Mitochondrial depolarization and ATP loss during high frequency nanosecond and microsecond electroporation.

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE - European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h  after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca2+ electrochemotherapy in vitro. We provide experimental proof that 7.5-12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca2+ electrochemotherapy, ensuring excellent cytotoxic efficacy.

Sensitization profiles to house dust mite Dermatophagoides pteronyssinus molecular allergens in the Lithuanian population: Understanding allergic sensitization patterns.

BACKGROUND: House dust mite (HDM) allergy is a prevalent global health concern, with varying sensitization profiles observed across populations. We aimed to provide a comprehensive assessment of molecular allergen sensitization patterns in the Lithuanian population, with a focus on Dermatophagoides pteronyssinus (Der p), and investigate patterns of concomitant reactivity among different allergens to enhance the accuracy of HDM allergy diagnostics. METHODS: A comprehensive analysis of 1520 patient test results in Lithuania from 2020 to 2022 was performed. Sensitization patterns to major (Der p 1, Der p 2, and Der p 23) and minor (Der p 5, Der p 7, and Der p 21) Der p allergen components were described using molecular-based diagnostics. Additionally, we investigated sensitization to allergen components from other allergen sources, including tropomyosins (Der p 10, Per a 7, Pen m 1, Ani s 3, Blo t 10) and arginine kinases (Pen m 2, Bla g 9, Der p 20). RESULTS: This study reveals a high prevalence of HDM sensitization in Lithuania - 481 individuals (45.38% of the sensitized group) exhibited sensitization to at least one Der p allergen component. Importantly, within the sensitized group, 37.21% of patients were sensitized to Der p 5, Der p 7, or Der p 21 in addition to major allergenic components. Distinct sensitization patterns were observed across different age groups, indicating the influence of age-related factors. Furthermore, we confirmed cross-reactivity between Der p 5 and Blo t 5 as well as between Der p 21 and Blo t 21, emphasizing the clinical relevance of these associations. We also highlighted the complexity of sensitization patterns among tropomyosins and arginine kinases. CONCLUSION: This study provides valuable insights into HDM allergy sensitization profiles in Lithuania, emphasizing the importance of considering major and minor HDM allergen components for accurate diagnosis and management of HDM-related allergic diseases. Differences between populations and age-related factors impact sensitization patterns. Understanding concomitant reactivity among allergens, such as Der p 5 and Blo t 5, Der p 21 and Blo t 21, tropomyosins, and arginine kinases, is crucial for improving diagnostic strategies and developing targeted interventions for allergic individuals.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols.

Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (µsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4+ and CD8+ central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.

Understanding the Immunomodulatory Effects of Bovine Colostrum: Insights into IL-6/IL-10 Axis-Mediated Inflammatory Control.

Bovine colostrum (COL), the first milk secreted by lactating cows postpartum, is a rich source of bioactive compounds that exert a significant role in the survival, growth, and immune development of neonatal calves. This study investigated the immunomodulatory effects of COL on cytokine production in vitro using a Caco-2/THP-1 macrophage co-culture model stimulated with Phorbol 12-myristate 13-acetate (PMA). COL pretreatment significantly reduced IL-6 (241.3 pg/mL) production induced by PMA (p < 0.05), while increasing IL-10 production (45.3 pg/mL), in comparison to PMA control (441.1 and 12.5 pg/mL, respectively). Further investigations revealed that the IL-6 suppressive effect of colostrum was heat-sensitive and associated with components of higher molecular mass (100 kDa). Moreover, colostrum primarily influenced THP-1 macrophages rather than Caco-2 epithelial cells. The effects of colostrum on IL-6 production were associated with reduced NF-κB activation in THP-1 macrophages. In calf-FMT transplanted C57BL/6 murine model, colostrum decreased intestinal permeability, reduced immune cell infiltration and intestinal score, and suppressed IL-6 (142.0 pg/mL) production during S. typhimurium infection, in comparison to control animals (215.2 pg/mL). These results suggest the immunomodulatory activity of bovine colostrum and its potential applications in inflammatory disorders. Further studies are needed to elucidate the underlying mechanisms and validate the findings in bovine models.

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.

The Influence of Feeding with Colostrum and Colostrum Replacer on Major Blood Biomarkers and Growth Performance in Dairy Calves.

Bovine colostrum (BC) is the first milk produced by lactating cows after parturition. BC is rich in various amino acids, proteins, and fats essential for the nutrition of the neonate calves. Despite the evident beneficial effect of BC on calves, the effect of BC on blood biomarkers is poorly understood. Calves that received BC showed significantly higher body mass at days 7 and 30 (38.54 kg and 43.42 kg, respectively) compared to the colostrum replacer group (p = 0.0064). BC induced greater quantities of blood neutrophils (0.27 × 109/L) and monocytes (4.76 × 109/L) in comparison to the colostrum replacer (0.08 and 0.06 × 109/L, respectively) (p = 0.0001). Animals that received BC showed higher levels of total serum protein (59.16 g/L) and albumin (29.96 g/L) in comparison to the colostrum replacer group (44.34 g/L and 31.58 g/L, respectively). In addition, BC induced greater intestinal mucus production in the Wistar rat model. Collectively, these results demonstrate that BC is important for the growth of calves and that it provides a significant beneficial effect on morphological and biochemical blood parameters.

High-Frequency Nanosecond Bleomycin Electrochemotherapy and its Effects on Changes in the Immune System and Survival.

In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4+ T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4+ Tr1 cells, increased the expansion of splenic CD8+ T, CD4+CD8+ T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Lin- population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy.

Bioluminescent calcium mediated detection of nanosecond electroporation: Grasping the differences between 100 ns and 100 µs pulses.

Electroporation is a phenomenon of transient or irreversible permeabilization of the cell membrane after pulsed electric field treatment. Fluorescent probes are frequently used to assess the extent of permeabilization, however, as an alternative, a D-luciferin oxidation-based method can be used. In this work, we have used sequences of a microsecond (1.3 kV/cm × 100 µs) and nanosecond (12.5 kV/cm × 100 ns) pulses to trigger various levels of cell permeabilization and assessed the differences in the response using a conventional fluorescent probe (YO-PRO-1 (YP)) and D-luciferin oxidation methodology. The nanosecond pulses (n = 5-100) have been delivered with 1 kHz repetition frequency, and the results were compared with 1 MHz protocols. Additionally, the effects of extracellular Ca2+ have been assessed. Various concentrations of CaCl2 (2, 5, and 10 mM) have been used, and it was shown that the bioluminescence of the cells after electroporation depends on extracellular calcium concentration. It was shown that the changes in bioluminescence signal could be used as a marker of cell membrane permeabilization on par with YP assay when calcium is added and thus, effectively employed for analysis of electroporation phenomenon in vitro both for nanosecond and microsecond pulses.

Effects of Time Delay Between Unipolar Pulses in High Frequency Nano-Electrochemotherapy.

OBJECTIVE: this work focuses on bleomycin electrochemotherapy using new modality of high repetition frequency unipolar nanosecond pulses. METHODS: As a tumor model, Lewis lung carcinoma (LLC1) cell line in C57BL mice (n = 42) was used. Electrochemotherapy was performed with intertumoral injection of bleomycin (50 µL of 1500 IU solution) followed by nanosecond and microsecond range electrical pulse delivery via parallel plate electrodes. The 3.5 kV/cm pulses of 200 and 700 ns were delivered in a burst of 200 at frequencies of 1 kHz and 1 MHz. For comparison of treatment efficiency, a standard 1.3 kV/cm x 100 µs x 8 protocol was used. RESULTS: It was shown that it is possible to manipulate the efficacy of unipolar sub-microsecond electrochemotherapy solely by the time delay between the pulses. SIGNIFICANCE: the results suggest that the sub-microsecond range pulses can be as effective as the protocols in European Standard Operating Procedures on Electrochemotherapy (ESOPE) using 100 µs pulses.

Bioluminescence as a sensitive electroporation indicator in sub-microsecond and microsecond range of electrical pulses.

The cell membrane permeabilization in electroporation studies is usually quantified using fluorescent markers such as propidium iodide (PI) or YO-PRO, while Chinese Hamster Ovary cell line frequently serves as a model. In this work, as an alternative, we propose a sensitive methodology for detection and analysis of electroporation phenomenon based on bioluminescence. Luminescent mice myeloma SP2/0 cells (transfected using Luciferase-pcDNA3 plasmid) were used as a cell model. Electroporation has been studied using the 0.1-5 µs × 250 and 100 µs × 1-8 pulsing protocols in 1-2.5 kV/cm PEF range. It was shown that the bioluminescence response is dependent on the cell permeabilization state and can be effectively used to detect even weak permeabilization. During saturated permeabilization the methodology accurately predicts the losses of cell viability due to irreversible electroporation. The results have been superpositioned with permeabilization and pore resealing (1 h post-treatment) data using PI. Also, the viability of the cells was evaluated. Lastly, the SP2/0 tumors have been developed in BALB/C mice and the methodology has been tested in vivo using electrochemotherapy with bleomycin.

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study.

Pulsed electric field (PEF) is frequently used for intertumoral drug delivery resulting in a well-known anticancer treatment-electrochemotherapy. However, electrochemotherapy is associated with microsecond range of electrical pulses, while nanosecond range electrochemotherapy is almost non-existent. In this work, we analyzed the feasibility of nanosecond range pulse bursts for successful doxorubicin-based electrochemotherapy in vivo. The conventional microsecond (1.4 kV/cm × 100 µs × 8) procedure was compared to the nanosecond (3.5 kV/cm × 800 ns × 250) non-thermal PEF-based treatment. As a model, Sp2/0 tumors were developed. Additionally, basic current and voltage measurements were performed to detect the characteristic conductivity-dependent patterns and to serve as an indicator of successful tumor permeabilization both in the nano and microsecond pulse range. It was shown that nano-electrochemotherapy can be the logical evolution of the currently established European Standard Operating Procedures for Electrochemotherapy (ESOPE) protocols, offering better energy control and equivalent treatment efficacy.

Sub-microsecond electrotransfection using new modality of high frequency electroporation.

Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300-700 ns) high frequency pulsed electric fields of 2-15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2-5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 µs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.

Predicting electrotransfer in ultra-high frequency sub-microsecond square wave electric fields.

Measurement of cell transmembrane potential (TMP) is a complex methodology involving patch-clamp methods or fluorescence-based potentiometric markers, which have limited to no applicability during ultrafast charging and relaxation phenomena. In such a case, analytical methods are applied for evaluation of the voltage potential changes in biological cells. In this work, the TMP-based electrotransfer mechanism during ultra-high frequency (≥1 MHz) electric fields is studied and the phenomenon of rapid membrane charge accumulation, which is non-occurrent during conventional low-frequency electroporation is simulated using finite element method (FEM). The influence of extracellular medium conductivity (0.1, 1.5 S/m) and pulse rise/fall times (10-50 ns) TMP generation are presented. It is shown that the medium conductivity has a dramatic influence on the electroporation process in the high-frequency range of applied pulsed electric fields (PEF). The applied model allowed to grasp the differences in polarization between 100 and 900 ns PEF and enabled successful prediction of the experimental outcome of propidium iodide electrotransfer into CHO-K1 cells and the conductivity-dependent patterns of MHz range PEF-triggered electroporation were determined. The results of this study form recommendations for development and pre-evaluation of future PEF protocols and generators based on ultra-high frequency electroporation for anticancer and gene therapies.

Antitumor Response and Immunomodulatory Effects of Sub-Microsecond Irreversible Electroporation and Its Combination with Calcium Electroporation.

In this work, we have investigated the feasibility of sub-microsecond range irreversible electroporation (IRE) with and without calcium electroporation in vivo. As a model, BALB/C mice were used and bioluminescent SP2/0 myeloma tumor models were developed. Tumors were treated with two separate pulsed electric field (PEF) pulsing protocols PEF1: 12 kV/cm × 200 ns × 500 (0.006 J/pulse) and PEF2: 12 kV/cm × 500 ns × 500 (0.015 J/pulse), which were delivered with and without Ca2+ (168 mM) using parallel plate electrodes at a repetition frequency of 100 Hz. Both PEF1 and PEF2 treatments reduced tumor growth and prolonged the life span of the mice, however, the PEF2 protocol was more efficient. The delay in tumor renewal was the biggest when a combination of IRE with calcium electroporation was used, however, we did not obtain significant differences in the final mouse survival compared to PEF2 alone. Anti-tumor immune responses were also investigated after treatment with PEF2 and PEF2+Ca. In both cases the treated mice had enlarged spleens and increased spleen T cell numbers, lower percentages of suppressor cell subsets (conventional CD4+CD25+ Treg, CD4+CD25-DX5+ Tr1, CD8+DX5+, CD4+CD28-, CD8+CD28-), changed proportions of Tcm and Tef/Tem T cells in the spleen and increased amount of tumor cell specific antibodies in the sera. The treatment based on IRE was effective against primary tumors, destroyed the tumor microenvironment and induced an anti-tumor immune response, however, it was not sufficient for complete control of tumor metastasis.

sICAM-1 as potential additional parameter in the discrimination of the Sjögren syndrome and non-autoimmune sicca syndrome: a pilot study.

OBJECTIVE: Both Sjögren's syndrome (SS) and non-autoimmune sicca syndrome (nSS) can show symptoms of dry eyes and a dry mouth, and objective reductions in tear and saliva production. Dry eyes and dry mouth are frequent but they are distinct pathological entities that require diagnostic discrimination. METHODS: The aim of present study was to compare the serum levels of sICAM-1, TFF3, RANTES, adiponectin, and FGF in primary (pSS), secondary due to rheumatoid arthritis (sSS), non-autoimmune sicca syndrome (nSS), and healthy groups. The serum levels of selected molecules were determined by enzyme-linked immunosorbent assay (ELISA) in 29 patients with pSS, 30 with sSS, 17 with nSS, and 15 healthy subjects. RESULTS: sICAM-1 was significantly elevated in pSS and sSS patients compared with nSS group. Levels of FGF, TFF3, and RANTES were significantly increased in pSS, sSS, and nSS patients compared with healthy controls. No significant correlations were found between the levels of measured molecules and the clinical parameters. CONCLUSIONS: Our study showed that sICAM-1 might be useful as an additional parameter for differential diagnosis of SS and nSS, and TFF could be additional diagnostic marker for SS diagnosis. KEY POINTS: • sICAM-1 was significantly elevated in Sjögren syndrome patients compared with non-autoimmune sicca syndrome group. • TFF was significantly elevated in Sjögren syndrome patients compared with healthy controls. • They might be useful as additional parameters for differential diagnosis.

Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields.

BACKGROUND: Skin infections, particularly caused by drug-resistant pathogens, represent a clinical challenge due to being a frequent cause of morbidity and mortality. The objectives of this study were to examine if low concentrations of acetic and formic acids can increase sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa to pulsed electric field (PEF) and thus, promote a fast and efficient treatment methodology for wound treatment. RESULTS: We have shown that the combination of PEF (10-30 kV/cm) with organic acids (0.1% formic and acetic acids) increased the bactericidal properties of treatment. The effect was apparent for both acids. The proposed methodology allowed to reduce the energy of electrical pulses and the inhibitory concentrations of acids, while still maintain high efficiency of bacteria eradication. CONCLUSIONS: Application of weak organic acids as bactericidal agents has many advantages over antibiotics because they do not trigger development of drug-resistance in bacteria. The combination with PEF can make the treatment effective even against biofilms. The results of this study are particularly useful for the development of new methodologies for the treatment of extreme cases of wound infections when the chemical treatment is no longer effective or hinders wound healing.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.

Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study.

Invasive infections caused by drug-resistant bacteria are frequently responsible for fatal sepsis, morbidity and mortality rates. In this work, we propose a new methodology based on nanosecond high frequency electric field bursts, which enables successful eradication of bacteria in vivo. High frequency (15 kHz) 15-25 kV/cm 300-900 ns pulsing bursts were used separately and in combination with acetic acid (0.1-1%) to treat Pseudomonas aeruginosa in a murine model. Acetic acid 1% alone was effective resulting in almost 10-fold reduction of bacteria viability, however combination of nanosecond electric field and acetic acid 1% treatment was the most successful showing almost full eradication (0.01% survival compared to control) of the bacteria in the contaminated area. The short duration of the pulses (sub-microsecond) and high frequency (kHz range) of the burst enabled reduction of the muscle contractions to barely detectable level while the proposed applicators ensured predominantly topical treatment, without electroporation of deeper tissues. The results of our study have direct application for treatment of wounds and ulcers when chemical treatment is no longer effective.

Induction of Different Sensitization Patterns of MRSA to Antibiotics Using Electroporation.

Treatment of bacteria-associated infections is complicated and antibiotic treatment alone is often inadequate to overcome biofilm infections. Physical methods allow overcoming this problem and propose solutions that are non-dependent on drug resistance. In this work, we investigated the feasibility of pulsed electric fields for sensitization of MRSA to common antibiotics. We analyzed the efficacy of inactivation of methicillin-resistant Staphylococcus aureus in 5⁻20 kV/cm electric field separately and in combination with gentamicin, doxycycline, ciprofloxacin, sulfamethoxazole, and vancomycin. Combined treatment allowed using up to 1000-fold smaller concentrations of antibiotics to induce the same inactivation of S. aureus.

Different permeabilization patterns of splenocytes and thymocytes to combination of pulsed electric and magnetic field treatments.

Genetic manipulation of T cells is frequently inefficient, however, when combined with physical methods (i.e. electroporation) a promising alliance with immunotherapy can be formed. This study presents new data on permeabilization of murine thymocytes and splenocytes as a T cell model using pulsed electric (PEF) and electromagnetic field (EMF). The 300ns, 500ns, 2µs and 100µs pulse bursts in a broad range of PEF 0-8kV/cm were applied separately and in combination with 3.3T, 0.2kV/cm EMF pulses. The permeabilization efficiency was evaluated using fluorescent dye (YO-PRO-1) and flow cytometry. It was shown that a >14% increase in thymocytes permeabilization is achieved when electroporation is applied in combination with EMF, however splenocytes responded in a different manner - a statistically significant (P<0.05) reduction in permeabilization was observed. The cytokine secretion patterns were mainly unaltered independently on the applied treatment parameters determined by secretion of IFNγ, IL-4 and IL-17 - the main cytokines of Th1, Th2 and Th17 cells. The results of this study are useful for development of pulsed power protocols for effective genetic modification of T cells.