Mechanism Of Synergistic Activity Research Articles

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin’s N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin’s Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin’s core function in branch stabilization.

Degradation of atrazine by electroactivation of persulfate using FeCuO@C modified composite cathode: Synergistic activation mechanism

In sulfate radical-based advanced oxidation processes (SR-AOPs), high-efficiency and perdurable materials have drawn considerable interest for use as cathodes, which can effectively degrade refractory organic contaminants through the synergistic electro-activation and transition metal activation of persulfate (PS). Here, the FeCuO@C modified composite cathode (FeCuO@C/AGF) was synthesized via the solvothermal and thermal treatment method based on the CuFe-MOF-74 structure, and the electro-activation PS process (EC/FeCuO@C/AGF/PS) was developed to effectively remove atrazine (ATZ). The surface morphology, electrochemical characteristics, chemical composition, crystal structure, and electrode surface wettability of FeCuO@C/AGF were investigated. It was found that the proposed EC/FeCuO@C/AGF/PS process can successfully remove 100% of ATZ in 20 min at a low current density (2 mA cm−2) and a low PS concentration (0.4 mM), and PS is successfully activated by combining the electrical and transition metal synergistic activation. The FeCuO@C/AGF cathode exhibits outstanding catalytic functionality over a broad pH range (2–9) and remains stable over five successive cycles. Additionally, the active species involved in the reaction as well as the potential ATZ degradation reaction mechanisms and pathways are discussed. Electrochemical oxidation is a process in which both radicals (SO4·−, ·OH, and O2·−) and non-radical (1O2) participate in the degradation of ATZ. The intermediates of the ATZ degradation process were studied upon the toxicity changing, and the toxicity of the intermediates was found to be reduced during degradation. These results present a novel approach toward the establishment of an effective and reliable electrode in SR-AOPs that can efficiently treat pesticide wastewater.

Preclinical validation of a novel therapeutic strategy for choroid plexus carcinoma

Choroid plexus carcinoma (CPC) is a rare infantile brain tumor with an aggressive clinical course that often leaves children with debilitating side effects due to aggressive and toxic chemotherapies. Development of novel therapeutical strategies for this disease have been extremely limited owing to the rarity of the disease and the paucity of biologically relevant substrates. We conducted the first high-throughput screen (HTS) on a human patient-derived CPC cell line (Children Cancer Hospital Egypt, CCHE-45) and identified 427 top hits highlighting key molecular targets in CPC. Furthermore, a combination screen with a wide variety of targets revealed multiple synergistic combinations that may pave the way for novel therapeutical strategies against CPC. Based on in vitro efficiency, central nervous system (CNS) penetrance ability and feasible translational potential, two combinations using a DNA alkylating or topoisomerase inhibitors in combination with an ataxia telangiectasia mutated and rad3 (ATR) inhibitor (topotecan/elimusertib and melphalan/elimusertib respectively) were validated in vitro and in vivo. Pharmacokinetic assays established increased brain penetrance with intra-arterial (IA) delivery over intra-venous (IV) delivery and demonstrated a higher CNS penetrance for the combination melphalan/elimusertib. The mechanisms of synergistic activity for melphalan/elimusertib were assessed through transcriptome analyses and showed dysregulation of key oncogenic pathways (e.g. MYC, mammalian target of rapamycin mTOR, p53) and activation of critical biological processes (e.g. DNA repair, apoptosis, hypoxia, interferon gamma). Importantly, IA administration of melphalan combined with elimusertib led to a significant increase in survival in a CPC genetic mouse model. In conclusion, this study is, to the best of our knowledge, the first that identifies multiple promising combinatorial therapeutics for CPC and emphasizes the potential of IA delivery for the treatment of CPC.

Alkali synergistic sulfide-modified nZVI activation of persulfate for phenanthrene removal

Sulfide-modified nano zero-valent iron (S-nZVI) and persulfate (PS) have been widely used in advanced oxidation processes, whereas their performance in alkaline solutions is rarely discussed. In this study, the synergistic activation mechanism of S-nZVI with alkali is proposed and verified by removal experiments and activator characterization. nZVI and S-nZVI with different sulfidation degrees were prepared and used to enhance PS oxidation for phenanthrene (PHE) removal. Results indicate that the S/Fe ratio significantly affects PHE degradation, with the optimal ratio at 0.28. S-nZVI exhibits superior degradation rates to nZVI over a wide pH range (3−11), especially in alkaline solutions with a maximum removal rate of 94.7% at pH = 9. At the same time, the relative synergistic enhancement ratio shows a maximum value of 1.80. Hydroxyl radicals are identified as the main active groups. X-ray photoelectron spectroscopy results suggest that the alkaline solutions promote the conversion of S2-, which increases from 24.7% to 33.1% compared to neutral solutions. Furthermore, the electrochemical tests of S-nZVI and nZVI reveal that S-nZVI has lower electron transfer impedance and higher oxidation peak current density at pH = 9, which probably elucidates that the alkali accelerates the electron conductivity of S-nZVI. These findings, for the first time, demonstrate the alkali synergistic activation mechanism of S-nZVI/PS systems and provide a deeper insight into the efficient degradation of persistent organic pollutants in complex pH environments.

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

SUMMARYKinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Microtubule-associated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

Equisetin Restores Colistin Sensitivity against Multi-Drug Resistant Gram-Negative Bacteria.

The overuse of antibiotics and the scarcity of new drugs have led to a serious antimicrobial resistance crisis, especially for multi-drug resistant (MDR) Gram-negative bacteria. In the present study, we investigated the antimicrobial activity of a marine antibiotic equisetin in combination with colistin against Gram-negative bacteria and explored the mechanisms of synergistic activity. We tested the synergistic effect of equisetin in combination with colistin on 23 clinical mcr-1 positive isolates and found that 4 µg/mL equisetin combined with 1 µg/mL colistin showed 100% inhibition. Consistently, equisetin restored the sensitivity of 10 species of mcr-1 positive Gram-negative bacteria to colistin. The combination of equisetin and colistin quickly killed 99.9% bacteria in one hour in time-kill assays. We found that colistin promoted intracellular accumulation of equisetin in colistin-resistant E. coli based on LC-MS/MS analysis. Interestingly, equisetin boosted ROS accumulation in E. coli in the presence of colistin. Moreover, we found that equisetin and colistin lost the synergistic effect in two LPS-deficient A. baumannii strains. These findings suggest that colistin destroys the hydrophobic barrier of Gram-negative bacteria, facilitating equisetin to enter the cell and exert its antibacterial effect. Lastly, equisetin restored the activity of colistin in a G. mellonella larvae infection model. Collectively, these results reveal that equisetin can potentiate colistin activity against MDR Gram-negative bacteria including colistin-resistant strains, providing an alternative approach to address Gram-negative pathogens associated with infections in clinics.

Urea‐Functionalized Swelling Poly(ionic liquid)s as Efficient Catalysts for the Transesterification and Hydrolysis of Ethylene Carbonate

AbstractUrea‐functionalized poly(ionic liquid)s (PILs) were synthesized through polymerization of urea tethered imidazolium ionic liquid monomers (urea‐IL) with sodium acrylate, and N,N′‐methylenebisacrylamide (MBA) as a crosslinker. Close‐packed and interconnected pores (1–4 ) under swollen state could be observed from the cryogenic scanning electron microscopy (cryo‐SEM) images. The promising catalytic activity of the PILs was illustrated for the transesterification reaction of ethylene carbonate with methanol. High activity and selectivity could be achieved by using poly(urea‐IL)‐n catalysts, which was similar to that of corresponding homogeneous ionic liquid catalysts. The urea tethered imidazolium in PILs acted as hydrogen‐bonding donor to activate ethylene carbonate and intermediate 2‐hydroxyethyl methyl carbonate (HEMC) for enhancing catalytic activity. The swelling ability of urea‐functionalized PILs in methanol enabled active urea sites accessible for substrates. However, the complete conversion of ethylene carbonate was limited by reversible reaction between ethylene carbonate and HEMC. A possible synergistic activation mechanism for the transesterification reaction was proposed and supported by NMR titrations. The catalyst can be reused and recycled five times with stable activity. Furthermore, urea‐functionalized swelling PILs also exhibited high catalytic activity for the hydrolysis of ethylene carbonate.

Halogen‐Bonding‐Donor Catalyzed Cyanosilylation of Aldehydes

AbstractAlthough halogen‐bonding has been utilized as an emerging means for activation of substrates, the use of the interaction for activation of relatively low Lewis basic electrophiles such as carbonyl compounds is still limited. Herein, we developed the halogen‐bonding‐donor donor catalyzed cyanosilylation of aldehydes via a synergistic activation mechanism, which were supported by a combined computational and experimental approach. The substrate scope for the reaction and the chemoselectivity of cationic XB‐donors are described.

Synergistic Nanocomposites of Different Antibiotics Coupled with Green Synthesized Chitosan-Based Silver Nanoparticles: Characterization, Antibacterial, in vivo Toxicological and Biodistribution Studies.

PurposeThe present study reports chitosan functionalized green synthesized CS-AgNPs, conjugated with amoxicillin (AMX), cefixime (CEF), and levofloxacin (LVX) for safe and enhanced antibacterial activity.MethodsThe CS-AgNPs and conjugates CS-AgNPs+AMX CS-AgNPs+CEF, and CS-AgNPs+LVX were characterized by UV–Vis, FTIR, SEM, TEM, EDX spectroscopy. The size distribution and zeta potential were measured using the dynamic light scattering (DLS) technique. The interaction between CS-AgNPs and antibiotic molecules was also investigated using UV–Vis spectroscopy at the concentrations of 5, 50, 500, and 5000 µM for each antibiotic. Antibacterial activity and synergism were assessed by the Fractional Inhibitory Concentration (FIC) index. The mechanism for synergistic activity was investigated by the detection of hydroxyl species based on the chemiluminescence of luminol. The biocompatibility index (BI) was calculated from IC50 using the HeLa cell line. In vivo toxicity and tissue distribution of silver ions were evaluated on Sprague Dawley rats. Physical interactions of antibiotics and significant (P<0.05) antibacterial activity were observed after loading on CS-AgNPs surfaces.ResultsThe spherical shape nanocomposites of CS-AgNPs with different antibiotics were prepared with mean size ranges of 80–120 nm. IC50 of antibiotics-conjugated CS-AgNPs decreased compared to CS-AgNPs. The biocompatibility (BI) index showed that antibiotics-conjugated CS-AgNPs have high antibacterial potential and low toxicity. Highly significant (P<0.005) increase in the generation of hydroxyl species indicated the radical scavenging mechanism for synergistic activity of CS-AgNPs after combined with different antibiotics. Biochemical analysis and histopathological examinations confirmed low toxicity with minor hepatotoxicity at higher doses. After oral administration, extensive distribution of Ag ion was observed in spleen and liver.ConclusionThe study demonstrates positive attributes of antibiotics-conjugated CS-AgNPs, as a promising antibacterial agent with low toxicity.

Trichloroethene degradation by nanoscale CaO2 activated with Fe(II)/FeS: The role of FeS and the synergistic activation mechanism of Fe(II)/FeS

The performance of trichloroethene (TCE) degradation was significantly improved with the addition of FeS in nanoscale calcium peroxide (nCP) environment and the enhancement of HO generation in nCP/Fe(II)/FeS system was confirmed by comparison with nCP/Fe(II) and nCP/FeS systems. The role of FeS in nCP/Fe(II)/FeS system demonstrated that the majority of reactive oxygen radicals (ROSs) was generated on the catalyst surface due to the heterogeneous reaction through FeS activated nCP in one hand, and the reductive FeS could promote TCE degradation by regenerating Fe(II) from Fe(III) in the other hand. The probe as well as scavenger test results suggested that the major ROSs was HO rather than O2−. Over 91.5% TCE could be degraded at the molar ratio of 2/2/4/1 of nCP/Fe(II)/FeS/TCE, in which more than 95.5% TCE was dechlorinated. The effect of surfactants on TCE degradation was evaluated. In contrast, further investigation found that BTEX removal was inhibited with the addition of FeS. In conclusion, this study revealed the well function and fundamental synergistic mechanism of FeS. Moreover, the degradation of TCE in actual groundwater confirmed that nCP/Fe(II)/FeS technique has a great advantage and potential in TCE contaminated groundwater remediation in ISCO practice.

Development of a Synergistic Activation Strategy for the Pilot-Scale Construction of Hierarchical Porous Graphitic Carbon for Energy Storage Applications.

Pursuing scalable production of porous carbon with facile and environmentally friendly synthesis methodology is a global goal. Herein, a unique hierarchical porous graphitic carbon (HPGC) with outstanding textural characteristics is achieved by a special synergistic activation mechanism, in which the low-temperature molten state of polymorphisms can induce a high-rate liquid phase porous activation. HPGC with high specific surface area (SSA, ∼2571 m2 g-1) and large pore volume (PV, ∼2.21 cm3 g-1) can be achieved, which also possesses the capability to tune textural characteristics (i.e., SSA, PV, pore size distribution, etc.) within a wide range. Furthermore, the pilot-scale production of HPGC is accomplished, which shows similar textural characteristics to the lab-scale HPGC. Due to the unique structure of HPGC and the capability of the textural control, it can be applicable in a variety of energy storage, energy conversion, and catalysis applications. The applications of pilot-scale HPGC products in supercapacitors and lithium sulfur batteries are highlighted in this work. Furthermore, the synergistic activation strategy can be promoted to other alkali-based carbon activation routes, which can open up new possibilities for the activated carbon production and lead to more widespread industrialized applications of HPGC.

Further characterization of the synergistic activation mechanism of cationic channels by M2 and M3 muscarinic receptors in mouse intestinal smooth muscle cells.

In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.

Structural mechanism of synergistic activation of Aurora kinase B/C by phosphorylated INCENP

Aurora kinases B and C (AURKB/AURKC) are activated by binding to the C-terminal domain of INCENP. Full activation requires phosphorylation of two serine residues of INCENP that are conserved through evolution, although the mechanism of this activation has not been explained. Here we present crystal structures of the fully active complex of AURKC bound to INCENP, consisting of phosphorylated, activated, AURKC and INCENP phosphorylated on its TSS motif, revealing the structural and biochemical mechanism of synergistic activation of AURKC:INCENP. The structures show that TSS motif phosphorylation stabilises the kinase activation loop of AURKC. The TSS motif phosphorylations alter the substrate-binding surface consistent with a mechanism of altered kinase substrate selectivity and stabilisation of the protein complex against unfolding. We also analyse the binding of the most specific available AURKB inhibitor, BRD-7880, and demonstrate that the well-known Aurora kinase inhibitor VX-680 disrupts binding of the phosphorylated INCENP TSS motif.

PARP inhibitors synergize with gemcitabine by potentiating DNA damage in non-small-cell lung cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors have demonstrated great promise in the treatment of patients with deficiencies in homologous recombination (HR) DNA repair, such as those with loss of BRCA1 or BRCA2 function. However, emerging studies suggest that PARP inhibition can also target HR-competent cancers, such as non-small-cell lung cancer (NSCLC), and that the therapeutic effect of PARP inhibition may be improved by combination with chemotherapy agents. In our study, it was found that PARP inhibitors talazoparib (BMN-673) and olaparib (AZD-2281) both had synergistic activity with the common first-line chemotherapeutic gemcitabine in a panel of lung cancer cell lines. Furthermore, the combination demonstrated significant in vivo antitumor activity in an H23 xenograft model of NSCLC compared to either agent as monotherapy. This synergism occurred without loss of HR repair efficiency. Instead, the combination induced synergistic single-strand DNA breaks, leading to accumulation of toxic double-strand DNA lesions in vitro and in vivo. Our study elucidates the underlying mechanisms of synergistic activity of PARP inhibitors and gemcitabine, providing a strong motivation to pursue this combination as an improved therapeutic regimen.

Cooperative Multifunctional Organocatalysts for Ambient Conversion of Carbon Dioxide into Cyclic Carbonates

A series of pincer-type compounds possessing an N-heterocyclic carbene precursor and a carboxyl group as proton transfer agent were synthesized and used as organocatalysts for the cycloaddition of epoxides with CO2. In this context, we have demonstrated the high activity of these one-component organocatalysts in the CO2 transformation to cyclic carbonates under ambient conditions (room temperature, 1 bar of CO2). The catalytic potential of these multifunctional organocatalysts on challenging internal epoxides is particularly deserving of mention because organocatalysts that are able to mediate the cycloaddition reaction of internal epoxides with CO2 under mild conditions remain scarce. The intramolecular synergistic activation mechanism was elucidated by control experiments and DFT calculations.

The inhibitory mechanisms by mixtures of two endophytic bacterial strains isolated from Ginkgo biloba against pepper phytophthora blight

Two endophytic bacterial strains, Fy11 and Zy44 (Bacillus amyloliquefaciens), isolated from Ginkgo biloba leaves, were tested as a single application and in combination for their abilities to suppress pepper phytophthora blight under greenhouse conditions. When both strains were applied in a mixture, the disease severity was more reduced. The objective of this study was to find out the inhibitory mechanisms of synergistic activity against pepper phytophthora blight through a mixture of Fy11+Zy44. Firstly, the colonization ability of the two endophytic bacterial strains was tested in pepper tissues. The result indicated that both endophytic strains were efficient colonizers of pepper, and could vertically transfer from roots to shoots and leaves. After inoculation for 7days, the population densities of Fy11-gfp and Zy44-gfp were 24.7×103 CFU/g FW and 13.0×103CFU/g FW in pepper roots, respectively. The cell counts of co-inoculated Fy11+Zy44 were not significant different comparing to a single strain inter pepper tissues. Secondly, the direct antagonism of antifungal compounds produced by the endophytic bacteria were investigated against pepper phytophthora blight. The crude lipopeptides produced by strain Zy44 highly effectively reduced disease index, and the control efficacy reached 84.8% and 67.2% in 7d and 15d after inoculation with the pathogen, respectively. The control effect of the secondary metabolites produced by strain Fy11 was not obvious. In the end, the induced systemic resistance (ISR) mediated by strains Fy11 and Zy44 on pepper were further analysed using priming effect of the defense-related genes after pathogen challenge. The expressions of CaPR4 (Capsicum annuum pathogenesis-protein 4) and CaBGLU (C. annuum β-1,3-glucanase) were strongly induced in the plants treated with strain Fy11 in 24h after pathogen challenge, but CaPR4 and CaBGLU transcripts were not increased in the plants treated with strain Zy44. These results indicated that the mechanisms of additive effect through combined strains Zy44 and Fy11 might be due to direct antagonism of antifungal lipopeptides produced by strain Zy44, as well as indirect inhibition through ISR induced by strain Fy11. Therefore, the synergistic interactions were due to the summation of biocontrol mechanisms of both biocontrol agents. This work provides a theoretical explanation for our findings of reduced disease severity with a mixture of endophytic bacteria.

A base–conjugate-acid pair for living/controlled ring-opening polymerization of trimethylene carbonate through hydrogen-bonding bifunctional synergistic catalysis

The ring-opening polymerization (ROP) of trimethylene carbonate (TMC) using a base–acid binary organocatalyst system of 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD) and Bronsted acid trifluoromethanesulfonic acid (TFA) with benzyl alcohol (BnOH) as the initiator has been investigated. The MTBD and its conjugate-acid pair H-bonding bifunctional synergistic activation mechanism was demonstrated by NMR measurements, and the living/controlled nature of MTBD/TFA catalyzed ROP of TMC was confirmed by the kinetic and chain extension experiments. The controlled ROP of TMC with the optimal molar ratio of MTBD/TFA = 2/1 proceeded to afford well-defined PTMC with narrow molecular weight distributions (Mw/Mn ∼ 1.1). The molecular weights determined by 1H NMR are in precise agreement with the theoretical values. 1H NMR, SEC, and MALDI-ToF MS measurements confirmed the structure of the obtained homopolymer PTMC. In addition, a diblock copolymer consisting of poly(trimethylene carbonate) and poly(L-lactide) (PTMC-b-PLLA) was synthesized successfully by the MTBD/TFA binary catalyst system.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP

Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation. DOI:http://dx.doi.org/10.7554/eLife.00884.001.

Cytosine Arabinoside Promotes Cytotoxic Effect of T Cells on Leukemia Cells Mediated by Bispecific Antibody

Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3×anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19(+) human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19(+) Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3×anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3×anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1-ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3×anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects.

AFM Probing the Mechanism of Synergistic Effects of the Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate (EGCG) with Cefotaxime against Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli

BackgroundExtended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae poses serious challenges to clinicians because of its resistance to many classes of antibiotics.Methods and FindingsThe mechanism of synergistic activity of a combination of (−)-epigallocatechin-3-gallate (EGCG) and β-lactam antibiotics cefotaxime was studied on Extended-spectrum β-lactamase producing Escherichia coli (ESBL-EC), by visualizing the morphological alteration on the cell wall induced by the combination using atomic force microscopy (AFM). Cells at sub-MICs (sub-minimum inhibitory concentrations) of cefotaxime were initially filamentated but recovered to the normal shape later, whereas cells at sub-MICs of EGCG experienced temporal disturbance on the cell wall such as leakage and release of cellular debris and groove formation, but later recovered to the normal shape. In contrast, the combination of cefotaxime and EGCG at their respective sub-MICs induced permanent cellular damages as well as continuous elongation in cells and eventually killed them. Flow cytometry showed that intracellular oxidative stress levels in the cell treated with a combination of EGCG and cefotaxime at sub-MICs were higher than those in the cells treated with either cefotaxime or EGCG at sub-MICs.ConclusionsThese results suggest that the synergistic effect of EGCG between EGCG and cefotaxime against ESBL-EC is related to cooperative activity of exogenous and endogenous reactive oxygen species (ROS) generated by EGCG and cefotaxime, respectively.