Actinomycin-X2-Immobilized Silk Fibroin Film with Enhanced Antimicrobial and Wound Healing Activities.

Actinomycin is a family of chromogenic lactone peptides that differ in their peptide portions of the molecule. An antimicrobial peptide, actinomycin X2 (Ac.X2), was produced through the fermentation of a Streptomyces cyaneofuscatus strain. Immobilization of Ac.X2 onto a prepared silk fibroin (SF) film was done through a carbodiimide reaction. The physical properties of immobilized Ac.X2 (antimicrobial films, AMFs) were analyzed by ATR-FTIR, SEM, AFM, and WCA. The findings from an in vitro study showed that AMFs had a more broad-spectrum antibacterial activity against both S. aureus and E. coli compared with free Ac.X2, which showed no apparent strong effect against E. coli. These AMFs showed a suitable degradation rate, good hemocompatibility, and reduced cytotoxicity in the biocompatibility assay. The results of in vivo bacterially infected wound healing experiments indicated that wound inflammation was prevented by AMFs, which promoted wound repair and improved the wound microenvironment. This study revealed that Ac.X2 transformation is a potential candidate for skin wound healing.

Synergistic binding of actinomycin D and echinomycin to DNA mismatch sites and their combined anti-tumour effects.

Combination cancer chemotherapy is one of the most useful treatment methods to achieve a synergistic effect and reduce the toxicity of dosing with a single drug. Here, we use a combination of two well-established anticancer DNA intercalators, actinomycin D (ActD) and echinomycin (Echi), to screen their binding capabilities with DNA duplexes containing different mismatches embedded within Watson-Crick base-pairs. We have found that combining ActD and Echi preferentially stabilised thymine-related T:T mismatches. The enhanced stability of the DNA duplex-drug complexes is mainly due to the cooperative binding of the two drugs to the mismatch duplex, with many stacking interactions between the two different drug molecules. Since the repair of thymine-related mismatches is less efficient in mismatch repair (MMR)-deficient cancer cells, we have also demonstrated that the combination of ActD and Echi exhibits enhanced synergistic effects against MMR-deficient HCT116 cells and synergy is maintained in a MMR-related MLH1 gene knockdown in SW620 cells. We further accessed the clinical potential of the two-drug combination approach with a xenograft mouse model of a colorectal MMR-deficient cancer, which has resulted in a significant synergistic anti-tumour effect. The current study provides a novel approach for the development of combination chemotherapy for the treatment of cancers related to DNA-mismatches.

Effect of SDS Micelles on Actinomycin D - DNA Complexes.

DNA thermal denaturation was evaluated as a measure of the effect of antitumor drug actinomycin D on the stability of the double helix and also the effect of SDS micelles on actinomycin D - DNA complexes. The results indicated that the melting temperature of DNA was dependent on drug concentration, increasing with actinomycin D concentration. High thermal stabilization (about 10 °C) of the DNA helix after the association with actinomycin D clearly demonstrates the intercalative binding mode. The presence of SDS micelles leads to the release of intercalated actinomcyin D molecules from DNA double helix and their further relocation in surfactant micelles. These results highlighted that the drug release can be controlled in time and by varying the concentration and nature of surfactant.

Discovery of actinomycin L, a new member of the actinomycin family of antibiotics.

Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L1 and L2 produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L1 and L2 are diastereomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X2 producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.

Separation and characterization of two series of unknown degradation impurities caused by light irradiation and autoclaving in xinfujunsu injection using liquid chromatography tandem mass spectrometry.

RATIONALE: A series of photodegradation impurities and a series of degradation impurities produced in autoclaving in xinfujunsu injection were discovered, and these unknown impurities were separated and characterized thoroughly using liquid chromatography tandem quadrupole time-of-flight mass spectrometry. METHODS: The column was a Platisil 5 µm ODS (4.6 × 250 mm, 5 µm). For the analysis of degradation impurities caused by light irradiation and autoclaving, the mobile phase was composed of 0.01 M ammonium formate aqueous solution and acetonitrile/isopropanol (90:10, V/V). Full scan LC-MS and LC-MS2 was carried out to obtain as much structural information as possible. The fragmentation behavior of actinomycin D, actinomycin S3 , and its impurities was studied and used to obtain information about the structures of these impurities. RESULTS: Based on MS2 spectral data and exact mass measurements, the chemical structures of two series of degradation impurities were characterized, among which five unknown impurities were photodegradation impurities and seven unknown impurities were degradation impurities produced in autoclaving of xinfujunsu injection. CONCLUSIONS: Based on characterization of impurities, this study also revealed the cause of impurity production and provided guidance for enterprises to improve the process and drug packaging material to reduce impurity content. Furthermore, this study also provided scientific basis for further improvement of official monographs in pharmacopoeias.

Multi-Omics Analysis Reveals Anti-Staphylococcus aureus Activity of Actinomycin D Originating from Streptomyces parvulus.

Staphylococcus aureus (S. aureus) is a common pathogen that causes various serious diseases, including chronic infections. Discovering new antibacterial agents is an important aspect of the pharmaceutical field because of the lack of effective antibacterial drugs. In our research, we found that one anti-S. aureus substance is actinomycin D, originating from Streptomyces parvulus (S. parvulus); then, we further focused on the anti-S. aureus ability and the omics profile of S. aureus in response to actinomycin D. The results revealed that actinomycin D had a significant inhibitory activity on S. aureus with a minimum inhibitory concentration (MIC) of 2 µg/mL and a minimum bactericidal concentration (MBC) of 64 µg/mL. Bacterial reactive oxygen species (ROS) increased 3.5-fold upon treatment with actinomycin D, as was measured with the oxidation-sensitive fluorescent probe DCFH-DA, and H2O2 increased 3.5 times with treatment by actinomycin D. Proteomics and metabolomics, respectively, identified differentially expressed proteins in control and treatment groups, and the co-mapped correlation network of proteomics and metabolomics annotated five major pathways that were potentially related to disrupting the energy metabolism and oxidative stress of S. aureus. All findings contributed to providing new insight into the mechanisms of the anti-S. aureus effects of actinomycin D originating from S. parvulus.

Actinomycin V Induces Apoptosis Associated with Mitochondrial and PI3K/AKT Pathways in Human CRC Cells.

Actinomycin (Act) V, an analogue of Act D, presented stronger antitumor activity and less hepatorenal toxicity than Act D in our previous studies, which is worthy of further investigation. We hereby report that Act V induces apoptosis via mitochondrial and PI3K/AKT pathways in colorectal cancer (CRC) cells. Act V-induced apoptosis was characterized by mitochondrial dysfunction, with loss of mitochondria membrane potential (MMP) and cytochrome c release, which then activated cleaved caspase-9, cleaved caspase-3, and cleaved PARP, revealing that it was related to the mitochondrial pathway, and the apoptotic trendency can be reversed by caspase inhibitor Z-VAD-FMK. Furthermore, we proved that Act V significantly inhibited PI3K/AKT signalling in HCT-116 cells using cell experiments in vitro, and it also presented a potential targeted PI3Kα inhibition using computer docking models. Further elucidation revealed that it exhibited a 28-fold greater potency than the PI3K inhibitor LY294002 on PI3K inhibition efficacy. Taken together, Act V, as a superior potential replacement of Act D, is a potential candidate for inhibiting the PI3K/AKT pathway and is worthy of more pre-clinical studies in the therapy of CRC.

Actinomycin X2, an Antimicrobial Depsipeptide from Marine-Derived Streptomyces cyaneofuscatus Applied as a Good Natural Dye for Silk Fabric.

Actinomycins as clinical medicine have been extensively studied, while few investigations were conducted to discover the feasibility of actinomycins as antimicrobial natural dye contributing to the medical value of the functional fabrics. This study was focused on the application of actinomycin X2 (Ac.X2), a peptide pigment cultured from marine-derived Streptomyces cyaneofuscatus, in the dyeing and finishing of silk fabric. The dyeing potential of Ac.X2 with silk vs. cotton fabrics was assessed. As a result, the silk fabric exhibited greater uptake and color fastness with Ac.X2. Through Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction (XRD) analyses, some changes of chemical property for the dyed fabric and Ac.X2 were studied. The silk fabric dyed with Ac.X2 exhibited good UV protection ability. The antibacterial properties of dyed and finished silk were also evaluated, which exhibited over 90% antibacterial activity even after 20 washing cycles. In addition, the brine shrimp assay was conducted to evaluate the general toxicity of the tested fabric, and the results indicated that the dyed silk fabrics had a good biological safety property.

p-Terphenyls and actinomycins from a Streptomyces sp. associated with the larva of mud dauber wasp.

In the course of searching for cytotoxic metabolites from insects associated actinomyces, two new natural p-terphenyl glycosides, strepantibin D (1) and strepantibin E (2), along with terferol (3), actinomycin D (4), actinomycin V (5) and actinomycin V0ß (6), were identified from the fermentation medium of a Streptomyces sp. which was obtained from the larva body of mud dauber wasp. Strepantibin D (1), previously reported as a synthetic derivative of terfestatin A, is firstly isolated as a natural p-terphenyl in this research. Strepantibin D (1) and terferol (3) showed medium cytotoxic activity against breast cancer cells MCF-7, MDA-MB-231 and BT-474. Actinomycins (4-6), especially actinomycin V (5), displayed remarkable cytotoxicity against breast cancer cells, with IC50 values ranging from 0.83 nM to 369.90 nM.

Exploring the Thermodynamics of 7-Amino Actinomycin D-Induced Single-Stranded DNA Hairpin by Spectroscopic Techniques and Computational Simulations.

NMR studies have indicated that the anti-tumor therapeutic agent actinomycin D (ACTD) can induce seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3GTGG-3' to form a hairpin structure with tandem GT mismatches at the stem region next to a loop of three stacked thymine bases. In an effort to uncover the preference of binding sequence and to elucidate the thermodynamics properties of the binding, a combination of spectroscopic techniques and computational simulation studies was performed with d(CCGTTnGTGG) and d(CCGAAnGAGG) (denoted as GTTn and GAAn, respectively; n = 3, 5, and 7) sequences. In the presence of 7-amino actinomycin D (7AACTD), all the six oligomers formed stable hairpin structures. The GTT5-7AACTD/GAA5-7AACTD hairpin structure was more stable than the corresponding GTTn-7AACTD and GAAn-7AACTD (n = 3, 7). No significant ΔG difference was observed between GTTn-7AACTD and GAAn-7AACTD complexes with the same loop length. In agreement with the 7AACTD-induced hairpin stability results, the binding affinity of GTTn and GAAn with 7AACTD increased from n = 3 to n = 5 and then decreased when n is 7. Moreover, GTTn and GAAn with the same loop length showed comparable binding affinities to 7AACTD. Furthermore, molecular dynamics simulations found that van der Waals interactions between GTTn/GAAn and 7AACTD were the primary attractive forces for 7AACTD binding, and the electrostatic interactions between the carbonyl groups of 7AACTD and bases in the hairpin were the major unfavorable forces. These findings furthered our understanding that 7AACTD is sensitive to the loop size and sequence as well as tandem GT/GA mismatches of their deoxyribonucleic acid (DNA) targets. A deep understanding of the thermodynamics and the molecular recognition mechanism of 7AACTD with ssDNAs would further the development of ACTD-like antitumor agents.

Single-Cell Analysis of Signaling Proteins Provides Insights into Proapoptotic Properties of Anticancer Drugs.

Single-cell DNA analysis technology has provided unprecedented insights into many physiological and pathological processes. In contrast, technologies that allow protein analysis in single cells have lagged behind. Herein, a method called single-cell Plasmonic ImmunoSandwich Assay (scPISA) that is capable of measuring signaling proteins and protein complexes in single living cells is described. scPISA is straightforward, comprising specific in-cell extraction and ultrasensitive plasmonic detection. It is applied to evaluate the efficacy and kinetics of cytotoxic drugs. It reveals that different drugs exhibit distinct proapoptotic properties at the single-cell level. A set of new parameters is thus proposed for comprehensive and quantitative evaluation of the efficacy of anticancer drugs. It discloses that metformin can dramatically enhance the overall anticancer efficacy when combined with actinomycin D, although it itself is significantly less effective. Furthermore, scPISA reveals that survivin interacts with cytochrome C and caspase-3 in a dynamic fashion in single cells during continuous drug treatment. As compared with conventional assays, scPISA exhibits several significant advantages, such as ultrahigh sensitivity, single-cell resolution, fast speed, and so on. Therefore, this approach may provide a powerful tool for wide, important applications from basic research to clinical applications, particularly precision medicine.

Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation.

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

Sensitive monitoring of RNA transcription by optical amplification of cationic conjugated polymers.

We reported a new strategy for sensitive monitoring in vitro RNA synthesis in real time based on fluorescence resonance energy transfer (FRET) from water-soluble conjugated polymer poly (9, 9-bis (6'-N, N, N,-trimethylammonium) hexyl) fluorene-co-alt-1,4-phenylene) bromide (PFP) to fluorogenic RNA aptamer/fluorophore (Spanich2/DFHBI and Broccoli/DFHBI) system. In this strategy, RNA of interest was transcribed accompanied by the Spanich2 or Broccoli. Then the 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) bound to the RNA aptamer sequence and thereby induced a fluorescence signal. PFP was used as the fluorescence energy donor, and Spanich2/DFHBI was the fluorescence energy acceptor. The fluorescence signal of Spanich2/DFHBI was amplified by light-harvesting and fluorescence amplification ability of PFP via FRET. And the limit of detection (LOD) (0.29 nM) was near 10-fold lower than that of RNA aptamer/DFHBI (LOD is 2.8 nM) alone by measuring the FRET ratio, which greatly reduced the variation of background signals. Most importantly, the addition of PFP did not interfere with RNA transcription in vitro, so this method was successfully applied to sensitively monitor RNA transcription and effect of T7 RNA polymerase inhibitor in real time, supplying a sensitive and simple method to study the modulation and inhibitor of RNA polymerase in vitro.

Escherichia coli as a Model for the Description of the Antimicrobial Mechanism of a Cationic Polymer Surface: Cellular Target and Bacterial Contrast Response.

In this study, we use Escherichia coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on monomethylether poly(ethylene glycol), methyl methacrylate, and 2-dimethyl(aminoethyl) methacrylate, whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups that can generate a charged surface by protonation when in contact with water. For this purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that the polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from large unilamellar vesicles at different phospholipid compositions indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that the polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of the outer membrane structure ( fabR, fadR, fabA, waaA, waaC, kdsA, pldA, and pagP), as well as regulators of cellular response to oxidative stress ( soxS), are more expressed when bacteria are exposed to the polymer surface. All together these results identified the outer membrane as the main cellular target of the antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In this perspective, data reported here could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.

Enantiomeric NMR discrimination of carboxylic acids using actinomycin D as a chiral solvating agent.

Actinomycin D (Act-D) is a biologically important polypeptide antibiotic clinically used to treat several malignant tumors. Herein, we extended its hitherto-unexplored application as an applicable chiral solvating agent (CSA) for the rapid enantiomeric determination of different chiral carboxylic acids in deuterated chloroform by 1H NMR spectroscopy. Notable enantiodiscrimination with well-splitting α-H or α-CH3 resonance signals of the enantiomers of carboxylic acids were achieved without significant interference from Act-D. To check its applicability for the determination of enantiomeric excess (ee) values, various mandelic acid (MA) samples were determined and compared with the observed ones, resulting in an excellent linear relationship. To our knowledge, this is the first example of using a natural antibiotic compound as a CSA to achieve chiral recognition for carboxylic acids.

Antimicrobial potential of haloalkaliphilic Nocardiopsis sp. AJ1 isolated from solar salterns in India.

Antagonistic haloalkaliphilic Nocardiopsis sp. AJ1 (GenBank JX575136.1), isolated and identified from the saline soil of Kovalam solar salterns was able to produce antimicrobial secondary metabolites and effectively suppressed several bacterial and fungal pathogens. The metabolite extracted from ethyl acetate precipitation suppressed the bacterial and fungal pathogens to the range between 2.14 and 20.14 mm and also controlled the shrimp killer virus WSSV by 83% than the control and significantly (p < 0.05) differed. GC-MS analysis revealed that, the ethyl acetate precipitation contains pyrrolo (1,2-A(pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-) and actinomycin C2. Non ribosomal peptide synthetase (NRPS) was amplified by PCR with the amplicon size of 750-800 bp length and further predicted the secondary structure by Iterative Threading Assembly Refinement (I-TASSER) bioinformatics approach. I-TASSER prediction helped to find out the secondary, 3-D structure, and ligand binding sites. The top ten modelling concluded that, the NRPS gene is closely similar to surfactin synthesizing gene, surfactin A synthetase C (SRFA-C). The findings revealed that, the active compounds from the secondary metabolites effectively suppressed the pathogenic bacteria, fungi, and virus and useful to develop antimicrobials.

A new actinomycin Z analogue with an additional oxygen bridge between chromophore and ß-depsipentapeptide from Streptomyces sp. KIB-H714.

Actinomycin Z6 (1), a new member of the actinomycin family, along with three congeners of the Z-type (Z1, Z3, Z5) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the ß-ring of the new compound actinomycin Z6 includes an additional ring linked between the actinoyl chromophore and ß-peptidolactone. In Z3 and Z5, the L-threonine in ß-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.

Comparison of actinomycin peptide synthetase formation in Streptomyces chrysomallus and Streptomyces antibioticus.

Actinomycin peptide synthetase genes constitute two oppositely oriented transcriptional units, acmADR, and acmBC, separated by a non-coding intergenic region. Gene constructs of the intergenic region together with its adjoining gene acmA or acmB from the actinomycin biosynthetic gene cluster of Streptomyces chrysomallus were transferred into Streptomyces lividans TK64. Each construct expressed the respective synthetase indicating divergent promoters. Primer extension revealed for both directions -10 and -35 boxes similar to σ70 -dependent promoters from Streptomyces and E. coli. No conspicuous regulatory sequences were detected. Accordingly, S. chrysomallus-grown in glucose-containing medium-produced the peptide synthetases AcmA and AcmB/C as well as actinomycin during logarithmic growth phase. Alignments with the corresponding intergenic region of the actinomycin biosynthetic gene cluster in Streptomyces antibioticus identified analogous -10 and -35 boxes of σ70 consensus sequence. However, in S. antibioticus-cultivated in the same conditions-AcmA and AcmB/C were at maximum activity in late log phase and actinomycin formation peaked in stationary phase. The different patterns of formation of actinomycin and its peptide synthetases encoded by the highly homologous actinomycin biosynthetic gene clusters in S. chrysomallus and S. antibioticus suggest strain-specific control of biosynthesis in agreement with absence of pathway-specific regulatory genes.

Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway.

A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.

Improved flow cytometry crossmatching in kidney transplantation.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.