitrate administration had viability criteria on201Tl studies, but 20 segments that were non-viable on99mTc-tetrofosmin. studies were viable on201Tl studies. Using a threshold value of >40% of peak activity, only seven segments remained non-viable on99mTc-tetrofosmin studies. Overall agreement between99mTc-tetrofosmin with nitrates and201Tl-reinjection regarding the presence of myocardial viability was 90%. Detection of myocardial viability with99mTc-tetrofosmin. was enhanced after nitrate administration, correlating with viability criteria observed on thallium studies.

The Role of a Positron- and High-energy Gamma Photon Probe in Intraoperative Localization of Recurrent Melanoma

Purpose: This preliminary study retrospectively evaluated the ability of intraoperative localization of recurrent melanoma using F-18 fluorodeoxyglucose (FDG) and a probe sensitive to both high-energy gamma rays and positrons to enable complete tumor resection and improved patient outcome. Materials and Methods: Three hours before surgery for resection of recurrent melanoma, 5 patients (mean age, 52 ± 22 years) with a history of local surgery, radiation therapy, and/or large habitus received 14.6 ± 3.2 mCi of F-18 FDG. Intraoperative tumor localization was performed with a radiation probe (PET-Probe; IntraMedical Imaging LLC, Los Angeles, CA). Intraoperative tumor tissue activities, background tissue activities, pathology results, and patient follow up (clinical/imaging) were recorded. Results: Eight of the 19 surgical specimens were identified by the probe as having increased FDG uptake when compared with the surrounding tissues before resection. All 8 specimens contained melanoma. Of the 11 specimens that were not identified using the probe, one contained melanoma, yielding a sensitivity of 89% (8 of 9) and a specificity of 100% (10 of 10). In 3 of the 5 cases, the probe allowed the identification of nonvisualized and nonpalpable tumor foci that were later confirmed pathologic. At an average follow up of 210 days (range, 30–515 days), 2 of 5 patients had no evidence of recurrent melanoma by clinical or radiographic evaluations. Conclusion: In the setting of recurrent melanoma, there appear to be potential benefits to intraoperative detection with FDG and a positron-detecting probe, particularly in cases with challenging or altered anatomy.

Zinc chloride–mediated reduction of apoptosis as an adjunct immunosuppressive modality in cardiac transplantation ☆

BACKGROUND Zinc (Zn) blocks caspase-3 activation in cardiac allografts and therefore may synergistically decrease apoptosis along with cyclosporine (CsA), which inhibits mitochondrial release of cytochrome c. Simultaneous treatment of rat recipients of heterotopic heart transplants with zinc chloride (ZnCl(2)) thus may allow lower doses of CsA for immunosuppression. METHODS PVG (RT1(c)) rat hearts were transplanted heterotopically into the abdomen of ACI (RT1(a)) rats. Group 1 (n = 15) rats received no treatment. Group 2 rats (n = 8) received 2 mg/kg/day CsA (sub-therapeutic dose) by oral gavage. Group 3 rats (n = 9) received 2 mg/kg/day oral CsA in addition to 1 mg/kg/day sub-cutaneous ZnCl(2) delivered by osmotic pump. All rats were imaged using Annexin V-bound (99m)Technetium ((99m)Tc-Annexin V) on post-operative Day 4 and subsequently killed. Annexin V avidly binds apoptotic cells in vivo. Region of interest per whole body (WB) data were calculated using the images. The allograft survival study was conducted with n = 11, 6, and 5 in control, CsA, and CsA+Zn groups, respectively. Finally, percentages of allografts that reached tolerance were measured in both CsA-only and CsA+Zn groups (n = 8 each). RESULTS Zinc chloride had an additive effect with CsA on apoptotic blockade and graft survival. The regions of interest per WB uptake of (99m)Tc-Annexin V were 2.43% +/- 0.37%, 2.08% +/- 0.52%, and 1.49% +/- 0.29%*, and acute survivals were 6.4 +/- 1.7, 7.2 +/- 2.1, and 11.2 +/- 2.5* days for control, CsA, and CsA+Zn groups, respectively (*p < 0.001 vs controls). In addition, 87.5% of allografts became tolerant and survived for 90 days in the CsA+Zn group compared with only 37.5% in the CsA-only group (p = 0.049). CONCLUSION Zinc-mediated reduction of apoptosis served as an effective adjunct immunosuppressive therapy to CsA in a rat model of cardiac transplantation.

Influence of exercise rehabilitation on myocardial perfusion and sympathetic heart innervation in ischaemic heart disease

Abstract.Exercise rehabilitation improves the clinical status in ischaemic heart disease. The purpose of this study was to assess the influence of exercise rehabilitation on myocardial perfusion and sympathetic heart innervation. Sixteen patients with ischaemic heart disease and previous myocardial infarction were investigated by means of exercise/rest tetrofosmin and metaiodobenzylguanidine (MIBG) exercise/rest single-photon emission tomography (SPET) studies, before and 6 months after starting an exercise rehabilitation programme. Tomograms were divided into 15 segments, and these were grouped into five myocardial anatomical regions. Regional uptake of both tracerswas quantified and expressed as a percentage of maximumpeak activity. The percentage ≤55% was chosen to evaluate defect size, and the results were expressed as a percentage of left ventricular mass. Areas with perfused and denervated myocardium and areas with ischaemic myocardium were calculated. In addition, regions with <75% of peak activity in the exercise perfusion study at baseline were divided into two groups according to whether there was an increase in peak activity of >10% (representing reversible regional defects) or an increase of <10% (representing fixed regional defects) in the rest study. These percentages were compared with the percentages obtained in the innervation study, and with the percentages obtained in exercise/rest perfusion and innervation studies performed 6 months after starting rehabilitation. Myocardial perfusion defects were significantly smaller than myocardial innervation defects before and 6 months after starting exercise rehabilitation. The area of ischaemia 6 months after starting exercise rehabilitation was significantly smaller than that before rehabilitation (0.31%± 1.4% vs 1.4%±1.6%, P<0.01). The size of innervation defects and the area of perfused and denervated myocardium did not show significant differences between the two studies performed before and 6 months after starting exercise rehabilitation. In reversible regional defects the percentage of peak activity was significantly increased 6 months after starting exercise rehabilitation in exercise and rest studies (P<0.001), while in fixed regional defects it was significantly increased only in exercise studies (P<0.001). There was no significant change in the regional MIBG percentages. We conclude that in ischaemic heart disease, exercise rehabilitation over a period of 6 months improves myocardial perfusion, but does not cause changes in sympathetic myocardial innervation.

Development of Radiocontrast Agents for Vascular Imaging

The revolution in molecular imaging techniques is profoundly changing the understanding of the pathophysiology and treatment of atherosclerosis. With these rapid changes there is an increasing demand for development of sensitive and well tolerated novel imaging agents that can be rapidly translated from small animal models into patients with atherosclerosis. Nuclear medicine and positron emission tomography techniques have the ability to detect and serially monitor a variety of biologic and pathophysiologic processes usually with tracer quantities of radiolabeled peptides, drugs, and other molecules at dosages free of pharmacologic adverse effects unlike the current generation of intravenous agents required for magnetic resonance imaging (MRI) and computed axial tomography (CT) scanning.A representative sampling of the wide array of radiopharmaceuticals developed specifically for radionuclide imaging of atherosclerosis, that have been approved for clinical use and those in pre-clinical trials, have been reviewed in this article.The presence of an inflammatory stimulus increases expression of CC (cysteine-cysteine motif) chemokine receptor (CCR)-2 on monocytes and macrophages, and somatostatin receptors on T lymphocytes. Radiolabeled monocyte chemoattractant protein (MCP)-1 binds with high affinity to CCR-2 and can be used to detect subacute and chronic inflammatory lesions. Similarly, radiolabeled octreotide or depreotide can be used to detect activated T lymphocytes which may identify the vulnerable plaque. Animal models indicate that 99mTc-annexin V, 125I-MCP-1 and [18F]-fluoro-2-deoxyglucose are effective in identifying apoptotic cell death, macrophage infiltration and metabolic activity in atheromatous lesions, respectively. Expression of αvβ3 integrin is increased in activated endothelial cells and vascular smooth muscle cells after vascular injury, and αvβ3 integrin is minimally expressed on smooth muscle cells and is not expressed on quiescent epithelial cells. Radiolabeled high-affinity peptides can be used to target the αvβ3 integrin and visualize areas of vascular damage. Advances in technology such as the micro-single photon emission computed tomography (microSPECT) have the potential to overcome the drawbacks of older CT and MRImethodologies, such as lack of biologically relevant ligands and compatible blood pool contrast agents for imaging. Despite these advances in imaging technology, the small size of atheromatous lesions makes it difficult to detect using external imaging techniques. Therefore, recently there has been renewed interest in the use of intravascular catheter-based radiation detectors.

Apoptosis in a rodent model of cranial suture fusion: in situ imaging and gene expression analysis.

Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m–labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor–mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.