Dvora Kidron

Molecular imaging of cell death in vivo by a novel small molecule probe

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an 18F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo.

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

PurposeAcute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia.MethodsUsing both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis.ResultsApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining.ConclusionApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.

Improved detectability of experimental allergic encephalomyelitis in excised swine spinal cords by high b-value q-space DWI

Experimental allergic encephalomyelitis (EAE) is the primary experimental model of multiple sclerosis (MS), which involves both inflammation and demyelination and is known to be species-dependent. Spinal cord abnormalities were found in more than 80% of postmortem specimens of MS patients. In the present study, T1, T2 and high b-value q-space diffusion-weighted magnetic resonance imaging (MRI) were used, for the first time, to characterize the EAE model in excised swine spinal cords. The MR images were compared with histological staining and clinical scoring. Although all spinal cords were excised from swine with severe or very severe (clinical score between 3 to 5 on a scale of 5) motor impairments, T1- and T2-weighted MRI revealed white matter (WM) abnormalities in only five of the ten EAE diseased spinal cords studied, while high b-value q-space diffusion weighted MRI (q-space DWI) detected WM abnormalities in all diseased spinal cords studied. Interestingly, high b-value q-space DWI was able to detect abnormalities in the normal appearing white matter (NAWM) even in spinal cords where no plaques were identified by the T1- and T2-weighted MR images. Good anatomical correlation was observed between the high b-value q-space MR images and histology. The extent of DWI abnormalities paralleled the clinical scoring and correlated with histology. In addition, areas classified as NAWM by the T1- and T2-weighted MR images that showed abnormalities in the q-space DWI were also found to have abnormal histology. This improved detection level of the EAE model by high b-value q-space DWI over conventional T1-, and T2-weighted MRI is briefly discussed.

Monitoring of chemotherapy-induced cell death in melanoma tumors by N,N'-Didansyl-L-cystine.

Early assessment of the efficacy of anticancer agents is a highly desirable and an unmet need in clinical oncology. Clinical imaging of cell-death may be useful in addressing this need, as induction of tumor cell-death is the primary mechanism of action of most anticancer drugs. In this study, we examined the performance of N,N′-Didansyl-L-cystine (DDC), a member of the ApoSense family of novel small molecule detectors of cell-death, as a potential tool for monitoring cell-death in cancer models. Detection of cell-death by DDC was examined in fluorescent studies on B16 melanoma cells both in vitro and ex vivo following its in vivo administration. In vitro, DDC manifested selective uptake and accumulation within apoptotic cells that was highly correlated with Annexin-V binding, changes in mitochondrial membrane potential, and caspase activation. Uptake was not ATP-dependent, and was inducible by calcium mobilization. In vivo, DDC selectively targeted cells undergoing cell-death in melanoma tumors, while not binding to viable tumor cells. Chemotherapy caused marked tumor cell-death, evidenced by increased DDC uptake, which occurred before a detectable change in tumor size and was associated with increased animal survival. These data confirm the usefulness of imaging of cell-death by DDC as a tool for early monitoring of tumor response to anti-cancer therapy.

p53 Regulates insulin-like growth factor-I receptor gene expression in uterine serous carcinoma and predicts responsiveness to an insulin-like growth factor-I receptor-directed targeted therapy.

The role of the insulin-like growth factors (IGF) in endometrial cancer has been well established. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I, is usually overexpressed in endometrial tumours. Uterine serous carcinoma (USC) constitutes a defined histological category among endometrial cancers. Mutation of the p53 gene appears early in the course of the disease and is considered a key event in the initiation of USC. The aim of the present study was to evaluate the potential interactions between p53 and the IGF-IR in USC. In addition, we investigated the role of p53 as a biomarker in IGF-IR targeted therapies. Immunohistochemical analysis in a collection of 35 USC specimens revealed that IGF-IR is highly expressed in primary and metastatic USC. Likewise, p53 was expressed in 85.7% of primary tumours and 100% of metastases. A significant negative correlation between p53 expression and survival was noticed. In addition, using USC-derived cell lines we provide evidence that p53 regulates IGF-IR gene expression via a mechanism that involves repression of the IGF-IR promoter. We show that the mechanism of action of p53 involves interaction with zinc finger protein Sp1, a potent transactivator of the IGF-IR gene. Finally, we demonstrate that USC tumours overexpressing p53 are more likely to benefit from anti-IGF-IR therapies. In summary, we provide evidence that p53 regulates IGF-IR gene expression in USC cells via a mechanism that involves repression of the IGF-IR promoter. The interplay between the p53 and IGF-I signalling pathways is of major basic and translational relevance.

CO2 laser-assisted sclerectomy surgery part I: concept and experimental models.

PurposeTo evaluate the safety and performance of a second-generation device for CO2 laser-assisted sclerectomy surgery system in experimental models. Materials and MethodsLaser-assisted deep sclerectomy using a modified CO2 laser system (OT-134—“IOPtiMate”; IOPtima Ltd, Israel) was performed in 3 experimental models: enucleated pig eyes, human cadaver eyes, and live rabbit eyes. A half-thickness scleral flap was created, and a CO2 laser with a beam-manipulating system was used to achieve deep scleral ablation over the Schlemm canal zone. Aqueous percolation and scleral perforation rates were recorded. Intraocular pressure was monitored in live rabbits up to 21 days postoperatively. The shape and location of the scleral ablation zone, thermal damage, and the healing process were examined by histopathological analysis. ResultsDeep scleral ablation and aqueous percolation were repeatedly achieved in all the models. Micro-perforations occurred in 4/18 human eyes (22.2%), in 4/23 rabbit eyes (17.4%), and in none of the 38 porcine eyes. Mean intraocular pressure in the rabbits was significantly decreased (by 6.3±3.6 mm Hg) on the first postoperative day (P<0.0001) and gradually returned to normal. In all but one of the cadaver eyes, effective fluid percolation was achieved. Histology in each case disclosed deep scleral craters with a thin intact sclero-corneal tissue layer at the ablation area. Mild thermal damage, limited to the ablated scleral walls was detected and resolved after 10 days. ConclusionsThe results in these experimental studies indicated that CO2 laser-assisted sclerectomy surgery using the OT-134 system is a safe and efficacious procedure for achieving effective fluid percolation.

Senescence in amniocytes and placentas from trisomy 21 pregnancies.

Abstract Objective: Senescence has been described as a stable cell proliferation arrest resulting from the progression of primary human fibroblasts through a finite number of population doublings in vitro. Accelerated telomere shortening was observed in pregnancies complicated by intrauterine growth restriction, in placentas of diabetic mothers and trisomy 21 amniocytes. We hypothesized that under conditions of stress, telomeres in placentas will be shorter and there will be more cells with the senescence phenotype. Methods: The two study groups included placental biopsies from 7 cases of trisomy 21 and amniocytes from 10 cases of trisomy 21. The control groups consisted of placental biopsies from 6 cases and amniocytes from 10 pregnancies with a normal karyotype. The samples were analyzed for the presence of senescent cells based on the number of fragments in each cell. Results: A significantly higher percentage of cells in the senescent state, based on a higher percentage of cells with more fragmentations, were found in the amniocytes (20.8%) and in trophoblasts (94.3%) from placentas with trisomy 21 compared to the control groups. Conclusion: Among other genetic instability parameters, trisomy 21 amniocytes and trophoblasts express a higher prevalence of senescent cells than were previously reported.

Decreased Pathological Placental Findings in Aspirin-Treated Pregnant Women at Risk of Hypertensive Complications

Objective: The small controlled trials reporting large reductions in the incidence of preeclampsia and intrauterine growth restriction (IUGR) in highrisk pregnant women treated with low-dose aspirin have recently been followed by large clinical trials suggesting less beneficial results. The effect of low-dose aspirin on placental lesions associated with preeclampsia and IUGR has not yet been studied.Methods: We participated in the large multicenter randomized collaborative low-dose aspirin study in pregnancy (CLASP) trial of low-dose aspirin for the prevention and treatment of preeclampsia and intrauterine growth restriction. As part of this study, we evaluated placentae submitted from 25 women treated with aspirin and 28 with placebo.Results: More of the pathological findings classically described in preeclampsia and IUGR were demonstrated in the placentae from the placebo group than from the aspirin group (54% vs. 16%, P = 0.02). The placental findings did not correlate with clinical pregnancy outcome o...

Abstract 5301: Blood vessel structure and expression of permeability related factors in advanced lung cancer complicated with pleural effusion

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background. Malignant pleural effusion (MPE) is a common and debilitating condition in patients with lung cancer. For unknown reasons, some tumors are more strongly associated with the condition than others. The pathophysiology of this condition has not been fully determined and to date no specific medical treatment is available. Our goal was to study the structure and assess the permeability related factors in MPE associated blood vessels to better understand the mechanisms of this condition. Methods. Immunohistochemical analysis of human NSCLC formalin-fixed paraffin-embedded specimens from patients who underwent lobectomy (MPE- cases) or pleural biopsy (MPE+ cases) was employed. Nine adenocarcinomas (AC) and eight squamous cell carcinomas (SCC) with and without MPE were studied. For endothelial cell (EC) staining and microvessel density (MVD) determination, the anti-CD31 Abs was employed. Pericytes were stained by aSMA Abs. Coexpression of some of receptor tyrosine kinases (PDGFR, VEGF/VEGFR, Tie-2) was also evaluated. Results. In SCC complicated with MPE a narrow, clogged and fibrotic vessels were seen in tumors located near the pleura (pleura involved). In MPE positive AC specimens there were regions of tumor vessels where ECs were irregularly laminated and hardly noticed. This observation was also confirmed by CD31 staining where lower number of positive vessels related to the total number of vessels was observed (88.4% in MPE+ vs. 94.8% in MPE- cases, p<0.05). Regarding the vessel coverage by pericytes, the pericyte expression (aSMA staining) was shown to be less extensive in cases with than without effusion (59.1% vs 96.2%, p<0.001) probably because of their thinning or even detachment. This clarification could also explain the finding that the PDGFR was more expressed on ECs on MPE - tumors than on MPE + ones. For VEGF and VEGFR staining, the staining index was more prominent in tumors with than without effusion, for both staining area and staining intensity (1.7 and 1.9, respectively). However, in distinction to the staining of tumor cells almost no vessel EC staining by VEGFR Abs was seen, while staining by anti-VEGF Abs was substantial. Similarly, the staining of Ang-2 receptor (Tie-2) was found in the cytoplasm of the tumor cells, but not in the vascular endothelium, regardless the pleura effusion positive or negative features. Conclusions. To our knowledge this is one of the first studies on structural abnormalities of blood vessels in human lung cancer malignant pleural effusion. For now, because of the relatively small number of cases analyzed no single mechanism was found able to explain the occurrence of a pleural effusion, and it is likely that multiple factors contribute to effusion formation. This formulation may also better explain why some patients with pleural metastases have malignant effusions while others do not. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5301. doi:1538-7445.AM2012-5301