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Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription.Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined.Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity.Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Clin Cancer Res; 18(3); 796–807. ©2011 AACR.

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CCR Translation for This Article from Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy

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PurposeTH-MYCN transgenic mice represent a valuable preclinical model of neuroblastoma. Current methods to study tumor progression in these mice are inaccurate or invasive, limiting the potential of this murine model. The aim of our study was to assess the potential of small animal positron emission tomography (SA-PET) to study neuroblastoma progression in TH-MYCN mice.ProcedureSerial SA-PET scans using the tracer 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) have been performed in TH-MYCN mice. Image analysis of tumor progression has been compared with ex vivo evaluation of tumor volumes and histological features.Results[(18)F]FDG-SA-PET allowed to detect early staged tumors in almost 100% of TH-MYCN mice positive for disease. Image analysis of tumor evolution reflected the modifications of the tumor volume, histological features, and malignancy during disease progression. Image analysis of TH-MYCN mice undergoing chemotherapy treatment against neuroblastoma provided information on drug-induced alterations in tumor metabolic activity.ConclusionsThese data show for the first time that [(18)F]FDG-SA-PET is a useful tool to study neuroblastoma presence and progression in TH-MYCN transgenic mice.

Stimulation of isolated rat Kupffer cells by viable Leptospira interrogans, Treponema pallidum and Borrelia garinii elicited cellular responses resulting in the release of different tumor necrosis factor alpha (TNF-alpha) levels, depending on the spirochetes. L. interrogans induced TNF-alpha levels higher than those achieved with B. garinii and T. pallidum (in this order), but lower than the levels achieved with lipopolysaccharide (LPS). In contrast to L. interrogans, pretreatment of borreliae and treponemes with polymyxin B did not substantially diminish the ability of B. garinii and T. pallidum to stimulate Kupffer cells. Purified T. pallidum lipoproteins TpN47, TmpA, TpN15-TpN17, and B. garinii OspA induced TNF-alpha responses comparable to that achieved by LPS. This response was almost insensitive to the action of polymyxin B.

Chlamydia trachomatis pgp3 DNA immunized (no. 300) and non-immunized (no. 300) C3H/HeN mice were infected by vaginal inoculation with infectious C. trachomatis serotype D elementary bodies (EBs) and the spread of infection to the salpinges was assessed by cell culture isolation from tissue homogenates 7, 14, 21, 28, 35 and 42 days post-infection (p.i.). Overall, the pgp3-DNA immunization prevented salpinx infection in 94 (56%) mice, if compared with the 168 positive animals found among the non-immunized animals (P < 0.001). A group of negative control animals (i.e. mice immunized with plasmid DNA containing an irrelevant insert) was not protected, whereas all the mice of a positive immune control group (mice that had resolved a primary genital C. trachomatis infection) were resistant to re-infection. Pgp3 DNA immunization induced both humoral and mucosal anti-pgp3 antibodies.

Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Translational Relevance Rhabdomyosarcomas are the most common pediatric soft tissue sarcoma. They are a leading cause of cancer death in children with few therapeutic options in highrisk categories. N-Myc is a transcription factor belonging to the MYC protein family that has a highly restricted expression pattern in non-fetal tissues. N-Myc protein expression in patient samples of rhabdomyosarcoma and an apoptotic response to sustained reduction of expression in rhabdomyosarcoma cell lines imply that N-Myc represents a tumor-specific therapeutic target in these tumors. Systemic treatment of mice with rhabdomyosarcoma cell line xenografts using an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide that inhibits transcription of MYCN shows promise by eliminating tumors. This direct antigene approach may have therapeutic potential in the treatment of other cancers and diseases.

Purpose The administration of new anticancer drugs in animal models is the first step from in vitro to in vivo preclinical protocols. At this stage it is crucial to ensure that cells are in the logarithmic phase of growth and to avoid vascular impairment, which can cause inhomogeneous distribution of the drug within the tumour and thus lead to bias in the final analysis of efficacy. In subcutaneous xenograft murine models, positivity for cancer is visually recognisable 2–3 weeks after inoculation, when a certain amount of necrosis is usually already present. The aim of this study was to evaluate the accuracy of FDG small animal PET for the early detection of malignant masses in a xenograft murine model of human rhabdomyosarcoma. A second goal was to analyse the metabolic behaviour of this xenograft tumour over time. Methods We studied 23 nude mice, in which 7×106 rhabdomyosarcoma cells (RH-30 cell line) were injected in the dorsal subcutaneous tissues. Each animal underwent four FDG PET scans (GE, eXplore Vista DR) under gas anaesthesia. The animals were studied 2, 5, 14 and 20 days after inoculation. We administered 20 MBq of FDG via the tail vein. Uptake time was 60 min, and acquisition time, 20 min. Images were reconstructed with OSEM 2D iterative reconstruction and the target to background ratio (TBR) was calculated for each tumour. Normal subcutaneous tissue had a TBR of 0.3. Necrosis was diagnosed when one or more cold areas were present within the mass. All the animals were sacrificed and histology was available to verify PET results. PET results were concordant with the findings of necropsy and histology in all cases. Results The incidence of the tumour was 69.6% (16/23 animals); seven animals did not develop a malignant mass. Ten of the 23 animals had a positive PET scan 2 days after inoculation. Nine of these ten animals developed a tumour; the remaining animal became negative, at the third scan. The positive predictive value of the early PET scan was 90% (9/10 animals) while the negative predictive value was 46% (6/13 animals). In the whole group of animals, mean TBR increased scan by scan. There was a statistically significant difference in TBR between 2 and 20 days after inoculation. Necrosis was present at the second scan in two animals, at the third scan in six animals and at the fourth scan in 11 animals. Conclusion The high positive predictive value of FDG PET 2 days after inoculation means that an animal with a first positive scan has a very high likelihood of developing a mass and can be treated at an early stage with an experimental drug. Animals negative at this point in time will never develop a mass or will eventually do so at a late phase. As 2 of the 16 (12.5%) positive animals had necrosis at the second scan, indicating a vascular mismatch, it may be argued that animals should be treated 2 days after inoculation to guarantee homogeneous vascularisation (thereby ensuring a good drug supply within the tumour) in all subjects.

The aim of this study was to develop a new experimental model of Chlamydophila pneumoniae infection in the hamster. Intraperitoneal injection of C. pneumoniae purified elementary bodies (EBs) in the hamsters caused a systemic infection, since it was possible to isolate viable chlamydiae from several organs up to 14 days after infection. In particular, spleen infection was detectable up to 7 days post infection in 100% of animals. In contrast, cultures of the organs obtained from intranasally infected animals were far less frequently positive. Systemic infection probably occurred via macrophages, as demonstrated by the presence of intracellular chlamydial inclusions in peritoneal macrophages of peritoneally inoculated animals four days after infection. Furthermore, by infecting LLC-MK2 cells with supernatant preparations obtained from these macrophages, it was possible to observe the development of chlamydial intra-cytoplasmic inclusions after 96 h. Immunization of 18 hamsters with heat-inactivated purified EBs completely protected 16 animals and substantially reduced infection levels in the remaining two. Sera obtained from immunized hamsters prior to challenge reacted mainly against two C. pneumoniae proteins of about 60 kDa, when tested by immunoblot.

The in vitro activity of six cathelicidin peptides against 25 strains of Chlamydia was investigated. SMAP-29 proved to be the most active peptide, reducing the inclusion numbers of all 10 strains of Chlamydia trachomatis tested by ≥50% at 10 μg/ml. This peptide was also active against C. pneumoniae and C. felis.

We report that intraperitoneal injection of Chlamydia pneumoniae purified elementary bodies (EBs) in the hamster causes a systemic infection allowing the isolation of viable chlamydiae from several organs for several days post-infection (p.i.). In particular, spleen infection occurred up to Day 7 p.i. in 100% of animals. Systemic infection probably occurs via macrophages as intraperitoneally injected chlamydiae which are taken up by the hamster macrophages remain viable and can infect in vitro cell cultures. Immunization of 18 hamsters with heat-inactivated purified EBs, completely protected the spleens of 16 animals and substantially reduced infection levels in the remaining two. This model, therefore, provides a robust screening tool for the assessment of the protective activity of potential vaccine candidates. In a pilot study on five recombinant antigens recently described as EB surface proteins, three gave results undistinguishable from non-immunized, or mock-immunized controls; however two antigens, derived, respectively, from the product of the lcrE gene (a component of the putative TTSS of C. pneumoniae) and the product of Cpn0498 open reading frame, proved to be capable of inducing protective immune responses.