MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the “compact”, InternalinB-bound conformation, but not when MET is in the “open” conformation. These findings provide further support for the importance of the “compact” conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the  In principle, each of these stages may be targeted in therapy, and the ability to target multiple stages simultaneously is an attractive prospect.A major challenge in the therapy of solid tumors, and notably of carcinomas that constitute over 85% of all human cancers, is the development of agents that inhibit metastasis, namely the growth of cells derived from the primary tumor at distant sites in the body. Metastasis is a multi-stage process in which cancer cells migrate into adjacent tissues (invasion), cross the wall of blood or lymphatic capillaries to be transported across the general circulation (intravasation), exit from the bloodstream into a secondary tissue/organ (extravasation) and finally give rise to secondary tumorsMET proto-oncogene, has multiple and essential physiological roles in vertebrate embryogenesis, where it is required for normal development of the liver parenchyma and the labyrinth layer of the placenta3 as well as distant migration of the myogenic precursor cells4. HGF/SF and MET have further and important physiological roles in postnatal life, where they control the regeneration after injury of several epithelial organs including skin5 and liver7. They also exert, however, multiple and crucial roles in the early stages of metastasis of epithelial cancers by controlling (i) delamination of epithelial cells and the process of epithelial-mesenchymal transition crucial for long distance epithelial cell migration8, (ii) degradation of the basement membrane and remodeling of the extracellular matrix via urokinase9 and matrix metalloproteinases10, (iii) integrin-dependent migration of cancer cells as a result of activation of focal adhesion kinase and paxillin11, (iv) formation of the pre-metastatic niche via tumor-derived exosomes12, (v) tumor lymphangiogenesis14, a process essential for lymphatic metastasis and, (vi) haemangiogenesis16. Further, there is growing evidence for a major role of HGF/SF and MET in the maintenance of cancer stem cells in colon17, breast18 and prostate19 carcinomas, and accumulating reports of involvement of the MET-HGF/SF axis in cancer cell resistance to targeted therapies both in vitro23 and in cancer patients26.The signaling system mediated by the growth and motility factor HGF/SF and its receptor MET, the tyrosine kinase encoded by the 27. The ectodomain is a heterodimer arising from furin cleavage of a single precursor chain and consists of an N-terminal 283 amino acid α-chain and the 625 extracellular amino acids of the β-chain. An N-terminal semaphorin homology domain (SEMA), which binds the ligand HGF/SF, is followed by a “stalk” comprising one cysteine rich (CR) domain and 4 immunoglobulin domains termed Ig1-Ig428. The MET ectodomain has been expressed recombinantly as a series of truncations containing the SEMA, CR and 0, 2 or 4 Ig domains, which we will refer to as MET567, MET741 and MET928, respectively. HGF/SF consists of an amino terminal domain, 4 kringle domains (K1 - K4) and a C terminal serine protease homology domain (SPH domain). The N terminal and first kringle domain (NK1) of the HGF/SF ligand drive a high affinity interaction with MET29, and the SPH domain also binds MET30. The SEMA domain has been reported as necessary and sufficient for HGF/SF binding28, although an interaction with the Ig3-Ig4 “lower stalk” region has also been reported31.The MET receptor is a single-pass transmembrane protein consisting of a large extracellular ectodomain, a transmembrane segment and an intracellular receptor tyrosine kinase domainin vitro and in in vivo. We also report a crystal structure of the 107_A07 Fab in complex with a MET receptor fragment containing amino acids 519–740, which includes the CR, Ig1 and Ig2 domains but not the ligand-binding SEMA domain. These data provide insights into the conformation of MET during ligand binding and cell signaling.Here, we report the isolation of a new human, phage-derived monoclonal antibody to MET (107_A07) displaying potent receptor antagonistic activity, and we describe the activity of 107_A07 on cancer cells 28 was produced from CHO Lec3.2.8.1 cells and purified by affinity chromatography  followed by cation exchange . 107_A07 and D1.3 Fabs and IgG –formatted antibodies were produced by transfection of suspension HEK293F cells (Invitrogen) with Valproic Acid (Sigma) added to 4 mM following transfection. Fab proteins were purified using affinity resins KappaSelect and/or GammaBind Plus (GE Life Sciences). IgG formatted antibodies were purified by Protein A affinity chromatography. For cell cycle analysis, 107_A07 and D1.3 Fabs were further purified by gel filtration chromatography (Superdex 200 10/300 (GE Life Sciences). Unless stated otherwise, 7A2 scFv was produced in Pichia pastoris and purified by Ni-NTA chromatography followed by gel filtration. Anti-MET antibody 5D5 sequences were obtained from US Patent No. 7,476,724 B2. Heavy and light chains were synthesized  with restriction sites that allowed cloning into Fab vectors pBIOCAM1-3F and pBIOCAM3-3F, expressed in HEK293F cells and the Fab purified by Ni-NTA chromatography.Soluble MET741 protein32.Recombinant Fab were partially reduced with TCEP and PEGylated with maleimide-activated PEG . PEG-Fab and free PEG were monitored by SDS-PAGE and barium chloride & iodine staining33 was performed on solid-phase recombinant MET928 and light chain shuffling performed on the output by cloning the resulting VH gene pool back into the original scFv phage library34. Biopanning with the chain-shuffled library (109 clones) was performed with biotinylated MET928 in solution and streptavidin-coated dynabeads. Phage pools were cloned into expression vector35 and small-scale expressions performed in BL21 (DE3) bacteria in 96-well format. Approximately 960 supernatants were screened directly for inhibition of HGF/SF-induced scatter of BxPC-3 human pancreatic cancer cells. Affinity maturation was performed by diversification of the CDR3 regions of 7A2 VH and VL using oligonucleotide-directed mutagenesis and stringent selection of the resulting phage library with biotinylated MET928 in solution.Biopanning with a scFv phage libraryIn vitro angiogenesis assay was performed using the modified co-culture assay as described previously36. Briefly, fibroblast cells were seeded in gelatin-coated chamber slides. Human umbilical vein endothelial cells (HUVECs) were seeded on to the confluent fibroblasts and D1.3 and 107_A07 antibodies (200 nM) were added to the cells. The co-cultures were fixed and stained for CD31. Number of tubules was counted manually from 10 fields for each well and the field area was measured using AngioSys 1.0 imaging software.HGF/SF-induced cell migration across a porous membrane coated with 100 µg/ml collagen  was analyzed using a modified Boyden chamber assay. Cells (SKOV-3 or U87MG) were labeled with the fluorescent dye Calcein AM (Life Technologies) and migration assessed by quantification of fluorescence on the underside of the membrane using a Typhoon instrument (GE Life Sciences). Data were analysed with ImageQuant software and background fluorescence subtracted. For cell cycle analysis, U87MG human glioblastoma cells were serum-starved for 48 hours prior to a 24 hour incubation with 300 pM HGF/SF with or without 0.9 µM 107_A07 FAb or 1 µM D1.3 FAb. Cells were trypsinised, fixed, stained with propidium iodide in the presence of RNAse and analyzed by flow cytometry according to standard procedures. nu/nu mice (Crl:NMRI-Foxn1nu) were obtained from Charles River Laboratories . U87MG cells were obtained from ECACC and identity checked at the DSMZ. Mice were injected subcutaneously with 107 U87MG human glioblastoma cells and antibody administered every 3–5 days between day 7 and 33, when treatment stopped. Groups of 8 mice were treated with 107_A07 IgG at either 2 mg/kg or 10 mg/kg. Control animals were treated with either PBS or D1.3 IgG at 10 mg/kg. All animal experiments were carried out in accordance with the United Kingdom coordinating committee on cancer research regulations for the welfare of animals and the German Animal Protection Law, and were also approved by the local responsible authorities Landesamt für Gesundheit und Soziales Berlin (LAGeSo).NMRI D was determined using a Biacore instrument and represents the mean of three experiments using 107_A07 FAb purified sequentially on both KappaSelect and GammaBind Plus chromatographic resins. For competition analysis by SPR, a CM5 chip coated with MET928 was exposed to: 7A2 scFv , NK1  or a mixture of the two .KFabs were mixed with 100 nM MET928 one hour before addition to microplates coated with HGF/SF or the fragment NK1. After one hour bound MET928 was detected with anti-5xHis (Qiagen) followed by DELFIA® Eu-N1 rabbit anti-mouse-IgG. DELFIA® enhancement solution was then added and signal quantitated using time-resolved fluorescence with a Fusion instrument (Perkin Elmer).Fab and MET proteins were co-incubated at a 3:2 molar ratio for 140 minutes at room temperature in 25 mM Tris pH 7.4, 150 mM NaCl, centrifuged and analyzed by size exclusion chromatography (Superdex 200 10/300).Briefly, Fab 107_A07 and his-tagged MET741 were co-incubated, then digested with Pepsin and EndoHf deglycosidase. The remaining complex was purified by Ni-NTA affinity resin and size exclusion chromatography, concentrated to 5.9 mg/ml and crystallized by the sitting-drop vapor diffusion method at 19 °C.free values reached 21.5% and 25.7% respectively. The coordinates of the structure have been deposited to the Protein Data Bank under accession ID 5LSP. Additional method information is provided in the Supplementary Information file.X-ray data collection experiments were performed at the European Synchrotron Radiation Facility (Grenoble France), beamline ID29. The crystals diffracted to a maximum resolution of 2.6 Å. The crystals contained two molecules of the MET/Fab complex in the asymmetric unit. The crystal structure was solved using the Molecular Replacement (MR) method. After several rounds of manual rebuilding and refinement the R/RD.D.C., A.P., J.M.C. and E.G. are co-inventors on a patent application relating to the antibodies described in this manuscript. D.D.C. is currently an employee of Genentech, Inc.10 clones formatted as single chain Fvs (scFvs)33. The selected population was affinity matured en masse by chain-shuffling the selected population of VH genes34. A chain-shuffled scFv library of 109 clones was constructed and was subjected to stringent selection using biotinylated MET928 to allow the emergence of new VH/VL combinations with potentially superior binding properties to the original. In addition to affinity-based approaches, a “competitive elution” selection was performed in parallel in which MET-bound phage populations were incubated with high (micromolar) concentrations of the ligand HGF/SF in an effort to elute HGF/SF-competitive antibodies. Selected populations were cloned into a bacterial expression vector35 and individual clones were screened directly for functional activity using a colony scatter assay. Hits emerging from this screen were assessed in a more quantitative cell migration assay, based on migration of fluorescently labeled SKOV3 human ovarian cancer cells using a Boyden chamber. A number of clones were identified that inhibited HGF/SF-induced migration of SKOV3 cells, including clone 7A2 . Fab are much less likely than scFv to form higher-order species, and so we converted 7A2 to a Fab format.Recombinant antibodies recognizing MET928 were generated by phage display using two rounds of panning on immobilized antigen. Binders were generated using a previously described human antibody phage display library of ~10D in the nanomolar range .Conversion of 7A2 to a Fab format diminished the agonistic activity as expected but also resulted in reduced affinity and potency, therefore an additional round of affinity maturation was conducted using oligonucleotide directed mutagenesis to create phage display libraries where the CDR3 region of the V gene heavy chain (VH) was diversified. Mutagenic primers with contiguous blocks of 5–6 randomized amino acids using 3 overlapping primers were used to randomize the 11 amino acids of the VH CDR3 and 9 amino acids from VL CDR3. Stringent selections were carried out using limiting amounts of biotinylated MET928 down to 10 pM. DNA from the output of the resulting selections was subcloned into a bacterial expression vector and 1152 clones were screened by ELISA and sequenced. 146 clones with unique sequences that were positive in ELISA were identified for further study. VL mutants were not identified, suggesting this library was ineffective or was dominated by the parental sequence. The selected clones were expressed, purified and the off-rates compared to the parental 7A2 clone using surface plasmon resonance (SPR). The top 8 clones showing the greatest improvement in off-rate were identified and produced as Fab fragments. Sequence analysis revealed that the top group of clones came from VH CDR3 mutagenesis and all carried a Y105W mutation. In addition, 6/8 of these clones retained G106 and M107 despite the complete diversification within this region of the selected clones. Based on affinity and potency rankings, the affinity-matured clone 107_A07 was selected for further biological and biochemical analysis. SPR experiments yielded a K38. We assessed 107_A07 Fab for inhibition of these processes. Inhibition of cell migration was compared with 7A2, the parental antibody of 107_A07, and as a control we also included a Fab-formatted version of 5D5.v1, the monoclonal antibody from which onartuzumab was derived37. 107_A07 Fab dose-dependently inhibited HGF/SF-induced cell migration of SKOV-3 human ovarian cancer cells , followed by DNA content analysis by propidium iodide staining. U87MG cells express HGF/SF40 and divide rapidly in culture, however addition of exogenous HGF/SF caused a further and measurable increase in DNA synthesis  chains to the C-terminal cysteine residues41. The majority of unconjugated PEG was removed by size exclusion chromatography and ultrafiltration. In SDS-PAGE, PEG-107_A07 Fab chains displayed reduced migration and insensitivity to reducing agents, consistent with removal of the inter-chain disulphide bond  tubulogenesis (a process that recapitulates in vitro the sprouting of new capillaries in vivo)  Fig. , PEG-107vo) Fig. .The VH and VL domains of 107_A07 were expressed as an intact human IgG and the activity of 107_A07 IgG and control (D1.3) IgG were assessed in cell migration assays where agonism was found with 107_A07 IgG Fig. . The pea28. Complex formation was assessed by size exclusion chromatography and was readily detectable with constructs MET741 and MET928 but not with MET567 . In contrast, a Fab-formatted version of the anti-Met antibody 5D5 was found to bind all 3 fragments (not shown), consistent with binding to a distinct site within the ligand binding SEMA domain37. We next investigated whether 7A2/107_A07 competes with HGF/SF or fragments of HGF/SF for binding to MET using SPR. Due to the rapid binding kinetics observed for 7A2, we compared binding of 7A2 and NK1 to immobilized MET extracellular domain (MET928) individually and when mixed. Clear competition for binding was observed with the mixture of 7A2 and NK1  domain, MET741 containing the SEMA, CR and Ig1-2 domains and MET928 containing the SEMA, CR and Ig1-4 domains567 Fig. , indicatNK1 Fig. . BiochemNK1 Fig. .Figure 3519–741 complex were obtained and diffracted to a maximum resolution of 2.6 Å at the European Synchrotron Radiation Facility . The structure was solved by the Molecular Replacement method43 and refined to Rcryst and Rfree values of 21.5% and 25.7% respectively . Initial crystallization experiments with complexes formed by the 107_A07 Fab and MET741 failed to yield diffraction grade crystals. We overcame this by exploiting a pepsin cleavage site (NGL|GCR) located in the linker connecting the SEMA and CR domains of MET, generating a receptor fragment containing the CR, Ig1 and Ig2 domains of MET, which contains MET residues 519–741. Crystals of the 107_A07 Fab – METxes Fig.  that, whxes Fig.  is descrxes Fig. . MALDI a2 and 243 Å2 respectively. The VH domain binds a discontinuous epitope including the tip of the β-hairpin , residues connecting the β-hairpin and the β-strand C (K599 and K600) and the tip of β-strand C (R602)  Fig.  and invo02) Fig. .2 group creates an extensive hydrogen bonding network with the carbonyl groups of the backbone . InlB321 binds with high affinity to a primary site in the Ig1 domain of MET with its leucine-rich repeat central domain. It also binds with lower affinity to a secondary site in the SEMA domain with its interdomain repeat. Binding to both sites is essential for receptor activation45.Within the 107_A07 Fab - MET complex, the conformation of the CR domain of MET relative to the Ig1-Ig2 fragment matches closely the conformation of the corresponding region of MET observed in the crystal structure of MET741 in complex with InlB321. InlB321 is a truncated fragment of InternalinB (InlB), a surface protein of 45, the structure of the MET CR-Ig1-Ig2 section reported here is rigid, due to extensive interactions between the top of Ig1 (notably the β-hairpin structure) and the CR domain  analysis of the soluble MET ectodomain42 and several crystal structures45 have clearly established that the first four extracellular domains of MET  can adopt a more compact or a more extended conformation as a result of rotation and translation of the CR domain relative to the SEMA domain ain Fig. . The pre37 or InlB45 have shown that the CR domain can adopt two main orientations relative to the SEMA resulting in a compact (PDB entry 2UZY) or extended (PDB entries 1SHY and 4K3J) arrangement of the SEMA, CR, Ig1 and Ig2 domains ) and shows that onartuzumab binds an area of the SEMA domain that overlaps with the secondary binding site of InlB42 and occurs in the crystal structure of the InlB-MET741 complex45. Our data further support a role for this conformation in HGF/SF-induced MET signaling.Structural alignment of the MET-InlB complex and the MET-107_A07 complex indicates that when MET is in the compact conformation, the binding sites for InlB on the MET SEMA and 107_A07 on the Ig1 domain are spatially proximate, despite their location on separate domains Fig. . While a48 but to our knowledge this is the first time that experimental evidence for this bond has been observed. Attachment of Cys584 to the N-terminal peptide of MET may have occurred before or after proteolysis; whether this disulphide bond exists in intact, full-length MET remains to be determined. The possibility is intriguing as such a bond, if it exists, may go some way towards explaining why the compact arrangement of the SEMA, CR, Ig1 and Ig2 domains has been reported to dominate over the extended conformation in SAXS experiments42.Our data also demonstrate that the Ig1 residue Cys584 is capable of forming a disulphide bond external to the Ig1 domain. A putative disulphide linkage between Cys584 (Ig1 domain) and Cys26  has been postulated and discussed previously49 as well as blocking antibodies directed against the ligand HGF/SF51 and the MET extracellular region56. Several anti-MET antibodies progressed to clinical trials, including ABT-70057, LY287535858, ARGX-111 and onartuzumab. The development of a number of anti-MET antibodies with antagonistic activity has highlighted diverse mechanisms for receptor inhibition. For instance, onartuzumab37 and anti-MET CE-35562154 inhibit HGF/SF binding, MET activation and xenograft growth. Anti-MET LMH 8753 does not inhibit ligand binding but promotes receptor degradation and anti-MET F46 inhibits ligand binding and promotes receptor degradation59. An anti-MET nanobody (a single VH domain) inhibits MET activation and DNA synthesis in myeloma cells60 and a recent collection of llama anti-MET antibodies highlights multiple mechanisms of receptor inhibition56. These multiple pathways of receptor inhibition reflect the complex structural basis of MET signaling45. An anti-MET anticalin (PRS-110) with MET antagonistic activity has also been developed61. Interestingly, PRS-110 binds both to a loop in the SEMA domain in close proximity to the K1 binding site as well as the β-wing of the Ig1 of MET suggesting that the PRS-110 and 107_A07 epitopes may be closely related. While crystallographic analysis will be required in order to define more accurately the epitopes of a number of other anti-MET antibodies under development for therapy, the structures of onartuzumab37 and 107_A07 (this work) in complex with MET offer initial insights into mechanisms of MET inhibition. The unusual mechanism of MET inhibition by 107_A07, involving biochemical competition for ligand binding despite clear separation of binding footprints, contrasts with that of onartuzumab and illuminates the variety of potential mechanisms through which antibody-mediated MET inhibition can be achieved.The pivotal roles of HGF/SF and MET in cancer progression and metastasis have led to considerable expectation that agents blocking HGF/SF-MET signaling could have a strong impact in the therapy of metastatic tumors and led to a major effort in the design, synthesis and development of small molecule inhibitors of the MET kinaseSupplementary Information


Pseudomonas aeruginosa. The DNA-delivered monoclonal antibodies exhibit indistinguishable potency compared to bioprocessed IgG and protect against lethal pneumonia in mice. The DNA-delivered monoclonal antibodies decrease bacterial colonization of organs and exhibit enhanced adjunctive activity in combination with antibiotics. These studies support DNA-delivered monoclonal antibodies delivery as a potential strategy to augment the host immune response to prevent serious bacterial infections, and represent a significant advancement toward broader practical delivery of monoclonal antibody immunotherapeutics for additional infectious pathogens.The impact of broad-spectrum antibiotics on antimicrobial resistance and disruption of the beneficial microbiome compels the urgent investigation of bacteria-specific approaches such as antibody-based strategies. Among these, DNA-delivered monoclonal antibodies (DMAbs), produced by muscle cells in vivo, potentially allow the prevention or treatment of bacterial infections circumventing some of the hurdles of protein IgG delivery. Here, we optimize DNA-delivered monoclonal antibodies consisting of two potent human IgG clones, including a non-natural bispecific IgG1 candidate, targeting  Pseudomonas aeruginosa proteins confer protection against lethal pneumonia in mice.DNA-delivered monoclonal antibodies (DMAbs) can be produced by muscle cells in vivo, potentially allowing prevention or treatment of infectious diseases. Here, the authors show that two DMAbs targeting  The lack of progress in the development of new antibiotic classes and new understanding of host-pathogen interactions is prompting interest in microbiome-friendly approaches that engage the immune system with minimal disruption of the normal microbiota. One such approach is the investigation of antibody technology for preventing and treating bacterial infections. Therapeutic monoclonal antibodies (mAbs) are now a mainstay to treat several diseases, including primary immunodeficiencies, autoimmune disorders, asthma, cancer, and graft transplantation (reviewed in refs.8). However, only two mAbs are approved for treatment of infectious diseases in humans, palivizumab  and bezlotoxumab (targeting Clostridium difficile toxin B). Raxibacumab and obiltoxaximab, both targeting inhalation anthrax, were approved under the Food and Drug Administration (FDA) animal rule because they could not be reasonably tested for efficacy in humans. Several other infectious disease mAb candidates are currently being evaluated in human clinical trials . Yet, there are several technological hurdles with cost-effective mAb-based delivery that impede a paradigm shift from broad-spectrum approaches to pathogen-specific, targeted prevention and treatment of bacterial infections. These limitations to protein IgG administration include dosage (in the range of mg/kg), mAb production challenges, and significant costs associated with current manufacturing technology.The impact of growing antimicrobial resistance (AMR) arising from broad-spectrum antibiotic exposure threatens the antibiotic cornerstone of modern medicine. The global rate of mortality from AMR infections is estimated to be >700 000/year and expected to rise over the next decade11.DNA plasmid delivery offers a novel approach to circumvent the hurdles of traditional mAb delivery through direct, in vivo immunoglobulin (Ig) production. DNA-delivered monoclonal antibodies (DMAb) are transiently expressed and secreted by skeletal muscle cells and directly enter the systemic circulation to prevent virulence while promoting bacterial clearance. This is facilitated though specific Ig heavy and light chain nucleotide sequence optimizations, formulation development, and by recent advances in in vivo electroporation (EP) technology that has directly lead to increased DNA uptake into cells, translating to significantly enhanced DNA expression in vivoPseudomonas aeruginosa, an environmentally ubiquitous bacterium that can cause life-threatening infections in critically ill and immunocompromised patients. In addition, intrinsic antibiotic resistance via elaborate drug efflux systems, the acquisition of antibiotic resistance through horizontal gene transfer, as well as the ability to form biofilms makes P. aeruginosa a formidable foe in seriously ill patients. Two promising mAb targets against P. aeruginosa include the Psl exopolysaccharide and the type 3 secretion (T3S) injectisome PcrV protein. Psl is a highly abundant serotype-independent polysaccharide important for host cell attachment, biofilm formation, and immune evasion14. The T3S injectisome induces intoxication by injection of multiple effectors directly into the host cell cytoplasm where they interact with a multitude of eukaryotic co-factors. PcrV is thought to sit at the apex of the molecular syringe and plays a prominent role, along with other bacterial proteins, in host cell membrane pore formation15. Protective mAbs to both targets were recently described, with each alone exhibiting potent activity in multiple P. aeruginosa infection models17. In addition, clinical candidate MEDI3902, a bispecific mAb targeting Psl and PcrV, was recently described and shown to exhibit synergistic protective activity when compared to the individual mAbs or a parental mAb mixture18. DMAb delivery as an alternative strategy to bypass the need for protein IgG could be highly beneficial for administration of pathogen-specific mAbs to at-risk target populations. Furthermore, the low cost of DMAb production, the favorable safety profile of DNA delivery in humans, and the ease of administration19 would benefit a wider population of individuals that are at risk for infection.Employing this pathogen-specific approach we utilized the DMAb platform to deliver two well-characterized mAbs targeting P. aeruginosa colonisation of major organs. We also demonstrate that skeletal muscle cells can support production and secretion of functional, engineered bispecific IgG. This finding highlights the flexibility of the host cellular machinery to form novel, non-natural Ig forms in vivo and expands the possibilities for encoding additional pathogenic-specific affinities. Furthermore, we show that DMAb-BiSPA exhibits enhanced protective activity with antibiotic treatment in a lethal pneumonia model. Taken together, our results suggest that DNA delivery of full length IgG mAbs is a promising strategy for prevention of serious bacterial infections and possibly for other therapeutic indications.Here, we describe the development and analysis of synthetic DMAbs consisting of a monospecific anti-PcrV IgG (DMAb-αPcrV) and clinical candidate bispecific antibody MEDI3902 (DMAb-BiSPA) for in vivo production and activity. We observe that DMAb production in vivo can rapidly yield functional and protective titers for both constructs. These DMAbs can persist and have similar potency to bioprocess-produced mAbs, along with comparable prevention of P. aeruginosa mAb genes to be re-encoded for optimal expression into a DNA expression vector system based on their previously described potent protective in vivo activity against lethal P. aeruginosa infection18. The nucleotide and amino acid sequences of the human immunoglobulin gamma 1 (IgG1) heavy and light chains (Fab and Fc portions) were optimized taking into consideration both human and mouse codon bias, and encoded as a single, polycistronic unit in the pGX0001 DNA plasmid backbone, resulting in two constructs: DMAb-αPcrV and DMAb-BiSPA  was performed on the cell lysates and in the medium, which verified DMAb IgG production and secretion Fig. . A WesteP. aeruginosa DMAb-αPcrV (100 μg), DMAb-BiSPA (100 μg), control DMAb-DVSF3 (100 μg), or control pGX0001 empty vector (100 μg) were administered to BALB/c mice by intramuscular (IM) injection in the tibialis anterior (TA), followed by intramuscular electroporation (IM-EP). Muscle tissue was harvested 3 days post injection and sections were probed with a goat anti-human IgG Fc antibody followed by detection with a donkey anti-goat IgG conjugated to AF488  that lack T cells and have non-functional B cells. Anti-P. aeruginosa DMAb-αPcrV (100 μg) or DMAb-BiSPA (100 μg) was administered to nude mice (n = 5/group) IM in the TA or quadriceps (quad) muscles, followed by IM-EP. Serum was collected to monitor long-term human IgG1 expression in circulation. Expression of both DMAbs was observed for 100–120 days post administration, supporting the hypothesis that these novel DNA-delivered mAbs can be produced in skeletal muscle in substantial amounts detectable in systemic circulation, with expression for several weeks  were administered 100 and 300 μg doses (three injection sites × 100 µg) of DMAb-αPcrV or DMAb-BiSPA by IM-EP. The following peak DMAb expression levels were observed at day 7 following injection for DMAb-αPcrV and DMAb-BiSPA, respectively: 7.1–17.1 and 2.9–7.2 μg ml−1 (at the 100 μg dose), and 31.2–49.7 and 3.2–12.7 μg ml−1 (at the 300 μg dose)  Fig. . Human Ise) Fig. , b. For se) Fig. . To confse) Fig. .P. aeruginosa strain 6077 (PA 6077), using a lethal mouse pneumonia infection model. Mice were injected 5 days before PA 6077 challenge with DMAb-αPcrV (300 μg), DMAb-BiSPA (300 μg), or an unrelated control DMAb-DVSF3 (300 μg) that targets dengue virus (DENV)20. We also included a positive control group in which mice received protein MEDI3902 IgG (2 mg kg−1) 1 day before challenge. Randomly selected animals from DMAb-αPcrV-treated and DMAb-BiSPA-treated animals were euthanized to monitor DMAb expression levels in serum at the time of challenge, as well as to evaluate the potency of the expressed DMAbs. As indicated in Fig. −1, respectively, when quantifying total human IgG from serum. We further evaluated the potency of in vivo expressed DMAb-αPcrV and DMAb-BiSPA by quantifying antibody expression based on the anti-cytotoxic activity from serum. No difference was observed in the quantification methods, indicating that in vivo expressed monospecific and bispecific DMAb-IgGs are fully functional and equivalent in activity in comparison to bioprocessed IgG . Animals receiving the control DMAb-DVSF3 succumbed to infection within 24–55 h. In contrast, approximately 94% of animals (15/16) that received either DMAb-αPcrV or DMAb-BiSPA survived challenge . As expected, all the positive control animals receiving MEDI3902 IgG (2 mg kg−1) survived challenge. In addition, treatment of mice with DMAb-BiSPA at 100 μg (1 site × 100 µg), 200 μg (2 sites × 100 µg), or 300 µg (3 sites × 100 µg) followed by infection with P. aeruginosa, yielded concentration-dependent survival  in each tissue. A significant reduction  in CFU lung burden was observed with DMAb-BiSPA but not DMAb-αPcrV-treated animals  to simulate the inadequate drug exposure encountered in patients infected with a resistant bacterium, and a subtherapeutic dose of DMAb-BiSPA  are administered when a Gram negative or 2. A large percentage of these infections are caused by ESKAPE pathogens and broad-spectrum antibiotics are frequently administered as last-resort treatment26. However, antibiotic usage is often associated with negative side effects, including disruption of the beneficial microflora and potential for the spread of cross-resistance in non-targeted bacteria. Furthermore, inappropriate usage of antibiotics in humans or for agricultural purposes is likely a contributing factor to global resistance rates27. This growing predicament and the lack of novel mechanism antibiotics in the pharmaceutical pipeline has fostered the development of alternative antimicrobial strategies such as pathogen-specific mAbs. We previously reported on the derivation of a monospecific anti-PcrV mAb (V2L2MD) and bispecific anti-PcrV/Psl mAb MEDI3902 that confer potent protective activity against one of the ESKAPE pathogens, P. aeruginosa17. Using these clones as model molecules, we encoded the IgG heavy and light chain sequences into a synthetic DNA vector to test the feasibility of plasmid DNA delivery (DMAb) as an alternative mode of direct delivery of mAb in vivo to overcome some of the limitations of traditional mAb manufacturing and delivery impeding broader use.The global health and economic impact of antimicrobial resistant infections is staggering and there is a compelling need to investigate novel strategies to prevent or treat these infections. Over 2 million cases of community-acquired and hospital-acquired resistant infections are reported each year in the United StatesP. aeruginosa can be encoded in synthetic DNA vectors, DMAbs, and produced in vivo by skeletal muscle. The anti-Pseudomonas DMAbs bound effectively to therapeutic targets and were protective in a mouse model of lethal pneumonia caused by an aggressive P. aeruginosa strain. We identified that a single dose of DMAb is transiently expressed for 3–4 months and protection against lethal infection is comparable to treatment of mice with purified IgG. This result supports the possibility of long-term mAb administration, as DMAbs can be continuously expressed from muscle until the plasmid is eventually lost. In addition to routine administration, another foreseeable advantage for anti-P. aeruginosa DMAbs would be for high-risk patients with recurring infections related to chronic illnesses or implanted devices, where DMAbs may reduce the need for extended antibiotic regimens. Furthermore, we demonstrated that DMAbs can also function synergistically with a commonly used antibiotic, MEM. The synergistic effect of DMAb and antibiotic combination suggests that this strategy could have potential in reducing antibiotic treatment regimens, thereby reducing the length of antibiotic exposure in patients. This adjunctive activity is equivalent to that observed with protein IgG in previous studies18. Although we did not evaluate DMAb activity against a panel of P. aeruginosa bacterial strains, the experiments outlined in this manuscript demonstrate that DMAb is functionally equivalent to protein IgG. All bioprocessed anti-Pseudomonal IgG mAbs  have been previously shown to be protective against P. aeruginosa clinical isolates derived from diverse serotypes, multiple T3S phenotypes , and multiple infection sites29.In the current study, we demonstrated that mAbs against 32. We believe that DMAb delivery represents a significant advancement not only for mAb therapy and DNA-delivery technology, but also for novel pathogen-specific treatment approaches to enhance host immunity.The field of mAb engineering is evolving dynamically and DMAb delivery offers an additional strategy to help transport biologically functional mAbs rapidly in vivo. In addition to obvious clinical benefits, in vivo expression of non-traditional bispecific mAb isoforms, as presented here, emphasizes the versatility of muscle to be engaged as protein production factories. Importantly, DMAb expression is transient, with similar efficacy to other therapeutic deliveries. It may be possible to develop an inducible system that will eliminate the DNA plasmid when it is no longer needed. Alternatively, DMAb DNA can potentially be re-administered indefinitely as there are no associated anti-vector responses, allowing for long-term therapy through repeat administration33 has been reported. This study demonstrated anti-tumor efficacy comparable to protein IgG, further supporting the concept that a gene-encoded mAb can have functionality. To our knowledge, our study is the first demonstration of DNA-encoded mAb delivery that is protective against a bacterial target and the first delivery of an engineered IgG isoform. Early studies with DNA plasmid-encoded antibodies demonstrated feasibility but exhibited low-IgG expression in serum35. Our group previously demonstrated the protective efficacy of DMAbs targeting viral infections, showing rapid protection against chikungunya36 and DENV infections20. The DMAb-targeting DENV did not promote antibody-dependent enhancement of disease. These two infectious disease models did not require high serum IgG levels; however, optimized DMAb formulations to increase expression levels are desirable so as to provide extended coverage after a single DMAb administration. Toward this end, we sought to optimize serum DMAb expression levels for our efforts against P. aeruginosa 38 is a cytotoxic (ExoU+) strain that was used for all infection experiments.Human embryonic kidney (HEK) 293 T cells (ATCC CRL-3216) were maintained in Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal bovine serum , in mycoplasma-free conditions. Routine testing was performed at the University of Pennsylvania. All cells were maintained at a low passage number. 18. The nucleotide sequence for each human IgG1 heavy and Igκ light chains were codon optimized for both mouse and human biases to enhance expression in mammalian cells40. Sequences were also RNA optimized for improved mRNA stability. This has been shown to result in more efficient translation on the ribosome42, leading to increase protein yield43. The optimized heavy and light chain genes were then inserted into the pGX0001 DNA expression vector, under the control of a human cytomegalovirus promoter and bovine growth hormone polyA.The sequences of the single specificity anti-PcrV IgG (clone V2L2MD) and engineered bispecific IgG  were obtained as previously describedcis, separated by a furin cleavage site (RGRKRRS) and P2A peptide. The result was two plasmids: DMAb-αPcrV and DMAb-BiSPA. HEK 293T cells were transfected with DMAb DNA using GeneJammer  transfection reagent. Cell supernatants and cell lysates were harvested 48 h post transfection and assayed for human IgG production by ELISA and Western blot.Both genes were encoded in −1 Sodium Azide (Sigma) in PBS). 50 µl of staining solution was added to each section and incubated for 2 h. Sections were washed 5 min in 1×PBS three times. Donkey anti-goat IgG AF488  was diluted 1:200 in incubation buffer and 50 µl was added to each section. Section were washed after 1 h incubation and mounted with DAPI-Fluoromount  and covered.BALB/c mice were injected with 100 μg of DMAb by IM injection in the TA muscle followed by IM-EP. Tissue was harvested 3 days post injection, fixed in 4% Neutral-buffered Formalin  and immersed in 30% (w/v) sucrose  in D.I. water. Tissues were then embedded into O.C.T. compound  and snap-frozen. Frozen tissue blocks were sectioned to a thickness of 18 µm. Muscle sectioned were incubated with Blocking-Buffer (0.3% (v/v) Triton-X (Sigma), 2% (v/v) donkey serum in PBS) for 30 min, covered with Parafilm. Goat anti-human IgG-Fc fragment antibody  was diluted 1:100 in incubation buffer (1% (w/v) BSA (Sigma), 2% (v/v) donkey serum, 0.3% (v/v) Triton-X (Sigma), and 0.025% (v/v) 1g mlIn vivo expression of DMAb constructs was imaged with a BX51 Fluorescent microscope (Olympus) equipped with Retiga3000 monochromatic camera (QImaging).−1 purified anti-human IgG-Fc  and incubated overnight at 4 °C. The following day, plates were washed and blocked at room temperature for at least 1 h with PBS containing 10% FBS. Samples were serially diluted two-fold and transferred to the blocked plate and incubated for 1 h at room temperature. Purified human IgGκ  was used as a standard. Following incubation, samples were probed with an anti-human IgGκ antibody conjugated to horseradish peroxidase  at a 1:20,000 dilution. Plates were developed using o-Phenylenediamine dihydrochloride substrate  and stopped with 2N H2SO4. A BioTek Synergy2 plate reader  was used to read the plates at OD450 nm. Alternatively, human IgG from serum was quantified as described above with the exception of using an anti-idiotype mAb  specific for V2L2MD or MEDI3902 as the capture reagent. Purified V2L2MD or MEDI3902 was used as a standard.Ninety-six-well, high-binding immunosorbent plates were coated with 10 µg ml−1 Goat anti-Human IgG (H + L) . Plates were washed and blocked for 1–2 h at room temperature with Blocker Casein in PBS (Thermo Fisher). After blocking, a standard containing MEDI3902 or V2L2MD was serially diluted 1:2 across the plate, while serum samples were diluted 1:20, 1:40, 1:80, and 1:160. Plates containing the samples were then incubated for 1 h at room temperature. After washing, plates were probed with Donkey anti-Human IgG-HRP  at a 1:4000 dilution and incubated for 1 h at room temperature. After washing, the immune reaction was developed by adding SuperSignal ELISA Pico Reagent (Thermo Fisher) and fluorescence was read on the Perkin Elmer Envision.DMAb was also quantified from serum using 384-well black MaxiSorp plates  coated with 10 μg mlDMAb was also quantified from serum based on anti-cytotoxic activity mediated by DMAb-αPcrV and DMAb-BiSPA that measures the protection of A549 cells (ATCC® CCL-185™) from the cytotoxic effects of PA 6077. The activity of mouse serum was compared to a standard curve of naïve mouse serum spiked with V2L2MD IgG.Pseudomonas PcrV protein  at 0.5 µg ml−1. The following day, serum samples from DMAb-administered animals were serially diluted two-fold and then transferred to the blocked plate. Samples were probed with an anti-human IgG H + L antibody conjugated to HRP  at a dilution of 1:5000 and developed with OPD substrate.Ninety-six-well plates immunosorbent plates were coated overnight with g and the supernatant containing the protein fraction was collected. The samples were quantified using a bicinchoninic acid assay  and 10 µg total lysate was loaded on a 4–12% Bis-Tris SDS-PAGE gel . The gel was transferred to a nitrocellulose membrane using the iBlot2 system (Thermo Fisher Scientific). The membrane was blocked in 5% powdered skim milk + 0.5% Tween-20 and then probed using a donkey anti-human H + L antibody conjugated to HRP . Bands were developed using a chemiluminescent system  and visualized on film.The cell lysates from DMAb-transfected cells were collected in cell lysis buffer . Samples were centrifuged at 16,000 × −1, Sigma Aldrich) 30 min–1 h before IM injection of 100–300 µg DMAb-αPcrV or DMAb-BiSPA in the TA or quad muscles, followed by electroporation (IM-EP). Serum levels of DMAbs were monitored following administration.Female, 6–8-week-old B6.Cg-Foxn1nuJ and BALB/c mice were purchased from The Jackson Laboratory  and housed in the animal facilities at the University of Pennsylvania or MedImmune, AstraZeneca. All animal protocols were approved by the institutional University of Pennsylvania and MedImmune Institutional Animal Care and Use Committees (IACUC), following guidelines from Association for Assessment and Accreditation of Laboratory Animal Care International. Further IACUC oversight was provided by The Animal Care and Use Review Office, required for DARPA-funded research. Animals received an IM pre-injection of hyaluronidase (400 U mln = 8/group) received 100 µg or 300 µg of DMAb-αPcrV or DMAb-BiSPA by IM-EP at day 5 before challenge. The unrelated DENV DMAb-DVSF320 was included as a control. A fourth group of animals received an intraperitoneal injection of purified protein IgG MEDI3902 (2 mg kg−1) on day −1 before challenge. On day 0, animals received an intranasal challenge of 9.75e5–1.0e6 CFUs of the aggressive, anti-microbial resistant P. aeruginosa strain 6077. Animals were monitored for 6 days following intranasal challenge for survival as described in ref. 16. Briefly, animals were anesthetized with ketamine and xylazine followed by intranasal administration of the bacterial inoculum contained in 0.05 ml. For organ burden analyses, lungs, spleens, and livers were harvested from DMAb-treated animals 24 h post infection followed by homogenizing and plating of Luria agar plates for enumeration of bacterial CFU. IL-1β, IL-6, and KC/GRO were quantified from the supernatant of lung homogenates using a multiplex kit , according to the manufactures instructions. For DMAb and MEM combination experiments, MEM was administered subcutaneously 4 h after infection.BALB/c mice  for a minimum of 48 h. Fixed tissues were then routinely processed and embedded in paraffin, sectioned at 3-µm thickness, and stained with Gill’s hematoxylin  and eosin  for histologic evaluation by a pathologist blinded to the experimental conditions.n = 5 mice was calculated to be needed in order to ensure adequate power. We performed student’s t-test or one-way analysis of variance (ANOVA) calculations, where necessary. Survival data was represented by a Kaplan–Meier survival curve and significance was calculated using a log-rank test and one-way ANOVA with correction for multiple comparisons. These data were considered significant if p < 0.05. The lines in all graphs represent the mean value and error bars represent the standard deviation. No samples or animals were excluded from the analysis. Randomization was not performed for the animal studies. Samples and animals were not blinded before performing each experiment.All statistical analyses were peformed using GraphPad Prism 6.0 software  or SPSS (IBM). Sample size calculations for two independent proportions were calculated with alpha 0.05 and power 0.90. A minimum of The nucleotide sequences for the optimized human IgG1 heavy and Igκ light chains have been deposited in the NCBI Nucleotide database with accession codes LQ055727.1, LQ055728.1, LQ055748.1, and LQ055750.1. The authors declare that all other relevant data supporting the findings of the study are available in this article and its Supplementary Information files, or from the corresponding author upon request.Supplementary Information


We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to  The light harvesting like membrane protein 3 (LIL3) is a member of the light-harvesting complex protein family (LHCP) and characterized by the presence of a common LHC sequence motif . LIL3 isHordeum vulgare) .The intrinsic fluorescence of Chl a/b, was monitored at a final concentration of 30 nM Chl diluted in reconstitution buffer containing 6 mM DDM, while non-fluorescent LIL3.2 was titrated in a 1:1 dilution series (concentrations between 20.000 and 0.63 nm) in reconstitution buffer containing 6 mM DDM. After a two hour incubation at RT, samples were loaded into MonolithThe solubilization of LIL3 (17.5 nM) in DDM was monitored by dynamic light scattering (DLS) at 25 °C in a N-Dodecyl β-D-Maltoside (DDM) concentration gradient ranging from 0–2 mM DDM using a quartz cuvette in the Zetasizer Nano ZSP instrument . The size of the 2 mM DDM detergent micelles was compared to 2 mM DDM micelles containing LIL3, Chl or affinity purified reconstituted LIL3/Chl a. The average value of the peak, weighted by the Y-axis parameter (mean hydrodynamic diameter) is given for all samples. Cumulative analysis is not valid for the polydisperse samples measured. Therefore, the distribution means in the linear range is reported, not Z-Average and PDI –19.4 nM) was tested upon solubilization of both molecules in DDM (6 mM). For reconstitution, the mixture of both micelles was heated and upon cooling to RT, the interaction of Chl a with increasing concentrations of LIL3 was investigated over a time-frame of 150 minutes using microscale thermophoresis  micelles using a process in which a temperature gradient induces a mass transport (thermophoresis). Typically, thermophoresis of molecules along a microscale temperature gradient is affected by molecular changes like size, charge or hydration shell, and a fluorescent reporter can be used to quantify a molecular interaction . Here, Csis MST,  inlet.At time point zero, and during the first 90 min, no significant change in the fluorescence difference analysis was recorded; however, Chl specific fluorescence showed high variability when the LIL3 concentration was increased . This vaThe LIL3 concentration dependent change in the fluorescence difference analysis continuously decreased with time and saturated at a LIL3 concentration of about 115 nM after 150 min . Data shIn vitro expressed LIL3.1, and LIL3.2, and a mixture of LIL3.1 and LIL3.2 (LIL3.1/2) (27 μM) were each reconstituted with either Chl a, Chl b, or a mixture of Chl a and b (Chl a/b) and isolated by native PAGE , changes in the mean hydrodynamic diameter and the poly-dispersity of DDM micelles were determined from the intensity distribution of particles with characteristic Brownian motion . The meaDDM micelles showed a distribution means of 7.53 nm relative to the 6.5 nm observed when LIL3 was solubilized by DDM. DLS analysis of both experiments shows that both distribution profiles overlap; however, while the intensity of both distribution profiles is maintained, solubilized LIL3 shows a sharper and less disperse distribution with a relative decrease in the means. Difference analysis  revealedSolubilization of Chl a in 2 mM DDM hardly affected the mean diameter of DDM micelles; whereas, solubilization of reconstituted LIL3 resulted in a small increase of the distribution means corresponding to an asymmetric change of the distribution profile . DLS anaDifference spectra between reconstituted LIL3 and DDM micelles . This inin vitro in the absence of carotenoids, lipids and of Chl b. A protocol for in vitro reconstitution of Chl binding to LIL3 is presented. The method has been established on the basis of protocols developed by Plumley and Schmidt [We show by mobility analysis of micelles that Chl a binding to LIL3 can be reconstituted  Schmidt  and Paul Schmidt  for Chl 8SO4]/[CTAB] [The determined size of DDM micelles corresponds well to literature –24. Howe]/[CTAB] .Mobility analysis of reconstituted LIL3 by DLS and native PAGE indicated that chlorophyll is bound to LIL3. However, neither of the methods differentiated between a binding of Chl a and Chl b. In contrast, thermophoretic mobility analysis showed that LIL3 interacted only with Chl a . FinallyHow can these findings be explained? In DLS, changes in the mean hydrodynamic diameter of dissolved molecules can be determined , 26. RecA higher charge density cannot explain the effect observed. LIL3.1 and LIL3.2 micelles contain 7.8 and 6.8 negative charges at pH 7, respectively; whereas, micelles containing Chl are uncharged. Micelles containing LIL3 were therefore retarded based on a larger hydrodynamic diameter despite a higher charge density. DLS analysis had also shown that LIL3 aggregated in the absence of detergent  27]. Th. Th27]. In thermophoresis, highly variable chlorophyll specific fluorescence is determined when the interaction of LIL3 and Chl was investigated in the absence of a pre-incubation at time point zero. Variability was especially noted when the ratio of Chl:LIL3 decreased . This maRhodopseudomonas viridis [1—keto, or 132—keto-methoxy tetrapyrrol side groups, or with the 173—ester carbonyl group can arrest rotation of Chl around the coordinated Mg2+ [Direct biochemical evidence for binding of bacteriochlorophyll (BChl) was first shown by crystallization of the bacterial reaction center of photosystem II from  viridis . Structu viridis , 33. Pol viridis . The oxy viridis –35, and  viridis . Cooperated Mg2+ –36.2+ ion and for formation of salt bridges in a homo- and hetero-dimer [In LIL3.1 and LIL3.2, AA pairs E170, N173, and E173, N176 fulfil the criteria for positional binding of Chl a; also, AAs pairs E170, R175, and E173, R178 fulfil the criteria for coordination of the central Mgro-dimer . Additioro-dimer . Chl molro-dimer , 38. ThiIsolation of fluorescent native complexes upon de-etiolation of barley identified co-migration of LIL3 with CHS, POR and GGR. All proteins, except LIL3, had been described to be of central importance for the regulation of Chl synthesis. However, these proteins had not been identified in interaction with Chl. This indicated a structural and/or functional significance for binding of Chl to LIL3 , 10. Sevin vivo; however, we do not find evidence that carotenoids are required for binding Chl a to LIL3 in vitro. The in-vitro reconstitution protocol makes it now possible to investigate the Chl a binding site in LIL3 using genetic screens. It may also be investigated whether the incubation period of more than 90 min was required to saturate Chl binding sites in vitro or reflected the lack of assembly factors in the in vitro approach.LIL3 has been described to stabilize and anchor GGR to the membrane via the LHC motif . AlthougS1 FigLil3 was solubilized at increasing concentrations 0.305 nM –10 μM in the presence of a constant concentration (120 nM) of Chl in DDM micelles (6 mM). Normalized fluorescence difference from three independent MST measurements at time point zero was plotted against the LIL3.2 concentrations (A). The time course for binding of Chl a was investigated by determination of Kd values upon initiation of reconstitution assays. Determined stable Kd values were plotted against the delay time after reaction onset .(TIF)Click here for additional data file.S2 FigThe distribution profile of DDM micelles  and of solubilized LIL3.2  was analyzed. Difference analysis (A–B) shows how the intensity (Intensity (%) of the DDM micelle distribution profile (Diameter (d. nm)) changes upon solubilization of LIL3.(TIF)Click here for additional data file.S3 FigRecombinant LIL3.2 bound to Chl a (A), Chl a/b (B) and Chl b (C) was affinity purified at CMC (0.2 mM DDM) and analysed by dynamic light scattering at 25 °C in 2 mM DDM . As controls, the dynamic light scattering of LIL3 in 2 mM DDM (LIL3), Chl in 2 mM DDM  and the micelle in a concentration of 2 mM DDM (DDM) were measured.(TIF)Click here for additional data file.S1 TableLil3 was solubilized at increasing concentrations (0.305 nM– 10 μM) in the presence of a constant concentration of Chl (120 nM) in DDM micelles (6 mM). The time course for binding of Chl a was investigated by determination of Kd values upon initiation of reconstitution assays. Determined stable Kd values were plotted against the delay time after reaction onset.(TIF)Click here for additional data file.


Soil urease, alkaline phosphatase and hydrogen peroxidase activities decreased with increasing years of continuous potato cropping. On average, the P–B rotation significantly increased (p <0.05) tuber yield by 19% and 18%, compared to P–C and P–L rotation respectively. P–L rotation also increased potato tuber yield compared to P–C, but the effect was lesser relative to P–B rotation. These results suggest that adopting potato–legume rotation system has the potential to improve soil biology environment, alleviate continuous cropping obstacle and increase potato tuber yield in semi–arid region.This study was conducted to explore the changes in soil microbial populations, enzyme activity, and tuber yield under the rotation sequences of Potato–Common vetch (P–C), Potato–Black medic (P–B) and Potato–Longdong alfalfa (P–L) in a semi–arid area of China. The study also determined the effects of continuous potato cropping (without legumes) on the above mentioned soil properties and yield. The number of bacteria increased significantly (p < 0.05) under P–B rotation by 78%, 85% and 83% in the 2, 4 and 7–year continuous cropping soils, respectively compared to P–C rotation. The highest fungi/bacteria ratio was found in P–C (0.218), followed by P–L (0.184) and then P–B (0.137) rotation over the different cropping years. In the continuous potato cropping soils, the greatest fungi/bacteria ratio was recorded in the 4–year (0.4067) and 7–year (0.4238) cropping soils and these were significantly higher than 1–year (0.3041), 2–year (0.2545) and 3–year (0.3030) cropping soils. Generally, actinomycetes numbers followed the trend P–L>P–C>P–B. The P–L rotation increased aerobic azotobacters in 2–year (by 26% and 18%) and 4–year (40% and 21%) continuous cropping soils compared to P–C and P–B rotation, respectively. Generally, the highest urease and alkaline phosphate activity, respectively, were observed in P–C (55.77 mg g Solanum tuberosum L.) is one of the staple food crops in the world. In northern China, potato has been the dominant crop, providing the main food source for the increasing human population 33]. Numb 14 days –36.4 titration method; urease activity was measured using phenol sodium hypochlorite colorimetric method; and phosphatase activity was measured using disodium phenyl phosphate colorimetric method [Hydrogen peroxidase activity was determined using KMnOc method .–1) was obtained by determining the tuber yield of each plot in each year at harvest. The tuber yield in the continuous potato cropping fields (CK) was determined in October 2011 and that of potato–legume rotation was determined in October, 2013.The tuber yield per hectare  with the rotation treatment as the fixed effect and replicate as random effect. Means comparison based on Duncan’s multiple range test (p<0.05). Due to significant year by treatment interactions for most of the variables evaluated in the study, the treatment effect was assessed separately for each year.The effect of continuous potato cropping on bacteria, fungi and actinomycetes are presented in Potato–legumes rotation had significant effect (p <0.05) on bacteria, fungi and actinomycetes numbers (ANs) depending on treatment . The numContinuous cropping had significant effect (p <0.05) on fungi/bacteria ratio . The resThe trend of fungi/ bacteria ratio under various crop rotation systems was not consistent in all the years of this study . The P–B4 CFU /g), which was significantly higher than the other years of continuous cropping by 505% on average. Response of both nitrifying and denitrifying bacteria to continuous potato cropping was not consistent  depending on treatment and cropping year . The treSoil urease, alkaline phosphatase and hydrogen peroxidase activities decreased gradually with increased years under continuous potato cropping . The lowSoil urease, hydrogen peroxidase and alkaline phosphatase activities were significantly increased in the 2–year cropping soils under the potato-legume rotation . The rot–1, which was significantly greater by 62% (13220 kg ha–1), 45% (14706 kg ha–1) and 37% (15553 kg ha–1) compared to 4, 4 nd 7 years, respectively. However, no significant differences (p <0.05) were observed in tuber yield between 3–7 years continuous cropping plots  in potato tuber yield depending on the treatment . The tubResults of the present study indicated significant positive correlations between BN and AA, NB (p<0.05), ANA, UR, AP (p<0.01), FN and AA, ANA, and AP (p<0.05) . SignifiSoil microorganisms are important components of the terrestrial ecosystems and their quantities can reflect not only the degree of energy metabolism, but also soil fertility status . HoweverReasonable rotation system is an important basis for preventing continuous cropping obstacles and improving crop production . As repoSoil microbial floras were shifted from fungi type to bacteria type by rotation of Common vetch and Medicago lupulina for 1 and 2–year continuous cropping, but fungi numbers did not increase by rotation of Longdong alfalfa at the same continuous cropping years. The amelioration was significant by rotation of Medicago lupulina and Longdong alfalfa for potato soil in 3 and 7–year continuous cropping and the improvement effect of Medicago lupulina was the best. For 4–year continuous cropping field, soil microbial floras were also shifted to bacteria type by rotation of these three legumes. However, the actinomycetes mycelium decreased for up to 4–year continuous cropping field by rotation of three legumes . TherefoMicrobial physiological group of nitrogen play an important role in nitrogen transformation in soil, including aerobic azotobacter, anaerobic azotobacter, nitrifying bacteria, denitrifying bacteria and ammonifying bacteria . In receAfter rotation with legumes, there was difference in physiological groups of microorganisms in potato continuous cropping soil. The aerobic azotobacters of 1 and 2–year potato continuous cropping soil have a very sensitive response to legumes and its number increases greatly by rotation of legumes compared with the continuous cropping soil. However, similar results were not observed in 3–year continuous potato cropping soil. Soil anaerobic nitrogen–fixing bacteria were significantly improved in all potato continuous cropping fields by rotation with Medicago lupulina and Common vetch. Ammonification can produce ammonia in the soil, which can be converted to nitrate by nitrifying bacteria. Denitrifying bacteria usually participate in the revivification of nitrate in the soil. The existence and activities of nitrifying and denitrifying bacteria in the soil play an important role in soil fertility and plant nutrition . Our resSoil enzymes have a close relation with soil microorganisms and its biodiversity, both of which can drive soil biological metabolic processes . The res3––N [Current study indicated a significant positive correlation between BN and AA, NB (p<0.05), ANA, UR, AP (p<0.01), FN and AA, ANA, AP (p<0.05). These findings showed that fungi and bacteria are the major soil microorganisms responsible for N cycling processes . Crensha3––N , 59. TheSoil microbial communities increased significantly after rotation with legumes. Number of bacteria and fungi/bacteria ratio was greatly improved by rotation of potato–Longdong alfalfa and potato–Black medic. Thus, obstacles to sustainable production of potato in the semi–arid region of Northwestern China due to continuous cropping can be reduced by adopting potato–legume rotation cropping.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(XLSX)Click here for additional data file.


Children are the most vulnerable population exposed to the use of antibiotics often incorrectly prescribed for the treatment of infections really due to viruses rather than to bacteria. We designed the MAREA study which consisted of two different studies: i) a surveillance study to monitor the safety/efficacy of the antibiotics for the treatment of pneumonia (CAP), pharyngotonsillitis and acute otitis media in children younger than 14 yrs old, living in Liguria, North-West Italy and ii) a pre−/post-interventional study to evaluate the appropriateness of antibiotic prescription for the treatment these infections. In this paper, we show only results of the appropriateness study about the antibiotic prescription for the treatment of pneumonia.Patients included in this study met the following inclusion criteria: i) admission to the Emergency/Inpatient Dpt/outpatient clinic of primary care pediatricians for pneumonia requiring antibiotics, ii) informed written consent. The practice of prescribing antibiotics was evaluated before-and-after a 1 day-educational intervention on International/National recommendations.Global adherence to guidelines was fulfilled in 45%: main reason for discordance was duration (shorter than recommended). Macrolide monotherapy and cephalosporins were highly prescribed; ampicillin/amoxicillin use was limited. 61% of patients received >1 antibiotic; parenteral route was used in 33%. After intervention, i) in all CAP, cephalosporin prescription decreased (−23%) and the inappropriate macrolide prescriptions was halved and, ii) in not hospitalized CAP (notH-CAP), macrolides were prescribed less frequently (−25%) and global adherence to guidelines improved (+39%); and iii) in H-CAP antibiotic choice appropriateness increase.Prescribing practices were sufficiently appropriate but widespread preference for multidrug empirical regimens or macrolide in monotherapy deserve closer investigation. Also the Regional Health Plan of Liguria, promotes the reduction of the use of antibiotics and especially of last generation antibiotics when appropriate therapies equally effective against the etiologic agents are available. Educational interventions seem to be effective in improving a more rationale use of antibiotics  than if they had milder pneumonia [1.75 (SD: 1.49) days]  was appropriate in all cases.p = .17) (Table p < .001) (Table p = .001) and in phase 2 . In addition, in H-CAP, we found a significant increase after the educational intervention and the adherence to guidelines reached 100% (p = .03) (Table p = .26)  Table . Adherenp = .034). In phase II, the frequency of inappropriate prescriptions was further reduced and became similar being 7.81% (5/64 prescriptions) for macrolides and 3.33% (3/90 prescriptions) for other antibiotics (p = .22). This was mainly due to a 45% reduction in inappropriate macrolide prescription in phase II even though not statistically significant (p = .26), (data not shown).Comparing the frequency of inappropriateness in antibiotic choice for the different antibiotic classes, we found that in phase I the frequency of inappropriate prescriptions was higher for macrolides than for other antibiotics   (Table p = 0.27). In notH-CAP, a 36% increase in guideline adherence of duration of treatment was observed in phase II (p = .025) . In notH-CAP, a 5-fold increase in adherence also for duration was found after the educational intervention being 4.76% (1/21 prescriptions) before and 27.27% (6/22 prescriptions) after but this enhancement was not statistically significant (p = .095).Analyzing only macrolide prescriptions appropriate for choice, we found that only 13.19% of them were concordant to the guidelines (19/144 prescriptions) also for duration with an about 30% increase in adherence in phase II but without reaching statistically significance  of the 399 evaluated doses (1 case missing) Table  and in np < .001) (Table p = .0003) and in phase II (p = .0003) (data not shown).Concordant doses were found more frequently in H-CAP than in notH-CAP being 88.01% and 67.42%, respectively (1) Table ; this wafor all parameters was fulfilled in 45% of the cases. Age did not affect the frequency of concordant prescriptions (not shown). No statistically significant difference between phase I and phase II was found both in all CAP and in H-CAP (Tables p = .19)  Table .Analyzing inappropriate prescriptions (No = 220), the main reason for discordance was duration of treatment , followed by wrong dose  and antibiotic choice . Similar results were obtained analyzing the two phases separately (not shown).p = .039).Since the most common deviation from the guidelines was duration, we re-evaluated “global appropriateness” only considering concordance/discordance for indication, antibiotic choice and dose. We found an almost 60% of adherence to guidelines . Again, in notH-CAP, after educational intervention, appropriateness significantly increased from 46.88% to 64.71%  the decision making regarding use/nonuse of antibiotics was appropriate in all cases but global adherence to guidelines was fulfilled in less than a half of the cases and the main reason for discordance was duration of treatment; ii) cephalosporins and macrolide monotherapy were highly prescribed whereas use of unprotected ampicillin/amoxicillin was very limited; iii) a high percentage of patients received more than one antibiotic and parenteral route was used in over one third of prescriptions; iv) . About 50% of antibacterial prescriptions for children are inappropriate [Despite availability of guidelines, overprescription/abuse of antibiotics in the treatment of respiratory infections is still a worldwide problem , 10. Aboropriate , 16 and,ropriate .There are several reasons for these unjustified prescriptions: i) to be conditioned by parental pressure to receive antibiotics , 11, 18;In our studied population, the decision making regarding use/nonuse of antibiotics was appropriate in all cases. However, a rational drug use occurs not only when an appropriate drug is prescribed but also when it is administered in correct doses and for an adequate duration of therapy . SeveralAssessing individual parameters, antibiotic choice was adherent to the recommendations in over 90% of the cases and was higher for H-CAP. In phase I, macrolides were inappropriately prescribed more frequently than other antibiotics (13.5% vs 5.6%). In a retrospective analysis of 1 year-ambulatory pediatric visits, Saleh et al.  found siStreptococcus pneumonia (the resistance of Streptococcus pneumoniae could be overcome by high dose of ampicillin (up to 80 mg/kg) rather than by adding of clavulanate), also pneumonia due to Mycoplasma pneumoniae is not very unusual in preschool children [Of note that more than 60% of patients received more than one antibiotic similarly to the results reported by DeLuca et al.  who founchildren .Despite the fact that there are no evidence supporting the superiority of intravenous/intramuscular treatment of pneumonia as compared to the oral one, in our study parenteral route accounted for about one third of prescriptions and among them ampicillin alone or added to sulbactam was the most frequently administered , followed by third-generation cephalosporins (25.0%). An abuse of parenteral cephalosporins in Italian hospitalized children was already denounced in a study conducted last decade by Esposito et al. . ParenteWe also reported that duration of use and dose of antibiotics was concordant with the guidelines in over 60% and in over 80%, respectively. Inappropriate duration was seen only for macrolides that were used shorter than recommended . - the guidelines we refer suggested to use macrolides for 14 days but other recommendations such as guidelines by the Pediatric Infectious Diseases Society and the Infectious Diseases Society of America suggests to prescribe a shorter course of antibiotic treatment at least in milder pneumonia . In our On the basis of these results, guidelines alone seem to have a limited efficacy in changing physician behavior in the management of pediatric pneumonia, probably because primary care pediatricians have been faced with a variety of Italian/international guidelines, sometimes with contrasting key messages, requiring substantial time and effort to be analyzed and followed. Therefore, paediatricians knowledge and approach to antibiotic use require corrections to improve prescribing practices –14, 38. Global adherence to guidelines was similar in the two phases of the study. However, analyzing antibiotic prescriptions in notH-CAP, the educational intervention determined an about 44% improvement in overall adherence to guidelines. Parameters were also evaluated separately, so that nonadherence to one parameter did not preclude assessment of the others. Antibiotic choice appropriateness rate was not modified by the educational intervention being very high already in phase I (91%). However, analyzing only H-CAP, we found a significant increase in antibiotic choice appropriateness rate after the educational intervention and the adherence to guidelines reached 100%. Particularly, macrolide prescriptions were prescribed inappropriately in 13.46% and this rate was halved after educational intervention.Regarding the duration of use, educational intervention only slightly increase adherence to guidelines from 60.59% to 64.71%. However, in notH-CAP, a 36% increase in adherence to guidelines was observed in phase II. Inappropriate duration was seen only for macrolides that were used shorter than recommended.The dose of antibiotics was concordant with the guidelines in 81.20% of the 400 evaluated doses. In the discordant doses, the dose per kilogram was lower than that recommended in 67.57% of the cases. Concordant doses were found more frequently in H-CAP than in notH-CAP being 88.01% and 67.42%. Educational intervention did not modify this rate.In our study, no ADR was reported. The lack of compliance in reporting ADRs is one of the main weakness of the paediatric pharmacovigilance system . EducatiEven if involving a single Italian region , we regard the external validity of our findings to be high, since our data represent unselected and consecutive patients. However, due to a high heterogeneity of drug use among Italian regions  our findAmong limitations, data on microbiological investigation were not available in all cases, therefore some patients diagnosed with pneumonia may have had viral etiologies. However, even with the understanding that some pneumonias in our study population were likely viral, the misdiagnosis of viral pneumonias as bacterial should not have affected antibiotic selection once the physician’s intent was to treat bacterial pneumonia.In the surveillance part of the MAREA study, a needed sample size of 840 patients (420 patients each phase) was calculated assuming an expected frequency of hospitalization of about 10% in pediatric population with a diagnosis of pneumonia, AOM and/or pharyngotonsillitis. We enrolled only 620 patients but this only slightly decreased the statistical power (from 80% to 75%) because the observed frequency of hospitalization for the three indications was higher than expected . Alternatively, to determine the hospitalization percentage more accurately, we should have conducted a pilot study. Being the appropriateness study strictly depend on the surveillance study (i.e. the number antibiotics prescriptions are related to the number of patients enrolled), the same sample size was kept also for the appropriateness study.Our study shows that prescribing antibiotic practices were globally sufficiently appropriate, but certain aspects deserve closer investigation, above all the widespread preference for multidrug empirical regimens or macrolide in monotherapy which in some cases seemed difficult to justify.In addition, findings from this study underline that priority actions for improving the rational use of antibacterials in children should concentrate on education activities such as active participation of the pediatricians to training courses or availability of electronic decision support systems that may facilitate guideline-concordant antibiotic prescription.


Chrysanthemum indicum, an edible flower, possesses abundant bioactivities; the supercritical-carbon dioxide fluid extract from flowers and buds of C. indicum (CISCFE) have strong anti-inflammatory, anti-oxidant, and lung protective effects. However, the role of CISCFE combined with BLM treatment on tumor-bearing mice remains unclear. The present study aimed to investigate the potential synergistic effect and the underlying mechanism of CISCFE combined with BLM in the treatment of hepatoma 22 (H22) tumor-bearing mice. The results suggested that the oral administration of CISCFE combined with BLM could markedly prolong the life span, attenuate the BLM-induced pulmonary fibrosis, suppress the production of pro-inflammatory cytokines (interleukin-6), tumor necrosis factor-α, activities of myeloperoxidase, and malondiadehyde. Moreover, CISCFE combined with BLM promoted the ascites cell apoptosis, the activities of caspases 3 and 8, and up-regulated the protein expression of p53 and down-regulated the transforming growth factor-β1 by activating the gene expression of miR-29b. Taken together, these results indicated that CISCFE could enhance the anti-cancer activity of BLM and reduce the BLM-induced pulmonary injury in H22 tumor-bearing mice, rendering it as a potential adjuvant drug with chemotherapy after further investigation in the future.Bleomycin (BLM), a family of anti-tumor drugs, was reported to exhibit severe side effects limiting its usage in clinical treatment. Therefore, finding adjuvants that enhance the anti-tumor effect and reduce the detrimental effect of BLM is a prerequisite.  Streptomyces verticillus [Bleomycin (BLM), a glycopetide originally isolated from ticillus , is a clticillus . Severalticillus . Howeverticillus ,4. ThereThe integration of different signaling pathways plays a critical role in the normal development and tissue homeostasis of metazoans. When one or more signals fail to integrate, the entire signaling network might collapse resulting in diseases, especially cancer . The losChrysanthemum indicum (C. indicum) Linné, a traditional medicinal and edible flower, is widely used as herbal tea, alcoholic beverage, and food additive or directly used to treat several infectious diseases and ailments, such as headache, eye diseases, and various immune-related disorders with high efficacy and low toxicity [C. indicum (CISCFE) have been extensively applied not only in many classical prescription, but also used in daily life as functional foods, cosmetics, and beverages [SCFE has a strong ability to protect against the chemotherapy-induced renal cell damage [SCFE has a protective effect against lipopolysaccharide-induced lung injury and UV-induced skin injury [SCFE combined with BLM in vivo remains unclear.toxicity ,13,14. Mtoxicity . Importaeverages . Pongjitl damage . In addin injury ,19. HoweSCFE combined with BLM and investigate the underlying mechanism in the treatment of cancer.In the present study, we used the classical H22 ascites tumor-bearing mice model ,21 to exSCFE, BLM, and their combination, we evaluated the life span of the H22 tumor-bearing mice model. As shown in SCFE  alone groups exhibited no significant influence on the life-span of the tumor-bearing mice (p > 0.05), the BLM alone group could prolong the survival time (p < 0.05), while the mice treated with BLM + CISCFE  for seven days could significantly prolong the life span as compared to BLM alone (p < 0.05). These data suggested that CISCFE-M, H doses could improve the BLM anti-tumor effect. Thus, CISCFE at a middle dose of 360 mg/kg was used in the subsequent studies.To better understand the anti-tumor activities of CISCFE combined with BLM on tumor growth. The weight, abnormal diameter, as well as ascites of mice in the model group increased rapidly compared to the control group. On the contrary, the weights, abnormal diameters, and ascites of mice significantly decreased in the BLM alone and BLM + CISCFE-M groups (M: 360 mg/kg), compared to the model group during seven days (p < 0.05). The mice in the treated groups showed greater vitality and were in good order, while the combination was more effective than individual treatment. Additionally, no obvious differences were observed in body weight and abnormal diameter between CISCFE-M alone group and model group (p > 0.05). These results demonstrated that CISCFE had little or no effect on tumor-bearing mice; however, it significantly enhanced the anti-tumor activity of BLM.SCFE and BLM can potentially enhance the efficacy of BLM on H22 ascites cell apoptosis, flow cytometry was utilized to assess the rate of apoptosis. As shown in SCFE-M combined group, the rate of apoptotic cells (Annexin V+/PI− + Annexin V+/PI+) was notably increased as compared to the model group ; the rate of apoptotic cells was obviously increased in the BLM + CISCFE-M combined group as compared to BLM alone (p < 0.05). All results suggested that CISCFE combined with BLM could remarkably increase the H22 ascites cell apoptosis.To evaluate whether the combination of CISCFE-M combined group as compared to the model group . Furthermore, the combination of BLM + CISCFE-M had a statistically stronger effect than BLM alone . These results suggested that CISCFE combined with BLM enhanced the effect of BLM on caspase 3 and caspase 8 activities.Previous studies have demonstrated that caspase families play a vital role in tumor cell apoptosis, including caspase 3 and caspase 8. SCFE alone group .As shown in ne group C,H showene group D,I, haemne group E,J. In ap < 0.05, respectively). Conversely, the combination group dramatically decreased the production of these cytokines as compared to the BLM alone group . No significant differences were observed between the model and CISCFE alone groups.To evaluate the extent of inflammation in lung tissues, the productions of tumor necrosis factor-alpha (TNF-α) and interleukin (IL-6) were measured. SCFE against BLM-induced lung injury was related to anti-oxidative effect, the activities of myeloperoxidase (MPO) and malondialdehyde (MDA) were assayed. As shown in p < 0.05, respectively). However, coupling BLM with CISCFE remarkably decreased the MPO and MDA activities when compared with BLM alone , indicating that CISCFE could decrease the BLM-induced oxidative stress.To explore whether the protection effect of CIp < 0.05), whereas the combination of BLM with CISCFE significantly increased the p53 expression as compared to the BLM alone group (p < 0.05). On the other hand, the combination of BLM with CISCFE could significantly inhibit the TGF-β1 (B) expression in lungs when compared with the BLM alone group (p < 0.05).As shown in SCFE could modulate the miR-29b expression of BLM-treated tumor-bearing mice. As shown in SCFE or BLM alone did not exhibit any distinct effect on miR-29b expression in the ascites cells and lung tissues as compared to the model group . However, the combination of BLM with CISCFE significantly enhanced the expression of miR-29b in ascites cells and lung tissues as compared to BLM alone (p < 0.05).miR-29b is a well-established vital tumor suppressor and fibrosis modulator ,23, playC. indicum, a traditional medicinal and edible flower, possesses heat clearing and toxin-removal activities. Modern pharmacological research displayed that CISCFE has anti-bacterial, anti-virus, anti-inflammation, anti-sympathetic, anti-oxidant, and anti-neoplastic functions [SCFE alone exerted no apparent anti-tumor effect in the mice. However, CISCFE combined with BLM groups markedly prolonged the life-span and significantly decreased the change of weight, abnormal diameter, as well as ascites fluid compared to the BLM alone group. These results suggested that CISCFE could be a potential adjuvant for BLM to enhance the anti-cancer effect in tumor-bearing mice.The clinical usage of chemotherapeutics is well-known to exert severe side-effects . Therefounctions ,28. ThesSCFE enhanced anti-cancer activity of BLM, we sought to investigate the tumor cell apoptosis. Apoptosis is involved in many physiological processes, which can exclude the abnormal or damaged cells, promoting cancer cell apoptosis as an efficient method to control the tumor growth [SCFE can induce apoptosis and inhibit cell proliferation through signal transducers and activators of transcription factors-3 (STAT-3) and NF-κB signaling pathways in different cancer cell lines [SCFE combined with BLM could remarkably increase the H22 ascites cell apoptosis. Caspase 8 as a critical pro-apoptotic molecule, which, once activated, can trigger the downstream caspase cascade, including the major cell apoptosis executor caspase 3 [SCFE. Furthermore, p53 is a crucial apoptotic protein that directly mediates the downstream caspase 3 and caspase 8 to exert its effect on cell apoptosis. Furthermore, p53 is also a vital tumor suppressor, inactivated in most human cancers with high mutations [SCFE combined with BLM groups, the expression of p53 was notably enhanced as compared to BLM alone, with the markedly increased promotion of apoptosis in cancer cells, accompanied by enhanced activities of caspases 3 and 8. These findings imply that the mechanism of CISCFE combined with BLM in inducing apoptosis of H22 ascites tumor cells might also involve the activation of the p53 apoptotic pathway.In order to further understand the mechanism of CIr growth . Previoull lines ,31. Our aspase 3 , Thus, tutations , and canSCFE alone group had no obvious effect on cells apoptosis; however, when coupled with BLM, CISCFE remarkably promoted the survival rate of H22 tumor-bearing mice. Could it also be caused by reducing the detrimental effect of BLM? It is well-known that one of the most serious side-effects of BLM is pulmonary fibrosis, which in turn increases the mortality rate of BLM-treated patients [SCFE. Pulmonary fibrosis is a chronic fibrosis interstitial lung disease with poor prognosis and unknown etiology. The pro-inflammatory cytokines are known to play a significant role in the processing of pulmonary fibrosis, including TNF-α and IL-6. Furthermore, the excessive free radicals will result in oxidative stress or chronic inflammation [SCFE combined with BLM could distinctly decrease the levels of inflammatory cytokines  and oxidant enzymes . Moreover, previous data reported that TGF-β1 suppressed the release of pro-inflammatory cytokines including TNF-α, IL-6, and itself. In turn, TNF-α and IL-6 stimulated the activity of the TGF-β1 [SCFE combined BLM group as compared to BLM alone, thereby proving that CISCFE could relieve the side-effects of BLM.Based on above findings, CIpatients . A pulmoammation ,2,35, the TGF-β1 . Consecue TGF-β1 . In the P53 and TGF-β1 signaling pathways are equally important in the initiation of cancer, due to abundant cross-talk . In the SCFE or BLM alone had no obvious effect on miR-29b expression in the ascites cells and lung tissues. Interestingly, the expression of miR-29b levels was dramatically enhanced when CISCFE was coupled with BLM. These results indicated that CISCFE combined with BLM affected the miR-29b expression, and regulated the balance between p53 and TGF-β1 signaling pathways in H22 tumor-bearing mice.miR-29b, a vital tumor suppressor, can suppress the tumor cell growth by regulating the expression of p53 , therebyBleomycin (BLM) hydrochloride was purchased from Haizheng Pharmaceuticals . Mouse tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) the enzyme-linked immunosorbent assay (ELISA) reagents were purchased from eBioscience ; Myeloperoxidase (MPO) and malondiadehyde (MDA) Colorimetric Activity Assay Kits were obtained from Jiancheng Institution of Biotechnology . Medium RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco ; Penicillin-Streptomycin were obtained from Hyclone ; The Annexin V-fluorescein isothiocyanate (FITC) apoptosis kit was offered by Keygen Biotech ; TRIzol reagent was offered by Invitrogen Life Technologies . All other chemicals and reagents used in the study were of analytical grade.2 extract of Chrysanthemum indicum (CISCFE) was prepared and offered by the Institute of New Drug Research & Development Guangzhou University of Chinese Medicine (Lot. 20121104) [. According to our previous report, the composition analysis was done by combining Gas Chromatography-Mass Spectrometer (GC-MS) and high-performance liquid chromatography with Photodiode Array Detector (HPLC-PAD). Thirty compounds were detected by GC-MS, four compounds were identified by HPLC-PAD  . Accordi2. The cells were incubated in RPMI1640 medium until they reached approximately 2 × 106 cells/mL and 80% viability.The mouse H22 hepatocellular carcinoma cells used in this study were purchased from American Type Culture Collection  and were revived at 37 °C, then maintained at Dulbecco’s Modified Eagle’s medium (obtained RPMI-1640 medium supplemented with 10% FBS and 1% Penicillin-Streptomycin) in a humidified atmosphere with 5% COMale Kunming (KM) mice (18–22 g) were purchased from the Experimental Animal Center, Institute of Guangzhou University of Chinese Medicine . The animals were housed in a 12-h light/dark cycle under a constant temperature of 24 °C and relative humidity of 65% ± 15%, and fed with standard diet and tap water. The animal experiments were conducted according to the guidelines established by the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Welfare Committee of Guangzhou University of Chinese Medicine.6 cells/mL) were inoculated through abdomen into male KM mice and the ascites cells were passaged three times in the mice, after seven days, the ascites fluid was extracted and diluted with normal saline; the cell concentration was adjusted to 2 × 106 cells/mL and injected into each animal. After five days, 90 mice were randomly divided into nine groups with 10 mice in each group: the control group  injection), model group , BLM alone group , CISCFE-L, M, H doses alone group  administration), and BLM  combined with CISCFE-L, M, H doses group . After 24 h, the control and model were intraperitoneally injected normal saline, and BLM alone group with BLM. The CISCFE alone group were respectively gavaged CISCFE-L, M, H solvent; the BLM combined with CISCFE groups were respectively intraperitoneally injected BLM and gavaged CISCFE-L, M, H solvent once per day for a total of seven consecutive days. All the mice were allowed free access to water and food until death, and the survival rate was calculated.H22 cells , and BLM  combined with CISCFE-M dose group . After 24 h, the control and model groups were intraperitoneally injected with normal saline and the BLM alone group was intraperitoneally injected with BLM; the CISCFE alone group was gavaged CISCFE-M solvent; the BLM combined with CISCFE groups was intraperitoneally injected BLM and gavaged CISCFE-M solvent once per day for a total of seven consecutive days. At day 8, 10 mice of each group were executed and ascites were collected with lung tissues for the subsequent tests. A portion of the ascites was solubilized in the TRIzol reagent for the extraction of total RNA, and the other portion was used for Western blotting analysis. The lung tissue was rapidly removed and washed in ice-cold normal saline, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis.Another 50 mice were randomly divided into five groups with 10 mice in each group: the control and model group , BLM alone group , CILung tissues were fixed in 10% neutral buffered formalin and embedded in paraffin wax, cut into 5 μm thick slices, and subjected to haematoxylin-eosin (H&E) staining and Masson’s trichrome staining to detect inflammation or collagen deposition, respectively. The lung injuries were ranked from 0  to 4 (severe) for four categories: congestion, edema, interstitial inflammation, and inflammatory cell infiltration; the overall lung injury score was calculated by adding up the individual scores of each category .g for 10 min at 4 °C. The total supernatant was extracted, and subsequently, the enzyme-linked immunosorbent assay (ELISA) kits  were utilized for the estimation of activities of MPO and MDA in the lung tissues.Lung tissues (0.2 g) were homogenized  with steel shot in nine volumes of cold normal saline (4 °C), followed by centrifugation at 3000× g for 10 min at 4 °C. The total supernatant was extracted to estimate the secreted TNF-α as well as IL-6 by the operation sequences utilizing ELISA kits.Other lung tissues (0.3 g) were homogenized  with steel shot in 9 volumes of cold PBS (4 °C) and centrifuged at 3000× 6 cells/mL PBS. Then, 400 µL Annexin V-FITC and 5 µL Annexin V-FITC were integrated with the cells, respectively, and incubated for 15 min in the dark (4 °C). Subsequently, 10 µL propidium iodide (PI) was lightly mixed with the cells and incubated for 5 min (4 °C), followed by FACS . The apoptotic cells were quantified as the percentage of sub-G1 DNA content in each sample.The ascites fluid was centrifuged  and washed twice using the pre-chilled PBS. The cell concentration was adjusted to 1 × 10g for 5 min at 4 °C, the total supernatant was aspirated and 200 µL radio immunoprecipitation assay buffer (RIPA) lysis buffer added and agitated to completely to crack the cells on the ice, followed by centrifugation at 12,000× g for 5 min at 4 °C. The total protein was collected in the supernatant. Next, the nuclear and cell plasma protein extraction kits were utilized to isolate the respective protein fractions. The protein concentration was estimated by bicinchoninic acid assay method, an equivalent amount separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 1 h with PBS containing 5% dried milk powder and incubated overnight at 4 °C with rabbit anti-Mcl-1 , rabbit anti-BAG3 (1:1000), or mouse anti-MMP2 . Then, the membranes were washed in TBST, and the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in TBST were added. The lung tissues and ascites cells were washed two to three times with cold PBS to remove the blood and then homogenized. Approximately, 10 volumes of RIPA lysis buffer was added and mixed vigorously complete cracking of the cells on ice, followed by centrifugation at 12,000× g for 5 min at 4 °C; the total protein was collected in the supernatant.The ascites cells were washed with cold PBS three times and centrifuged at 1000× The activities of caspase 3 and caspase 8 in the H22 ascites cells were evaluated by the respective caspase activity assay kit purchased from Keygen Biotech . Both activities were correspondingly assayed by spectrophotometry (λ = 405 nm), according to the manufacture’s protocols.Ct−ΔΔ, ΔΔCt = (CtSample − CtGAPDH) − (CtControl − CtGAPDH). The primers sequences of gene including miR-29b, GAPDH was synthesized by Invitrogen, the siRNA sequences were listed as follows: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCTAAACC-3′; 5′-ACACTCCAGCTGGGGCTGGTTTCATATGGTGG-3′ for miRNA-29b and 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′ for GAPDH control.H22 ascites fluid, as well as lung tissues, were washed with PBS; the ascites fluid was extracted with TRIzol reagent  to isolate the total RNA. Total RNA (1.5 μg) was reverse transcribed using Kit  to yield cDNA. The reaction was run at 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min, the solubility curve 75–95 °C, heat up 1 °C each 20 s on Applied Biosystems Step-One Fast Real-Time PCR system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Fold change = 2p < 0.05 was considered statistically significant. The comparison of survival curves was determined by the log-rank (Mantel-Cox) test. All data were analyzed using the statistical analysis software .All data were assessed by one-way ANOVA method, Least Significant Difference (LSD), and Dunnett’s T3 (3) test for comparison between any two means; SCFE could enhance the anti-cancer activity of BLM in H22 tumor-bearing mice. The synergistic effect of BLM was related to CISCFE by inducing apoptosis of H22 ascites tumor cells and reducing the pulmonary injury induced by BLM. Thus, the possible underlying mechanism was associated with the regulation of the balance of p53 and TGF-β1 signaling pathways. These results indicated that CISCFE could serve as a putative adjuvant drug with chemotherapy in the future.Our studies confirmed that CI