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Human Gene Therapy
Hum Gene Ther. 2009 July; 20(7): 751–758.
Published online 2009 April 16. doi:  10.1089/hum.2008.145
PMCID: PMC2829288

This article has been retractedRetraction in: Hum Gene Ther. 2010 March; 21(3): 363    See also: PMC Retraction Policy

The Oncopathic Potency of Clostridium perfringens Is Independent of Its α-Toxin Gene


Hypoxia in solid tumors is a major obstacle in conventional treatment because of inefficient delivery of therapeutic agents to the lesions, but offers the potential for anaerobic bacterial colonization that can lead to tumor destruction. We have previously reported a recombinant Clostridium perfringens (Cp) strain constructed by deletion of the superoxide dismutase (sod) gene and insertion of the Panton–Valentine leukocidin (PVL) gene, Cp/sod/PVL, which showed elevated oxygen sensitivity, tumor selectivity, and oncopathic potency in an orthotopic model of pancreatic cancer in immune-competent and syngeneic mice, and that led to substantial prolongation of animal survival. A major limitation to Cp/sod/PVL in clinical applications is that it expresses phospholipase C (plc), the α-toxin and the major virulence determinant in Cp that is causative in the development of gas gangrene. In this study, the plc gene in Cp/sod/PVL was knocked out to create Cp/plc/sod/PVL, which was shown to be incapable of inducing gas gangrene in mice. Intravenous injection of Cp/plc/sod/PVL spores led to a significant survival advantage in tumor-bearing mice with the same efficacy as Cp/sod/PVL, indicating that the oncopathic potency of Cp is independent of a functional plc gene. The treatment also did not lead to an attenuated immune response to a subsequent pathogen challenge, indicating that a systemic immune-suppressive effect in the host is absent. Consequently, Cp/plc/sod/PVL is a novel oncopathic bacterial agent for the effective treatment of pancreatic cancer and other poorly vascularized tumors, with a substantially enhanced safety profile, which is essential for the development of translational studies in the future.


Pancreatic carcinoma is currently the fourth leading cause of cancer-related death in the United States (Greenlee et al., 2001; Jemal et al., 2004; Lebedeva et al., 2006). Conventional treatment options for advanced or metastatic pancreatic cancer are limited (Casper, 1993; Storniolo et al., 1999; Stephens, 1998). Median survival time is 6 to 10 months for patients with locally advanced disease, and 3 to 6 months for those with metastatic disease (Evans et al., 1997; Sener et al., 1999). Pancreatic cancer contains large, poorly vascularized areas that limit the efficacy of radiation and chemotherapeutic drugs (Kondoh et al., 2003). Hypoxia is a characteristic of tumors that is potentially exploitable with bioreductive drugs or gene therapy (Theys et al., 2003; Pawelek et al., 2003). In the 1960s, Clostridium was shown to be effective in causing tumor regression in rodent models, but a subsequent clinical trial failed to demonstrate any benefit that would outweigh the toxicities (Engelbart and Gericke, 1964). More recently, Vogelstein, Dang, and colleagues investigated the tumor colonization properties of various anaerobic bacteria, including eight Clostridium strains (Dang et al., 2001).

Clostridium perfringens (Cp) is an obligate anaerobic, spore-forming, gram-positive bacterium, which requires anaerobic conditions to grow. The wild-type Cp strain retains residual oxygen tolerance and can cause toxicities in animals. The major oxygen tolerance gene in Cp is one that encodes superoxide dismutase (sod) (Lehmann et al., 1996; Geissmann et al., 1999; Briolat and Reysset, 2002), and we have reported that the oxygen sensitivity of Cp in vitro and its safety profiles in vivo could be substantially elevated by a deletion of this gene (Li et al., 2008). However, Cp and Cp/sod were only minimally effective as oncopathic agents, as microbial replication in immune-competent hosts is inhibited by inflammatory cellular responses (Cote et al., 2004). We hypothesized that the oncopathic potency of anaerobic bacteria could be substantively enhanced by vector-mediated expression of inflammation-evasive genes from heterologous bacteria, such as the Panton–Valentine leukocidin (PVL) gene from Staphylococcus aureus (Kato, 1981; Okumoto, 1985; Genestier et al., 2005). Indeed, the intratumoral titers were logarithmically amplified in Cp/sod/PVL, which led to substantively enhanced oncopathic potency and survival prolongation in tumor-bearing mice without additional systemic and organ toxicities (Li et al., 2008).

However, Cp/sod/PVL expresses the phospholipase C (plc) gene, which is the major virulence determinant in Cp and is known to be causative in the development of gas gangrene (MacLennan, 1962; Smith, 1975). plc cleaves lecithin, the major phospholipid in eukaryotic cell membranes, into phosphorylcholine and diacylglycerol (Mollby and Wadstrom, 1973). The plc deletion mutants of Cp showed a complete loss of virulence and gas gangrene formation in mice, which could be reconstituted by reinsertion of the plc gene (Awad et al., 1995; Stevens, 1997). In the present study we constructed a novel agent, Cp/plc/sod/PVL, by deleting the plc gene from Cp/sod/PVL and tested its safety and effectiveness as an oncopathic agent to treat pancreatic cancer in mice.

Materials and Methods

Bacterial growth and spore purification of Cp

All bacteria used in this study were derived from wild-type Clostridium perfringens, type A, subgroup 1 (strain 13124 purchased from the American Type Culture Collection [ATCC], Manassas, VA). The growth, sporulation, and spore isolation have been described (Li et al., 2008). Briefly, Cp vegetative bacteria were transferred to sporulation medium and grown for 5 days to obtain the maximal yield of spores. The sporulating bacterial culture was incubated at 90°C for 10 min, suspended in 70% ethanol for 20 min, and centrifuged at 5000 rpm. The bacterial spores were extracted with 58% Renografin solution (Renocal-76 diluted in water) (Renocal-76; Bracco Diagnostics, Princeton, NJ) by centrifugation at 18,000 rpm, and then incubated at 37°C for 45 min with saline–EDTA containing lysozyme (100 μg/ml), washed five times with phosphate-buffered saline (PBS), and stored at −20°C at 1 × 108 spores/ml.

Construction of the plc knockout strains of Cp

plc knockout strains (Cp/plc, Cp/plc/sod, and Cp/plc/sod/VPL) were constructed by replacing it with a 46-bp random and noncoding sequence by homologous recombination in the parental Cp, Cp/sod, and Cp/sod/VPL strains (Li et al., 2008). To construct a homologous recombination fragment, the random sequence and flanking regions (0.7 kb each) of the plc gene were amplified by polymerase chain reaction (PCR). The three DNA fragments were used to generate, by overlapping PCR, one homologous recombination fragment that was then inserted into pUC19 and cloned in Escherichia coli. The fragment was released from the plasmid and transfected into the parental Cp strains by electroporation. The transformants were screened by colony hybridization, using the random sequence probe, and confirmed by DNA sequencing analysis (Ezaki et al., 2000).

Experimental model of gas gangrene in mice

Female C57BL/6 mice (10 per group) were injected intramuscularly via the right upper thigh with either PBS or 1.0 × 109 colony-forming units (CFU) of vegetative Cp in a volume of 100 μl of PBS, using a 26-gauge needle. The lethal gas gangrene infection induced by wild-type Cp strains produced swollen hemorrhagic thighs with crepitus, extensive tissue necrosis, and mortality within 24 hr. All treated animals were observed carefully twice daily for 14 days for signs and symptoms of gas gangrene, and mortalities were also noted.

Orthotopic pancreatic cancer model in immune-competent and syngeneic mice

The PANC02 murine pancreatic cancer cell line was established from a murine pancreatic ductal adenocarcinoma (Block et al., 1997) and employed to evaluate the oncopathic potency of Cp in a previous study (Li et al., 2008). Briefly, 1 × 105 PANC02 cells were suspended in 10 μl of Earle's balanced salt solution (EBSS) buffer and directly injected into the pancreas of 6- to 8-week-old immune-competent and syngeneic female C57BL/6 mice under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the Mount Sinai School of Medicine (New York, NY). Palpable tumors of 5 × 5 mm2 in size or greater developed in >90% of the animals after about 18 days. All tumor-bearing mice were determined to have palpable tumors before use in the experiments.

Evaluation of intratumoral bacterial proliferation, content of inflammatory cells, tumor response, and antitumor efficacy

The maximal tolerated doses (MTDs) for various Cp strains in tumor-bearing mice were determined by intravenous injection of escalating doses of the respective spores, ranging from 104 to 108 at half-log increments (n = 10). The end point was survival as defined by the time of death or by sacrifice when the animals appeared distressed as defined by substantial weight loss, lethargy, or ruffled fur. Statistical significance was analyzed by one-tailed Fisher exact probability test. For kinetics analysis, the treated animals were killed by compressed CO2 gas at various time points including day 3 after spore injection. Tumors, major organs (pancreas, liver, spleen, heart, lung, kidney, and bone marrow), and blood were collected from the killed animals. Bacterial titers were quantified by agar plating. To assess bacterial biodistribution and tumor response, tumor and organ samples were fixed overnight in 4% paraformaldehyde and paraffin-embedded tissue sections were subjected to Gram and hematoxylin–eosin (H&E) staining. To determine the number of inflammatory cells in tumors, tumor sections were subjected to immunohistochemical staining for neutrophils, using rat anti-mouse Gr-1 monoclonal antibodies (BD Biosciences, San Jose, CA) or for macrophages, using rat anti-mouse F4/80 monoclonal antibodies (AbD Serotec, Raleigh, NC) in addition to H&E and Gram staining, followed by morphometric and statistical analysis. For Gram, H&E, and each of the immunohistochemical stainings, one central section was analyzed for each tumor or tissue sample from each mouse. For the immunohistochemical stains, positive staining in tissue sections was quantified with Image-Pro software (MediaCybernetics, Silver Spring, MD), and a positive staining cell index was calculated as the ratio of tumor-infiltrating cells per unit tumor area (10,000 pixels as one unit tumor area). The percentage of tumor necrosis was also quantified with Image-Pro software. The results were statistically analyzed by two-sided unpaired Student t test. The antitumor efficacy of various Cp strains in tumor-bearing mice was assessed by survival after intravenous administration of bacterial spores at their respective maximal tolerated doses. Survival data were analyzed by the Kaplan–Meier method and comparisons of survival curves between different groups were made by the log-rank test.

Intravenous pathogen challenge in tumor-bearing mice treated with Cp spores

Stationary bacterial cells were obtained from Pseudomonas aeruginosa (ATCC 33351) grown in trypticase soy broth containing 5% defibrinated sheep's blood (Brown et al., 1981) and from Listeria monocytogenes (ATCC 43251) grown in trypticase soy broth (Seki et al., 2002). Pseudomonas aeruginosa (1 × 105) or L. monocytogenes (5 × 105) bacterial cells were washed and diluted in 0.2 ml of PBS and then intravenously injected into tumor-bearing mice that had been treated with PBS control, Cp/plc/sod/PVL spores, or Cp/plc/sod spores for 3 days (five mice per group) (Brown et al., 1981; Wagner et al., 1994; Seki et al., 2002). To determine bacterial titers, a 0.1-ml blood sample was taken from each animal on day 0 (5 min after injection of pathogens) and 6 hr, 1 day, 7 days, and 14 days after pathogen infection, and the major organs including pancreas, spleen, liver, heart, lung, and kidney were harvested from killed animals on day 14. The numbers of colony-forming units of P. aeruginosa or L. monocytogenes in animal blood and organs were counted by plating on trypticase soy agar.


The plc gene knockout Cp strains Cp/plc, Cp/plc/sod, and Cp/plc/sod/PVL were constructed by homologous recombination and confirmed by DNA sequencing. The bacterial proliferation, sporulation, and germination profiles of these strains were determined in vitro. There was no statistically significant difference in bacterial growth rates (Fig. 1A), spore yields (Fig. 1B), and germination efficiencies (Fig. 1C) between the three plc strains and their respective parental strains, Cp, Cp/sod, and Cp/sod/PVL. These results suggest that knockout of the plc gene in Cp strains has no negative impact on bacterial characteristics.

FIG. 1.
In vitro characterization of the plc+ and plc Cp strains. Bacterial proliferation, sporulation, and germination efficiencies in plc+ strains (Cp, Cp/sod, and Cp/sod/PVL) and plc strains (Cp/plc, Cp/plc ...

To examine the gas gangrene-causing capabilities of the various Cp strains, PBS control or 1 × 109 vegetative bacterial cells of plc+ strains and plc strains in 0.1 ml were injected intramuscularly into the hind legs of normal mice. Mice developed gas gangrene and exhibited marked swelling in about 6–8 hr, followed by blackening of the foot, sloughing of fur, crepitus beneath the skin, and severe muscle destruction with extension of tissue necrosis onto the abdominal wall and thorax, and mortality in 24 hr. These pathologic findings are in agreement with previous studies using this animal model (Stevens et al., 1987) and with clinical reports describing gas gangrene in humans (McNee and Dunn, 1917). Of mice injected with Cp, Cp/sod, and Cp/sod/PVL, 70, 20, and 20% developed gas gangrene, respectively, which was not detected in mice injected with Cp/plc, Cp/plc/sod, and Cp/plc/sod/PVL (Fig. 2). The experiment was repeated once, with 70, 30, and 20% of mice injected with plc+ strains developing gas gangrene, which again was not detected in any of the plc strains. The results confirm that plc is a causative gene in Cp for gas gangrene development.

FIG. 2.
Induction of gas gangrene in normal mice after intramuscular injection of plc+ and plc Cp strains. Normal C57BL/6 mice were intramuscularly injected with PBS or 1 × 109 vegetative bacterial cells of Cp, Cp/sod ...

The maximal tolerated dose (MTD) for the newly constructed recombinant strain Cp/plc/sod/PVL in PANC02 tumor-bearing C57/BL6 mice was determined to be 1 × 107 spores (results not shown), which was the same as that of its parental strain, Cp/sod/PVL (Li et al., 2008). To evaluate its oncopathic potency and effectiveness in cancer treatment, 1 × 107 spores of Cp/sod/PVL and Cp/plc/sod/PVL were injected into PANC02 tumor-bearing C57BL/6 mice, which were killed on day 3 after spore administration. Sections of tumor lesions in mice were analyzed by Gram staining and bacterial titers in tumor extracts from the killed animals were determined by quantitative bacterial culture. There was no statistically significant difference between mice treated with Cp/sod/PVL and Cp/plc/sod/PVL (p > 0.7) (Fig. 3A), suggesting that the plc strain is equally efficient in intratumoral replication as the plc+ strain. The major organs including brain, heart, lung, spleen, liver, kidney, pancreas, and bone marrow were also harvested for determination of bacterial titer and there was no Cp found in any of those organs. Tumor sections were then analyzed by histological and immunohistochemical staining followed by morphometric and statistical analyses. As shown in Fig. 3B and C, the content of neutrophils and macrophages in Cp/sod/PVL and Cp/plc/sod/PVL-treated mice did not show a statistically significant difference (p > 0.5 and 0.6, respectively). Importantly, whereas there were significant increases in the extent of tumor necrosis in the lesions of both Cp/sod/PVL and Cp/plc/sod/PVL spore-treated mice versus PBS control (p < 0.01 and 0.01, respectively), there was no significant difference between the Cp/sod/PVL- and Cp/plc/sod/PVL-treated mice (p > 0.8) (Fig. 3D). Those experiments were repeated once, and the same results and significant differences were observed. These results suggested that there was no negative impact in tumor response after knocking out the plc gene in Cp/sod/PVL.

FIG. 3.
Intratumoral bacterial titers, inflammatory cell contents, and quantification of tumor necrosis after Cp/sod/PVL and Cp/plc/sod/PVL treatments. Tumor samples in mice treated with PBS, Cp/sod/PVL, or Cp/plc ...

To evaluate antitumor efficacy, a long-term survival study was performed in PANC02 tumor-bearing mice that were injected with 1 × 107 spores of Cp/sod/PVL or Cp/plc/sod/PVL (n = 16, respectively), or PBS (n = 10). The survival curves were plotted according to the Kaplan–Meier method as shown in Fig. 4. A statistically significant survival prolongation over the PBS control group was observed in animals treated with either Cp/sod/PVL or Cp/plc/sod/PVL. The median survival time was extended to 64 days with Cp/sod/PVL treatment and to 71 days with Cp/plc/sod/PVL treatment, versus 22 days for the PBS control group (p < 0.001 for both). All PBS-treated mice died of tumor growth within 40 days, whereas 8 of 16 (50%) of the Cp/sod/PVL-treated animals and 7 of 16 (44%) of the Cp/plc/sod/PVL-treated animals remained alive at 120 days, when all animals were killed and found to be tumor free (p > 0.8). The survival study was repeated once, and demonstrated similar patterns and statistics (p > 0.66, Cp/plc/sod/PVL vs. Cp/sod/PVL). These results suggest that the plc gene knockout strain Cp/plc/sod/PVL is equally effective as the parental Cp/sod/PVL strain in intratumoral bacterial replication, induction of tumor necrosis, and prolongation of animal survival.

FIG. 4.
Kaplan–Meier survival curves for tumor-bearing mice after Cp/sod/PVL and Cp/plc/sod/PVL treatment. PANC02 tumor-bearing C57/BL6 mice were injected intravenously with 1 × 107 spores of Cp/sod ...

To evaluate whether host immunity is compromised, 5 × 105 L. monocytogenes or 1 × 105 P. aeruginosa were intravenously injected into tumor-bearing mice that had been treated with PBS or with Cp/plc/sod or Cp/plc/sod/PVL spores for 3 days. Titers of the injected pathogens in blood were determined by quantitative bacterial culture on day 0 and 6 hr, 1 day, 7 days, and 14 days after pathogen challenge. As shown in Fig. 5, the maximal bacterial titers for both L. monocytogenes and P. aeruginosa injection were found on day 0, which was collected at 5 min after pathogen infection. The microorganisms disappeared completely after 7 days in both Cp/plc/sod/PVL- and Cp/plc/sod-treated mice, as well as in PBS-treated controls, whereas a lower number of bacterial cells was found 6 hr and 1 day after pathogen injection. There was no statistically significant difference in pathogen titers at any time point among Cp/plc/sod/PVL, Cp/plc/sod, and PBS-treated groups, in both the L. monocytogenes (Fig. 5A) and the P. aeruginosa (Fig. 5B) challenge. The major organs including pancreas, spleen, liver, heart, lung, and kidney were found to be histologically normal and pathogen negative (data not shown).

FIG. 5.
Serum bacterial titers of Listeria monocytogenes (A) and Pseudomonas aeruginosa (B) in tumor-bearing mice treated with Cp spores and pathogen challenge. Tumor-bearing mice were treated with Cp/plc/sod/PVL (circles, n = 5) ...


Hypoxic cores in poorly vascularized tumors are a major hindrance in cancer therapy, as they inhibit the effective delivery of therapeutic medications. The presence of hypoxia in solid tumors however, also offers the potential for anaerobic bacterial colonization and tumor destruction (Critchley et al., 2004). A number of oncopathic bacteria have been described including gram-positive and obligate anaerobes such as clostridia and bifidobacteria, which were shown to selectively germinate and grow in the hypoxic regions of solid tumors after intravenous injection (Yazawa et al., 2001). Vogelstein and coworkers constructed a strain of Clostridium novyi devoid of its lethal toxin gene (C. novyi-NT), which was shown to germinate and grow within the avascular regions of tumors and destroy surrounding tumor cells in mice (Dang et al., 2001). More recently, the same group showed that treatment of mice bearing large tumors with C. novyi-NT plus a single dose of liposome-encapsidated doxorubicin led to eradication of the tumors (Cheong et al., 2006). Clostridia have also been genetically engineered to selectively deliver prodrug-activating enzymes such as E. coli cytosine deaminase (Theys et al., 2001) and nitroreductase (Lemmon et al., 1997) that enhance treatment efficacy via a bystander effect.

We have reported the development of a recombinant strain of Clostridium perfringens, Cp/sod/PVL, that exhibited reduced cytotoxicity and substantially enhanced oncopathic potency in immune-competent mice bearing orthotopic and syngeneic pancreatic cancer (Li et al., 2008). The major gene associated with oxygen tolerance in Cp is the superoxide dismutase gene and its knockout strain (Cp/sod) exhibited reduced oxygen tolerance in vitro and limited growth in normal tissues in vivo, leading to a 1-log elevation in its MTD in animals. In addition, we have constructed Cp/sod/PVL, a recombinant strain that also expresses the Panton–Valentine leukocidin (PVL) gene, which could directly damage membranes of phagocytes including monocytes, macrophages, and neutrophils. Cp/sod/PVL was shown to lead to a significant reduction in intratumoral content of inflammatory cells, logarithmically elevated intratumoral bacteria titers, enhanced tumor necrosis, and substantially prolonged animal survival, with about half the animals remaining alive after 120 days without apparent systemic and organ toxicities (Li et al., 2008). It is the first time that a genetically modified clostridia bacterial strain provided significant survival advantage by treatment with itself, without being combined with an anticancer chemotherapeutic drug or prodrug substrate.

Although treatment with Cp/sod/PVL spores is effective, the presence of the plc gene could constitute a risk. Wild-type Clostridium perfringens is the primary causative agent of gas gangrene or clostridial myonecrosis (Stevens, 1997). It invades traumatized and/or ischemic tissue and although the infection is relatively localized, the bacteria produce extracellular toxins that are responsible for the extensive tissue destruction and necrosis seen in classical cases of gas gangrene. Bacterial growth is accompanied by the production of extracellular toxins and leads to gas production, extensive necrosis, and tissue damage. Left untreated, the disease progresses rapidly and leads to systemic toxemia, profound shock, and death (Darke et al., 1977; Stevens, 1997). The lethal dermonecrotic α-toxin is the most toxic extracellular enzyme produced by C. perfringens type A strains and is essential for its virulence (Awad et al., 1995; Ellemor et al., 1999). α-Toxin is a phospholipase C that hydrolyzes both phosphatidylcholine and sphingomyelin (Rood, 1998; Titball and Rood, 2000), both of which are important constituents of eukaryotic cell membranes. Genetic studies have shown that a C. perfringens mutant in which the plc structural gene had been inactivated by homologous recombination was unable to cause clostridial gas gangrene in mice, whereas the virulence was restored when the plc mutation was complemented by a plasmid carrying the wild-type plc gene (Awad et al., 1995), indicating that phospholipase C is the causative agent for the disease process.

In this study, we genetically knocked out the plc gene in Cp/sod/PVL to generate a recombinant strain, Cp/plc/sod/PVL, and showed that its ability to induce gas gangrene induction was eliminated. Importantly, deletion of the plc gene in Cp strains showed no negative impacts in bacterial growth characteristics and oncopathic potency. Cp/plc/sod/PVL spore treatment led to equivalent antitumor efficacy compared with Cp/sod/PVL in a long-term survival study, without any statistically significant difference between the two strains. Taking together, these results indicate that the plc gene in Cp is not essential for its oncopathic activity, and that knocking out this gene does not reduce its antitumor efficacy.

Another issue in treatment safety was that the immune-suppressing PVL gene knocked into a mutant strain might, despite increasing bacterial survival and proliferation in tumor lesions, compromise the host immune defense system, which is important to defend against other infectious bacteria. It has been demonstrated that intravascular injections of defined doses of several pathogenic bacterial strains including Listeria, Staphylococcus, Pseudomonas, and pneumococci would be rapidly and efficiently cleared in the circulation, with the culturable bacteria disappearing swiftly from the blood stream (Rogers, 1960; Brown et al., 1981; Wagner et al., 1994; Seki et al., 2002). In this study, we challenged mice treated with Cp/plc/sod and Cp/plc/sod/PVL with defined safe doses of two pathogenic bacterial strains, gram-positive Listeria monocytogenes and gram-negative Pseudomonas aeruginosa, by intravenous injection, and demonstrated efficient clearance for both. These results suggest that treatment with inflammation-evasive Cp/plc/sod/PVL does not compromise or attenuate the host immune response to infectious pathogens. Thus, Cp/plc/sod/PVL, with its tumor selectivity and oncopathic potency the same as that of the parental Cp/sod/PVL strain but with a substantially enhanced safety profile, may be developed into an effective and safe oncopathic agent for the future treatment of pancreatic cancer and other poorly vascularized tumors in patients.


The authors thank Drs. Tian-gui Huang, Lan Wu, and Marcia Meseck for helpful discussions, and Ms. Yafang Wang and Mr. Boxun Xie for technical assistance. This research was supported by NIH grant CA-120017.

Author Disclosure Statement

As coinventors in a patent application submitted in March 2008, Zhiyu Li, James Wetmur, and Savio Woo have financial conflicts of interest in this manuscript, which is managed in accordance with institutional policies.


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