Hypochlorous Acid-Modified Serum Albumin Causes NETosis in the Whole Blood Ex Vivo and in Isolated Neutrophils.

Type 2 diabetes mellitus (T2DM) is accompanied by halogenative stress resulting from the excessive activation of neutrophils and neutrophilic myeloperoxidase (MPO) generating highly reactive hypochlorous acid (HOCl). HOCl in blood plasma modifies serum albumin (Cl-HSA). We studied the formation of neutrophil extracellular traps (NETs) in the whole blood and by isolated neutrophils under the action of Cl-HSA. It was found that Cl-HSA induces neutrophil priming and NETosis. MPO-containing as well as MPO-free NETs were found. These NETs with different composition can be a product of NETosis of one and the same neutrophil. NET formation in neutrophils with vacuolated cytoplasm was detected. In the presence of Cl-HSA, acceleration of NET degradation was observed. Accelerated NET degradation and neutrophil priming can be the factors contributing to the development of complications in T2DM.

Molecular mimicry of the receptor-binding domain of the SARS-CoV-2 spike protein: from the interaction of spike-specific antibodies with transferrin and lactoferrin to the antiviral effects of human recombinant lactoferrin.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R2 > 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by KD to be 23 nM and 16 nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1-4 mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.

Structure-biological activity relationships of myeloperoxidase to effect on platelet activation.

Myeloperoxidase (MPO), an oxidant-producing enzyme of neutrophils, has been shown to prime platelet activity promoting immunothrombosis. Native MPO is a homodimer, consisting of two identical protomers (monomer) connected by a single disulfide bond. But in inflammatory foci, MPO can be found both in the form of a monomer and in the form of a dimer. Beside MPO can also be in complexes with other molecules and be modified by oxidants, which ultimately affect its physicochemical properties and functions. Here we compared the effects of various forms of MPO as well as MPO in complex with ceruloplasmin (CP), a physiological inhibitor of MPO, on the platelet activity. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. MPO was modified with HOCl in a molar ratio of 1:100 (MPO-HOCl). Using surface-enhanced Raman scattering (SERS) spectroscopy we showed that peaks at about 510 and 526 cm-1 corresponded to disulfide bond was recognizable in the SERS-spectra of dimeric MPO, absent in the spectrum of hemi-MPO and less intense in the spectra of MPO-HOCl, which indicates the partial decomposition of dimeric MPO with a disulfide bond cleavage under the HOCl modification. It was shown hemi-MPO to a lesser extent than dimeric MPO bound to platelets and enhanced their agonist-induced aggregation and platelet-neutrophil aggregate formation. MPO modified by HOCl and MPO in complex with CP did not bind to platelets and have no effect on platelet activity. Thus, the modification of MPO by HOCl, its presence in monomeric form as well as in complex with CP reduces MPO effect on platelet function and consequently decreases the risk of thrombosis in inflammatory foci.

The Level of LINE-1 mRNA Is Increased in Extracellular Circulating Plasma RNA in Patients with Colorectal Cancer.

We performed a comparative quantitative analysis of LINE-1 mRNA levels in extracellular total plasma RNA in patients with colon cancer and practically healthy donors. Quantitative multiplex PCR with reverse transcription was used to assess the level of LINE-1 and 18S rRNA mRNA in extracellular total plasma RNA. The median of LINE-1 mRNA values in colon cancer patients (4.95) was significantly higher than in healthy donors (2.3) (p=0.037). It was shown for the first time that the level of LINE-1 mRNA in total RNA from blood plasma can be determined in the format of a liquid biopsy and serve as a new potential non-invasive marker of colon cancer.

Binding of lactoferrin to the surface of low-density lipoproteins modified by myeloperoxidase prevents intracellular cholesterol accumulation by human blood monocytes.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

Injection-seeded single-longitudinal-mode Ti:Sapphire laser with no active stabilization.

We report on the first injection-seeded nanosecond Ti:Sapphire laser that demonstrates a stable single-longitudinal-mode operation with no feedback loop for active cavity stabilization. The short cavity generates 6-mJ transform-limited pulses at a wavelength of 807 nm and with a slope efficiency of 43%. An intracavity dispersive prism makes a novel cavity design for injection-seeded lasers and provides pre-selection of the emission wavelength. In support of these experiments, we perform numerical simulations that include extra cavity losses. The simulation results are in good agreement with the outcome of the experiment and reveal the formation scenario of the laser mode.

The Role of Halogenative Stress in Atherogenic Modification of Low-Density Lipoproteins.

This review discusses formation of reactive halogen species (RHS) catalyzed by myeloperoxidase (MPO), an enzyme mostly present in leukocytes. An imbalance between the RHS production and body's ability to remove or neutralize them leads to the development of halogenative stress. RHS reactions with proteins, lipids, carbohydrates, and antioxidants in the content of low-density lipoproteins (LDLs) of the human blood are described. MPO binds site-specifically to the LDL surface and modifies LDL properties and structural organization, which leads to the LDL conversion into proatherogenic forms captured by monocytes/macrophages, which causes accumulation of cholesterol and its esters in these cells and their transformation into foam cells, the basis of atherosclerotic plaques. The review describes the biomarkers of MPO enzymatic activity and halogenative stress, as well as the involvement of the latter in the development of atherosclerosis.

Myeloperoxidase-Induced Oxidation of Albumin and Ceruloplasmin: Role of Tyrosines.

Neutrophil myeloperoxidase (MPO) plays an important role in protecting the body against infections. MPO products - hypohalous acids and phenoxyl radicals - are strong oxidants that can damage not only foreign intruders but also host tissues, including blood plasma proteins. Here, we compared the MPO-induced oxidation of two plasma proteins with antioxidant properties - human serum albumin (HSA) and ceruloplasmin (CP). Incubation of both proteins with hypochlorite (NaOCl) or catalytically active MPO (MPO + H2O2), which synthesizes hypochlorous acid (HOCl) in the presence of chloride ions, resulted in the quenching of protein tryptophan fluorescence. Oxidation-induced changes in the structures of HSA and CP were different. HSA efficiently neutralized MPO-generated oxidants without protein aggregation, while CP oxidation resulted in the formation of large aggregates stabilized by strong covalent bonds between the aromatic amino acid residues. Tyrosine is present in the plasma as free amino acid and also as a component of the polypeptide chains of the proteins. The number of tyrosine residues in a protein does not determine its propensity for aggregate formation. In the case of CP, protein aggregation was primarily due to the high content of tryptophan residues in its polypeptide chain. MPO-dependent oxidation of free tyrosine results in the formation of tyrosyl radicals, that do not oxidize aromatic amino acid residues in proteins because of the high rate of recombination with dityrosine formation. At the same time, free tyrosine can influence MPO-induced protein oxidation due to its ability to modulate HOCl synthesis in the MPO active site.

Capacity of ceruloplasmin to scavenge products of the respiratory burst of neutrophils is not altered by the products of reactions catalyzed by myeloperoxidase.

CP is a copper-containing ferroxidase of blood plasma, which acts as an acute phase reactant during inflammation. The effect of oxidative modification of CP induced by oxidants produced by MPO, such as HOCl, HOBr, and HOSCN, on its spectral, enzymatic, and anti-inflammatory properties was studied. We monitored the chemiluminescence of lucigenin and luminol along with fluorescence of hydroethidine and scopoletin to assay the inhibition by CP of the neutrophilic respiratory burst induced by PMA or fMLP. Superoxide dismutase activity of CP and its capacity to reduce the production of oxidants in respiratory burst of neutrophils remained virtually unchanged upon modifications caused by HOCl, HOBr, and HOSCN. Meanwhile, the absorption of type I copper ions at 610 nm became reduced, along with a drop in the ferroxidase and amino oxidase activities of CP. Likewise, its inhibitory effect on the halogenating activity of MPO was diminished. Sera of either healthy donors or patients with Wilson disease were co-incubated with neutrophils from healthy volunteers. In these experiments, we observed an inverse relationship between the content of CP in sera and the rate of H2O2 production by activated neutrophils. In conclusion, CP is likely to play a role of an anti-inflammatory factor tempering the neutrophil respiratory burst in the bloodstream despite the MPO-mediated oxidative modifications.

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

Structural Study of the Complex Formed by Ceruloplasmin and Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine. Inhibitors of tautomerase activity of MIF are perspective antiinflammatory compounds. Ceruloplasmin, the copper-containing ferroxidase of blood plasma, is a noncompetitive inhibitor of tautomerase activity of MIF in the reaction with p-hydroxyphenylpyruvate. Small-angle X-ray scattering established a model of the complex formed by MIF and ceruloplasmin. Crystallographic analysis of MIF with a modified active site supports the model. The stoichiometry of 3 CP/MIF trimer complex was established using gel filtration. Conformity of novel data concerning the interaction regions in the studied proteins with previous biochemical data is discussed.

[The significance of detection of nitrites in oral fluid of healthy people.]

The nitric oxide is a signal molecule in human organism characterized by a wide spectrum of biological effects. The exogenous nitric oxide is formed from food nitrates received with such green leafy vegetables as spinach, parsley, sorrel and also beet, cucumbers and tomatoes. The bacteria in oral cavity metabolize received with food nitrates up to nitrites. The nitrites as some intermediate metabolites of nitric oxide sufficiently exact reflect concentration of nitric oxide. The purpose of study is to examine analytical dependence and efficiency of express-device for detecting nitrites in saliva. The article presents the results of confirmation of testsystem for half-quantitative detection of content of nitrites in saliva. The device represents a hollow tube with a sensorial element within functioning by the principle of "dry chemistry". The concentration of nitrites in oral cavity was measured in 100 healthy people of both genders aged from 16 to 45 years. In case of consuming vegetables every day or 3-5 times a week higher levels of nitrite-anions (14,9-15,7 mg/l) are registered than in case of consuming vegetables and juices 1-2 times a week and rarely (9,9 mg/l). The concentrations of nitrites in saliva both in cases of regular training and low-activity life-style are within the limits of standards (11,9-14,9 mg/l). The average level of nitrites in saliva of participants of experiment with normal pressure made up to 16.5 mg/l that is significantly higher than in individuals from groups with increased and decreased arterial pressure (10,2 and 10,4 mg/l correspondingly).

[Methods for detection the indicators of metabolism in the oral liquid (a rewiew).]

The search for new ways to diagnose diseases of different etiologies and their introduction into practical health care remains one of the priority areas of modern medicine. Among the known methods for the analysis of biological fluids, a special place is occupied by the methods of express diagnostics of various pathological conditions by markers found in the oral fluid. This article presents a critical review of the latest developments of domestic and foreign researchers (56 sources are analyzed) concerning both existing and widely used devices and those that are at the development stage. The prospects of using oral fluid as a diagnostic medium, as well as various methods for the rapid detction of markers of pathological conditions, are discussed. The main principles, advantages and disadvantages of immunochromatographic tests, electrochemical, microfluidic analysis, isothermal amplification, and devices based on smartphones for express diagnostics of various markers in oral fluid are presented.

Comparison of Interaction between Ceruloplasmin and Lactoferrin/Transferrin: to Bind or Not to Bind.

The year 2016 marked the 50th anniversary of the discovery by S. Osaki who first showed that ceruloplasmin (CP, ferro:O2-oxidoreductase or ferroxidase) is capable of oxidizing Fe(II) to Fe(III) and favors the incorporation of the latter into transferrin (TF). However, much debate remains in the literature concerning the existence of a complex between the enzyme oxidizing iron and the protein facilitating its transport in plasma. We studied CP in exocrine fluids and demonstrated its high-affinity interaction with transferrin found in breast milk and in lacrimal fluid, i.e. with lactoferrin (LF). Here we present data obtained by comparing the interaction of CP with LF and TF using surface plasmon resonance and Hummel-Dreyer chromatography. Binding of apo-LF within the range of concentrations 1.6-51.3 µM with CP immobilized on a CM5-chip is characterized by KD = 1.07 µM. Under similar conditions, the KD for apo-TF was measured and appeared to be higher than 51.3 µM. Hummel-Dreyer chromatography of CP with 51 µM apo-LF/apo-TF in the effluent demonstrated the absence of interaction between apo-TF and CP in solution, contrary to efficient interaction between apo-LF and CP. In contrast to LF, the interaction of apo-TF with CP is probably not stable within the physiological range of concentrations of TF.

[Treatment for ventricular arrhythmias in the absence of structural heart disease: from guidelines to clinical practice]. / Lechenie zheludochkovykh aritmii v otsutstvie strukturnoi patologii serdtsa: ot rekomendatsii k klinicheskoi praktike.

AIM: To determine criteria for choosing management tactics in patients with ventricular arrhythmias (VA) in the absence of structural heart disease from the point of view of physicians and patients in clinical practice and to compare the immediate results of antiarrhythmic drug therapy (ADT) and radiofrequency ablation (RFA) with the trends in arrhythmic syndrome in the non-treatment group. SUBJECTS AND METHODS: Examinations were made in 90 patients (23 men and 67 women) (mean age, 44 (31; 57) years) with VA in the absence of structural heart disease. Preference was given to RFA (n = 32 (36%)), ADT (n = 37 (41%)), and follow-up tactics (n = 21 (23%)). At baseline and 1 month, Holter ECG monitoring was done; quality of life (QOL) was assessed; and anxiety and depression levels were detected using the SF-36 and HADS questionnaires. In addition, 71 physicians were surveyed about their preferences to the treatment of VA in individuals without structural heart disease. RESULTS: In the total group of patients, VA was unambiguously accompanied by the symptoms only in 47%. The signs of anxiety and depression were identified in 41 and 14% of cases, respectively. The efficiency of RFA was comparable to that of ADT (p > 0.1): a positive antiarrhythmic effect was observed in 71.9% of the patients in the RFA group and in 67.6% in the ADT group. During one month, 38.1% of the patients in the follow-up group showed a spontaneous substantial reduction in the number of ventricular premature beats (VPBs) or disappearance of unstable ventricular tachycardia (UVT), which met the criteria for a positive effect. At baseline, the QOL indicators on a social functioning scale in the RFA group were worse than those in the ADT group. At the same time, most QOL indicators in the patients who have chosen a wait-and-see tactic were significantly higher than those in the RFA and ADT subgroups. The patients treated with ethacyzin in the ADT group more frequently achieved a positive effect. In the interviewed physicians' opinion, the choice of a tactic depended on the impact of arrhythmia on health status (68%), the number of VPBs per day (61%), and the presence of UVT (56%). RFA or ADT was most often recommended when there were 10,000-15,000 or more VPBs per day ((49 and 35% of the respondents, respectively). 46.5% of the respondents stated that ß-blockers were the drug of choice for idiopathic frequent VPBs. Only 30% of the respondents considered it appropriate to restrict to a follow-up in the presence of asymptomatic VPBs. CONCLUSION: Patient management in clinical practice generally complies with the current guidelines; however, much importance is attached to the severity of arrhythmia (the number of VPBs per day, the presence of UVT) in addition to the presence of symptoms. In the opinion of most physicians, the initiation of treatment is justified when there are 10,000-15,000 and more per day. QOL assessment may be promising in choosing the optimal management tactics for these patients. Treatment should not be initiated immediately in patients with a high level of QOL, especially in those with arrhythmia lasting less than 12 months, by taking into account that there can be a spontaneous improvement in 38% of cases within the next month. The immediate results of ADT and RFA are comparable in patients with VA in the absence of structural heart disease. The Class IC antiarrhythmic drug ethacyzin is the most effective agent that ensures positive changes in arrhythmic syndrome in 66.7% of cases with the rate of side effects being in 17.8%.

[The development of express-test for semiquantitative detection of thiocyanate ions in saliva.]

The article presents the results of approbation of the express test-system for semi-quantitative detection of content of thiocyanate ions in saliva as markers of tobacco smoking. The device corresponds to hollow tube with sensory element inside functioning on the principle of "dry chemistry". At saliva intake, the analyzing component (thiocyanate ion) interacts with reagents of sensory element with formation of colored thiocyanate complex. The intensity of color of formed complex permits to judge about content of thiocyanates in saliva by comparison with standard scale. The concentration of of thiocyanate ions in oral fluid of 100 health people of both genders aged from 16 to 45 years was analyzed. The questionnaire survey of respondents was carried out to establish smoking and non-smoking contingents. The analysis established that 30% were active tobacco smokers. The everyday smokers made up to 50% out of them, non-regular smokers (several cigarettes per week or month) - 50%. The reliable relationship between intensity of smoking and concentration of thiocyanate ions in saliva is established. The level of thiocyanate ions in saliva is significantly higher (2.5 mmol/l) in the group of everyday smokers than in saliva of non-smokers of periodically using tobacco articles (0.3-0.5 mmol/l) The increased concentrations of thiocyanate ions in saliva (≥ 1,5 mmol/l) were established in 7% of nonsmokers and are possible related to consumtion of food containing glucosinolates. The test is efficient for detecting smokers using from 1 to 10 cigarettes per day. However, it is of no use in case of sporadic type of smoking.

White-light generation control with crossing beams of femtosecond laser pulses.

We investigated the variations in generated white-light when crossing two femtosecond laser beams in a Kerr medium. By changing the relative delay of two interacting intense femtosecond laser pulses, we show that white-light generation can be enhanced or suppressed. With a decrease of the relative delay an enhancement of the white-light output was observed, which at even smaller delays was reverted to a suppression of white-light generation. Under choosen conditions, the level of suppression resulted in a white-light output lower than the initial level corresponding to large delays, when the pulses do not overlap in time. The enhancement of the white-light generation takes place in the pulse that is lagging. We found that the effect of the interaction of the beams depends on their relative orientation of polarization and increases when the polarizations are changed from perpendicular to parallel. The observed effects are explained by noting that at intermediate delays, the perturbations introduced in the path of the lagging beam lead to a shortening of the length of filament formation and enhancement of the white-light generation, whereas at small delays the stronger interaction and mutual rescattering reduces the intensity in the central part of the beams, suppressing filamentation and white-light generation.

Myeloperoxidase Stimulates Neutrophil Degranulation.

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

The biodegradation of fullerene C60 by myeloperoxidase.

It is shown for the first time that the mammalian enzymes can cause the degradation of the C60 fullerene molecules. This biodegradation is caused by the action of а hypochlorite generated neutrophil enzyme myeloperoxidase of fullerene molecule and leads to the loss of the topology of the fullerene core.

Dendritic Cells Transfected with a DNA Construct Encoding Tumour-associated Antigen Epitopes Induce a Cytotoxic Immune Response Against Autologous Tumour Cells in a Culture of Mononuclear Cells from Colorectal Cancer Patients.

Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour-associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA-transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic-assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA-transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin-positive lymphocytes. In addition, DNA-transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate-loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC-based vaccines.