Ethaar El-Emir

Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression

Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

A One-Pot Three-Component Radiochemical Reaction for Rapid Assembly of 125I-labeled Molecular Probes

Nuclear imaging in conjunction with radioactive tracers enables noninvasive measurements of biochemical events in vivo. However, access to tracers remains limited due to the lack of methods for rapid assembly of radiolabeled molecules with the prerequisite biological activity. Herein, we report a one-pot, three-component, copper(II)-mediated reaction of azides, alkynes, and [(125)I]iodide to yield 5-[(125)I]iodo-1,2,3-triazoles. Using a selection of azides and alkynes in a combinatorial approach, we have synthesized a library of structurally diverse (125)I-labeled triazoles functionalized with bioconjugation groups, fluorescent dyes, and biomolecules. Our preliminary biological evaluation suggests that 5-[(125)I]iodo-1,2,3-triazoles are resistant to deiodination in vivo, both as small molecular probes and as antibody conjugates. The ability to incorporate radioactive iodide into triazoles directly from the parent azides and alkynes makes the method broadly applicable and offers the potential to rapidly assemble molecular probes from an array of structurally diverse, and readily available, building blocks.

SYNERGY BETWEEN VASCULAR TARGETING AGENTS AND ANTIBODY-DIRECTED THERAPY

PURPOSE Tumor heterogeneity necessitates the use of combined therapies. We have shown that combining antibody-directed therapy with antivascular agents converts a subcurative to a curative treatment. The purpose of this study was to investigate, by radioluminographic and microscopic techniques, the regional effects of the two complementary therapies. METHODS AND MATERIALS Nude mice bearing colorectal tumors were injected with 125I-labeled anti-carcinoembryonic antigen antibody, and images were obtained for antibody distribution and modeling studies using radioluminography. For therapy studies, the mice were given radioimmunotherapy alone (131I-A5B7 anti-carcinoembryonic antigen antibody), the antivascular agent combretastatin A-4 3-0-phosphate (200 mg/kg), or both. Extra mice were used to study the regional tumor effects of these therapies over time: relevant histochemical procedures were performed on tissue sections to obtain composite digital microscopic images of apoptosis, blood vessels, perfusion, hypoxia, and morphology. RESULTS Antibody distribution, modeling, and immunohistochemistry showed how radioimmunotherapy (7.4 MBq/40 microg antibody) effectively treated the outer, well-oxygenated tumor region only. Combretastatin A-4 3-0-phosphate treated the more hypoxic center, and in doing so altered the relationship between tumor parameters. CONCLUSION The combined complementary therapies produced cures by destroying tumor regions with different pathophysiologies. Relating these regional therapeutic effects to the relevant tumor parameters microscopically allows optimization of therapy and improved translation to clinical trials.

Microdistribution of targeted, fluorescently labeled anti carcinoembryonic antigen antibody in metastatic colorectal cancer: Implications for radioimmunotherapy

Purpose: Most radioimmunotherapy studies on radiolabeled antibody distribution are based on autoradiographic and radioluminographic data, which provide a lack of detailed information due to low resolution. We used fluorescently labeled anti–carcinoembryonic antigen (CEA) antibody (A5B7) to investigate quantitatively the kinetics and microdistribution of antibody in a clinically relevant orthotopic colorectal cancer model (LS174T) using high-resolution digital microscopy. Experimental Design: Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to morphologic images of the same or adjacent tumor sections. Results: Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with tumor size at 10 minutes and 1 hour (P ≤ 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were positively related to tumor size (P ≤ 0.001). Conclusion: The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.

The Nonuniformity of Antibody Distribution in the Kidney and its Influence on Dosimetry

Abstract Flynn, A. A., Pedley, R. B., Green, A. J., Dearling, J. L., El-Emir, E., Boxer, G. M., Boden, R. and Begent, R. H. J. The Nonuniformity of Antibody Distribution in the Kidney and its Influence on Dosimetry. Radiat. Res. 159, 182–189 (2003). The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with 125I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from 14C, 131I, 186Re, 32P and 90Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.

Characterisation and radioimmunotherapy of L19-SIP, an anti-angiogenic antibody against the extra domain B of fibronectin, in colorectal tumour models

Angiogenesis is a characteristic feature of tumours and other disorders. The human monoclonal antibody L19- SIP targets the extra domain B of fibronectin, a marker of angiogenesis expressed in a range of tumours. The aim of this study was to investigate whole body distribution, tumour localisation and the potential of radioimmunotherapy with the L19-small immunoprotein (SIP) in colorectal tumours. Two colorectal tumour models with highly different morphologies, the SW1222 and LS174T xenografts, were used in this study. Localisation and retention of the L19-SIP antibody at tumour vessels was demonstrated using immunohistochemistry and Cy3-labelled L19-SIP. Whole body biodistribution studies in both tumour models were carried out with 125I-labelled L19-SIP. Finally, 131I-labelled antibody was used to investigate the potential of radioimmunotherapy in SW1222 tumours. Using immunohistochemistry, we confirmed extra domain B expression in the tumour vasculature. Immunofluorescence demonstrated localisation and retention of injected Cy3-labelled L19-SIP at the abluminal side of tumour vessels. Biodistribution studies using a 125I-labelled antibody showed selective tumour uptake in both models. Higher recorded values for localisation were found in the SW1222 tumours than in the LS174T (7.9 vs 6.6 %ID g−1), with comparable blood clearance for both models. Based on these results, a radioimmunotherapy study was performed in the SW1222 xenograft using 131I-Labelled L19-SIP (55.5 MBq), which showed selective tumour uptake, tumour growth inhibition and improved survival. Radio- and fluorescence-labelled L19-SIP showed selective localisation and retention at vessels of two colorectal xenografts. Furthermore, 131I-L19-SIP shows potential as a novel treatment of colorectal tumours, and provides the foundation to investigate combined therapies in the same tumour models.

Community-based recruitment of patients with COPD into clinical research

Abstract Identifying subjects for clinical trials is difficult and the evidence base for recruitment strategies is limited, particularly in the field of COPD. We compared the efficiency and patient characteristics of different community-based recruitment strategies during a non-commercial COPD trial in the UK. Recruiting from general practice COPD registers was less efficient and identified patients with significantly milder disease than recruiting through pulmonary rehabilitation and patient groups. We report our experience and propose that pulmonary rehabilitation and patient groups may represent an enriched pool of COPD patients to recruit into clinical trials. Trial registration number: EudraCT 2011-001063-43

Spatial accuracy of 3D reconstructed radioluminographs of serial tissue sections and resultant absorbed dose estimates.

Many agents using tumour-associated characteristics are deposited heterogeneously within tumour tissue. Consequently, tumour heterogeneity should be addressed when obtaining information on tumour biology or relating absorbed radiation dose to biological effect. We present a technique that enables radioluminographs of serial tumour sections to be reconstructed using automated computerized techniques, resulting in a three-dimensional map of the dose-rate distribution of a radiolabelled antibody. The purpose of this study is to assess the reconstruction accuracy. Furthermore, we estimate the potential error resulting from registration misalignment, for a range of beta-emitting radionuclides. We compare the actual dose-rate distribution with that obtained from the same activity distribution but with manually defined translational and rotational shifts. As expected, the error produced with the short-range 14C is much larger than that for the longer range 90Y; similarly values for the medium range 131I are between the two. Thus, the impact of registration inaccuracies is greater for short-range sources.