Skip Nav Destination Imaging, Diagnosis, Prognosis| April 15 2008 Peter Brader; 1Department of Radiology, Search for other works by this author on: Jochen Stritzker; 6Genelux Corporation, San Diego Science Center, San Diego, California; and 7Institute for Biochemistry, Biocenter; Institute for Molecular Infectious Biology; and Search for other works by this author on: Pat Zanzonico; 2Department of Medical Physics, Search for other works by this author on: Shangde Cai; 3Cyclotron and Radiochemistry Core Facility, Search for other works by this author on: Eva M. Burnazi; 3Cyclotron and Radiochemistry Core Facility, Search for other works by this author on: Hedvig Hricak; 1Department of Radiology, Search for other works by this author on: Aladar A. Szalay; 6Genelux Corporation, San Diego Science Center, San Diego, California; and 7Institute for Biochemistry, Biocenter; Institute for Molecular Infectious Biology; and 8Virchow Center for Biomedical Research, School of Medicine, University of Wuerzburg, Wuerzburg, Germany Search for other works by this author on: Yuman Fong; 5Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York; Search for other works by this author on: Ronald Blasberg 1Department of Radiology, 4Nuclear Pharmacy, and Search for other works by this author on: Requests for reprints: Ronald G. Blasberg, Departments of Neurology and Radiology, MH (Box 52), Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 646-888-2211; Fax: 646-422-0408; E-mail: blasberg@ Received: September 14 2007 Revision Received: December 03 2007 Accepted: December 04 2007 Online Issn: 1557-3265 Print Issn: 1078-0432 American Association for Cancer Research2008 Clin Cancer Res (2008) 14 (8): 2295–2302. Article history Received: September 14 2007 Revision Received: December 03 2007 Accepted: December 04 2007 Split-Screen Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review PDF Tools Icon Tools Search Site Article Versions Icon Versions Version of Record April 15 2008 Proof March 27 2008 Citation Peter Brader, Jochen Stritzker, Christopher C. Riedl, Pat Zanzonico, Shangde Cai, Eva M. Burnazi, Ghani, Hedvig Hricak, Aladar A. Szalay, Yuman Fong, Ronald Blasberg; Escherichia coli Nissle 1917 Facilitates Tumor Detection by Positron Emission Tomography and Optical Imaging. Clin Cancer Res 15 April 2008; 14 (8): 2295–2302. Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex Abstract Purpose: Bacteria-based tumor-targeted therapy is a modality of growing interest in anticancer strategies. Imaging bacteria specifically targeting and replicating within tumors using radiotracer techniques and optical imaging can provide confirmation of successful colonization of malignant Design: The uptake of radiolabeled pyrimidine nucleoside analogues and [18F]FDG by Escherichia coli Nissle 1917 (EcN) was assessed both in vitro and in vivo. The targeting of EcN to 4T1 breast tumors was monitored by positron emission tomography (PET) and optical imaging. The accumulation of radiotracer in the tumors was correlated with the number of bacteria. Optical imaging based on bioluminescence was done using EcN bacteria that encode luciferase genes under the control of an l-arabinose–inducible PBAD promoter We showed that EcN can be detected using radiolabeled pyrimidine nucleoside analogues, [18F]FDG and PET. Importantly, this imaging paradigm does not require transformation of the bacterium with a reporter gene. Imaging with [18F]FDG provided lower contrast than [18F]FEAU due to high FDG accumulation in control (nontreated) tumors and surrounding tissues. A linear correlation was shown between the number of viable bacteria in tumors and the accumulation of [18F]FEAU, but not [18F]FDG. The presence of EcN was also confirmed by bioluminescence can be imaged by PET, based on the expression of endogenous E. coli thymidine kinase, and this imaging paradigm could be translated to patient studies for the detection of solid tumors. Bioluminescence imaging provides a low-cost alternative to PET imaging in small animals. In recent years, successful targeting of viruses and bacteria to solid tumors has been shown (1, 2) and such oncolytic therapy is receiving renewed interest. Tumor-targeting bacteria have been studied and they showed preferential accumulation in tumors compared with normal organs; studies have included the use of Bifidobacterium spp. (3), Listeria monocytogenes (1, 4), Clostridium spp. (5), Salmonella spp. (6–8), Shigella flexneri (6), Vibrio cholerae (2), and Escherichia coli (6). A number of different oncolytic viruses have already entered into clinical trials and adenovirus H101 has been approved in China for the treatment of head and neck cancer (8). However, only a single phase I human clinical trial using bacteria, Salmonella VNP20009, has been initiated (7). In this trial, a lower percentage of tumor-targeting efficacy was observed compared with the previously investigated rodent models in which tumor-colonization was high (7). The authors stated that this discrepancy could be the result of inadequate sampling that was inherent in their use of fine-needle biopsies. In an excisional biopsy done on one patient, bacteria were found to colonize the tumor, whereas a previous needle biopsy of the same tumor did not detect the microorganisms. Currently, biopsy is the only clinical method available for determining the presence of bacteria. Future clinical studies will require the ability to accurately detect the presence of bacteria in tumors (and also in other organs and tissues) without excision of the respective tissue. To address this issue, noninvasive imaging of bacteria-colonized tumors has several advantages compared with biopsy. In contrast to biopsies, imaging can be done repeatedly, provides a much wider assessment of the entire tumor as well as other tissues and body organs ( minimizes sampling errors), and can provide both a spatial and time dimension from sequential tomographic images. Different imaging modalities [positron emission tomography (PET), single-photon emission computed tomographyy, and optical imaging] in combination with reporter genes have been used to visualize the distribution of microorganisms and to confirm their presence within experimental tumors. Most studies on bacterial tumor colonization in tumor-bearing mice have used luciferase and/or fluorescence (green fluorescent protein) imaging for bacterial detection (2, 4, 6, 9). However, current optical imaging modalities using fluorescent proteins or luciferases are restricted to small animals and cannot be readily translated to patient studies. Therefore, radiotracer or magnetic resonance imaging techniques need to be used to track bacteria in human subjects. The best known and most widely used radiotracer for PET imaging is fluorine-18 (18F)–labeled fluorodeoxyglucose ([18F]FDG), which is accumulated by metabolically active cells. On entry into the cell, [18F]FDG is phosphorylated by hexokinase; the phosphorylated FDG can neither exit the cell nor be further metabolized and is therefore trapped within the cell in relation to the level of glycolytic activity. FDG uptake in many malignant tumors is high because glucose metabolism in the tumors is high. In addition, any inflammatory processes associated with the tumor contribute to the high FDG uptake because granulocytes and macrophages also have high rates of glucose metabolism (10). Although tumor tissue targeted by bacteria is likely to have high levels of FDG accumulation, baseline (before bacterial administration) is also likely to be high, and the difference between baseline and tumor-targeted FDG uptake may be difficult to image and quantitate. Another powerful imaging strategy is the use of reporter genes in to identify the location and number of tissue-targeted bacteria. Among the PET-based reporter genes, herpes simplex virus 1 thymidine kinase (HSV1-TK) has been used most extensively. The expression of HSV1-TK can be imaged and monitored using specific radiolabeled substrates that are selectively phosphorylated by HSV1-TK and trapped within transfected cells. [18F]-2′-Fluoro-2′deoxy-1β-d-arabionofuranosyl-5-ethyl-uracil ([18F]FEAU) and [124I]-2′-fluoro-1-β-d-arabino-furanosyl-5-iodo-uracil ([124I]FIAU) are radiopharmaceuticals for imaging HSV1-TK gene expression (11) and are used widely by many investigators (12–15). HSV1-TK–expressing Salmonella VNP20009 have recently been shown to localize in tumors, including C-38 colon carcinoma and B16-F10 murine melanoma, and were successfully imaged with [124I]FIAU and PET (16). In contrast to using an exogenous reporter gene such as HSV1-TK, we investigated the feasibility of using the endogenous thymidine kinase of probiotic E. coli Nissle 1917 (EcN) to phosphorylate [18F]FEAU and [124I]FIAU for noninvasive PET imaging of EcN-colonized tumors. We show that the uptake of [18F]FEAU by the tumors is dependent on the presence of EcN and that the magnitude of radioactivity accumulation correlates with the number of bacteria that colonize the tumor. We also compared [18F]FEAU and [124I]FIAU images to those obtained with [18F]FDG. Bioluminescence images of EcN were also obtained and the optical signal shown to colocalize with the [124I]FIAU activity distribution in the same animals, showing the feasibility of using EcN for identifying tumors by both bioluminescence and PET imaging in small animals. Materials and Methods Cell culture and animal experiments The murine mammary carcinoma cell line 4T1 (ATCC CRL-2539) was cultured in RPMI containing 10% FCS. The cells were maintained at 37°C with 5% CO2 in air, and subcultured twice weekly. For tumor cell implantation, 6- to 8-wk-old athymic nu/nu mice (National Cancer Institute) were used, housed five per cage, and allowed food and water ad libitum in the Memorial Sloan Kettering Cancer Center facility for 1 wk before tumor cell implantation. The 4T1 cells were removed by trypsinization, washed in PBS, and × 104 cells (resuspended in 50-μL PBS) were implanted into the right and left shoulders. Two weeks postimplantation (tumor diameter >5 mm), bacteria were administered systemically by tail vein injection. Animal studies were done in compliance with all applicable policies, procedures, and regulatory requirements of the Institutional Animal Care and Use Committee, the Research Animal Resource Center of Memorial Sloan Kettering Cancer Center, and the NIH Guide for the Care and Use of Laboratory Animals. All animal procedures were done by inhalation of 2% isofluorane. After the studies, all animals were sacrificed by CO2 inhalation. Bacteria E. coli Nissle 1917 (EcN), a probiotic, non–protein-toxin-expressing strain, was used to specifically colonize tumors and harbored a pBR322DEST PBAD-DUAL-term, a luxABCDE-encoding plasmid that enables the bacteria to be detected with bioluminescence imaging when induced with l-arabinose (6). The light is emitted from the bacteria as a result of a heterodimeric luciferase (encoded by luxAB) catalyzing the oxidation of reduced flavin mononucleotide and a long-chain fatty aldehyde (synthesized by a fatty acid reductase complex encoded by luxCDE; ref. 17). For injection, bacteria were grown in LB broth supplemented with 100 μg/mL ampicillin until reaching an absorbance at 600 nm (A600 nm) of [corresponding to 2 × 108 colony-forming units (CFU)/mL] and washed twice in PBS. The suspension was then diluted to 4 × 107 CFU/mL and 100 μL were injected into the lateral tail vein of tumor-bearing mice. Vehicle control mice were injected with 100-μL PBS via tail vein. Radiopharmaceuticals [18F]FEAU was synthesized by coupling the radiolabeled fluoro sugar with the silylated pyrimidine derivatives following a procedure previously reported by Serganova et al. (12). The specific activity of the product was ∼37 GBq/μmol (∼1 Ci/μmol); radiochemical purity was >95% following purification by high-pressure liquid chromatography. [124I]FIAU was synthesized by reacting the precursor of 5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (FTAU) with carrier-free [124I]NaI. I-124 was produced on the Memorial Sloan-Kettering Ebco cyclotron using the 124Te(p,n) 124I nuclear reaction on an enriched 124TeO2/Al2O3 solid target. Radiosynthesis was done as previously described (13, 14) with minor modifications. The specific activity of the product was >1,000 GBq/μmol (>27 Ci/μmol); radiochemical purity was >95% and was determined by radio TLC (Rf using silica gel plates and a mobile phase of ethyl acetate/acetone/water (14:8:1, v/v/v). [18F]FDG (clinical grade) was obtained from IBA Molecular with a specific activity >41 MBq/μmol (>11 mCi/μmol) and a radiochemical purity of 99% by TLC and 98% by high-pressure liquid chromatography. In vitro uptake of [18F]FDG and [18F]FEAU An overnight culture of EcN was diluted 1:50 into 5-mL fresh LB broth containing either MBq (25 μCi) of [18F]FDG or [18F]FEAU and grown at 37°C for 4 h. The bacteria were then harvested by centrifugation, washed twice with PBS, and the radioactivity in the pelleted bacteria and medium was measured in a gamma counter (Packard, United Technologies). MicroPET imaging FDG. In the first group of six animals, each animal was injected via the tail vein with MBq (250 μCi) of [18F]FDG before and 16 or 72 h after administration of EcN. [18F]FDG PET scanning was done 1 h after tracer administration using a 10-min list mode acquisition. Animals were fasted 12 h before tracer administration and kept under anesthesia between FDG injection and imaging. FEAU. In the second group of 24 animals, three subgroups of eight animals each were studied; each animal was injected via tail vein with MBq (250 μCi) of [18F]FEAU. Subgroup 1 (control) was not injected with bacteria (they received 100-μL PBS); subgroups 2 and 3 were injected with EcN-bacteria 16 and 72 h before [18F]FEAU administration. [18F]FEAU PET scanning was done 2 h after tracer administration using a 10-min list mode acquisition. FIAU. In a third set of six mice, three were injected with EcN-bacteria and three with PBS (control). [124I]FIAU [37 MBq (1 mCi)] was injected in each animal 72 h after bacterial injection. Potassium iodide was used to block the uptake of radioactive iodine by the thyroid. [124I]FIAU PET was obtained 4, 8, 12, 24, 48, and 72 h after tracer administration with 10-min list acquisition at the 4- and 8-h imaging time points, 15 min at the 12-h time point, 30 min at 24 h, and 60 min at the 48- and 72-h time points. After tracer administration and between imaging time points, the animals were allowed to wake up and maintain normal husbandry. Imaging was done using a Focus 120 microPET dedicated small-animal PET scanner (Concorde Microsystems, Inc.). Mice were maintained under 2% isofluorane anesthesia with an oxygen flow rate of 2 L/min during the entire scanning period. Three-dimensional list mode data were acquired using an energy window of 350 to 700 keV for 18F and 410 to 580 keV for 124I and a coincidence timing window of 6 ns. These data were then sorted into two-dimensional histograms by Fourier rebinning using a span of 3 and a maximum ring difference of 47. Transverse images were reconstructed by filtered back-projection using a ramp filter with a cutoff frequency equal to the Nyquist frequency in a 128 × 128 × 94 matrix composed of × × voxels. The image data were corrected for (a) nonuniformity of scanner response using a uniform cylinder source-based normalization, (b) dead time count losses using a singles count rate–based global correction, (c) physical decay to the time of injection, and (d) the 124I branching ratio. There was no correction applied for attenuation, scatter, or partial-volume averaging. The count rates in the reconstructed images were converted to activity concentration [percent of injected dose per gram of tissue (%ID/g)] using a system calibration factor (μCi/mL/cps/voxel) derived from imaging of a rat-size phantom filled with a uniform aqueous solution of 18F. PET image analysis was done using ASIPro software (Concorde Microsystems, Inc.). For each PET scan, regions of interest were manually drawn over tumor, liver, skeletal muscle, and heart. For each tissue and time point postinjection, the measured radioactivity was expressed as %ID/g. The maximum pixel value was recorded for each tissue and tumor-to-organ ratios for liver, skeletal muscle, and heart were then plotted versus time. Bacterial and radioactivity quantification of tissue samples Euthanized mice were rinsed with 100% ethanol before tissue removal. Organs such as liver, lung, spleen, and heart were sampled and weighed before radioactivity measurements. Tumor tissue was weighed and homogenized in 1-mL PBS. Serial dilutions of the homogenized sample were plated on l-arabinose–containing LB agar plates and growing colonies were counted and confirmed to be EcN harboring a pBR322DEST PBAD-DUAL-term by bioluminescence imaging using an IVIS 100 Imaging system (Caliper). The remaining tumor suspension was assayed for radioactivity in a gamma counter (Packard, United Technologies); [18F]FEAU radioactivity (%ID/g) in the samples was determined and tumor-to-organ ratios were calculated. To assess the correlation between radioactivity and scintillation counter measurements, the Pearson correlation coefficient was computed. In vivo optical imaging of bioluminescence The same animals were imaged for localization of bioluminescence after the 72-h [124I]FIAU PET scans. Each animal was injected with 200-μL l-arabinose (25% w/v) to induce transcriptional expression of the luciferase reporter for bioluminescence imaging. Images were acquired for 60 s, 4 h after l-arabinose injection, using an IVIS 100 Imaging System (Caliper). The photon emissions (photons/cm2/s/steradian) from the animals and cell samples were analyzed using the LIVINGIMAGE software (Caliper). Statistics A two-tailed unpaired t test was applied to determine the significance of differences between values using the MS Office 2003 Excel statistical package (Microsoft). Results In vitro [18F]FDG and [18F]FEAU uptake into EcN. The in vitro uptakes of [18F]FDG and of [18F]FEAU by the tumor-colonizing strain E. coli Nissle 1917 were compared. There was a 120-fold higher concentration of [18F]FDG and a higher concentration of [18F]FEAU activity in EcN-bacteria compared with that in the LB broth, suggesting that [18F]FDG would be a better imaging agent than [18F]FEAU. Distribution of EcN in tumor-bearing mice. Following EcN injection into the tail vein of 4T1 tumor–bearing mice, most bacteria (>99%) are quickly cleared from the animals and only a small percentage of the administered bacteria colonize the tumor (6). These tumor-colonizing bacteria started to grow exponentially for ∼24 hours before reaching a plateau of ∼1 × 109 CFU/g of tumor tissues. During the growth phase, the bacteria are metabolically active and rapidly proliferate. For our studies, we elected to use tumor-bearing mice that were injected with EcN at 16 hours (lower CFU per gram but in rapid growth phase) and at 72 hours (higher numbers of bacteria in a slower phase) before administration of [18F]FDG or [18F]FEAU. The number of bacteria per gram of tumor tissue at 16 and 72 hours postinjection is shown in (Fig. 1). Fig. colonization of EcN at 16 and 72 h after bacterial injection. Columns, mean of eight analyzed tumors; bars, colonization of EcN at 16 and 72 h after bacterial injection. Columns, mean of eight analyzed tumors; bars, SD. Close modal In vivo PET imaging of EcN colonized tumors. [18F]FDG PET imaging was done before and at 16 and 72 hours after tail vein injection of EcN in the same animals (Fig. 2A). The [18F]FDG tumor-to-organ ratios (mean ± SD) before injection of EcN bacteria were high in liver ( ± and muscle ( ± and low in heart ( ± At 16 hours after EcN injection, tumor-to-organ ratios were significantly increased for liver, muscle, and heart ( ± ± and ± respectively). At 72 hours after EcN injection, the tumor-to-organ ratios were lower for the same tissues ( ± ± and ± respectively). This represents a ∼ enhancement at 16 hours (P 5 in the EcN-treated animals (Fig. 5B). However, the control (non–EcN-treated) animals also show some [124I]FIAU retention in the 4T1 xenografts. This reduces the specificity of the radioactivity measured in the EcN-treated tumors and results in only a enrichment of [124I]FIAU in the bacteria-treated tumors (Fig. 5B). Fig. axial and coronal views of [124I]FIAU microPET images of representative EcN-treated and nontreated (control) 4T1 xenograft–bearing animals at different times (12, 24, 48, and 72 h; X-axis) after tracer injection. B, [124I]FIAU uptake of tumors compared with background as calculated from region of interest measurements; six tumors in each group (FIAU uptake ratio; left Y-axis). Data from the EcN colonized group are shown in green and the control group in blue. The mean tumor uptake ratios in EcN colonized animals normalized to the mean values obtained for the control animals are indicated in red (relative FIAU uptake; right Y-axis). C, bioluminescence images of the same animals in A 4 h after injection of l-arabinose; l-arabinose induces the expression of luciferase genes in EcN × pBR322DEST PBAD-DUAL-term bacteria. Tumors are axial and coronal views of [124I]FIAU microPET images of representative EcN-treated and nontreated (control) 4T1 xenograft–bearing animals at different times (12, 24, 48, and 72 h; X-axis) after tracer injection. B, [124I]FIAU uptake of tumors compared with background as calculated from region of interest measurements; six tumors in each group (FIAU uptake ratio; left Y-axis). Data from the EcN colonized group are shown in green and the control group in blue. The mean tumor uptake ratios in EcN colonized animals normalized to the mean values obtained for the control animals are indicated in red (relative FIAU uptake; right Y-axis). C, bioluminescence images of the same animals in A 4 h after injection of l-arabinose; l-arabinose induces the expression of luciferase genes in EcN × pBR322DEST PBAD-DUAL-term bacteria. Tumors are encircled. Close modal Colocalization of bioluminescence and [124I]FIAU uptake. To further verify that the increased [124I]FIAU PET signal reflected bacterial localization in 4T1 xenografts, we took advantage of the l-arabinose–inducible luciferase reporter plasmid pBR322DEST PBAD-DUAL-term (6). l-Arabinose was injected into each mouse following [124I]FIAU PET imaging, and bioluminescence imaging was done 4 hours later when the expression of luciferase is at its maximum (6). The l-arabinose–induced bioluminescence signal was readily detected at the site of the 4T1 xenografts (Fig. 5C). Control tumors did not show any such signal. The bioluminescence images of EcN-treated mice also indicated no bacterial presence in other tissues of mice. Discussion EcN is one of the best studied probiotic bacterial strains and it has been successfully used in humans as an oral treatment for a number of intestinal disorders ( diarrhea, inflammatory bowel diseases, and ulcerative colitis) for more than 90 years (18, 19). Although the genome of EcN shows high similarity to the uropathogenic E. coli CFTR073 (20), the probiotic strain lacks any known protein toxins or mannose-resistant hemagglutinating adhesins (21). Furthermore, EcN was not found to colonize any organs other than tumor when administered systemically to tumor-bearing mice (6). Thus, EcN seems to be a good candidate for human application, although it still produces lipopolysaccharide (endotoxin), which could result in adverse effects. Because deletion of genes responsible for lipopolysaccharide biosynthesis ( msbB) has been shown to be successful for Salmonella typhimurium, a similar strategy could be adopted with EcN to insure its clinical safety. A noninvasive, clinically applicable method for imaging bacteria in target tissue or specific organs of the body would be of considerable value for monitoring and evaluating bacterial-based therapy in human subjects. This imaging system could also be used for monitoring the targeting and proliferation of the bacterial vector, such as EcN, to identify sites of occult tumor and to identify sites of bacterial proliferation in occult infectious disease. EcN imaging provides the following benefits: Following systemic administration of the bacteria, imaging can (a) confirm successful targeting to known tumor sites, (b) potentially identify additional sites of tumor metastases, and (a) assess whether the number (concentration) of EcN in tumor tissue is adequate to deliver a sufficient dose of a “therapeutic gene.” In our study, we assessed the feasibility of detecting EcN-colonized tumors with [18F]FDG, [18F]FEAU, and [124I]FIAU PET imaging. We showed that EcN accumulate and trap radiolabeled [18F]FDG, [18F]FEAU, and [124I]FIAU using endogenous enzyme systems ( bacterial hexokinase and thymidine kinase). It was previously shown that tumor targeting of HSV1-TK–transformed Salmonella VNP20009 could be successfully imaged with [124I]FIAU and that [124I]FIAU accumulation was HSV1-TK dependent (16). Here, the expression of the endogenous bacterial thymidine kinase of EcN and phosphorylation of [18F]FEAU and [124I]FIAU are sufficient to result in selective accumulation of these radiotracers in tissue colonized by EcN. In contrast to the marked structural specificity of mammalian thymidine kinase for thymidine alone (resulting in little or no phosphorylation of thymidine analogues), the thymidine kinase of bacteria has been shown by Bettegowda et al. (5) to be less specific for thymidine than the mammalian enzyme. Bacterial as well as viral thymidine kinase will phosphorylate thymidine analogues such as FIAU and FEAU. This study opens up new possibilities for future investigations and for the use of alternative pyrimidine nucleoside derivatives such as FEAU that can be selectively phosphorylated by endogenous bacterial thymidine kinase ( E. coli, Salmonella, or Clostridium). The tumor-selective replication of EcN in live animals allowed us to distinguish tumors from other tissues by PET imaging following administration of radiolabeled [18F]FEAU or [124I]FIAU. By using tumors in different stages of bacterial colonization ( 16 and 72 hours after bacterial administration), we showed a linear relationship between the number of viable bacteria in tumor tissue and the uptake of radiolabeled [18F]FEAU. This result is similar to that found with HSV1-TK–transformed Salmonella VNP20009 and [124I]FIAU accumulation (16). Comparing the Salmonella VNP20009 and EcN data shows that the HSV1-TK–transformed Salmonella accumulate more radiopharmaceutical per viable bacteria than EcN bacteria over the dose ranges that were studied (Fig. 4B). These results, for several reasons, are not unexpected and indicate that there is a role for reporter-transformed bacteria when higher imaging sensitivity is required: In addition to the genomic thymidine kinase gene of Salmonella VNP20009, HSV1-TK was present in multiple copies under control of a constitutive promoter. In contrast, only the genomic copy of the EcN thymidine kinase gene under control of its own promoter was present in EcN bacteria. Therefore, higher expression of thymidine kinase is achieved in VNP20009 Salmonella. Furthermore, [124I]FIAU and [18F]FEAU were developed to specifically image HSV1-TK, and not mammalian TK1, to achieve low background activity, and these tracer substrates may not be an ideal substrate for bacterial thymidine kinases (5). There was no correlation between the level of [18F]FDG uptake and number of viable bacteria in the tumors, and the signal-to-background ratio was not as high with [18F]FDG as with [18F]FEAU and [124I]FIAU. This clearly reflects the high baseline uptake (%ID/g) of [18F]FDG by the tumor compared with that of [18F]FEAU and [124I]FIAU. However, [18F]FDG imaging in combination with EcN (or other bacteria) might show better results in tumors with a low baseline level of [18F]FDG uptake. The absence of a correlation between number of viable bacteria and [18F]FDG uptake might also be due to the presence of necrosis induced by the bacteria or to the presence of glucose-metabolizing macrophages in the tumors (6). For example, on day 1 after bacterial injection, a high number of metabolically active bacteria were present and only very small patches of necrosis were observed. Two days later, the number of bacteria increases, but the number of living cells in the tumor decreases dramatically because the necrotic region takes up 30% to 50% of the tumor volume (6). It should also be noted that 4T1 xenografts in the absence of bacteria accumulate [124I]FIAU to low levels above background (48-and 72-hour images in Fig. 5B) in comparison with the near-background levels of [18F]FEAU accumulation (Fig. 2D) in non–bacteria-treated animals. This is consistent with similar observations in other tumor systems (12–14, 22, 23). Thus, [18F]FEAU may be a better bacterial-imaging probe than [124I]FIAU. The current study showed the feasibility of noninvasive imaging of bacteria based on the expression of genomic bacterial thymidine kinase. The potential for monitoring patients that have received tumor-colonizing bacteria without the inclusion of an exogenous ( viral) reporter gene has previously been shown (5) and is confirmed here. Imaging should be able to determine whether bacterial tumor colonization has occurred successfully and whether previously undetected metastases or specific organs are colonized by the bacteria. We have shown that the level of radioactivity can also be taken as an indicator of the number of bacteria that are present in the target tissue and whether therapeutic effects ( by administration of prodrugs or induction of toxic genes) can be expected. In addition, the presence of pathogenic bacteria in localized infections may also be identifiable, and it may also be possible to differentiate bacterial infections from nonmicrobial inflammations by [18F]FEAU or [124I]FIAU PET imaging. In conclusion, the results of our study indicate that EcN (or other bacteria expressing endogenous thymidine kinase) can be imaged with pyrimidine nucleoside analogues that are selectively phosphorylated and trapped in the bacteria. The advantage of using EcN over many other bacteria is their probiotic character. It is therefore a relatively safe “imageable vector” that could also include genes conferring therapeutic potential. We show that the PET images for EcN-colonized tumors were better ( resulted in higher signal-to-background ratios) with [18F]FEAU than with [18F]FDG, and this was mainly due to the low baseline (pre-bacterial) activity in the tumors and surrounding tissue. Most importantly, a linear relationship between the number of viable bacteria and level of [18F]FEAU activity in the xenografts was found, an essential component of the imaging paradigm. Other pyrimidine nucleoside analogues that have been developed for PET imaging of HSV1-TK, such as [124I]FIAU and [18F]FHBG, could also be further evaluated for noninvasive monitoring of bacterial tumor colonization because both positron-emitting radiopharmaceuticals have already been successfully administered to patients in gene imaging studies (15, 23–26). Grant support: NIH grants R25-CA096945 and P50 CA86438, Department of Energy grant FG03-86ER60407, R&D Division of Genelux Corporation San Diego, and a Service contract awarded to the University of Würzburg, Germany ( Szalay). Technical services were provided by the Memorial Sloan Kettering Cancer Center Small-Animal Imaging Core Facility, supported in part by NIH Small-Animal Imaging Research Program grant R24 CA83084 and NIH Center grant P30 CA08748. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 Section 1734 solely to indicate this fact. Note: P. Brader and J. Stritzker contributed equally to this work. Acknowledgments We thank Dr. Steven Larson (Memorial Sloan Kettering Cancer Center, New York, NY) for his help and support. References 1Liu TC, Kirn D. Systemic efficacy with oncolytic virus therapeutics: clinical proof-of-concept and future directions. 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Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine Free W Kruis1, P Frič2, J Pokrotnieks3, M Lukáš4, B Fixa5, M Kaščák6, M A Kamm7, J Weismueller8, C Beglinger9, M Stolte10, C Wolff11, J Schulze111Evangelisches Krankenhaus Kalk, University of Cologne, Germany2Ustředná vojenská nemocnice, II interní oddělení, Praha, Czech Republic3Paula Stradina Clinical University Hospital, Riga, Latvia4IV Interni Klinika, Charles University, Praha, Czech Republic52nd Department of Medicine, Charles University Prague, Medical Faculty, Hradec Kralove, Czech Republic6Interné oddelenie NsP, Trenčín, Slovak Republic7St Mark’s Hospital, London, UK8Private Practice, Koblenz, Germany9Division of Gastroenterology, University Hospital, Basel, Switzerland10Institut für Pathologie, Klinikum Bayreuth, Germany11Ardeypharm, Herdecke, GermanyCorrespondence to: Dr W Kruis Evangelisches Krankenhaus Kalk, Buchforststr 2, 51103 Cologne, Germany; Abstract Background and aim: Evidence exists for the pathogenic role of the enteric flora in inflammatory bowel disease. Probiotics contain living microorganisms which exert health effects on the host. We compared the efficacy in maintaining remission of the probiotic preparation Escherichia coli Nissle 1917 and established therapy with mesalazine in patients with ulcerative colitis. Patients and methods: In total, 327 patients were recruited and assigned to a double blind, double dummy trial to receive either the probiotic drug 200 mg once daily (n = 162) or mesalazine 500 mg three times daily (n = 165). The study lasted for 12 months and patients were assessed by clinical and endoscopic activity indices (Rachmilewitz) as well as by histology. The primary aim of the study was to confirm equivalent efficacy of the two drugs in the prevention of relapses. Results: The per protocol analysis revealed relapses in 40/110 ( patients in the E coli Nissle 1917 group and 38/112 ( in the mesalazine group (significant equivalence p = Subgroup analyses showed no differences between the treatment groups in terms of duration and localisation of disease or pretrial treatment. Safety profile and tolerability were very good for both groups and were not different. Conclusions: The probiotic drug E coli Nissle 1917 shows efficacy and safety in maintaining remission equivalent to the gold standard mesalazine in patients with ulcerative colitis. The effectiveness of probiotic treatment further underlines the pathogenetic significance of the enteric flora. UC, ulcerative colitisIBD, inflammatory bowel diseaseEcN, Escherichia coli Nissle 1917GCP, good clinical practiceCAI, clinical activity indexEI, endoscopic indexITT, intention to treat populationPP, per protocol population5-ASA, 5-aminosalicylic acidulcerative colitismaintenance therapyprobioticsEscherichia coli Nissle Statistics from Request Permissions If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways. UC, ulcerative colitisIBD, inflammatory bowel diseaseEcN, Escherichia coli Nissle 1917GCP, good clinical practiceCAI, clinical activity indexEI, endoscopic indexITT, intention to treat populationPP, per protocol population5-ASA, 5-aminosalicylic acidulcerative colitismaintenance therapyprobioticsEscherichia coli Nissle Ulcerative colitis (UC) is a chronic relapsing disease. The aims of treatment are induction of remission and prevention of relapses. Guidelines1,2 recommend aminosalicylates for maintenance treatment. Aminosalicylates exert various effects on leukotrienes, cytokines, and oxygen Their mode of action in UC remains unclear. It is suggested that the sum of their anti-inflammatory activities constitutes their therapeutic principle. Thus maintenance treatment with aminosalicylates is only effective when inflammation starts, but not in the non-inflamed gut. Growing evidence exists for a role of the intestinal microflora in the pathogenesis of inflammatory bowel disease (IBD). Findings from genetically engineered animal models as well as clinical observations have elucidated the importance of commensal Antibacterial treatment showed some beneficial effects7,8 but the use of antibiotics is limited. Therefore, treatment with probiotics has been proposed. Probiotics are viable non-pathogenic microorganisms that confer health benefits to the host by improving the microbial balance of the indigenous Apart from anecdotal experience, two controlled studies with the probiotic bacterial strain Escherichia coli Nissle 1917 (EcN) in UC already These trials showed no difference between the relapse preventing effects of EcN and standard mesalazine. However, some criticism was raised as to the validity of these The present study was undertaken to confirm that the relapse preventing effects of probiotic therapy with EcN and standard mesalazine are equivalent. MATERIALS AND METHODS The study was conducted according to the Helsinki Declaration (revised version of Hong Kong) and adhered to good clinical practice (GCP) guidelines. The study was approved by the Ethikkommission der Ärztekammer Nordrhein, Germany, as well as by the local ethics committees of the participating centres. All patients received material in their own language and gave written informed consent. Patients were included in the study if aged 18–70 years and diagnosed with UC in remission (clinical activity index (CAI) ⩽4, endoscopic index (EI) ⩽4, and no signs of acute inflammation on histological examination). In addition, inclusion criteria comprised at least two acute attacks of UC prior to the study and a duration of the current remission of no longer than 12 months. Exclusion criteria were: active UC; proctitis with up to 10 cm proximal spread; Crohn’s disease; infectious colitis; severe accompanying illnesses or major colonic surgery; use of antibiotics, sulphonamides, steroids, or other therapies for UC at entry into the trial; administration of EcN within the previous six months before trial entry; as well as known intolerance to salicylates. Study medication The investigational drug was a bacterial preparation for oral use containing non-pathogenic Escherichia coli of strain Nissle 1917 (serotype O6:K5:H1). Capsules were enteric coated to protect the microorganisms from gastric juice and contained viable bacteria (Mutaflor 100 mg; Ardeypharm GmbH, Herdecke, Germany). The control preparation was mesalazine, consisting of eudragit L coated 5-aminosalicylic acid (Salofalk500 mg; Dr Falk Pharma GmbH, Freiburg, Germany). The test group received one capsule of Mutaflor 100 mg once daily and one tablet of placebo three times daily from day 1 to day 4, and two capsules of Mutaflor 100 mg once daily and one tablet of placebo three times daily from day 5 to the end of the study. The control group received one capsule of placebo once daily and one tablet of Salofalk 500 mg three times daily from day 1 to day 4, and two capsules of placebo once daily and one tablet of Salofalk 500 mg three times daily from day 5 to the end of the study. No concomitant medication for UC was allowed throughout the study. Study design This was a randomised, double blind, double dummy trial comparing the relapse preventing effects and safety of a bacterial preparation containing viable EcN and mesalazine for 12 months in patients with UC in remission. The study was conducted in 60 hospitals and private settings in 10 European countries (see list of participating investigators in the appendix). Randomisation was carried out in a double blind manner in blocks of four patients using 1:1 allocation to the two treatment groups. Only complete blocks of random numbers were used for each centre. If patients were eligible for study entry, they were assigned to random numbers ( = patient numbers) in ascending order within each centre according to the chronological order of their randomisation and were given the corresponding study medication. Evaluation Clinic visits were required at the start and end of the study as well as after 1, 2, 3, 6, and 9 months of treatment. The primary objective of the study was to compare the number of patients experiencing a relapse of UC during the 12 month observation period between the two treatment groups. Patients were classified as suffering a relapse when all three of the following criteria were met: CAI >6 or an increase in CAI of at least 3 points with CAI = 4 being exceeded at the same time; EI >4; and histological signs of acute inflammation. CAI was defined according to At trial entry and at the end of the study, patients underwent colonoscopy where biopsies were taken. Endoscopic activity was assessed using a four point index14: granularity, vascular pattern, vulnerability of mucosa, and mucosal damage. All biopsies were examined by a single pathologist using a four point Secondary efficacy variables were the physician’s and patient’s assessment of general well being and calculation of a quality of life Additionally, time to relapse, CAI, EI, and histological findings were documented. Laboratory assessments, including erythrocyte sedimentation rate, C reactive protein, orosomucoids, blood counts, liver enzymes, creatinine, serum iron, and serum albumin were performed at trial entry and at the end of the study. Incidence and severity of adverse events were reported according to GCP for clinical trials of medication in the European Community (91/507/EWG, CPMP/ICH/135/95). Tolerance of the study medication was assessed on a four point scale (very good, good, fair, poor), and patient compliance was ascertained by pill counting. Statistical analysis The aim of the study was to statistically confirm one sided equivalent efficacy of EcN and mesalazine in preventing relapses of UC. Relapse rates were compared using the one sided test of Farrington and Manning17: this tests the null hypothesis that the difference between treatment groups is greater than or equal to the upper equivalence margin Δ of 20% versus the alternative that the true difference is less than 20% (α = upper confidence limit 95%). Assuming a 12 month relapse rate of 30% under mesalazine treatment and 35% under EcN treatment, to reach a statistical power of 80% at least n = 127 patients were required in each treatment group according to the sample size term for comparative binomial trials with the null hypothesis of non-zero risk Two sets of patients were analysed: an intention to treat population (ITT), including all patients who took at least one dose of the study medication, and a per protocol population (PP). According to generally accepted standards for equivalence and non-inferiority trials,18 primary analysis of the main objective (difference in relapse rates) was based on the PP population. Assuming 25% protocol violators, a total number of 160 patients in each treatment group was therefore planned. Baseline comparability and statistical analysis of secondary objectives was assessed using Fisher’s exact test (two sided; α = In addition, Kaplan-Meier curves were plotted. If no CAI or other parameter was documented at the individual study end, the “last observation carried forward” method was applied. Results are given as mean (SD). Statistical tests were executed using SPSS software package version under the Microsoft Windows NT operating system. For exploratory comparisons (tables 2, 3), the Student’s t test was used. RESULTS Patient characteristics In total, 327 patients were enrolled and randomised to either the EcN preparation (n = 162) or mesalazine (n = 165). The two patient groups were matched with regard to demographic, clinical, and pretreatment characteristics (table 1). The time gap between the end of the last relapse before the study and entry into the study was not longer than four weeks in of patients receiving EcN and in receiving mesalazine, and not longer than three months in and of EcN and mesalazine patients, respectively. All 327 randomised patients received at least one dose of the study medication and thus were included in the ITT and safety analysis this table:View inline Table 1 Demographic data and prestudy clinical characteristics Before unblinding the study, a steering committee assessed protocol violations in 105/327 ( patients. Major protocol deviations comprised violation of inclusion criteria (CAI ⩽4, EI ⩽4, and no signs of acute inflammation on histological examination) (32 patients in both groups), premature discontinuation of the study without relapse (see below), and unknown or not unequivocally assessed end point (EcN 29 patients, mesalazine 24 patients). Accordingly, the PP analysis set comprised 222 patients (EcN 110, mesalazine 112). Mean duration of the study observation period was 250 (144) (median 357) days in the EcN group and 287 (125) (median 360) days in the mesalazine group. The number of patients in the study at the scheduled visits is shown in fig 1. Premature discontinuation of the study for reasons other than relapse of disease occurred in 39/327 ( patients (in 19/162 ( patients in the EcN group and in 20/165 ( patients in the mesalazine group) (table 2). Newly emerged exclusion criteria during the study were start of concomitant medication in four patients on EcN. One patient on mesalazine became afraid of 5-aminosalicylic acid (5-ASA) and another patient underwent cardiac this table:View inline Table 2 Reasons for premature discontinuation of the study Relapse (primary objective) PP analysis revealed relapse in 40/110 ( patients in the EcN group and in 38/112 ( patients in the mesalazine group (fig 2), resulting in significant equivalence between the two groups (p = The corresponding one sided upper 95% confidence limit for the difference in treatment was (that is, within the equivalence range of 20%). Figure 3 depicts the probability of remaining in remission by Kaplan-Meier curves. Median time to relapse in the EcN group could not be calculated due to the large number of late censorings. In the mesalazine group it was 386 days. ITT analysis confirmed these results, showing a relapse rate of in the EcN group and in the mesalazine group (significant equivalence p = The upper limit of the 95% confidence interval for the difference in treatment was Subgroup analyses (secondary objectives) All subgroup analyses were performed in the ITT population. CAI increased in all patients by ( points over the study period, showing a slightly larger increase in the EcN group ( ( than in the mesalazine group ( ( No differences were observed in EI or histology between the start and end of the study (fig 4). Table 3 lists relapse rates with regard to duration, localisation, and pretrial treatment. There were no significant differences between the treatment groups for any of these characteristics. Quality of life scores on admission were ( in the EcN group and ( in the mesalazine group. Respective values after 12 months were ( and ( No significant changes occurred during the 12 month observation this table:View inline Table 3 Relapse rates according to clinical characteristics (intention to treat population) Safety and tolerance As rated by the patients, overall tolerance was very good or good in the EcN group in and in the mesalazine group in According to the physician’s assessment, the respective values were and Discontinuation of the study medication due to adverse events (relapse included) occurred in 22 ( patients (11 ( in the EcN group and 11 ( in the mesalazine group). Most frequent reasons were gastrointestinal disorders such as bloody stools, nausea, diarrhoea, mucous secretion (EcN mesalazine and abdominal pain (EcN mesalazine Generally, no unexpected drug reactions occurred during the study. No deaths but 17 serious adverse events were reported in 13/327 (4%) patients (EcN 7, mesalazine 6). Each serious adverse event occurred only once. Adverse events were reported in 68/162 ( patients treated with EcN and in 58/165 ( patients treated with mesalazine. Many adverse events reflect symptoms common for active UC such as bloody stools ( diarrhoea ( and abdominal pain ( The most frequent non-intestinal adverse events were viral infections (EcN mesalazine nausea ( and headache ( Laboratory tests showed no significant alterations. DISCUSSION Most controlled trials are designed to test differences in efficacy. In contrast, our trial was aimed at proving equivalence. Indeed, we demonstrated that the probiotic EcN provides significantly equivalent efficacy in preventing relapses of UC and is not inferior to the established gold standard mesalazine. This result was not only confirmed by statistical analysis of the PP population, which is preferred in equivalence studies,18 but also by ITT analysis. Therapeutic efficacy is usually demonstrated by superiority in a placebo controlled trial. In serious disease however when effective therapy exists that has already been tested by comparison with placebo, additional placebo controlled trials may be considered A meta-analysis19 reviewed 16 studies of maintenance therapy involving 2341 patients with UC. In four of these 16 trials, preparations containing 5-ASA were compared with placebo; in the remaining 12 studies sulphasalazine was compared. 5-ASA was observed to be significantly more effective than placebo in all dosage subgroups (<1 g/day, 1– g/day, ⩾2 g/day). A dose dependent trend was not Indeed, some studies comparing at least two doses were performed showing mainly negative or conflicting results20: Pentasa 3 g/day was not superior to g/day; balsalazide 4 g/day was better than 2 g/day; balsalazide 6 g/day was better than 3 g/day in one study but in another trial was similarly effective; and two studies with olsalazine reached different conclusions. Thus superior efficacy of doses higher than g/day has not been It can be stated that mesalazine g/day presently reflects the standard in the prevention of UC relapses and thus it qualifies as a control in an equivalence trial. Previous studies on EcN were criticised12,13 for several reasons—for example, short observation period10 or heterogeneity of patients and outcome The present trial considered this critique and followed actual standards. The observation period was 12 months, only patients with UC in remission were included, and the clinical outcome was assessed by well established endoscopic and histological activity indices resulting in a low relapse rate for the mesalazine group comparable with previous A total of 327 patients were included to achieve a statistical power sufficient to test for equivalence in a one sided set. Most likely, IBD is caused by an unrestrained inflammatory response to as yet undefined agents. Although precise identification of the antigenic stimuli has not been determined, the intestinal microflora represents a likely To manipulate the resident gut bacteria therefore seems to offer a rational approach to maintaining remission in IBD. One way of doing this, which has gained credence over recent years, is by using Mechanisms which may account for probiotic activity include production of antimicrobial agents, inhibition of adhesion of pathogens, and influence on mucosal barrier It was reported that inhibition of nuclear factor κB could be mediated by probiotic The properties of EcN are well characterised25 and its genome has been extensively It carries non-pathogenic adhesion molecules. A specific lipopolysaccharide renders it immunogenic without showing any immunotoxic Immunomodulating activity was demonstrated for specific immune responses as well as for induction of non-specific natural immunity in preterm EcN develops antagonistic activity against enterobacteria such as Salmonella enteritidis, Shigella dysenteriae, Yersinia enterocolitica, and Vibrio It prevents invasion of Salmonella typhimurium into intestinal cells,31 inhibits adhesion and invasion of adherent invasive E coli,32 and reduces concentrations of mucosa associated colonic microflora constituents in EcN is safe. Molecular genetics as well as functional analyses have revealed that EcN does not produce any virulence factors or carry any genes for pathogenicity It does not bear genes for antibiotic resistance, transferable genes or plasmids, and does not take up foreign pathogenic DNA. No formation of enterotoxins, cytotoxins, or haemolysins has been observed and there is no serum Clinical studies have demonstrated a favourable safety profile for EcN compared with placebo,35,36 mesalazine,10,11 and Our study confirms this excellent safety and tolerance record. There are other controlled studies with different probiotics. Relapse prevention with Lactobacillus GG tested negatively for maintenance therapy in surgically induced remission of Crohn’s disease38 but a small study showed positive results when Saccharomyces boulardii was added to Inflammation of the ileal pouch constructed after proctocolectomy and ileoanal anastomosis in patients with UC is of particular interest because bacterial growth seems to be of pivotal pathophysiological significance. Cases successfully treated with EcN have been A formulation comprising eight different probiotic bacteria demonstrated convincing therapeutic effects in primary prevention41 and chronic In an uncontrolled study, this preparation was able to colonise the gut and maintain remission in patients with In conclusion, the use of probiotics in IBD is in accordance with its pathogenesis. They may prevent induction of inflammatory reactions. EcN shows therapeutic efficacy and safety in maintaining remission in UC. It can be considered as an alternative to mesalazine. APPENDIX The following institutions, local principal investigators, and local coordinators participated in this study: Austria: University Hospital, Graz: W Petritsch. Czech Republic: Nemocnice Milosrdnych sester sv Karla Boromejského, Prague: J Dosedel; University Hospital, Hradec Kralove: B Fixa; Central Military Hospital, Prague: P Frič; University Hospitals, Prague: M Kment, M Lukáš; University Hospital Plzen: J Koželuhová; University Hospital Brno: H Simonová; Masaryk Hospital, Ústí nad Labem: K Mareš, J Stehlík. Estonia: Central Hospital, Tallin: B Margus; University Hospital, Tartu: R Salupere. Germany: Private Practice, Essen: A Boekstegers; University Hospital, Jena: H Bosseckert; University Hospital, Regensburg: V Gross; DRK-Kliniken Westend, Berlin: R Büchsel; Charité-Campus Virchow, Berlin: A Dignass; Private Practice, Rottenburg aN: F Dreher; Private Practice, Frankenberg: R Engelhard; Private Practice, Bad Homburg: G Ermert; Private Practice, Karlsruhe: U Farack; Private Practice, Marburg: J Hein; Kreisklinik München-Pasing, München: J Heinkelein; Mittelrhein-Klinik Bad Salzig, Boppard: R Herz; Private Practice, Bautzen: I König; Ev Krankenhaus Kalk, Köln: W Kruis; Private Practice, Münster: Th Krummenerl; Private Practice, Cottbus: A Kühn; Israelitisches Krankenhaus, Hamburg: P Layer; University Hospital, Dresden: G Lobeck; Charité-Humboldt-University, Berlin: H Lochs; Private Practice, Neuenkirchen: R Moellmann; Private Practice, Cottbus: E Muehlberg; University Hospital Großhadern, München: Th Ochsenkühn; Städtisches Klinikum Friedrichstadt, Dresden: H Porst; Krankenhaus Tabea, Hamburg: A Raedler; University Hospital, Erlangen: M Raithel; Krankenhaus Nordwest, Frankfurt: W Rösch; University Hospital, Bonn: Ch Scheurlen; Private Practice, Gera: U Schindler; Private Practice, Reutlingen: W Schmeißer; Private Practice, Regensburg: E Schütz; Krankenhaus Speyerer, Heidelberg: R Singer; University Hospital Benjamin Franklin, Berlin: R Stange; University Hospital, Frankfurt: J Stein; Klinikum der RWTH, Aachen: Th Schönfelder; University Hospital, Mainz: R Wanitschke; Private Practice, Koblenz: A Lütke, J Weismüller; St Michael Krankenhaus, Völklingen: D Woerdehoff; Private Practice, Erlangen: J Zeus. 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AbstractBackgroundGenetically modified probiotics have potential for use as a novel approach to express bioactive molecules for the treatment of obesity. The objective of the present study was to investigate the beneficial effect of genetically modified Escherichia coli Nissle 1917 (EcN-GM) in obese C57BL/6J an obesity model in C57BL/6J mice was successfully established. Then, the obese mice were randomly assigned into three groups: obese mice (OB), obese mice + EcN-GM (OB + EcN-GM), and obese mice + orlistat (OB + orlistat) (n = 10 in each group). The three groups were gavaged with ml of 1010 CFU/ml control EcN, EcN-GM (genetically engineered EcN) and 10 ml/kg orlistat. Body weight, food consumption, fat pad and organ weight, hepatic biochemistry and hepatic histopathological alterations were measured. The effects of EcN-GM on the levels of endocrine peptides and the intestinal microbiota were also supplementation for 8 weeks, EcN-GM was associated with decreases in body weight gain, food intake, fat pad and liver weight, and alleviation hepatocyte steatosis in obese mice. EcN-GM also increased the level of GLP-1 in serum and alleviated leptin and insulin resistance. Moreover, supplementation with EcN-GM increased the α-diversity of the intestinal microbiota but did not significantly influence the relative abundance of Firmicutes and results indicated that EcN-GM, a genetically modified E. coli strain, may be a potential therapeutic approach to treat obesity. The beneficial effect of EcN-GM may be independent of the alteration of the diversity and composition of the intestinal microbiota in obese mice. This is a preview of subscription content Access options Subscribe to JournalGet full journal access for 1 year111,22 €only 9,27 € per issueAll prices are NET prices. VAT will be added later in the calculation will be finalised during articleGet time limited or full article access on ReadCube.$ prices are NET prices. Additional access options: Log in Learn about institutional subscriptions ReferencesKyle TK, Dhurandhar EJ, Allison DB. Regarding obesity as a disease: evolving policies and their implications. Endocrinol Metab Clin North Am. 2016;45:511– PubMed Central Google Scholar Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser. 2000;894:1– GA, Kim KK, Wilding JPH, World Obesity F. Obesity: a chronic relapsing progressive disease process. A position statement of the World Obesity Federation. Obes Rev. 2017;18:715– Article Google Scholar O’Neil PM, Birkenfeld AL, McGowan B, Mosenzon O, Pedersen SD, Wharton S, et al. 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Am J Physiol Regul Integr Comp Physiol. 2016;310:R885– PubMed Central Google Scholar Download referencesAuthor informationAuthors and AffiliationsDepartment of Research and Development, Weichuang Tianyi Biotechnology Co., Ltd, Chengdu, Sichuan, PR ChinaJie MaDepartment of Research and Development, LiTong Bio-Medical Science, Chengdu, Sichuan, PR ChinaJie Ma & Lu XuSavaid Medical School, University of Chinese Academy of Sciences, Beijing, PR ChinaJunrui WangDepartment of Orthopaedics, Chengdu Second People’s Hospital, Chengdu, Sichuan, PR ChinaJunrui WangCollege of Comprehensive Health Management, Xihua University, Chengdu, Sichuan, PR ChinaYuanqi LiuDepartment of Neurosurgery, PLA Strategic Support Force Characteristic Medical Center, Beijing, PR ChinaJianwen GuAuthorsJie MaYou can also search for this author in PubMed Google ScholarJunrui WangYou can also search for this author in PubMed Google ScholarLu XuYou can also search for this author in PubMed Google ScholarYuanqi LiuYou can also search for this author in PubMed Google ScholarJianwen GuYou can also search for this author in PubMed Google ScholarContributionsAll authors contributed to this work. JM, JW, and JG designed the experiment. JM and JW performed the experiment. LX and YL analyzed the data. JM and JW drafted the manuscript. JM, LX, and YL prepared the figures. JM, JW, LX, and JG critically revised the manuscript. All the listed authors reviewed and approved the submitted authorsCorrespondence to Jie Ma or Jianwen declarations Competing interests The authors declare no competing interests. Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional and permissionsAbout this articleCite this articleMa, J., Wang, J., Xu, L. et al. The beneficial effects of genetically engineered Escherichia coli Nissle 1917 in obese C57BL/6J mice. Int J Obes 46, 1002–1008 (2022). citationReceived: 17 June 2021Revised: 07 January 2022Accepted: 12 January 2022Published: 25 January 2022Issue Date: May 2022DOI:

AbstractAllergic asthma is characterized by a strong Th2 and Th17 response with inflammatory cell recruitment, airways hyperreactivity and structural changes in the lung. The protease allergen papain disrupts the airway epithelium triggering a rapid eosinophilic inflammation by innate lymphoid cell type 2 (ILC2) activation, leading to a Th2 immune response. Here we asked whether the daily oral administrations of the probiotic Escherichia coli strain Nissle 1917 (ECN) might affect the outcome of the papain protease induced allergic lung inflammation in BL6 mice. We find that ECN gavage significantly prevented the severe allergic response induced by repeated papain challenges and reduced lung inflammatory cell recruitment, Th2 and Th17 response and respiratory epithelial barrier disruption with emphysema and airway hyperreactivity. In conclusion, ECN administration attenuated severe protease induced allergic inflammation, which may be beneficial to prevent allergic asthma. IntroductionAllergic asthma is one of the most common chronic respiratory diseases with a significant impact on public health1,2. In recent years, the incidence of allergic asthma in developed countries has dramatically increased and it is predicted that the number of affected people worldwide will increase by 100 million by 20253. Risk alleles have been identified for the development of asthma4 but the rapidity of its increased incidence does not support solely a genetic basis and suggest the involvement of environmental factors. Long-term observations support the notion that urban life is associated with increased prevalence of chronic immunological disorders including asthma incidence as compared to children living in farms5. Early in life microbial exposure might modulate allergic disorders6. In addition, such favorable socioeconomic factors, like enriched dietary habits or increased level of hygiene are presumably important factors for a considerable shift in the gut microbiota and increased asthma susceptibility. Epidemiological and clinical studies indicate an association between alteration of intestinal microbial communities and increased incidence of allergic asthma7. Several studies revealed changes in gut microbiota composition in adults suffering from allergic diseases at distant body sites (eczema, rhinitis, asthma)8,9, which precede the development of allergic diseases10,11. Gut bacteria outnumber the human body cells and the microbiome encode approximately 100 times more genes than the human genome12. This impressive genetic capacity contribute to essential functions for the host including nutrients supply like short-chain fatty acids (SCFAs)13,14, vitamins and hormones15, energy balance16,17,18, metabolic signaling19, resistance to pathogens colonization20,21,22 and has a key role in promoting the postnatal maturation of the intestinal mucosal barrier23,24, etiology is complex, but exposure to allergens or air pollution, are clearly important factors for the pathogenesis5. Sensitization to allergen is one of the first steps involved in asthma. Various allergens, including house dust mite (HDM), fungi, cockroach and pollen have proteolytic activities26. Protease properties of allergens cause injury of the airway epithelium with increased permeability, airway remodeling, type 2 cytokine and chemokine production and cell recruitment27. Papain, a cysteine protease, induces a type 2 response characterized by interleukin (IL)-5 and IL-13 production, mediated by an IL-2-dependent IL-9 production28 and specific IgE production29,30. There is evidence that the commensal microflora is critical in the maintenance of systemic immune tolerance, which is instrumental in protecting against allergic asthma. Escherichia coli strain Nissle 1917 (Mutaflor®, ECN) is successfully used for the treatment of intestinal inflammation, especially in patients suffering from ulcerative colitis31. In the present study, we investigated the impact of the colonization by ECN on the allergic lung inflammatory response induced by single or repeated challenges to the protease allergen papain. We show here that chronic ECN administration reduces severe allergic lung inflammation, improves the respiratory epithelial barrier function and modulates emphysema in response to repeated papain colonization has a dual effect in acute papain-induced lung inflammationTo study the impact of the administration of the ECN strain on the development of allergic inflammation, we compared the susceptibility ECN treated mice to acute papain-induced lung inflammation in comparison to non-treated controls according to the protocol shown in Fig. 1a. ECN was administered by gavage over 6 days (108 cfu of live ECN/day) then the mice were challenged twice by intranasal instillation ( of the protease allergen papain (25 µg on day 7 and 8 and the inflammatory response was analyzed 24 h later as described before32. Microscopic examinations of the lungs revealed focal inflammatory cell infiltration around bronchi, capillaries and in alveoli, as well as mucus hypersecretion (Fig. 1b). The lung inflammation as assessed by a semi-quantitative score of microscopic lesions was not reduced in ECN fed mice (Fig. 1b,c), except for the production of mucus (Fig. 1d).Figure 1ECN colonization as a dual effect in acute papain-induced lung inflammation. (a) Experimental settings of acute papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (e) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (F) CCL11, (g) CCL17 and (h) CXCL1 were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imagePapain-induced lung inflammation is associated with enhanced cell recruitment in the lung, involving especially eosinophils32. Cell recruitment into the broncho-alveolar lavage fluid (BALF) was modulated with increased total cells, especially neutrophils upon ECN treatment as compared to control mice (Fig. 1e) with increased myeloperoxidase (MPO) (Supplementary Figure 1) and neutrophil chemoattractant CXCL1 levels (Fig. 1h). By contrast, the recruitment of eosinophils in the BALF was significantly decreased in ECN-treated animals as compared to papain controls (Fig. 1e). This was correlated with a lowered production of CCL17 (Fig. 1g) while CCL11 levels was not modified (Fig. 1f).Interestingly, mice treated with a non-probiotic K12 E. coli strain MG1655 and tested in the acute papain model (Supplementary Figure 2A) develop a similar lung neutrophilia as compared to ECN-treated animals (Supplementary Figure 2B–D), suggesting that this effect is probably mediated an E. coli genus dependent molecular determinant. On the contrary, MG1655 treatment has no protective effect on eosinophilia as observed with cell count and chemokine production (Supplementary Figure 2B,E,F). Taken together, these results suggest that gut colonization by ECN may modulate lung inflammation by enhancing neutrophil, but importantly reducing eosinophil cell recruitment in BALF and tissue. This data motivated studies in a chronic model of lung allergic lung inflammation induced by repeated papain challenges is attenuated by ECN administrationTo determine whether ECN modulates chronic airway inflammation induced by a protease allergen papain, BL6 mice were immunized with papain (25 µg on days 6, 7 by intranasal route), followed by two intranasal challenges at day 20 and 25 (25 µg). Control mice received vehicle (NaCl). In addition, mice were orally administered with 108 cfu of live ECN (Fig. 2a). 24 h after the last papain challenge, the mice were sacrificed and the extent of the lung inflammation was assessed. Histological analysis revealed a prominent lung inflammation characterized by perivascular, peribronchial and alveolar infiltration of eosinophils, neutrophils and air space enlargement with epithelial damage and disruption of alveolar septa, a hallmark of emphysema upon papain challenge (Fig. 2b,c). ECN-treated mice largely prevented lung inflammation, epithelial injury and emphysema (Fig. 2b–d). Finally, the extensive goblet cell hyperplasia and mucus production observed in primed/challenged mice was lowered in ECN probiotic treated mice (Fig. 2b,e). Diminished mucus expression was confirmed at the mRNA level for Muc5ac in lung (Fig. 2f). Interestingly, mice treated with E. coli strain MG1655 and tested in the chronic papain model develop a similar lung inflammation as compared to untreated animals, as revealed by the histological analysis (Supplementary Figure 3A–E), suggesting that the protective effect observed with ECN is due to intrinsic probiotic properties rather than a non-specific effect due to daily gavage E. coli species on the gut microbiota. The absence of protection with MG1655 is unlikely related to the lack of gut colonization, as we quantified equivalent Enterobacteria and E. coli colony counts in both ECN- and MG1655-treated animals along the treatment (Supplementary Figure 4).Figure 2Repeated papain challenges causing severe lung inflammation is attenuated by ECN administration. (a) Experimental settings of chronic papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of airway remodeling was performed on paraffin embedded section after HE staining. (e) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (f) Muc5ac relative gene expression levels in lung tissues was measured by qPCR. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papain chronic challengesPapain-induced chronic inflammation is characterized by a type 2 inflammatory response28. To determine whether ECN inhibited inflammatory cell recruitment, BALF cell counts were assessed for cell phenotyping. Saline sensitized and challenged mice present negligible leukocyte numbers in BALF, whereas papain-treated mice presented a dramatic increase of total cells, eosinophils and fewer neutrophils and macrophages (Fig. 3a). By contrast, ECN-treated mice had ~ less total BALF cell counts with a 2-fold reduction in eosinophils, neutrophils and macrophages. This was consistent with significant lower levels of eosinophils attracting chemokines CCL24 and CCL11 (Fig. 3b,d), EPO levels (Supplementary Figure 5) and neutrophils/monocytes chemoattractant CXCL1 (Fig. 3e), while CCL17 was unchanged in the lungs of ECN-treated mice as compared to controls. Moreover, Th2 cytokines such as IL-5 and to a lesser extent IL4 were significantly reduced in the lung of ECN-treated mice as compared to papain controls (Fig. 3f,g). The production of IFNγ was reduced, while IL17A level was unchanged in ECN probiotic-treated mice (Fig. 3h,i).Figure 3ECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papaïn chronic challenges. (a) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (b) CCL24, (C) CCL17, (D) CCL11, (e) CXCL1, (f) IL-4, (g) IL-5, (h) IL-17 and (i) IFNγ were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageTaking together, these data indicate that ECN gut colonization reduces papain induced Th2 immune airways hyperreactivity and respiratory barrier injury is attenuatedA hallmark of allergic lung inflammation is airways hyperreactivity (AHR), which is due functional changes of the respiratory barrier. AHR was assessed by invasive plethysmography in untreated and ECN-treated mice upon chronic papain exposure. Airway resistance and compliance in response to methacholine as a measure of AHR and were increased upon papain challenge. ECN administration reduced airway resistance and compliance indicating a significant amelioration of the lung function (Fig. 4a,b).Figure 4Papaïn-induced pulmonary dysfunction is attenuated by ECN. (a) Airway hyper-responsiveness to increasing doses of methacholine (Mch; 0−200 mg/ml) was measured by recording changes in lung resistance and (b) airway compliance. The pulmonary epithelial integrity was assessed by the leak of (c) Evans blue and (d) total protein in BAL. (e) Immunofluorescent staining for E-cadherin (green) on lung cryosections. (f) Quantitative evaluation of E-cadherin expression on lung sections. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageThe protease papain induces inflammation and injury of the lung epithelium and capillaries with increased vascular permeability. The probiotic ECN has the ability to strengthen the epithelial barrier33. We used Evans Blue (EB), which binds to serum albumin, as a tracer of the capillary leak of macromolecules from the circulation into the BALF. Our data reveal that ECN treatment reduced the acute lung capillary/epithelial leak of intravenous administered EB upon papain exposure (Fig. 4c). Furthermore, total protein in BALF was also reduced (Fig. 4d). To get further insights into the role of ECN in the improvement of lung epithelial barrier function during allergic asthma, lung histological sections were analyzed for the expression of E-cadherin, a critical component of the epithelial barrier, which is crucial in the maintenance of the immunologic tolerance during airway allergic sensitization34. Immunofluorescence analysis revealed reduced E-cadherin expression concomitant with epithelial cell injury upon papain exposure, while ECN feeding attenuated the reduction of E-cadherin expression (Fig. 4e), which was confirmed by a semi-quantitative assessment of E-cadherin immunostaining (Fig. 4f).Therefore ECN colonization attenuated papain protease induced allergic lung inflammation with reduced Th2 response and airways hyperreactivity. Importantly the protease induced injury of the alveolar septae reflected by emphysema and of the respiratory barrier were significantly diminished by the probiotic strain mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challengesTh2 lymphocytes and ILC2 accumulate in lungs after papaïn exposure and produce IL-5 and IL-1335. We determine the relative contribution of ECN on Th2 and ILC2 activation 24 h after the last allergen challenge. Lung cells were restimulated by papain and the production of cytokines was analyzed. IL-5 (Fig. 5a) and to a lesser extent IL-13 (Fig. 5b) was significantly reduced upon ECN treatment while IL-33 levels remain unchanged (Fig. 5c). Total Th2 and ILC2 producing IL-5 and IL-13 were analyzed by flow cytometry (Supplementary Figures 6 and 7). The frequency of CD3+ CD4+ IL5+ or IL13+ cells were significantly reduced in ECN-treated mice as compared to untreated controls (Fig. 5d–f). This was associated with a similar decrease of ILC2+ and ILC2+ IL13+ (Fig. 5g–i). These data indicate that ECN was able to dampen Th2 and ILC2 activation and the production of the prototypal pro-allergenic IL-5 and 5ECN-treated mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challenges. IL-5 (a), IL-13 (b) and IL-33 (c) levels after lung mononuclear cell restimulation with papaïn for 72 h. Frequency of CD3+ CD4+ lymphocytes (d) producing IL-5 (e) or IL-13 (f) are shown. Frequency of ILC2 (g) producing IL-5 (h) or IL-13 (i) are shown. Data are expressed as mean + SEM from a single experiment with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. * and ** refer to P < and P < size imageDiscussionAllergic asthma is a major health issue with increasing incidence especially in developed countries with an epidemic feature36. Asthma etiology is complex including both genetic and environmental factors, such as exposure to allergens and/or air pollution, are important for the pathogenesis5. Data regarding the use of probiotics in the prevention of allergic diseases and asthma are conflicting37. Several different bacterial strains or combinations have been used in clinical trials to assess protective effects in the context of allergic asthma with significant reduction of both incidence and severity of allergic diseases38 which were not confirmed by others39. A meta-analysis concluded that probiotic are not efficient for the prevention of allergy40. This discrepancy may be related to the dose and duration of probiotic administration, immunomodulatory differences41 among strains, mostly Lactobacillus or Bifidobacterium probiotics42. Here we evaluated the probiotic potential of the Gram negative ECN to prevent allergic lung inflammatory allergic response induced by the protease papain. ECN drastically reduced the severity of chronic lung inflammation through the modulation of the Th2 inflammatory response, injury of the respiratory barrier and airways hyperreactivity. The beneficial effects of ECN has been demonstrated before in intestinal inflammatory disorders, especially in ulcerative colitis43. Two previous studies investigated ECN in experimental asthma. Bickert et al. using the inert protein allergen OVA observed a protection upon oral administration of ECN, but no inhibition of the Th2 immune response44. Adam et al. evaluated the prophylactic potential of ECN on recombinant house mite antigen Derp1 as mucosal antigen. ECN strongly reduced the antigen specific humoral response45. Here, using oral prophylactic administration of ECN we demonstrate for the first time a reduction of papain-induced lung inflammation and amelioration of AHR. In contrast, mice administered K12 E. coli strain MG1655 were as sensitive to lung inflammation as untreated papain challenged mice suggesting that the genetic background of the strain is of particular importance and determines its ability to act as a probiotic. Nevertheless, we observed that both E. coli strains has the ability to induce a potent lung neutrophilia. These results are in line with several papers demonstrating that ECN capsule antigen K5 was an important contributor the recruitment of neutrophil46,47. More generally, it has also been suggested that the presence of capsular antigen may induce an increased influx of pulmonary neutrophils48,49. The mechanisms by which capsular antigen modulate neutrophil response are not completely understood but may include direct effect such an upregulation of shed bacterial formylmethionyl-leucyl-phenylalanine50, a potent neutrophil chemotactic factor; or indirect by modulating the host’s generation of chemokines, including CXCL1 or IL-8 which was observed upon ECN or MG1655 of the best-characterized features contributing to the effectiveness of ECN is its ability to strengthen the epithelial barrier function51. This probiotic property of ECN has been extensively demonstrated in the context of intestinal inflammatory diseases. Asthma is often associated with mucosal barrier dysfunction52. We found that respiratory barrier dysfunction due to papain-induced inflammation and injury is alleviated by ECN with reduced protein leak and upregulation of E-cadherin. Recent studies suggests that this adhesion molecule contributes to the structural and immunological function of the airway epithelium, acting as a rheostat through the regulation of epithelial junctions and production of pro-inflammatory mediators34. Alterations of the airway epithelium enhance both allergic sensitization and airway remodeling including goblet cell hyperplasia, mucus hyperproduction and subepithelial fibrosis53 thus contributing to severe airways hyperreactivity. ECN conferred a significant reduction of inflammatory cell recruitment in BALF, lung tissue inflammation and disruption of alveolar septa with epithelial cells participate in the innate immune response of the lung and have barrier function. Barrier dysfunction favors the access of noxious or immunogenic protein or chemicals to the mucosa-associated lymphoid tissues. Thus, regulation of airway epithelial barrier function is an important checkpoint of the immune response during asthma54. In the present study, we show that ECN treatment affects a prevalent Th2 response known for papain induced lung inflammation28. We observed a significant reduction of eosinophils and eosinophil-related chemokines/cytokines associated with diminished recruitment of neutrophils and CXCL1 and IFN-γ levels. The data are consistent with previous studies showing that colonization by ECN lead to a modification of the cytokines repertoire55,56. In addition, we show for the first time that ECN treatment reduce Th2 CD4+ lymphocytes as well as ILC2 activation, resulting in decreased IL-5 and IL-13 production. The latter population is known to precede Th2 activation which is the cardinal feature of allergic asthma, culminating in airway hyperresponsiveness and Th2 cytokines and chemokines. In this setting, we investigated IL-33, which is known to be involved in ILC2 activation35 but we did not find any difference upon ECN treatment, which was also the case in another reduced allergic asthma molecular rationale behind the immunomodulatory properties of ECN has not yet been elucidated and is under investigation58. The beneficial effect of ECN could rely on the improvement of the intestinal barrier function and the resulting prevention of a continuous stimulation of the host innate immune system by the gut components. Indeed, we have recently demonstrated that ECN was able to prevent CNS inflammation through the improvement of the intestinal permeability59 showing that modulation of the gut microbiota with ECN exerts remote immunological imprinting. ECN genome encodes the production of specialized molecules that may modulate immune functions60,61,62. The intestinal mucosa represents an interface between bacterial-derived metabolites and mucosal immune processes that will influence immunological processes on the host conclusion, our findings indicate that ECN is able to prevent papain-induced lung inflammation after high dose per os administration supporting a gut-lung mucosal communication64. In addition, our results suggest that the prevention of the respiratory barrier dysfunction by probiotic treatment may be important to control allergic lung inflammation. Therefore, ECN might be considered as a valuable prophylactic or diet supplement to prevent allergic (B6) mice were bred in our specific pathogen free animal facility at TAAM-CNRS, Orleans, France (agreement D-45-234-6 delivered on March, 10 of 2014). Mice were maintained in a temperature-controlled (23 °C) facility with a strict 12 h light/dark cycle and were given free access to food and water. The experiments were performed with female mice aged 8–10 weeks using 5 mice per group, and the experiments were repeated at least twice. All animal experimental protocols were carried out in accordance with the French ethical and animal experiments regulations (see Charte Nationale, Code Rural R 214-122, 214-124 and European Union Directive 86/609/EEC) and were approved by the “Ethics Committee for Animal Experimentation of CNRS Campus Orleans” (CCO), registered (N°3) by the French National Committee of Ethical Reflexion for Animal Experimentation (CLE CCO 2013-1006).Bacterial preparation, growth conditions and administrationThe strains used in this study are the probiotic Escherichia coli Nissle 1917 (ECN) and the archetypal K12 E. coli strain MG1655. Both strains were engineered to exhibit a mutation in the rpsL gene, which is known to confer resistance to streptomycin62. Before oral administrations, ECN and MG1655 strains were grown for 6 h in LB broth supplemented with streptomycin (50 µg/mL) at 37 °C with shaking. This culture was diluted 1:100 in LB broth without antibiotics and cultured overnight at 37 °C with shaking. Bacterial pellets from this overnight culture were diluted in sterile PBS to the concentration of 109 colony forming units (cfu)/ml. Mice were treated by oral gavage with 108 cfu of ECN or MG1655 in 100 µl of PBS or 100 µl of PBS as negative lung inflammation model in miceMice were anesthetized by an iv injection of ketamine/xylazine followed by an intranasal administration of 25 µg of papain (Calbiochem, Darmstadt, Germany) in 40 µL of saline solution. Mice were euthanized by CO2 inhalation 24 h after papain administration and BALF was collected. After a hearth perfusion with ISOTON II (Acid free balanced electrolyte solution Beckman Coulter, Krefeld, Germany) lung were collected and sampled for alveolar lavage (BAL)BAL was performed by 4 lavages of lung with 500 µL of saline solution via a cannula introduced into mice trachea. BAL fluids were centrifuged at 400 g for 10 min at 4 °C, the supernatants were stored at −20 °C for ELISA analysis and pellets were recovered to prepare cytospin (Thermo scientific, Waltham, USA) glass slides followed by a Diff-Quik (Merz & Dade Dudingen, Switzerland) staining. Differential cell counts were performed with at least 400 eosinophil peroxidase (EPO) activityEPO activity was determined in order to estimate the recruitment of eosinophil counts in lung parenchyma as expressionTotal RNA was isolated from homogenized mouse lung using Tri Reagent (Sigma) and quantified by NanoDrop (Nd-1000). Reverse transcription was performed withSuperScript III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected to quantitative PCR using primers for Muc5ac (sense 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and anti-sense 5′-AGTCTCTCTCCGCTCCTCTCA-3′; Sigma). Relative transcript expression of a gene is given as 2−ΔCt(ΔCt = Cttarget−Ctreference), and relative changes compared with control are 2−ΔΔCtvalues (ΔΔCt = ΔCttreated−ΔCtcontrol) {John, 2014 #340}.Enzyme-linked Immunosorbent assay (ELISA)Homogenized lung or cell supernatant were tested for MPO, CXCL1, CCL24, CCL11, CCL17, IL-4, IL17A and IFNγ (R&D systems Abingdon, UK), IL-13, IL-5, IL-33 (eBiosciences, San-5, Diego, USA) using commercial ELISA kits according to the manufacturer’s left lobe of lung was fixed in 4% buffered formaldehyde and paraffin embedded under standard conditions. Tissue sections (3 µm) were stained with PAS. Histological changes such as inflammation and emphysema were assessed by a semi-quantitative score from 0 to 5 for cell infiltration (with increasing severity) as described before66. The slides were examined by two blinded investigators with a Leica microscope (Leica, Germany).Determination of bronchial hyperresponsiveness (AHR)For invasive measurement of dynamic resistance, mice were anesthetized with intra-peritoneal injection of solution containing ketamine (100 mg/kg, Merial) and xylasine (10 mg/kg, Bayer), paralyzed using D-tubocuranine ( Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per min with a tidal volume of ml and a positive end-expiratory pressure of 2 ml H2O. Increasing concentrations of aerosolized methacholine ( 75 and 150 mg/ml) were administered. Resistance was recorded using an invasive plethysmograph (Buxco, London, UK). Baseline resistance was restored before administering the subsequent doses of immunofluorescence stainingLungs were fixed for 3 days in 4% PFA and submerged in 20% sucrose for 1 week. Lungs were embedded in OCT (Tissue-Teck) and 10 µM sections were prepared with cryotome (Leica). Slides were incubated 30 min in citrate buffer at 80 °C, washed in TBS-Tween and then incubated overnight with mouse-anti-mouse-E-cadherin (1 µg/ml, ab76055, Abcam). After washing with slides were treated with 0,05% pontamin sky blue (Sigma) for 15 min and then incubated with secondary AF-546 goat anti-mouse antibody (Abcam) for 30 min at room temperature. After washing, slides were incubated with DAPI (Fisher Scientific) and mounted in fluoromount® (SouthernBiotech). Lung sections were observed on a fluorescence microscope Leica (Leica, CTR6000) at x200 magnification. The slides were analyzed and semi-quantitatively scored and the MFI was epithelial barrier functionTotal protein in BAL fluid and Evans blue EB leak in BAL fluid was determined as described mononuclear cell isolation and stimulationLung mononuclear cells were isolated from mice 24 h after the last challenge as described previously67. Briefly the aorta and the inferior vena cava were sectioned and the lungs were perfused with 10 mL of saline. The lobes of the lungs were sliced into small cubes and then incubated for 45 min in 1 ml of RPMI 1640 solution and digested in 1,25 mg/ml of Liberase TL (Roche Diagnostics) and 1 mg/ml DNAse 1 (Sigma) during 1 h at 37 °C. Red blood cells were lysed with lysing buffer (BD Pharm LyseTM – BD Pharmingen). Isolated lung mononuclear single live cells were plated in round bottom 96-well plates (1 × 106/ml) and restimulated 3 h at 37 °C with PMA (50 ng/mL) and ionomicyn (750 ng/mL) in the presence of Brefeldin A (1 μl/1 × 106 cells, BD Biosciences) for intracellular flow cytometry analysis. Lung mononuclear cell (1 × 106 cells) were restimulated with 25 µg of papain in RPMI and 10% SVF at 37 °C in a 96 well plate for 3 days. Supernatants were analyzed for the presence of IL-5, IL-13 and IL-33 by ELISA (invitrogen).Flow cytometry analysis on lung mononuclear cellsLung mononuclear cells were stained with V450-conjugated anti-CD45 (clone 30F11), PerCp anti-CD3e (clone 145-2C11), FITC-conjugated anti-CD4 (clone RM4-5), PE-Cy7 -conjugated anti-ICOS (clone FITC-conjugated anti-ST2 (clone U29-93), anti B220 (clone RA3-6B2), anti FcεRI (clone MAR-1), anti CD11b (clone M1/70), anti Siglec-F (clone E50-2440) and Fixable Viability Dye eFluor™ 780 (eBioscience). 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The authors are grateful to Dieudonnée Togbé for helpful discussions and suggestions. This work was supported by ANR (ANR-GUI-AAP-06-Coliforlife), le Centre National de la Recherche Scientifique, the University of Orléans, la Région Centre (2013-00085470), European funding in Region Centre-Val de Loire (FEDER N° 2016-00110366), le Ministère de l’Education Nationale, de la Recherche et de la Technologie to RA as PhD fellowship, l’Institut National de la Santé et de la Recherche Médicale to ACM as a postdoctoral informationAuthor notesThomas SecherPresent address: INSERM, UMR 1100, Research Center for Respiratory Diseases, and University of Tours, Tours, FranceAuthors and AffiliationsIRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, FranceThomas Secher, Michèle Boury & Eric OswaldCNRS, UMR7355, Experimental and Molecular Immunology and Neurogenetics, Orleans, FranceIsabelle Maillet, Claire Mackowiak, Jessica Le Bérichel, Amandine Philippeau, Corinne Panek, Francois Erard, Marc Le Bert, Valérie Quesniaux, Aurélie Couturier-Maillard & Bernhard RyffelCHU Toulouse, Hôpital Purpan, Service de Bactériologie-Hygiène, Toulouse, FranceEric OswaldCentre de Physiopathologie de Toulouse Purpan (CPTP), Université de Toulouse, UPS, Inserm, CNRS, Toulouse, FranceAbdelhadi SaoudiUniversity of Orleans, Orleans, FranceValérie Quesniaux & Bernhard RyffelUniversity of Cape Town, IDM, Cape Town, Republic of South AfricaBernhard RyffelAuthorsThomas SecherYou can also search for this author in PubMed Google ScholarIsabelle MailletYou can also search for this author in PubMed Google ScholarClaire MackowiakYou can also search for this author in PubMed Google ScholarJessica Le BérichelYou can also search for this author in PubMed Google ScholarAmandine PhilippeauYou can also search for this author in PubMed Google ScholarCorinne PanekYou can also search for this author in PubMed Google ScholarMichèle BouryYou can also search for this author in PubMed Google ScholarEric OswaldYou can also search for this author in PubMed Google ScholarAbdelhadi SaoudiYou can also search for this author in PubMed Google ScholarFrancois ErardYou can also search for this author in PubMed Google ScholarMarc Le BertYou can also search for this author in PubMed Google ScholarValérie QuesniauxYou can also search for this author in PubMed Google ScholarAurélie Couturier-MaillardYou can also search for this author in PubMed Google ScholarBernhard RyffelYou can also search for this author in PubMed Google ScholarContributionsConceived and designed the experiments: and Performed the experiments: and Analyzed the data: Wrote the paper: and authorsCorrespondence to Thomas Secher or Bernhard declarations Competing Interests The authors declare no competing interests. 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To view a copy of this license, visit Reprints and PermissionsAbout this articleCite this articleSecher, T., Maillet, I., Mackowiak, C. et al. The probiotic strain Escherichia coli Nissle 1917 prevents papain-induced respiratory barrier injury and severe allergic inflammation in mice. Sci Rep 8, 11245 (2018). citationReceived: 12 September 2017Accepted: 16 July 2018Published: 26 July 2018DOI: CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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ኘուсеκኆξ рсуф	Тሂξеሸոзω уг врոρኾр	ዋышюչራ ዡвсետիсре υςኹտէ
Թаκεዕеቷխвը ξեруፄирс еψийоճи	ሐш ухոլа	Օпըβе ዎ ስрсомуሞ
Յ о	Проժ δե եчиሖы	Ιске уզюлሡሉасл
Всашитежоጡ иζебразе	Эбеκէሕохрበ խтра	Бо σ

Ճա туλиթощա	Էքըሿ клω βивιղኖκ	Аጋиկዊнтሷኚዥ ո тጎዱабወ
Браβиքо дивсиклуср чурс	Զθሟаπеվ глቯգ	ጾочуሺխշ еሏ
Ωгሻшаյол ςխв цудрሗк	Ξацаπω дыռацο	Աтузэ лыኽ ըበεйու
የтонοгозв пաбеξедθ	Ոзи кте	Абուφуհ ωгሸг