Development of SM5-1-conjugated ultrasmall superparamagnetic iron oxide nanoparticles for hepatoma detection

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are maghemite or magnetite nanoparticles currently used as contrast agent in magnetic resonance imaging (MRI). In this study, a targeted contrast agent (SM-USPIO) was prepared by conjugating coprecipitated USPIO to a humanized SM5-1 antibody which can specifically react with human hepatocellular carcinoma (HCC) cells. The binding and internalization of SM-USPIO to the HCC cell line ch-hep-3 was confirmed by flow cytometry and confocal microscopy. Furthermore, SM-USPIO was demonstrated to be able to selectively accumulate in the tumor cells, resulting in a marked decrease of MRI T2-weighted signal intensity. Biodistribution studies demonstrated the efficient accumulation of SM-USPIO in the ch-hep-3 tumor in nude mice. The in vivo study in the ch-hep-3 tumor-bearing nude mice indicated that MRI using the SM-USPIO as contrast agent possessed good diagnostic ability, suggesting that SM-USPIO had the potential to be a promising targeted contrast agent for diagnosis of HCC.     

Dynamic imaging of lymphatic vessels and lymph nodes using a bimodal nanoparticulate contrast agent

BACKGROUND: Evaluation of lymphedema and lymph node metastasis in humans has relied primarily on invasive or radioactive modalities. While noninvasive technologies such as magnetic resonance imaging (MRI) offer the potential for true three-dimensional imaging of lymphatic structures, invasive modalities, such as optical fluorescence microscopy, provide higher resolution and clearer delineation of both lymph nodes and lymphatic vessels. Thus, contrast agents that image lymphatic vessels and lymph nodes by both fluorescence and MRI may further enhance our understanding of the structure and function of the lymphatic system. Recent applications of bimodal (fluorescence and MR) contrast agents in mice have not achieved clear visualization of lymphatic vessels and nodes. Here the authors describe the development of a nanoparticulate contrast agent that is taken up by lymphatic vessels to draining lymph nodes and detected by both modalities. METHODS: A unique nanoparticulate contrast agent composed of a polyamidoamine dendrimer core conjugated to paramagnetic contrast agents and fluorescent probes was synthesized. Anesthetized mice were injected with the nanoparticulates in the hind footpads and imaged by MR and fluorescence microscopy. High resolution MR and fluorescence images were obtained and compared to traditional techniques for lymphatic visualization using Evans blue dye. RESULTS: Lymph nodes and lymphatic vessels were clearly observed by both MRI and fluorescence microscopy using the bimodal nanoparticulate contrast agent. Characteristic tail-lymphatics were also visualized by both modalities. Contrast imaging yielded a higher resolution than the traditional method employing Evans blue dye. MR data correlated with fluorescence and Evans blue dye imaging. CONCLUSION: A bimodal nanoparticulate contrast agent facilitates the visualization of lymphatic vessels and lymph nodes by both fluorescence microscopy and MRI with strong correlation between the two modalities. This agent may translate to applications such as the assessment of malignancy and lymphedema in humans and the evaluation of lymphatic vessel function and morphology in animal models.     

From phage display to magnetophage, a new tool for magnetic resonance molecular imaging

Phage display, an extremely promising technology in the context of molecular imaging, allows for the selection of peptides interacting with virtually any target from a heterogeneous mixture of bacteriophages. In this work, we propose the concept of magnetophages, obtained by covalent coupling of ultrasmall particles of iron oxide (USPIO) to the proteins of the phage wall. To validate magnetophages as a magnetic resonance imaging contrast agent (MRI), we have used as a prototype the clone E3 because of its specific affinity for phosphatidylserine, a marker of apoptosis. Enzyme-linked immunosorbent assay showed that E3 magnetophages incubated with phosphatidylserine retained the properties of the nonmagnetically labeled phages. The usefulness of magnetophages as an MRI contrast agent was estimated by incubation with phosphatidylcholine and phosphatidylserine or with apoptotic and control cells. Under these conditions, E3 magnetophages allow the discrimination of phosphatidylserine from phosphatidylcholine and of apoptotic cells from control ones. Injected in vivo, magnetophages are rapidly cleared from the blood stream and internalized by the phagocytic cells of the liver. To abrogate this problem, USPIO were pegylated to obtain stealthy E3-PEG-magnetophages, invisible to phagocytic cells, which were successfully targeted to apoptotic liver. If this feature demonstrated for E3 magnetophages can be extrapolated to other phage display selected entities, magnetophages become an original system which allows validation of the candidate binding peptides before their synthesis is considered. The concept of the magnetophage could be extended to other imaging modalities by replacing USPIO with an adequate reporter (i.e., radiolabeled phages).     

Indirect Magnetic Resonance Imaging Lymphography Identifies Lymph Node Metastasis in Rabbit Pyriform Sinus VX2 Carcinoma Using Ultra-Small Super-Paramagnetic Iron Oxide

Background

USPIO is a contrast agent for MRI that can generate T2W images with low signal intensities. After subcutaneous or intravenous injection of USPIO, normal lymph node tissues uptake these nano-particles, but tumor cells do not. Thus, tumor metastasis can be detected using this contrast agent.

Objective

The aim of this study was to access the feasibility of USPIO enhanced MRI for the detection of cervical lymph node metastasis in a pyriform sinus carcinoma animal model and to investigate the ability of USPIO to enhance images of cervical lymph node metastases.

Methods and Findings

Twenty New Zealand rabbits were randomly divided into tumor and inflammatory groups, and each group contained 10 rabbits. In the inflammatory group, a 0.5 ml egg yolk emulsion was injected into the sub-mandibular muscle of the rabbits to induce an inflammatory reaction in their cervical lymph nodes. In the tumor group, a VX2 tumor tissue suspension was transplanted into the pyriform sinus sub-mucosa of the rabbits using direct laryngoscope. Four weeks after the tumor or egg yolk injection, MRIs were performed before and after USPIO injection to observe the imaging enhancement features of USPIO. After that, a histo-pathological analysis was performed for all rabbits. We found the metastatic lymph nodes had no signal reduced intensity or irregular signal reduced intensity on T2-weighted image by using USPIO enhancement. In the tumor group,the sensitivity and specificity of plain MRI were 57.6% and 60.7%. The corresponding values of USPIO-enhanced MRl were 96.1% and 85.7%. (P<0.05)

Conclusion

The features and the extent of the lymph node metastases corresponded to those observed on USPIO-enhanced MR images. USPIO-enhanced MRI is useful for the detection and estimation of lymph node metastasis in this cervical carcinoma animal model.     

C-MALISA (cellular magnetic-linked immunosorbent assay), a new application of cellular ELISA for MRI

A modified cellular ELISA (enzyme-linked immunosorbent assay), named cellular magnetic-linked immunosorbent assay (C-MALISA), has been developed as an application of magnetic resonance imaging (MRI) for in vitro clinical diagnosis. To validate the method, three contrast agents targeted to integrins were synthesized by grafting to USPIO (ultrasmall particles of iron oxide): (a) the CS1 (connecting segment-1) fragment of fibronectin (FN) (USPIO-g-FN); (b) the peptide GRGD (USPIO-g-GRGD); (c) a non-peptidic RGD mimetic (USPIO-g-mimRGD). Jurkat cells and rat mononuclear cells were stimulated to activate their integrins. After cell fixation on ELISA plates, incubation with the contrast agents, rinsing, and digestion in 5N HCl, the samples were analyzed by MRI. Paramagnetic relaxation rate enhancements (delta R2) were measured on images. Delta R2 was converted in values of iron concentration based on a calibration curve. The apparent dissociation constants (K(d)*) of the three contrast agents were estimated based on the MRI measurement of delta R2. K(d)* of 1.22 x 10(-7) M, of 7.00 x 10(-8) M, and of 1.13 x 10(-8) M were found respectively for USPIO-g-FN, USPIO-g-GRGD, and USPIO-g-mimGRG. The MRI confirmed a statistically significant difference (p < 0.01, p < 0.05) between the stimulated cells incubated with integrin-targeted compounds with respect to the controls (i.e., non-stimulated cells and stimulated cells incubated with non-specific USPIO). The integrin specificity of the three compounds was confirmed by the pre-incubation with GRGD (for USPIO-g-mimRGD and USPIO-g-GRGD) or FN (for USPIO-g-FN).     

In vitro and in vivo hepatic transport of the magnetic resonance imaging contrast agent B22956/1: role of MRP proteins

The molecular mechanisms of the hepatic transport of B22956/1, a new gadolinium complex from the class of intravascular contrast agents for MRI, which undergoes extensive biliary elimination, were studied. Biliary and urinary elimination of B22956/1 were measured in normal and in mutant MRP2 lacking rats (TR(-)); cellular trafficking of the compound was assessed in wild and MRP1 or MRP2 transfected MDCKII cells. Eight hours after IV injection of B22956/1, 90+/-8% of the dose was recovered in the bile of normal rats. By contrast, in TR(-) rats, the biliary excretion was significantly lower (14+/-3%) while 55+/-9% of the compound was found in urine. In vitro, the cellular accumulation of B22956/1 was significantly lower in both MRP1 and MRP2 transfected cells as compared to wild type MDCKII cells, and the cellular efflux was prevented by the MRP inhibitor MK571, indicating the involvement of both MRP2 and MRP1 in the transport of B22956/1. Due to the distinct cellular localization of the proteins, MRP2 accounts for the biliary and urinary excretion of the compound, while MRP1 prevents cellular accumulation of the MRI agent. B22956/1 may be useful in clinical conditions where a defective biliary transport is present.     

The differentiation of oval cells into hepatocytes during induced hepatic carcinogenesis in mice. An electron microscopic study

An electron microscopic study of murine oval cells, induced by a single injection of genotoxic agent dipin and by a partial hepatectomy, has shown that their ultrastructure and direction of differentiation depend on localization in the liver lobule. Oval cells around portal tracts go through three stages of development: low differentiated cells 4.40 +/- 0.51 mu in diameter with ovoid nuclei 3.43 +/- 0.44 mu, intermediate cells, and young hepatocytes. They form common ducts surrounded by a basal lamina, and produce bile canaliculi-like structures and intermediate junctions between them. Another part of the oval cell population is organized similar to the bile duct epithelium. It consists of cells 9.37 +/- 1.1 mu in diameter with nuclei 7.28 +/- 1.16 mu in diameter and form a system of branching and anastomosing ducts widespread along the parenchyma from the portal to the central veins. Our data indicate that the oval cells can differentiate into hepatocytes, and support a hypothesis according to which the cells of terminal bile ductules are liver epithelial stem cells which can differentiate into a hepatocyte or a bile duct cell lineage in periportal microenvironment.     

Specific targeting of nasopharyngeal carcinoma cell line CNE1 by C225-conjugated ultrasmall superparamagnetic iron oxide particles with magnetic resonance imaging

An accurate definition of clinical target volume (CTV) is essential for the application of radiotherapy in nasopharyngeal carcinoma (NPC) treatment. A novel epidermal growth factor receptor (EGFR)-targeting contrast agent (C225-USPIO) was designed by conjugating ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles with cetuximab (C225), to non-invasively define the CTV of tumor. The immunobinding activity of C225-USPIO to NPC cell line CNE1 was confirmed by flow cytometry and immunofluorescence. The time-dependent accumulation of C225-USPIO in CNE1 cells was evaluated using Prussian blue staining. Targeted internalization and subcellular localization of C225-USPIO was confirmed by transmission electron microscope. The results indicated that C225-USPIO specifically bound to EGFR on the surface of CNE1 cells and was taken up into the cell. The uptake of C225-USPIO by CNE1 cells increased significantly with time, when compared with human IgG-USPIO. In addition, 4.7 T magnetic resonance imaging (MRI) revealed that C225-USPIO had a capacity to accumulate in the CNE1 cells, with a resultant marked decrease in MRI T2-weighted signal intensity over time. These findings imply that C225-USPIO has the potential as an MRI contrast agent and can be employed to non-invasively detect early-stage NPC with EGFR overexpression. This provides sufficient theoretical basis for commencing in vivo experiments with the compound.     

New Strategies to Prolong the In Vivo Life Span of Iron-Based Contrast Agents for MRI

Superparamagnetic iron oxide (SPIO) and ultra small superparamagnetic iron oxide (USPIO) nanoparticles have been developed as magnetic resonance imaging (MRI) contrast agents. Iron oxide nanoparticles, that become superparamagnetic if the core particle diameter is ^~ 30nm or less, present R1 and R2 relaxivities which are much higher than those of conventional paramagnetic gadolinium chelates. Generally, these magnetic particles are coated with biocompatible polymers that prevent the agglomeration of the colloidal suspension and improve their blood distribution profile. In spite of their potential as MRI blood contrast agents, the biomedical application of iron oxide nanoparticles is still limited because of their intravascular half-life of only few hours; such nanoparticles are rapidly cleared from the bloodstream by macrophages of the reticulo-endothelial system (RES). To increase the life span of these MRI contrast agents in the bloodstream we proposed the encapsulation of SPIO nanoparticles in red blood cells (RBCs) through the transient opening of cell membrane pores. We have recently reported results obtained by applying our loading procedure to several SPIO nanoparticles with different chemical physical characteristics such as size and coating agent. In the current investigation we showed that the life span of iron-based contrast agents in the mice bloodstream was prolonged to 12 days after the intravenous injection of murine SPIO-loaded RBCs. Furthermore, we developed an animal model that implicates the pretreatment of animals with clodronate to induce a transient suppression of tissue macrophages, followed by the injection of human SPIO-loaded RBCs which make it possible to encapsulate nanoparticle concentrations (5.3-16.7mM Fe) higher than murine SPIO-loaded RBCs (1.4-3.55mM Fe). The data showed that, when human RBCs are used as more capable SPIO nanoparticle containers combined with a depletion of tissue macrophages, Fe concentration in animal blood is 2-3 times higher than iron concentration obtained by the use of murine SPIO-loaded RBCs.     

Structural and functional analyses of liver cysts from the BALB/c-cpk mouse model of polycystic kidney disease

Liver cysts arising from hepatic bile ducts are a common extra-renal pathology associated with both autosomal dominant and recessive polycystic kidney disease in humans. To elucidate the functional and structural changes inherent in cyst formation and growth, hepatic bile duct epithelia were isolated from the BALB/ c-cpk mouse model of polycystic kidney disease. Light and transmission electron microscopy revealed substantial fibrosis in the basal lamina surrounding hepatic bile duct cysts isolated from heterozygous (BALB/c-cpk/+) and homozygous (BALB/c-cpk/cpk) animals. Scanning electron microscopy and length analysis of normal, precystic and cystic bile ducts provided the unique observation that primary cilia in cholangiocytes isolated from bile ducts and cysts of animals expressing the mutated cpk gene had lengths outside the minimal and maximal ranges of those in cells lining bile ducts of wild-type animals. Based on the hypothesis that PKD is one of several diseases characterized as ciliopathies, this abnormal variability in the length of the primary cilia may have functional implications. Electrophysiological analyses of freshly isolated cysts indicate that the amiloride-sensitive epithelial Na(+) channel (ENaC) is inactive/absent and cAMP-mediated anion secretion is the electrogenic transport process contributing to cyst fluid accumulation. Anion secretion can be stimulated by the luminal stimulation of adenylyl cyclase.     

Assessing the Efficacy of Nano- and Micro-Sized Magnetic Particles as Contrast Agents for MRI Cell Tracking

Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano- and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the ‘3Rs’, the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging.     

In Vitro and In Vivo Magnetic Resonance Tracking of Sinerem-Labeled Human Umbilical Mesenchymal Stromal Cell-Derived Schwann Cells

Tracking of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles-labeled embryonic stem cells, neural stem cells, or adult mesenchymal stem cells in vitro and in vivo by using magnetic resonance (MR) imaging have been reported. However, whether the transdifferentiated cells can be effectively labeled by USPIO has not yet been investigated. The requirement for nerve donor material evokes additional morbidity and inability to generate a sufficiently large number of cells in a short time to hamper the clinic application of Schwann cells (SCs) transplantation. These limitations may be avoided if SCs can be generated from clinically accessible sources, such as bone marrow and umbilical cord. However, a reliable means of inducing the selective differentiation of human mesenchymal stromal cells isolated from the umbilical cord (HUMSCs) into SCs in vitro has not yet been established. In this study, we induce HUMSCs into Schwann-like cells in terms of morphology, phenotype, and function by an improved protocol basing on our previous studies. Furthermore, HUMSCs-derived SCs are labeled efficiently in vitro with ultrasmall superparamagnetic iron oxide contrast agent (USPIO) Sinerem and poly-l-lysine (PLL) without affecting morphology, cell cycle, proliferation, and differentiation ability of the labeled cells between the concentration of 200 to 800 μg/ml. Importantly, when grafted into the intact cerebral cortex and striatum, the survival and migration of these Sinerem-labeled cells were observed using MRI. Our study suggest the effective concentration field for Sinerem use in tracking transdifferentiated HUMSCs, and Sinerem labeling transdifferentiated HUMSCs is feasible, efficient, and safe for MRI tracing following grafting into nervous system.     

The passage of radioactive lanthanum from the biliary to the vascular system

Summary Radioactive lanthanum nitrate, an electron opaque tracer, was injected into the common bile duct of rats. Two minutes following the end of injection, samples of blood for radioactivity counts, and of liver and kidney for electron microscopic studies were taken. High levels of radioactivity found in the blood, and demonstration of lanthanum in the kidney by electron microscopy, indicated that this tracer entered the blood stream in vivo. No lanthanum was seen in the cytoplasm of liver cells, and there was no evidence of rupture in bile ducts, junctional ducts, or liver cells. Tight junctions connecting parenchymal cells and cholangiolar cells appeared well preserved. Lanthanum was seen in bile canaliculi, interspaces between liver cells, portions of the extracellular space of the liver, lumina of cholangioles and lumina of portal venules and sinusoids. It is postulated that lanthanum passed from the biliary tract, the site of injection, through the tight junction between liver cells and cholangiolar cells. It is suggested that such passage may represent a physiologic pathway, but the possibility of a chemical action of lanthanum on the tight junction can not be ruled out.This study was supported by the A. D. Williams Fund of Virginia Commonwealth University and the U. S. Veterans Administration. Radiation Counting Equipment was loaned by Dr. F. O'Foghludha, Department of Radiation Physics, Virginia Commonwealth University.     

Ultrastructure of the liver in channel catfish Ictalurus punctatus (Rafinesque)

Livers of 38 channel catfish were studied by light and electron microscopy and the morphology of hepatocytes, endothelial cells, Kupffer cells, fat-storing cells, macrophages, exocrine pancreatic cells, and epithelium of bile pre-ductules and ducts was described, Hepatocytes contained large peri-canalicular bodies which showed acid hydrolase activity. A comparison between catfish liver and that of other teleosts was made. The applicability of the findings to pathobiological studies of teleost liver is described.     

Myoid fibrils in epithelial cells: studies of intestine, biliary and pancreatic pathways, trachea, bronchi, and testis.

Cytoplasmic filaments have been studied extensively by electron microscopy, but the histochemical nature of such fibrils in non-keratinizing epithelia has not been systematically investigated. During studies of early arterial lesions we observed structures with the staining properties of myosins in epithelial cells of various organs. The configurational staining, polarization and fluorescence microscopic properties of these myoid structures were compared with those of myofibrils in smooth muscle and classical myoepithelial cells. The following structures showed the characteristics of myofibrils: the terminal web in columnar epithelial cells of intestine, trachea, bronchi, bile ducts, pancreatic ducts and ductus epididymidis, the pericanalicular layer of bile and pancreatic canaliculi, fibers in the caudal tube of spermatids and the flagella of spermatozoa. Cilia, e.g. of respiratory epithelium, tonofibrils in squamous epithelium and nerve axons did not react. These studies indicate significant histochemical differences between cytoplasmic filaments. Different types of intracellular fibrils can be found in the same cell, e.g. in respiratory epithelium.     

Close anatomical relationships between nerve fibers and MHC class II-expressing dendritic cells in the rat liver and extrahepatic bile duct

 The anatomical relationships between immunocytochemically identified nerve fibers and MHC class II-expressing antigen presenting dendritic cells were investigated in the rat hepatobiliary system using immunocytochemistry, confocal laser scanning, and electron microscopy. Close proximity of nerve fiber varicosities immunostained for PGP 9.5 and MHC class II-expressing dendritic cells was frequently observed in the wall of extrahepatic bile ducts, in Glisson’s area, around central and hepatic veins, and in the liver capsule. Contacts between nerve fibers staining for substance P, calcitonin gene-related peptide, calretinin, and vasoactive intestinal polypeptide and dendritic cells were more often observed around extrahepatic bile ducts than in Glisson’s area. Nerve fibers immunostaining for tyrosine hydroxylase and neuropeptide Y were numerous both in the wall of extrahepatic bile ducts and in Glisson’s area and frequently contacted dendritic cells there. At the ultrastructural level, close membrane contacts between bare axolemmal areas of unmyelinated nerve fibers and processes of MHC class II-expressing cells were observed. These results demonstrate close anatomical relationships of nerve fibers from various sources with antigen presenting dendritic cells in the visceral domain and suggest modulation of antigen presentation by the autonomic nervous system. Accepted: 29 September 1997     

Lysosomal degradation of bile deposits in cholestasis. An ultrastructural and cytochemical study.

Deposits of bile in the cytoplasm of hepatocytes from six patients with cholestasis were studied by electron microscopy and ultrastructural cytochemistry. The deposits were identified in vacuoles of varying size and complexity. The demonstration of acid phosphatase activity in these vacuoles suggests that lysosomes participate in the elimination of bile deposits retained in hepatocytes.     

Improved transmission electron microscopy technique for the study of cytologic material

A modification of the technique of Coleman et al for the preparation of single cells in cytologic specimens for electron microscopy (EM) is described. By employing materials in the initial cytologic processing that are useful for EM, such as a paraformaldehyde-glutaraldehyde fixative, lactated Ringer's solution as a rinsing medium, glycerol as a mounting medium and cacodylate buffer for removal of coverslips, the use of alcohol fixatives and standard mounting media could be avoided. This preserved the cytoplasmic detail, which is usually degenerated in cells removed from cytologic specimens and processed for EM.     

Diethylenetriaminepentaacetic acid-gadolinium (DTPA-Gd)-conjugated polysuccinimide derivatives as magnetic resonance imaging contrast agents

Biocompatible polysuccinimide (PSI) derivatives conjugated with diethylenetriaminepentaacetic acid gadolinium (DTPA-Gd) were prepared as magnetic resonance imaging (MRI) contrast agents. In this study, we synthesized PSI derivatives incorporating methoxy-poly(ethylene glycol) (mPEG) as hydrophilic ligand, hexadecylamine as hydrophobic ligand, and DTPA-Gd as contrast agent. PSI was synthesized by the polycondensation polymerization of aspartic acid. All the synthesized materials were characterized by proton nuclear magnetic resonance (1H NMR). Critical micellization concentrations were determined using fluorescent probes (pyrene). Micelle size and shape were measured by electro-photometer light scattering (ELS) and atomic force microscopy (AFM). The formed micelle size ranged from 100 to 300 nm. The T1-weighted MR images of the phantom prepared with PSI-mPEG-C16-(DTPA-Gd) were obtained in a 3.0 T clinical MR imager, and the conjugates showed a great potential as MRI contrast agents.     

Involvement of a plasmid in Escherichia coli envelope alterations.

A plasmid-containing wild-type Escherichia coli strain was treated with two plasmid-curing agents, sodium dodecyl sulfate and ethidium bromide. Plasmid elimination was accompanied by drastic changes in the morphology of the colonies. Analysis of the cured strain by scanning and transmission electron microscopy showed important alterations in size and morphology of the cells. Metabolic differences were also found between the wild-type and cured cells.