Imaging Cell Death in Vivo

A technique to image programmed cell death would be useful both in clinical care and in drug development. The most widely studied agent for the in vivo study of apoptosis is radiolabeled annexin V, an endogenous protein labeled with technectium-99m, now undergoing clinical trials in both Europe and the United States. While annexin V has been studied extensively in humans the precise mechanism(s) of uptake this agent in vivo is unclear and needs further study. Other agents are also under development, including radiolabeled forms of Z- VAD.fmk, a potent inhibitor of the enzymatic cascade intimately associated with apoptosis. In addition other technologies, such as diffusion weighted magnetic resonance imaging and magnetic resonance imaging with contrast agents, such as small paramagnetic iron oxide particles coated with peptides have also been advocated as methods to monitor apoptotic cell death. The potential applications of imaging apoptosis as a marker of early response to therapy in cancer, acute cerebral and myocardial ischemic injury and infarction, immune mediated inflammatory disease and transplant rejection are reviewed.

KEY WORDS: Apoptosis – Annexin V – Phosphatidylserine – Radionuclide imaging.

Beginning with embryology and organogenesis, and lasting through the final acts of cells responding to near lethal trauma, apoptosis, programmed cell death, is an integral component of cell activity. In the case of organogenesis, cells are signaled to commence apoptosis when the organ has reached an appropriate size or shape,1 while cells that recognize they cannot commit a specified activity, activate the programmed cell death program. Regulation of apoptosis is necessary to maintain health. Either an excess or a deficiency of apoptosis causes disease. For example, transplant rejection is associated with immune induced apoptosis of the transplanted tissue by the host lymphocytes, lupus is associated with a decrease in apoptosis of immune competent cells, and cancer is associated with genetic changes in the neoplastic tissue, that serve to abnormally reduce the incidence of apoptosis. Apoptosis is initiated either by the cell recognizing a problem in its structure or function, or by external signals, triggering the cell death program. Once the process is initiated, the cell manifests a stereotyped behavior characteristic of apoptosis. Because this behavior is similar across cell lines (and across species) techniques have been developed to recognize and quantitate the process. These techniques have utilized specific indicators that permit the definitive recognition of the process (Figures 1-5). One major characteristic of apoptosis is destruction of DNA by enzymatic degradation: DNA is cut into pieces of 50-300 kilobases by endonucleases. On Western blot, these pieces form the pattern of a ladder (hence the term DNA laddering). The endonucleases leave specific bases of DNA exposed, which stain with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). In addition to DNA destruction, a characteristic change takes place in the cell membrane. A anionic phospholipid, phosphatidylserine (PS), which is normally restricted to the inner leaflet of the cell membrane by active enzyme transport, is expressed on the outer leaflet of the cell membrane. This membrane change is associated with changes in the mobility of water associated with the membrane, as well as providing a basis for specific signaling adjacent cells that this cell has initiated apoptosis. Adjacent cell signaling occurs when a physiologic protein, annexin V, binds to this membrane bound PS. Annexin binding to membrane bound PS occurs earlier than DNA destruction, and provides a much greater signal to identify apoptotic cells. Annexin was initially labeled with fluorescein to identify cells expressing PS using fluorescence activated cell sorting, and in histologie specimens. Tait and Stratton developed a radiolabeled form of annexin V to identify activated platelets (which express large amount of PS on their cell surface) as a marker for atrial thrombi.2 Blankenberg et al., applied the radiolabeled annexin technique to identify apoptosis cells in vivo.3 In addition to the nuclear medicine techniques, there are other methods of identifying apoptotic cells. This review will address the strengths and weaknesses of each of the methods available.

Figure 1.-Normal cell.

MR techniques and tracers for the imaging of cell death

Diffusion-weighted MR imaging (DWI) is a technique that takes advantage of the differences between the extra-, intra-, and transcellular motion (diffusion) of water molecules in the local cellular environments within a region of interest or voxel.4 Water proton spins that move quickly out of a voxel (i.e. diffuse in a nonrandom fashion) will cause a loss of MR signal as compared to stationary spins that have a higher MR signal. Because the majority of DWI signal relates to the extracellular space and tissue perfusion any contraction of the extracellular environment, such as seen with cellular swelling (restricted diffusion), will cause a signal loss that can be displayed as a bright signal on DWI images (by convention) or as dark on a map of diffusion, (i.e., so called average diffusion coefficient [ADC] mapping). DWI can therefore be used to distinguish between viable and necrotic tumor, as the latter will exhibit more free (unbound) water and therefore higher signal than the former. The same effect can be used to non-invasively determine the efficacy of treatment of marrow or soft tissue tumor as again treated presumably necrotic tumor will have higher signal on DWI as compared to viable treatment resistant tumor.5, 6

Figure 2.-Early apoptosis.

Water suppressed lipid proton spectroscopy is another MR technique that has been used to detect cells undergoing apoptosis both in vitro 7 and in vivo.8 Cells undergoing apoptosis have an associated increase in cytoplasmic neutral mobile lipid droplets composed of polyunsaturated fatty acids, cholesterol esters, and triglycerides.9 The increased formation of neutral mobile lipids also occur with the activation of T-lymphoblasts by phorbol myristate acetate as well as drug and antibody induced apoptosis in vitro.10 Its is possible that neutral lipid formation may occur during an active phase of lipid synthesis as a consequence of phospholipase activation and/or loss of mitochondrial electrochemical potential.

Figure 3.-Late apoptosis.

In any event, the resonance signal from neutral mobile lipids can be observed with standard water suppressed proton MR spectroscopy, using existing clinical software and scanners.11 In tissues outside the brain, however, water suppressed lipid proton MR spectroscopy is challenging clue to physiologic motion of lipids and non-specific “bleeding in” of lipid signal from adipose tissue.

Proton MR spectroscopy can also be sensitized to molecular diffusion to study the motion of relatively abundant molecules as choline in vivo as suggested by Hakumaki et al.12 In his studies Hakumaki noted decreases in ADC values of up to 50% of intracellular choline metabolites that were accompanied by a significant increases in a rapidly diffusing water component (presumably extracellular).13 These results suggest that there is a decrease in cell size and number, as well as intracellular viscosity that accompanies apoptosis. It remains to be seen whether this type of MR spectroscopy will be sensitive enough to image apoptosis as this type of pulse sequence as with conventional MR spectroscopy is unforgiving of motion artifact and the relatively small changes in signal found outside the brain.

MR contrast agents, such as iron nanoparticulates (SPIO, superparamagnetic iron oxide particles) can be conjugated to peptides that bind to characteristic changes in apoptotic cells. SPIO’s act as small permanent magnets that dephase proton spins in the local microenvironment thereby causing a local loss of MR signal in target tissues and cells. One such peptide recognizes the C^sup 2^ domain of synaptotagmin I, and binds with micromolar affinity. Synaptotagmin I is a binding domain for anionic phospholipids that act as Ca^sup ++^ receptors for neurotransmitter release.14 (PS) are selectively expressed on the surface of apoptotic cells just after activation of the caspase cascade, but prior to the morphologic changes seen later on in programmed cell death.15 Iron labeled synaptotagmin I (C^sup 2^_SPIO) may therefore be of interest for the MR detection of apoptosis. The disadvantages of C^sup 2^_SPIOs for imaging is the extremely large quantities of material needed to see significant decreases in the signal of apoptotic tissues with MR (20 mg/kg of iron per Cilogram body weight).16 Newer more biocompatible iron based particles that have decreased non-specific liver uptake and improved clearance of iron are also under development for use in the clinic.17

Figure 4.-Packaging.

Imaging the enzymatic cascade associated with apoptosis

Multiple enzymes are activated sequentially as the cell initiates the apoptotic cascade. A series of cysteine proteases, caspases, have been studied in detail. Inhibiting the activity of caspases is a major therapeutic target for drug development to treat diseases with excess apoptosis. An alternative approach to detect programmed cell death 18 utilizes tracers which act as substrates for caspase\s. Benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone [Z-VAD-fmk], a pan-caspase inhibitor, was labeled with ^sup 131^I to identify apoptosis in vivo. However the slow cellular influx and lack of uptake saturation of [^sup 131^I]IZ-VAD-fmk in target tissues suggested that the localization of tracer was non- specific in vivo and the absolute uptake of [^sup 131^I]IZ-VAD-fmk was low.

Figure 5.-Apoptotic bodies.

Annexin V imaging of phosphatidylserine expression during apoptosis

Radiolabeled annexin V,19-23 a human protein commonly labeled with fluorescent markers for in vitro detection and quantification of apoptotic cells 24 can be used to image apoptosis in vivo. The ability of radiolabeled annexin V to image apoptotic cells and tissues rests on its high affinity for the constitutive membrane aminophospholipid, phosphatidylserine (PS). Annexin V has a reversible, calcium dependent, nanomolar affinity for PS. PS comprises 10-15% of the total phospholipid content of plasma cell membrane and is normally restricted to the inner leaflet of the plasma membrane lipid bilayer by an ATP-dependent enzyme “translocase”.25, 26 Translocase in concert with a 2nd ATP- dependent enzyme, “floppase”, that pumps cationic phospholipids such as phosphatidylcholine (PC) and sphingomyelin to the cell surface, maintains an asymmetric distribution of different phospholipids between the inner and outer leaflets of the plasma membrane.

With the onset of apoptosis, PS is rapidly redistributed onto the cell surface within 30 to 120 minutes of signaling in culture.27 The number of annexin V binding sites per cell with the onset of apoptosis increases 100 to 1 000-fold during apoptosis, reaching values of 3-4 million in some cell lines.28, 29 The redistribution of PS and PC across the cell membrane at the beginning of the execution phase of apoptosis is also facilitated by a calcium ion dependent deactivation of translocase and floppase and activation of a 3rd enzyme called, “scramblase”.26 The externalization of PS is a general feature of apoptosis occurring prior to membrane bleb formation and deoxyribonucleic acid (DNA) degradation.27

The lag time between exposure to the trigger(s) and the time of observable morphologic signs of apoptosis is highly variable depending heavily on cell type, type of trigger(s), intensity and exposure, duration of signal, and the local environmental conditions of the cell.27 Most apoptotic pathways, however, converge on a common enzymatic pathway known as the “caspase cascade” that includes the key enzyme, caspase-3. After activation of caspase-3 the morphologic events of apoptosis quickly follow resulting in the orderly breakdown of cellular proteins, including the cytoskeleton, nuclear matrix, and the activation of poly-ADP-ribose polymerase. These morphologic events are collectively called the “execution phase”. The hallmark of the start of the execution phase is the redistribution and exposure of PS on the cell surface. PS exposure on the cell surface closely follows caspase-3 activation and occurs well before DNA fragmentation.30 Annexin V, therefore, is a sensitive marker of the early to intermediate phases of apoptosis.

Expression of PS is also a major signaling event that marks an apoptotic cell ready for ingestion and physiologic clearance by neighboring cells or macrophages preventing the uncontrolled release of potentially toxic intracellular substances.31, 32 Immune cells such as macrophages actually have been found to express surface receptors that recognize PS underscoring its role in cellular signaling and immune recognition. As an aside there maybe a value in radiolabeling macrophage PS receptor protein as a tracer for apoptosis provided that the protein would be both water soluble and able to bind PS in its free form. To date however, no in vitro or imaging studies have been reported with radiolabeled macrophage PS receptor protein.

Despite being widespread intracellulary throughout the body the physiologic role(s) of annexin V is unclear but the protein is known to be a potent inhibitor of coagulation, and phospholipase A^sub 2^ and protein kinase C. Both of these target enzymes are involved with apoptosis.25, 26 Annexin V may also be critical in the formation of calcium ion channels thereby regulating calcium-dependent processes in cellular organelles and the nucleus.33, 34

Recombinant (rh)-annexin V can be readily produced by expression in E. coli as previously described.35 This material can be purified with out any detectable endotoxin while maintaining PS binding activity equivalent to that of native annexin V isolated from human placenta, the organ in which annexin V was first found. In prior human and animal studies, rh-annexin V has been radiolabeled with ^sup 99m^Tc using several different ligands.36-39 One method, referred to as N^sub 2^S^sub 2^, ^sup 99m^Tc is transchelated from ^sup 99m^Tc-gluconate to the active ester 2,3,5,6-tetrafluoro-4,5- bis-S-(lethoxyethyl)-mercaptoacetamidopentanoate.40 Subsequently, the ^sup 99m^Tc active ester is conjugated to lysine functional groups on the annexin V. Recent phase I/II oncology studies in patients with cardiac tumor, lung carcinoma, non Hodgkin’s lymphoma, and metastatic breast carcinoma have used annexin V labeled with this technique.20 Another more efficient method of labeling uses a different coupling group, HYNIC (succinimidyl [6-hydrazinopyridine- 3-carboxylic acid]) also known as (succinimidyl [6- hydrazinonicotinic acid]). HYNIC is covalently attached to rh- annexin V-, which can be labeled with ^sup 99m^Tc performed by reacting the conjugate with ^sup 99m^Tc pertechnetate in the presence of stannous tricine.3, 41 While annexin V has been labeled with technetium 99m for SPECT imaging in humans, in the near future it should be possible to label annexin V with ^sup 18^F, ^sup 68^Ga,42 or ^sup 124^I,43 allowing higher resolution PET imaging.

Annexin imaging of early tumor response to therapy

Animal investigations demonstrate that radiolabeled annexin V can visualize the cytotoxic effects of chemotherapy on the marrow of normal rats and mice with leukemia.44 Taken together these observations suggest that intramedullary and tumor apoptosis can be imaged. Annexin V imaging may therefore be helpful in clinical management as an early assessment of therapeutic efficacy of anti- tumor agents.

A seminal clinical imaging study has been performed with radiolabeled annexin V in 15 patients with non-Hodgkin’s lymphoma, metastatic breast carcinoma, or lung carcinoma undergoing primary chemotherapy.20 This pilot study showed that radiolabeled annexin V identified significant increases in tumor uptake of tracer over baseline scans in 7 patients within the first 72 hours after the start of treatment. All 7 patients showed clinical response to their therapy with in several weeks. In 6 of the 8 patients without increases in annexin V uptake there was no response to treatment and patients died within 6 months.

The conclusions that can be drawn from this study are quite important and include; a) annexin V imaging can be performed serially and safely, b) significant post-treatment increases in annexin V uptake above pretreatment levels predict at least a partial response to chemotherapy, c) the increases in annexin V uptake appear to be specific and are not seen even in large necrotic tumors where there would be possible non-specific localization of the tracer both at baseline and post-treatment, and d) the increase in annexin V uptake appears to be heavily dependent on the exact time after the start of chemotherapy.

While there are no definitive human or animal model data at this time there are some clues as to when to perform annexin V imaging following the initiation of chemotherapy. Two to 3-fold increases in annexin V uptake have been noted as early as 1 hour, lasting approximately 90 minutes, following a single injection of high dose cyclophosphamide (150 mg/kg i.p.) in Balb/c mice bearing luciferase expressing BCL-1 syngeneic murine lymphoma.45 A similar increase was seen starting at 5 hours following a single injection of doxorubicin (10 mg/kg i.p). Paradoxically, both of these early drug-dependent transient increases in annexin V uptake were not accompanied by the loss of bioluminescent signal produced by the engineered BCL-1 lymphoma cells. Apoptosis of the BCL-1 lymphoma cells, however, eventually occurred some 3-22 hours later, as confirmed by serial BLI in vivo and then after excision in vitro.

The significance of these extremely early peaks in annexin V uptake prior to tumor cell loss and shrinkage is unclear but may relate a) to low levels of apoptosis of cells at a particularly sensitive phase of the cell cycle or b) to a pre-apoptotic “stressing” of tumor cells causing a transient expression of PS that may or may not be predictive of the commitment of a cell to apoptosis later on.46 Until recently PS expression was thought to occur only with apoptosis or platelet activation. New data suggest that PS expression can also occur with non-lethal cell injury prior to the irreversible morphologic changes such as DNA fragmentation.47- 54 These studies showed that intermediate levels of PS exposure were noted in cells with no other morphologic features of apoptosis could be readily reversed upon removal of physiologic stressors such as nitric-oxide, p53 activation, allergic mediators and growth factor deprivation. If these observations play out in vivo, then PS expression may define tissues at risk for cell death that may recover or be amendable to therapeutic intervention. Radiolabeled annexin V imaging may therefore be vastly more sensitive to regions of cellular injury as both stressed and dying cells can bind the tracer.

A 2nd peak of annexin V uptake (PS expression) would also be expected to occur hours later just prior to the loss of the bulk of tumor cells clue to end-stage apoptotic cell dea\th (i.e., autodigestion of plasma membrane, DNA, and cytoplasm with the contents packaged into small membrane bound vesicles = apoptotic bodies).

In summary, the clinical evidence with annexin V assessment of chemotherapeutic efficacy to date suggests that there is at least one peak of annexin V uptake, occurring early within hours of the initiation of chemotherapy. There may also be a later 2nd peak 24 to 72 hours after the completion of treatment. Until more definitive data is acquired it will be necessaiy to design clinical trials using ^sup 99m^Tc-annexin V so that early imaging (within the 1st day of therapy [immediate] followed up by re-injection of tracer and scanning 24 to 72 hours later [delayed]) are both examined.

Annexin imaging of ischemia and hypoxic-ischemic injury

Reperfusion therapy is the most effective approach to salvage ischemic neurons or myocardium. In the case of the heart, thrombolytic therapy or rescue angioplasty are effective in patients with acute ischemic syndromes.55 While these therapies result in substantial tissue salvage, acute reperfusion is also known to precipitate apoptosis of some neurons and myocytes.56, 57 Although the net outcome of reperfusion therapy is improved function, it is difficult to define the fraction of lost function clue to apoptotic cell death induced by the treatment. An imaging technique that identifies cell damage rapidly would be helpful to select therapies that would preserve the tissues at risk of apoptosis. Fluorescent- labeled annexin V has been applied in a murine model of cardiac ischemic reperfusion injury in situ and in vivo.58, 59 In these experiments fluorescent annexin V localized to ischemic myocytes within minutes after reperfusion with a maximum uptake seen after 25 minutes and remained unchanged thereafter. Treatment with specific caspases inhibitors designed to block apoptosis decreased both the number of PS positive myocytes and also delayed the onset of PS expression by more than 2 hours. Annexin imaging has been used successfully to identify acute myocardial infarction in patients.60, 61

Hypoxic-ischemic injury of the brain following perinatal asphyxia in neonates 62 and stroke in adults 63 is also intimately associated with programmed cell death. Following acute hypoxic-ischemic injury there is often delayed loss of gray and white matter largely due to the programmed death of neurons in response to local release of excitatory amino acids and free radicals which act by increasing intracellular free Ca^sup 2+^ and activating Ca^sup 2+^-dependent catabolic enzymes.64-66 Inflammatory cells are also actively recruited to a lesion by a variety of chemokines including MCP-1 (monocyte chemo-attractant peptide) within hours of an insult.67-69 These cells then exacerbate the loss of cerebral tissue from apoptosis due to the regional release of toxic pro-apoptotic immune mediators. Diffusion weighted MR can identify some of the early metabolic effects following cerebral ischemia (i.e decreased ADC, restricted diffusion) in both animal models and human subjects.70, 71 Although changes in diffusion correlate with apoptosis, they are not direct markers of the process.

Apoptosis following both focal and global cerebral hypoxic- ischemic injury can however be imaged with radiolabeled annexin V in animal models of both focal and global hypoxic ischemic injury.72- 74 One striking observation from these studies was the marked multifocal uptake of annexin V observed immediately after reversal of relatively brief minor episodes of cerebral ischemia. Interestingly these brains were found to be normal on DWI or T2- weighted MR imaging after reversal of hypoxia just prior to injection of tracer. These studies also found that annexin V could cross the intact blood brain barrier, localize on the surface and be taken up in the cytoplasm of injured neurons. The mechanism(s) of annexin V uptake in the ischemic brain is unclear but argues for an active set of pumps that appear to scavenge annexin V from the vascular extracellular space.

There is great interest in directly monitoring the effects of newer neuroprotective therapies designed specifically to inhibit programmed cell death and promote cell growth in injured tissues.75- 79 Anti-inflammatory measures have also shown promise in ameliorating the delayed effects of ischemic injury occurring days after the onset of neurologic deficit 80-83 and may in part explain the marked increase in the annexin V uptake of focal ischemic lesions noted at day 3 as compared to that seen in the first 24 hours following insult (personal data). Radiolabeled annexin V may be useful to identify therapies that reduce the inflammatory response in these conditions.

Imaging chronic inflammation, autoimmune disorders, and transplant rejection

Chronic inflammation is now known to be an integral component of many seemingly unrelated pathologic processes, including rheumatologic and other autoimmune diseases,84 seasonal rhinitis,85 AIDS,86 atherosclerosis,87, 88 transplant rejection,89 encephalitis 90 and neoplasia.91 Attention therefore is now being redirected to the imaging of monocytes, macrophages, and lymphocytes, the major cell types involved in chronic inflammation. Within 48 hours of initiation of focal inflammation monocytes and their derivative cells, macrophages become the dominant cell types at sites of inflammation.

When monocytes arrive at the site of inflammation they work to resolve inflammation. One major activity of these cells is the (PS)- specific recognition and clearance of apoptotic granulocytes, cells that have outlived their useful function.92, 93 Monocytes and macrophage also accelerate the apoptosis of bystander (unwanted) granulocytes at sites of inflammation via the selective release of a variety of soluble proteins in concert with soluble Fas ligand; a peptide which binds to the Fas death receptor, that is expressed on granulocytes and other types of inflammatory cells.94 The degree of macrophage infiltration and its associated granulocytic apoptotic response has been successfully imaged in vivo in a rodent model of turpentine-induced sterile abscesses.95 The clearance of granulocytes by monocytes and macrophages also effectively reduces the sensitivity of radiolabeled white cell or granulocytic chemotactic peptide imaging at sites of subacute and chronic inflammation, suggesting an advantage for radiolabelect annexin V imaging over granulocytic specific markers.

Another potential use of radiolabeled annexin V is the study of autoimmune disease. The inflammation associated with RA is caused by abnormal collections of immune cells responding inappropriately to idiotypic antigens.96, 97 These inflammatory cells often induce apoptosis of target tissues such as the synovium,98-100 prior to their own apoptotic cell death. As a result annexin imaging in these patients may be useful to test new therapies, especially since plain film radiography, nuclear scintigraphy with bone scanning and nonspecific inflammatory radiopharmaceuticals, and contrast enhanced MR are either insensitive or impractical guides for the assessment of therapy or disease monitoring.101, 102

Annexin V imaging was recently tested for its ability to assess changes in experimental collagen-induced rheumatoid arthritis.103 In this study ^sup 99m^TC-HYNIC annexin V was used for an autoradiographic study of the extent and severity of apoptosis in the front and rear paws of DBA/1 mice with type II collagen induced RA. There was a significant (p<0.002) nearly 3 fold increase in the uptake of annexin V in the front foot pads, rear toes, rear foot pads, and heels at the time of maximal extremity swelling as determined by serial caliper measurements at 4 weeks after inoculation with type II bovine collagen (n=9) as compared with controls (n=10). The front toes had 5-6 fold increased in uptake as compared with control (p<0.001). Histologic analysis revealed only scattered rare lymphocytes in the periarticular soft tissues without joint destruction. Dual autoradiography with ^sup 125^I-bovine serum albumin as a control showed that annexin V localization was specific. Treatment with methylprednisilone for 1 week (n=8) at 4 weeks postimmunization with collagen type II decreased annexin V uptake by 3-6 fold compared to untreated animals (p<0.002). These data show that ^sup 99m^Tc-annexin V can detect collagen induced immune arthritis and its response to steroid therapy prior to joint destruction.

Another promising area of clinical investigation is the use of radiolabeled annexin V imaging for the screening of organ transplant recipients for acute rejection. Animal studies in rats receiving heart, lung, or liver transplants have shown the feasibility of serial non-invasive monitoring using this approach.104-106

Phase IIa clinical trials to screen for acute rejection in heart transplant recipients with ^sup 99m^Tc-annexin V have demonstrated initial success in a group of 18 patients studied by Narula et al.107 In 5 of the 18 patients moderate to severe acute rejection was found on right ventricular endomyocarclial biopsy (EMB). In these patients with acute rejection ^sup 99m^Tc-annexin V uptake was intense and was distributed either diffusely or focally in the myocardium as shown below. Furthermore, histochemical analyses of the EMB specimens showed both TUNEL (DNA fragmentation) and caspases- 3 positive cells within the myocardium. In the remaining 13 patients without histologic evidence of rejection there “was no myocardial uptake above blood activity and no histochemical evidence of apoptosis on the TUNEL or caspases-3 assays.

Conclusions

Several imaging modalities have shown promise in the non- invasive assessment of cell death including DW and Proton MR spectroscopy, and novel tracers such as Z-VAD.fmk, synaptotagmin I, and annexin V. Of these, radiolabeled annexin V is the best studied and is now entering phase II and \III clinical trials for the assessment of therapeutic efficacy in cancer patients, the extent and severity of myocardial infarction, and the screening for acute rejection in heart transplant recipients. As there are many potential features of the apoptotic cascade that could be exploited for purposes of imaging one should expect rapid changes in this exciting new area of clinical research.

References

1. Bronckers AL, Goei W, van Heerde WL, Dumont FA, Reutelingsperger CP, van den Eijnde SM. Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling. Cell Tissue Res 2000;301:267- 72.

2. Stratton JR, Dewhurst TA, Kasina S, Reno JM, Cerqueria MD, Baskin DG et al. Selective uptake of radiolabeled annexin V on acute porcine left atrial thrombi. Circulation 1995;92:3113-21.

3. Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K et al. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proceedings of the National Academy Science, USA 1998;95:6349-54.

4. Baur A, Reiser MF. Diffusion-weighted imaging of the musculoskeletal system in humans. Skeletal Radiol 2000;29:555-62.

5. Byun WM, Shin SO, Chang Y, Lee SJ, Finsterbusch J, Frahm J. Diffusion-weighted MR imaging of metastatic disease of the spine: assessment of response to therapy. Am J Neuroradiol 2002;23:906-12.

6. Geshwind JFH, Artemov D, Abraham S, Omdal D, Huncharek MS, McGee C et al. Chemoembolization of liver tumor in a rabbit model: assessment of tumor cell death with diffusion-weighted MR imaging and histologie analysis. J Vasc Interv Radiol 2000;11:1245-55.

7. Blankenberg FG, Katsikis PD, Storrs RW, Beaulieu C, Spielman D, Chen JY et al. Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy. Blood 1997;89:3778-86.

8. Hakumaki JM, Poptani FI, Sandmair AM, Yla-Herttuala S, Kauppinen RA. ^sup 1^H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: implications for the in vivo detection of apoptosis. Nat Med 1999;5:1323-7.

9. Al-Saffar NM, Titley JC, Robertson D, Clarke PA, Jackson LE, Leach MO et al. Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells. Br J Cancer 2002;86:963-70.

10. Di Vito M, Lenti L, Knijn A, Iorio E, D’Agostino F, Molinari A et al. ^sup 1^H NMR-visible mobile lipid domains correlate with cytoplasimic lipid bodies in apoptotic T-lymphoblastoid cells. Biochim Biophys Acta 2001;1530:47-66.

11. Kwock L, Brown MA, Castillo M. Extraneous lipid contamination in single-volume proton MR spectroscopy: phantom and human studies. AJNR Am J Neuroradiol 1997;18:1349-57.

12. Hakumaki JM, Poptani H, Puumalainen AM, Loimas S, Paljarvi LA, Yla-Herttuala S et al. Quantitative ^sup 1^H nuclear magnetic resonance diffusion spectroscopy of BT4C rat glioma during thymidine kinase-mediated gene therapy in vivo: identification of apoptotic response. Cancer Res 1998;58:3791-9.

13. Hortelano S, Garcia-Martin ML, Cerdan S, Castrillo A, Alvarez AM, Bosca L. Intracellular water motion decreases in apoptotic macrophages after caspase activation. Cell Death Differ 2001;8:1022- 8.

14. Zhao M, Beauregard DA, Loizou L, Davletov B, Brindle KM. Non- invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent. Nature Med 2001;7:1241-4.

15. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger CPM. A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 1995;184:39-51.

16. Fukucla M, Kojima T, Mikoshiba K. Phospholipid composition dependence of Ca^sup 2+^-dependent phospholipid binding to the C2A domain of synaptotagmin. J Biochem 1996;14:8430-4.

17. Weissleder R, Moore A, Mahmood U, Bhorade R, Benveniste H, Chiocca EA et al. In vivo magnetic resonance imaging of transgene expression. Nat Med 2000;6:351-4.

18. Haberkorn U, Kinscherf R, Krammer PH, Mier W, Eisenhut M. Investigation of a potential scintigraphic marker of apoptosis: radioiodinated Z-Val-Ala-DL-Asp(O-methyl)-fluoromethyl ketone. Nucl Med Biol 2001;28:793-8.

19. Hofstra L, Dumont EA, Thimister PW, Heidendal GA, DeBruine AP, Elenbaas TW et al. In vivo detection of apoptosis in an intracardiac tumor. JAMA 2001;285:1841-2.

20. Belhocine T, Steinmetz N, Hustinx R, Bartsch P, Jerusalem G, Seidel L et al. Increased uptake of the apoptosis imaging agent ^sup 9mm^Tc rh-Annexin V in human tumors after one course of chemotherapy as a predictor of tumor response and patient prognosis. Clin Cancer Res 2002;8:2766-74.

21. Blankenberg FG. “To scan or not to scan, it is a question of timing”: ^sup 9mm^Tc-annexm V radionuclide imaging assessment of treatment efficacy after one course of chemotherapy. Clin Cancer Res 2002;8:2757-8.

22. Hofstra L, Liem IH, Dumont EA, Boersma HH, van Heerde WL, Doevendans PA et al. Visualization of cell death in vivo in patients with an acute myocardial infarction. Lancet 2000;356:209-12.

23. Narula J, Acio ER, Narula N, Samuels LE, Fyfe B, Wood D et al. Annexin-V imaging for noninvasive detection of cardiac allograft rejection. Nat Med 2001;7:1347-52.

24. Van Heerde WL, de Groot PG, Reutelingsperger CPM. The complexity of the phospholipid binding protein annexin V. Thromb Haemost 1995;73:172-9.

25. Williamson P, Schlegel RA. Back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells. Mol Memb Biol 1994;11:199-216.

26. Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997;89:1121- 32.

27. Martin SJ, Reutelingsperger CPM, McGahon AJ. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by over-expression of Bcl-2 Abl. J Exp Med 1995;182:1545- 56.

28. Bennett MR, Gibson DF, Sehwartz SM, Tait JF. Binding and phagocytosis of apoptotic vascular smooth muscle cells is mediated in part by exposure of phosphatidylserine. Circ Res 1995;77:1136- 42.

29. Tait JF, Smith C, Wood BL. Measurement of phosphatidylserine exposure in leukocytes and platelets by whole-blood flow cytometry with annexin V. Blood Cells Mol Dis 1999;25:271-8.

30. Naito M, Nagashima K, Mashima T, Tsuruo T. Phosphatidylserine externalization is a downstream event of interleukin-1b-converting enzyme family protease activation during apoptosis. Blood 1997;89:2060-6.

31. Brown SB, Savill J. Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes. J Immunol 1999;162:480-5.

32. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RAB, Henson PM. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 2000;405:85-90.

33. Luckcuck T, Trotter PJ, Walker JH. Localization of annexin V in the adult and neonatal heart. Biochem Biophys Res Comm 1997;238:622-8.

34. Matteo RG, Schomisch Moravec C. Immunolocalization of annexins IV, V, and VI in the failing and non-failing human heart. Cardiovasc Res 2000;45:961-70.

35. Tait JF, Smith C. Site-specific mutagenesis of Annexin-V: role of residues from Arg-200 to Lys-207 in phospholipid binding. Arch Biochem Biophys 1991;288:141-4.

36. Stratton JR, Dewhurst TA, Kasina S, Reno JM, Cerqueira MD, Baskin DG et al. Selective uptake of radiolabeled annexin V on acute porcine left atrial thrombi. Circulation 1995;92:3113-21.

37. Verbeke KA, Kieffer DM, Vanderheyden JL, Steinmetz N, Green A et al. Optimization of the preparation of ^sup 99m^Tc_Hynic-Annexin V for human use. J Nucl Med 2002;43(5 Suppl):381P.

38. Steinmetz ND, Stopeck AT, Woolfenden JM, Heidendal GA, Hofstra L, Thimister P et al. Imaging cell death in untreated and treated human tumors with ^sup 99m^Tc Annexin V prepared using the Apomate(R) kit. J Nucl Med 2001;42(5 Suppl):83P-4.

39. Yang DJ, Azhdarinia A, Wu P, Yu DF, Tansey W, Kalimi SK et al. In vivo and in vitro measurement of apoptosis in breast cancer cells using ^sup 99m^Tc-EC-annexin V. Cancer Biother Radiopharm 2001;16:73-83.

40. Kasina S, Rao TN, Srinivasan A, Sanderson JA, Fitzner JN, Reno JM et al. Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent. J Nucl Med 1991;32:1445-51.

41. Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K et al. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proceedings of the National Academy Science, USA 1998;95:6349-54.

42. Smith-Jones PM, Afroze F, Soffing M, Zanzonico P, Tait J, Strauss HW. ^sup 68^Ga Labeling of Annexin V: comparison with ^sup 99m^Tc-apomate and initial MicroPET studies. J Nucl Med. In press 2003.

43. Glaser M, Collingridge DR, Aboagye EO, Bouchier-Hayes L, Hutchinson OC, Martin SJ et al. Iodine-124 labelled Annexin-V as a potential radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 2003;58:55-62.

44. Blankenberg FG, Naumovski L, Tait JF, Post AM, Strauss HW. Imaging cyclophosphamide-induced intramedullary apoptosis in rats using ^sup 99m^Tc-radiolabeled annexin V. J Nucl Med 2001;42:309- 16.

45. Blankenberg FG. “To scan or not to scan, it is a question of timing”: ^sup 99m^Tc-annexin V radionuclide imaging assessment of treatment efficacy after one course of chemotherapy. Clin Cancer Res 2002;8:2757-8.

46. Strauss HW, Narula J, Blankenberg FG. Radioimaging to identify myocardial cell death and probably injury. Lancet 2000;356:180-1.

47. Lejeune M, Ferster A, Cantinieaux B, Sariban E. Prolonged but reversible neutrophil dyfunctions differentially sensitive to granulocyte colony-stimulating factor in children with acute lymphoblastic leukemia. Br J Haematol 1998;102:1284-91.

48. Hammill AK, Uhr JW, Scheuermann RH. Annexin V staining due to loss of membrane asymmetry can be reversible an\d precede commitment to apoptotic death. Exp Cell Res 1999;251:16-21.

49. Furukawa Y, Bangham CRM, Taylor GP, Weber JN, Osame M. Frequent reversible membrane damage in peripheral blood B cells in human T cell lymphotrophic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Clin Exp Immunol 2000;120:307-16.

50. Martin S, Pombo I, Poncet P, David B, Arock M, Blank U. Immunologic stimulation of mast cells leads to the reversible exposure of phosphatidylserine in the absence of apoptosis. Int Arch Allergy Immunol 2000;123:249-58.

51. Lin SH, Vincent A, Shaw T, Maynard KI, Maiese K. Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death. J Cereb Blood Flow Metab 2000;20:1380-91.

52. Geske FJ, Lieberman R, Strange R, Gerschenson LE. Early stages of p53-induced apoptosis are reversible. Cell Death Differ 2001;8:182-91.

53. Maiese K, Vincent AM. Membrane asymmetry and DNA degradation: functionally distinct determinants of neuronal programmed cell death. J Neurosci Res 2000;59:568-80.

54. Yang MY, Chuang H, Chen RF, Yang KD. Reversible phosphatidylserine expression on blood granulocytes related to membrane perturbation but not DNA strand breaks. J Leukoc Biol 2002;71:231-7.

55. Bahit MC, Cannon CP, Amman EM, Murphy SA, Gibson CM, McCabe CH et al. Direct comparison of characteristics, treatment, and outcomes of patients enrolled versus patients not enrolled in a clinical trial at centers participating in the TIMI 9 Trial and TIMI 9 Registry. Am Heart J 2003;145:109-17.

56. Lee SH, Kim M, Kim YJ, Kim YA, Chi JG, Roh JK et al. Ischemic intensity influences the distribution of delayed infarction and apoptotic cell death following transient focal cerebral ischemia in rats. Brain Res 2002;956:14-23.

57. Scarabelli T, Stephanou A, Rayment N, Pasini E, Comini L, Curello S et al. Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury. Circulation 2001;104:253-6.

58. Dumont EA, Hofstra L, van Heerde WL, van den Eijnde S, Doevendans PA, DeMuinck E et al. Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model. Circulation 2000;102:1564-8.

59. Dumont EA, Reutelingsperger CP, Smuts JF, Daemen MJ, Doevendans PA, Wellens HJ et al. Real-time imaging of apoptotic cell- membrane changes at the single-cell level in the beating murine heart. Nat Med 2001;7:1352-5.

60. Hofstra L, Liem IH, Dumont EA, Boersma HH, Van Heerde WL, Doevendans PA et al. Visualization of cell death in vivo in patients with an acute myocardial infarction. Lancet 2000;356:209-12.

61. Green AM, Steinmetz ND. Monitoring apoptosis in real time. Cancer J 2002;8:82-92.

62. Pulera MR, Adams LM, Liu H, Santos DG, Nishimura RN, Yang F et al. Apoptosis in a neonatal rat model of cerebral hypoxia- ischemia. Stroke 1998; 9:2622-30.

63. Vexler ZS, Roberts TPL, Bollen AW, Derugin N, Arieff AI. Transient cerebral ischemia. Association of apoptosis induction with hypoperfusion. J Clin Invest 1997;99:1453-9.

64. Lee J-M, Grabb MC, Zipfel GJ, Choi DW. Brain tissue responses to ischemia. J Clin Invest 2000;106:723-31.

65. Hara H, Friedlander RM, Gagliardini V, Ayata C, Fink K, Huang Z et al. Inhibition of interleukin 1b converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci USA 1997;94:2007-12.

66. Graham SH, Chen J. Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 2001;21:99-109.

67. Kim JS, Gautam SC, Chopp M, Zaloga C, Jones ML, Ward PA et al. Expression of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 after focal cerebral ischemia in the rat. J Neuroimmunol 1995;56:127-34.

68. Gourmala NG, Buttini M, Limonta S, Sauter A, Boddeke HW. Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration. J Neuroimmunol 1997;74:35-44.

69. Yamagami S, Tamura M, Hayashi M, Endo N, Tanabe H, Katsuura Y et al. Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats. J Leukoc Biol 1999;65:744-9.

70. Rupalla K, Allegrini PR, Sauer D, Wiessner C. Time course of microglia activation and apoptosis in various brain regions after permanent focal cerebral ischemia in mice. Acta Neuropathol (Berl) 1998;96:172-8.

71. Neumann-Haefelin T, Kastrup A, cle Crespigny A, Yenari MA, Ringer T, Sun GH et al. Serial MRI after transient focal cerebral ischemia in rats: dynamics of tissue injury, blood-brain barrier damage, and edema formation. Stroke 2000;31:1965-72.

72. Blankenberg FG, Busch E, Yenari MA. In vivo imaging of apoptotic cell death associated with cerebral hemispheric ischemia using ^sup 99m^Tc radiolabeled annexin V. Stroke 1998;29:330.

73. Blankenberg FG, Tait JF, Strauss HW. Apoptotic cell death: its implications for imaging in the next millennium. Eur J Nucl Med 2000;27:359-67.

74. D’Arceuil H, Rhine W, de Crespigny A, Yenari M, Tait JF, Strauss HW et al. ^sup 99m^Tc annexin V imaging of neonatal hypoxic brain injury. Stroke 2000;31:2692-700.

75. Scheepens A, Sirimanne ES, Breier BH, Clark RG, Gluckman PD, Williams CE. Growth hormone as a neuronal rescue factor during recovery from CNS injury. Neuroscience 2001;104:677-87.

76. Ferrer I, Krupinski J, Goutan E, Marti E, Ambrosio S, Arenas E. Brain-derived neurotrophic factor reduces cortical cell death by ischemia after middle cerebral artery occlusion in the rat. Acta Neuropathol (Berl) 2001;101:229-38.

77. Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, Lewczuk P et al. Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acacl Sci U S A 2001;98:4044-9.

78. Ay I, Sugimori H, Finklestein SP. Intravenous basic fibroblast growth factor (bFGF) decreases DNA fragmentation and prevents downregulation of Bcl-2 expression in the ischemic brain following middle cerebral artery occlusion in rats. Brain Res Mol Brain Res 2001;87:71-80.

79. Korenkov AI, Pahnke J, Frei K, Warzok R, Schroeder HW, Frick R etal. Treatment with nimodipine or mannitol reduces programmed cell death and infarct size following focal cerebral ischemia. Neurosurg Rev 2000;23:145-50.

80. Kataoka K, Yanase H. Mild hypothermia: a revived countermeasure against ischemic neuronal damage. Neurosci Res 1998;32:103-17.

81. Liao SL, Chen WY, Raung SL, Ku? JS, Chen CJ. Association of immune responses and ischemic brain infarction in rat. Neuroreport 2001;12:1943-7.

82. Barone FC, Parsons AA. Therapeutic potential of anti- inflammatory drugs in focal stroke. Expert Opin Investig Drugs 2000;9:2281-306.

83. Vila N, Castillo J, Davalos A, Chamorro A. Proinflammatory cytokines and early neurological worsening in ischemic stroke. Stroke 2000;31:2325-9.

84. Robinson E, Keystone EC, Schall TJ, Gillett N, Fish EN. Chemokine expression in rheumatoid arthritis (RA): evidence of RANTES and macrophage inflammatory protein (MIP)-1 [beta] production by synovial T cells. Clin Exp Immnuol 1995;101:398-407.

85. Bachert C, Wagenmann M, Holtappels G. Cytokines and adhesion molecules in allergic rhinitis. Am J Rhinol 1998;12:3-8.

86. Lee B, Doranz BJ, Rana S, Yi Y, Mellado M, Frade JM et al. Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4. J Virol 1998;72:7450-8.

87. Takahashi M, Masuyama J, Ikeda U, Kasahara T1 Kitagawa S, Takahashi Y et al. Induction of monocyte chemoattractant protein-1 synthesis in human monocytes during transendothelial migration in vitro. Circ Res 1995;76:750-7.

88. Ohtsuki K, Hayase M, Akashi K, Kopiwoda S, Strauss HW. Detection of monocyte chemoattractant protein-1 receptor expression in experimental atherosclerotic lesions: an autoradiographic study. Circulation 2001;104:203-8.

89. Grandaliano G, Gesualdo L, Ranieri E, Monno R, Stallone G, Schena FP. Monocyte chemotactic peptide-1 expression and monocyte infiltration in acute renal transplant rejection. Transplantation 1997;63:414-20.

90. Rosier A, Pohl M, Braune HJ, Oertel WH, Gemsa D, Sprenger H. Time course of chemokines in the cerebrospinal fluid and serum during herpes simplex type 1 encephalitis. J Neurol Sci 1998;157:82- 9.

91. Zhang L, Khayat A, Cheng H, Graves DT. The pattern of monocyte recruitment in tumors is modulated by MCP-1 expression and influences the rate of tumor growth. Lab Invest 1997;76:579-90.

92. Savill J. Apoptosis in resolution of inflammation. J Leukoc Biol 1997;61:375-80.

93. Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RAB, Henson PM. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 2000;405;85-90.

94. Brown SB, Savill J. Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes. J Immunol 1999;162:480-5.

95. Blankenberg FG, Tait JF, Blankenberg TA, Post AM, Strauss HW. Imaging macrophages and the apoptosis of granulocytes in a rodent model of subacute and chronic abscesses with radiolabeled monocyte chemotactic peptide-1 and annexin V. Eur J Nucl Med 2001;28:1384- 93.

96. Wilson C, Tiwana H, Ebringer A. Molecular mimicry between HLA- DR alleles associated with rheumatoid arthritis and proteus mirabilis as the aetiological basis for autoimmunity. Microbes Infect 2000;2:1489-96.

97. Hyrich KL, Inman RD. Infectious agents in chronic rheumatic diseases Curr Opin Rheumatol 2001;13:300-4.

98. Firestein GS, Yeo M, Zvaifler NJ. Apoptosis in rheumatoid arthritis synovium. J Clin Invest 1995;96:1631-8.

99. Matsumoto S, Muller-Ladner U, Gay RE, Nishioka K, Gay S. Ultrastructural demonstration of apoptosis, Fas and Bcl-2 expression of rheumatoid synovial fibroblasts. J Rheumatol 1996;23:1345-52.

100. Ceponis A, Hieta\nen J, Tamulaitiene M, Partsch G, Patiala H, Konttinen YT. A comparative quantitative morphometric study of cell apoptosis in synovial membranes in psoriatic, reactive and rheumatoid arthritis. Rheumatology 1999;38:431-40.

101. Lassere MN, van der Heijde D, Johnson KR, Boers M, Edmonds JJ. Reliability of measures of disease activity and disease damage in rheumatoid arthritis: implications for smallest detectable difference, minimal clinically important difference, and analysis of treatment effects in randomized controlled trials. Rheumatology 2001;28:892-903.

102. Strand V, Lassere M, van der Heijde D, Johnson K, Boers M. J. Recent rheumatoid arthritis clinical trials using radiographic end-points: updated research agenda. Rheumatol 2001;28:887-9.

103. Post AM, Katsikis PD, Tait JF, Geaghan SM, Strauss HW, Blankenberg FG. Imaging cell death with radiolabeled annexin V in an experimental model of rheumatoid arthritis. J Nucl Med 2002;43:1359- 65.

104. Vriens PW, Blankenberg FG, Stoot JH, Ohtsuki K, Berry GJ, Tait JF et al. The use of technetium ^sup 99m^Tc annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J Thorac Carcliovasc Surg 1998;116:844-53.

105. Blankenberg FG, Robbins RC, Stoot JH, Vriens PW, Berry GJ, Tait JF et al. Radionuclide imaging of acute lung transplant rejection with annexin V. Chest 2000;117:834-40.

106. Ogura Y, Krams SM, Martinez OM, Kopiwoda S, Higgins JP, Esquivel CO et al. Radiolabeled annexin V imaging: diagnosis of allograft rejection in an experimental rodent model of liver transplantation. Radiology 2000;214:795-800.

107. Narula J, Acio ER, Narula N, Samuels LE, Fyfe B, Wood D et al. Annexin-V imaging for noninvasive detection of cardiac allograft rejection. Nat Mecl 2001;7:1347-52.

F. BLANKENBERG 1, C. MARI 1, H. W. STRAUSS 2

1 Department of Radiology Stanford University School of Medicine, Stanford, CA, USA

2 Section of Nuclear Medicine Memorial Sloan Kettering Cancer Center New York, NY, USA

Address reprint requests to: H. W. Strauss, MD, Memorial Sloan Kettering Cancer Center, Nuclear Medicine, Chief of the Clinical Service, Room S212, 1275 York Avenue, New York, NY 10021, USA.

E-mail: straussh@mskcc.org

Copyright Edizioni Minerva Medica Dec 2003