Tripeptides conjugated with thiosemicarbazones: new inhibitors of tyrosinase for cosmeceutical use.

The development of skin-care products is recently growing. Cosmetic formulas containing active ingredients with proven efficacy, namely cosmeceuticals, are based on various compounds, including peptides. Different whitening agents featuring anti-tyrosinase activity have been applied in the cosmeceutical field. Despite their availability, their applicability is often limited due to several drawbacks including toxicity, lack of stability, and other factors. In this work, we present the inhibitory effect on diphenolase activity of thiosemicarbazone (TSC)-peptide conjugates. Tripeptides FFY, FWY, and FYY were conjugated with three TSCs bearing one or two aromatic rings via amide bond formation in a solid phase. Compounds were then examined as tyrosinase and melanogenesis inhibitors in murine melanoma B16F0 cell line, followed by the cytotoxicity assays of these cells. In silico investigations explained the differences in the activity, observed among tested compounds. Mushroom tyrosinase was inhibited by TSC1-conjugates at micromolar level, with IC50 lower than this for kojic acid, a widely used reference compound. Up to now, this is the first report regarding thiosemicarbazones conjugated with tripeptides, synthesised for the purpose of tyrosinase inhibition.

Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance.

Nanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which oxidative stress is a principal component. In the present study, nsPEF-induced oxidative stress was investigated in two gastric adenocarcinoma cell lines (EPG85-257P and EPG85-257RDB) which differ by their sensitivity to daunorubicin. Cells were exposed to 200 pulses of 10 ns duration, with the amplitude and pulse repetition frequency at 1 kHz, with electric field intensity varying from 12.5 to 50 kV/cm. The electroporation buffer contained either 1 mM or 2 mM calcium chloride. CellMask DeepRed visualized cell plasma permeabilization, Fluo-4 was used to visualize internal calcium ions content, and F-actin was labeled with AlexaFluor®488 for the cytoskeleton. The cellular viability was determined by MTT assay. An alkaline and neutral comet assay was employed to detect apoptotic and necrotic cell death. The luminescent method estimated the modifications in GSSG/GSH redox potential and the imbalance of proteasomal activity (chymotrypsin-, trypsin- and caspase-like). The reactive oxygen species (ROS) level was measured by flow cytometry using dihydroethidium (DHE) dye. Morphological visualization indicated cell shrinkage, affected cell membranes (characteristic bubbles and changed cell shape), and the reorganization of actin fibers with sites of its dense concentration; the effect was more intense with the increasing electric field strength. The most significant decrease in cell viability and GSSG/GSH redox potential was noted at the highest amplitude of 50 kV/cm, and calcium ions amplified this effect. nsPEF, particularly with calcium ions, inhibited proteasomal activities, resulting in increased protein degradation. nsPEF increased the percentage of apoptotic cells and ROS levels. The EPG85-257 RDB cell line, which is resistant to standard chemotherapy, was more sensitive to applied nsPEF protocols. The applied nsPEF method disrupted the metabolism of cancer cells and induced apoptotic cell death. The nsPEF ability to cause apoptosis, oxidative stress, and protein degradation make the nsPEF methodology a suitable alternative to current anticancer pharmacological methods.

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable.

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Gal (Gb3 and the P1 antigen) and Galα1→4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants resulted from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

Halogenated aromatic thiosemicarbazones as potent inhibitors of tyrosinase and melanogenesis.

A set of 21 halogenated thiosemicarbazones (TSCs) have been synthesized and its inhibitory properties toward activity diphenolase of mushroom tyrosinase and their ability to inhibition of melanogenesis in B16F10 murine, melanoma cell line have been investigated. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. The obtained outcomes allowed us to perform SAR analysis. TSC 6, 12 and 21 exhibited the most potent inhibitory properties showing IC50 of 0.5, 0.9 and 0.8 µM, respectively. They revealed reversible and competitive manner of tyrosinase inhibition. According to SAR analysis, para-substituted acetophenone derivatives of thiosemicarbazones have the highest affinity to the enzyme among the investigated compounds. Melanin production in B16F10 cells was inhibited by all investigated compounds at the micromolar level. Suggested inhibition mechanism is based on the interaction between a sulfur atom of thiourea moiety of the thiosemicarbazones, and copper ions in the active site of the enzyme. These results might be useful in searching novel inhibitors of melanogenesis which could be used in the cosmetic and food industry.

Cepharanthine induces ROS stress in glioma and neuronal cells via modulation of VDAC permeability.

Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid. Molecular dynamics studies show that CEP interacts with Voltage-dependent anion channel (VDAC), inducing the voltage-independent channel narrowing. In the new conformation, transport between mitochondria and cytoplasm is altered, which leads to the dose-dependent cytotoxicity. The biological effects of the interaction were investigated on glioblastoma multiforme (SNB-19) and neuronal (PC-12 + NGF) cell lines. The cytotoxic potential of cepharanthine was determined by MTT assay and flow cytometry apoptosis/necrosis studies. T-type calcium channel and VDAC were labelled by the immunocytochemical method. Additionally, fluorescent labelling of reactive oxygen species and mitochondria was performed. Changes in the pore size of VDAC were calculated as well. Molecular dynamics simulations were carried out to examine the interactions of cepharanthine with VDAC. The obtained results prove that cepharanthine enhances the apoptosis in glioma and neuronal cells by the release of reactive oxygen species. Cepharanthine alters the mitochondria-to-cytoplasm transport and thus induces the cytotoxicity with no selectivity.

Cell Membrane Transport Mechanisms: Ion Channels and Electrical Properties of Cell Membranes.

Cellular life strongly depends on the membrane ability to precisely control exchange of solutes between the internal and external (environmental) compartments. This barrier regulates which types of solutes can enter and leave the cell. Transmembrane transport involves complex mechanisms responsible for passive and active carriage of ions and small- and medium-size molecules. Transport mechanisms existing in the biological membranes highly determine proper cellular functions and contribute to drug transport. The present chapter deals with features and electrical properties of the cell membrane and addresses the questions how the cell membrane accomplishes transport functions and how transmembrane transport can be affected. Since dysfunctions of plasma membrane transporters very often are the cause of human diseases, we also report how specific transport mechanisms can be modulated or inhibited in order to enhance the therapeutic effect.

MRP1 protein expression in leukemic stem cells as a negative prognostic marker in acute myeloid leukemia patients.

BACKGROUND: It is well established that expression of multi-drug resistance (MDR) proteins (MDR1, BCRP, MDR3, MRP1, and LRP) in leukemic blasts correlates with acute myeloid leukemia (AML) patients' clinical response. Assuming that leukemic stem cells (LSC) are resistant to chemotherapy and responsible for relapse, it might be clinically relevant to evaluate the expression level of MDR proteins in LSC and relate it to the clinical outcome. METHODS: Bone marrow samples from 26 patients with de novo AML were labeled with antibodies to distinguish CD34+CD38-CD123+ LSC population and with antibodies against MDR1, BCRP, MDR3, MRP1, or LRP proteins. Multicolor flow cytometry was applied to evaluate the expression of MDR proteins in blasts and LSC. RESULTS: Nine of 26 patients with AML attained CR (30%). High negative correlation was found between MDR1 and LRP expression in blasts and the patient's remission. MDR proteins were expressed more frequently in LSC than in leukemic blasts. High negative correlation was also observed between remission achievement and MRP1 expression in LSC. CONCLUSIONS: Our data present for the very first time the high negative correlation between MRP1 protein expression in LSC and AML patients' remission. It does strongly suggest that MRP1 expression in LSC is an adverse prognostic marker in patients with de novo AML.

Differentiated all-trans retinoic acid response of naive CD4+CD25- cells isolated from rats with collagen-induced arthritis and healthy ones under in vitro conditions.

AIM O THE STUDY: To compare the potential of CD4+CD25- cells, isolated from both healthy rats and rats with CIA (Collagen-Induced Arthritis), for differentiation into regulatory T cells in the presence of all-trans retinoic acid in order to learn more about the activation mechanisms and therapeutic potential of regulatory T cells. MATERIAL AND METHODS: Sorted CD4+CD25- cells were cultured in vitro with/without ATRA, and then the frequency of regulatory T cells and their ability to secrete IL-10 by CD4+ FOXP3+ cells was examined. Gene expression of the foxp3, rarα, rarß, rxrß, and ppar ß/δ and protein expression of the Rarα, Rarß, and Rxrß in cells after stimulation with ATRA were also investigated. RESULTS: CD4+CD25- cells isolated from healthy animals or from animals with CIA are characterised by different potential of the differentiation into CD4+CD25+ FOXP3+ cells. Retinoic acid receptor Rxrß is present in the CD4+CD25- cells isolated from rats with CIA. CONCLUSIONS: We showed that although ATRA did not increase the frequency of Treg in culture, it significantly increased expression of rarß and rxrß only in lymphocytes taken from diseased animals and foxp3 expression only in healthy animals. Moreover, after ATRA stimulation, the frequency of Treg-produced IL-10 tended to be lower in diseased animals than in the healthy group. The results imply that the potential of naïve cell CD4 lymphocytes to differentiate into Tregs and their putative suppressive function is dependent on the donor's health status.

The Effect of Millisecond Pulsed Electric Fields (msPEF) on Intracellular Drug Transport with Negatively Charged Large Nanocarriers Made of Solid Lipid Nanoparticles (SLN): In Vitro Study.

Drug delivery technology is still a dynamically developing field of medicine. The main direction in nanotechnology research (nanocarriers, nanovehicles, etc.) is efficient drug delivery to target cells with simultaneous drug reduction concentration. However, nanotechnology trends in reducing the carrier sizes to several nanometers limit the volume of the loaded substance and may pose a danger of uncontrolled access into the cells. On the other hand, nanoparticles larger than 200 nm in diameter have difficulties to undergo rapid diffusional transport through cell membranes. The main advantage of large nanoparticles is higher drug encapsulation efficiency and the ability to deliver a wider array of drugs. Our present study contributes a new approach with large Tween 80 solid lipid nanoparticles SLN (i.e., hydrodynamic GM-SLN-glycerol monostearate, GM, as the lipid and ATO5-SLNs-glyceryl palmitostearate, ATO5, as the lipid) with diameters DH of 379.4 nm and 547 nm, respectively. They are used as drug carriers alone and in combination with electroporation (EP) induced by millisecond pulsed electric fields. We evaluate if EP can support the transport of large nanocarriers into cells. The study was performed with two cell lines: human colon adenocarcinoma LoVo and hamster ovarian fibroblastoid CHO-K1 with coumarin 6 (C6) as a fluorescent marker for encapsulation. The biological safety of the potential treatment procedure was evaluated with cell viability after their exposure to nanoparticles and EP. The EP efficacy was evaluated by FACS method. The impact on intracellular structure organization of cytoskeleton was visualized by CLSM method with alpha-actin and beta-tubulin. The obtained results indicate low cytotoxicity of both carrier types, free and loaded with C6. The evaluation of cytoskeleton proteins indicated no intracellular structure damage. The intracellular uptake and accumulation show that SLNs do not support transport of C6 coumarin. Only application of electroporation improved the transport of encapsulated and free C6 into both treated cell lines.

Calcium ion delivery by microbubble-assisted sonoporation stimulates cell death in human gastrointestinal cancer cells.

Ultrasound-mediated cell membrane permeabilization - sonoporation, enhances drug delivery directly to tumor sites while reducing systemic side effects. The potential of ultrasound to augment intracellular calcium uptake - a critical regulator of cell death and proliferation - offers innovative alternative to conventional chemotherapy. However, calcium therapeutic applications remain underexplored in sonoporation studies. This research provides a comprehensive analysis of calcium sonoporation (CaSP), which combines ultrasound treatment with calcium ions and SonoVue microbubbles, on gastrointestinal cancer cells LoVo and HPAF-II. Initially, optimal sonoporation parameters were determined: an acoustic wave of 1 MHz frequency with a 50 % duty cycle at intensity of 2 W/cm2. Subsequently, various cellular bioeffects, such as viability, oxidative stress, metabolism, mitochondrial function, proliferation, and cell death, were assessed following CaSP treatment. CaSP significantly impaired cancer cell function by inducing oxidative and metabolic stress, evidenced by increased mitochondrial depolarization, decreased ATP levels, and elevated glucose uptake in a Ca2+ dose-dependent manner, leading to activation of the intrinsic apoptotic pathway. Cellular response to CaSP depended on the TP53 gene's mutational status: colon cancer cells were more susceptible to CaSP-induced apoptosis and G1 phase cell cycle arrest, whereas pancreatic cancer cells showed a higher necrotic response and G2 cell cycle arrest. These promising results encourage future research to optimize sonoporation parameters for clinical use, investigate synergistic effects with existing treatments, and assess long-term safety and efficacy in vivo. Our study highlights CaSP's clinical potential for improved safety and efficacy in cancer therapy, offering significant implications for the pharmaceutical and biomedical fields.

Inhibition of MC38 colon cancer growth by multicomponent chemoimmunotherapy with anti-IL-10R antibodies, HES-MTX nanoconjugate, depends on application of IL-12, IL-15 or IL-18 secreting dendritic cell vaccines.

Background: The tumor microenvironment (TME) provides a conducive environment for the growth and survival of tumors. Negative factors present in TME, such as IL-10, may limit the effectiveness of cellular vaccines based on dendritic cells, therefore, it is important to control its effect. The influence of IL-10 on immune cells can be abolished e.g., by using antibodies against the receptor for this cytokine - anti-IL-10R. Furthermore, the anticancer activity of cellular vaccines can be enhanced by modifying them to produce proinflammatory cytokines, such as IL-12, IL-15 or IL-18. Additionally, an immunomodulatory dose of methotrexate and hydroxyethyl starch (HES-MTX) nanoconjugate may stimulate effector immune cells and eliminate regulatory T cells, which should enhance the antitumor action of immunotherapy based on DC vaccines. The main aim of our study was to determine whether the HES-MTX administered before immunotherapy with anti-IL-10R antibodies would change the effect of vaccines based on dendritic cells overproducing IL-12, IL-15, or IL-18. Methods: The activity of modified DCs was checked in two therapeutic protocols - immunotherapy with the addition of anti-IL10R antibodies and chemoimmunotherapy with HES-MTX and anti-IL10R antibodies. The inhibition of tumor growth and the effectiveness of the therapy in inducing a specific antitumor response were determined by analyzing lymphoid and myeloid cell populations in tumor nodules, and the activity of restimulated splenocytes. Results and conclusions: Using the HES-MTX nanoconjugate before immunotherapy based on multiple administrations of anti-IL-10R antibodies and cellular vaccines capable of overproducing proinflammatory cytokines IL-12, IL-15 or IL-18 created optimal conditions for the effective action of these vaccines in murine colon carcinoma MC38 model. The applied chemoimmunotherapy caused the highest inhibition of tumor growth in the group receiving DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg at the level of 72.4%. The use of cellular vaccines resulted in cytotoxic activity increase in both immuno- or chemoimmunotherapy. However, the greatest potential was observed both in tumor tissue and splenocytes obtained from mice receiving two- or three-component vaccines in the course of combined application. Thus, the designed treatment schedule may be promising in anticancer therapy.

Nanoelectropulse delivery for cell membrane perturbation and oxidation in human colon adenocarcinoma cells with drug resistance.

Ultrashort electric pulses in the nanosecond range (nsPEF) can affect extra- and intracellular lipid structures and can also alternate cell functioning reversibly and irreversibly. Several of the nsPEF effects are due to the abrupt rise in intracellular free calcium levels and calcium ions influx from the outside. Calcium is one of the most important factors in cell proliferation, differentiation, and cell death (apoptosis or necrosis). Manipulating calcium levels using electroporation can have different effects on normal and malignant cells. This study aimed to examine the impact of nsPEFs, combined with 1 mM Ca2+ in human colon adenocarcinoma cell lines: sensitive- LoVo and drug resistant-LoVoDX. In this study 200 pulses of 10 ns and high voltage (12.5-50 kVcm-1) were used. Cell viability was determined by MTT and clonogenic assay. Proteasomal activity, GSH/GSSG assay, ROS production, and PALS-1 protein were evaluated as oxidative stress markers and protein damage. Cell morphology was visualized by AFM, SEM, and confocal microscopy imaging. The results revealed that nsPEF with 1 mM Ca2+ is cytotoxic, particularly for LoVoDX cells, and safe for normal cells. NsPEF provoked ROS release, altered cell polarity, and destabilized cell morphology. These results can be important for future protocols for colon adenocarcinoma using calcium nsPEF.

The modulation of local and systemic anti-tumor immune response induced by methotrexate nanoconjugate in murine MC38 colon carcinoma and B16 F0 melanoma tumor models.

Methotrexate (MTX) which is one of the longest-used cytostatics, belongs to the group of antimetabolites and is used for treatment in different types of cancer as well as during autoimmune diseases. MTX can act as a modulator enable to create the optimal environment to generate the specific anti-tumor immune response. A novel system for MTX delivery is its conjugation with high-molecular-weight carriers such as hydroxyethyl starch (HES), a modified amylopectin-based polymer applied in medicine as a colloidal plasma volume expander. Such modification prolongs the plasma half-life of the HES-MTX nanoconjugate and improves the distribution of the drug in the body. In the current study, we focused on evaluating the dose-dependent therapeutic efficacy of chemotherapy with HES-MTX nanoconjugate compared to the free form of MTX, and examining the time-dependent changes in the local and systemic anti-tumor immune response induced by this therapy. To confirm the higher effectiveness of HES-MTX in comparison to MTX, we analyzed its action using murine MC38 colon carcinoma and B16 F0 melanoma tumor models. It was noted that HES-MTX at a dose of 20 mg/kg b.w. was more effective in tumor growth inhibition than MTX in both tumor models. One of the main differences between the two analyzed tumor models concerned the kinetics of the appearance of the immunomodulation. In MC38 tumors, the beneficial change in the tumor microenvironment (TME) landscape, manifested by the depletion of pro-tumor immune cells, and increased influx of cells with strong anti-tumor activity was noted already 3 days after HES-MTX administration, while in B16 F0 model, these changes occurred 10 days after the start of therapy. Thus, the immunomodulatory potential of the HES-MTX nanoconjugate may be closely related to the specific immune cell composition of the TME, which combined with additional treatment such as immunotherapies, would enhance the therapeutic potential of the nanoconjugate.

Combined therapy with methotrexate nanoconjugate and dendritic cells with downregulated IL-10R expression modulates the tumor microenvironment and enhances the systemic anti-tumor immune response in MC38 murine colon carcinoma.

Background: Understanding the negative impact of the tumor microenvironment on the creation of an effective immune response has contributed to the development of new therapeutic anti-cancer strategies. One such solution is combined therapy consisting of chemotherapeutic administration followed by dendritic cell (DC)-based vaccines. The use of cytostatic leads to the elimination of cancer cells, but can also modulate the tumor milieu. Moreover, great efforts are being made to increase the therapeutic outcome of immunotherapy, e.g. by enhancing the ability of DCs to generate an efficient immune response, even in the presence of immunosuppressive cytokines such as IL-10. The study aimed to determine the effectiveness of combined therapy with chemotherapeutic with immunomodulatory potential - HES-MTX nanoconjugate (composed of methotrexate (MTX) and hydroxyethyl starch (HES)) and DCs with downregulated expression of IL-10 receptor stimulated with tumor antigens (DC/shIL-10R/TAg) applied in MC38 murine colon carcinoma model. Methods: With the use of lentiviral vectors the DCs with decreased expression of IL-10R were obtained and characterized. During in vivo studies MC38-tumor bearing mice received MTX or HES-MTX nanoconjugate as a sole treatment or combined with DC-based immunotherapy containing unmodified DCs or DCs transduced with shRNA against IL-10R (or control shRNA sequence). Tumor volume was monitored during the experiment. One week after the last injection of DC-based vaccines, tumor nodules and spleens were dissected for ex vivo analysis. The changes in the local and systemic anti-tumor immune response were estimated with the use of flow cytometry and ELISA methods. Results and conclusions: In vitro studies showed that the downregulation of IL-10R expression in DCs enhances their ability to activate the specific anti-tumor immune response. The use of HES-MTX nanoconjugate and DC/shIL-10R/TAg in the therapy of MC38-tumor bearing mice resulted in the greatest tumor growth inhibition. At the local anti-tumor immune response level a decrease in the infiltration of cells with suppressor activity and an increase in the influx of effector cells into MC38 tumor tissue was observed. These changes were crucial to enhance the effective specific immune response at the systemic level, which was revealed in the greatest cytotoxic activity of spleen cells against MC38 cells.

The Beneficial Effect of IL-12 and IL-18 Transduced Dendritic Cells Stimulated with Tumor Antigens on Generation of an Antitumor Response in a Mouse Colon Carcinoma Model.

The main purpose of our study was to determine the effect of dendritic cell (DC) transduction with lentiviral vectors carrying sequences of il18 and/or il12 genes on the level of antitumor activity in vitro and in vivo. We examined the ability of DCs to migrate to the tumor-draining lymph nodes and infiltrate tumor tissue and to activate the local and systemic antitumor response. On the 15th day, DCs genetically modified for production of IL-12 and/or IL-18 were administered peritumorally to C57BL/6 female mice with established MC38 tumors. Lymphoid organs and tumor tissue were collected from mice on the 3rd, 5th, and 7th days after a single administration of DCs for further analysis. Administration of DCs transduced for production of IL-12 alone and in combination with IL-18 increased the inflow and activity of CD4+ and CD8+ T lymphocytes in the tumor microenvironment and tumor-draining lymph nodes. We also found that even a single administration of such modified DCs could trigger a systemic antitumor response as well as inhibit tumor growth. Application of the developed DC-based vaccines may exert a favorable impact on stimulation of an antitumor immune response, especially if these DC vaccines are administered repeatedly.

"Endothelial Antibody Factory" at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases.

The failures of anti-ß-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-ß-amyloid and anti-TDP43 Fabs turning them into an "antibody fragment factory". When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

Immune Response to COVID-19 mRNA Vaccine-A Pilot Study.

Twenty individuals (17 females, 3 males, aged 31-65 years (range), median: 46) who received both doses of the BioNTech Pfizer mRNA vaccine were examined (11 to 31 days, median: 25) after the second dose for the presence of antibodies against peptides of SARS-COV-2 and some of MERS-CoV, SARS-CoV1, HCov229E, and HCoVNL63. Clinical evaluation revealed that six people had COVID-19 in the past. We found that: (i) Six people claimed the presence of unwanted effects of vaccination, which were more frequent in those with a history of COVID-19 (4 out of 6 vs. 2 out of 14, p = 0.037); (ii) All individuals independent of the past history of COVID-19 responded equally well in IgG but those who experienced the disease tended to do better in IgA class (729.04 vs. 529.78 U/mL, p = 0.079); (iii) All those who had experienced the disease had IgG antibodies against nucleocapsid antigens but also 5 out of 14 who had not had the disease (6/6 vs. 5/14, p = 0.014); (iv) Anti S2 antibodies were present in the patients having COVID-19 in the past but also were found in those who had not had the disease (6/6 vs. 8/14, p = 0.144); (v) All vaccinated people were highly positive in the IGRA and the level of released IFN gamma was correlated with the numbers of HLADR positive lymphocytes in the blood (R = 0.5766, p = 0.008).

Monosubstituted Acetophenone Thiosemicarbazones as Potent Inhibitors of Tyrosinase: Synthesis, Inhibitory Studies, and Molecular Docking.

A set of 12 monosubstituted acetophenone thiosemicarbazone derivatives (TSCs) were synthesized and their inhibitory properties toward tyrosinase activity were tested. Moreover, their ability to inhibit melanogenesis in the B16F10 murine melanoma cell line was studied. In order to investigate the nature of interactions between the enzyme and the inhibitors, molecular docking to the active site was performed. TSCs 5, 6, 8, and 9 revealed a half maximal inhibitory concentration (IC50) below 1 µM. Compound 6 turned out to be the most potent tyrosinase inhibitor. All investigated compounds showed reversible inhibition of competitive or mixed type. The para-substituted TSCs had higher affinity for the enzyme as compared to their ortho- and meta-analogues. All investigated compounds inhibited melanin production in B16F10 cells at the micromolar level. Molecular docking showed that the sulfur atom of the thiourea moiety penetrates the active site and interacts with copper ions. The above outcomes might be helpful in the design of new tyrosinase inhibitors in the food and cosmetic industries.

Treatment with lentiviral vectors encoding shRNA against interleukin 10 modulates the immunosuppressive activity of murine colon carcinoma-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity and their accumulation is often associated with poor prognosis. The aim of the present study was to determine the mechanisms of action of lentiviral vectors encoding short hairpin (sh)RNA against interleukin-10 (IL-10), with particular emphasis on their influence on the activity of tumor-derived MDSCs. Lentiviral vectors encoding shRNA against IL-10 (shIL-10 LVs) were utilized to silence the expression of IL-10 either in MDSCs that were generated ex vivo from bone marrow cells cultured in the presence of supernatant from MC38 colon carcinoma cells, or in situ in the MC38 murine colon carcinoma environment. Although monocytic MDSCs (M-MDSCs) transduced with shIL-10 LVs exhibited increased suppressor activity, transduction of polymorphonuclear MDSCs (PMN-MDSCs) appeared to reduce their ability to inhibit T lymphocyte functions. Analysis of EGFP expression in MC38 tumors revealed that intratumorally inoculated shIL-10 LVs transduced tumor-infiltrating myeloid cells with the highest efficiency and, led to a decreased IL-10 level in the tumor microenvironment. However, the effect was accompanied by increased influx of PMN-MDSCs into tumors observed both on the 6th and on the 10th day after shIL-10 LV injections. Nevertheless, it was noted that suppressor activity of myeloid cells isolated from tumors was dependent on the efficiency of tumor-derived PMN-MDSC transduction with shIL-10 LVs. The increased percentage of transduced PMN-MDSCs on the 10th day was associated with diminished immunosuppressive activity of tumor-derived myeloid cells and an elevated ratio of cytotoxic T lymphocytes to M-MDSCs. The obtained data indicated that treatment with shIL-10 LVs may result in modulation of the immunosuppressive activity of MC38 colon carcinoma-derived MDSCs.

Investigating the photodynamic efficacy of chlorin e6 by millisecond pulses in metastatic melanoma cells.

Melanoma is considered the most aggressive type of skin cancer, still without effective treatment. Thus, alternative therapeutic methods are still in demand, and electroporation-supported photodynamic therapy (EP-PDT) of cancer cells seems a promising approach. New developments in EP-PDT aim at enhanced tumor selectivity and biocompatibility by applying a second-generation photosensitizer, i.e., Chlorin e6 (Ce6). We have verified the improved photodynamic effect of Ce6 on skin cancer melanoma (Me45) cells and control (CHO-K1) cells. In this study, we applied 1 or 5 pulses of 10 ms duration and assessed the EP-PDT effect with a variety of tests, such as singlet oxygen scavenger (ABMDMA) photooxidation, oxidoreductive potential measurements, kinetic measurements with fluorescent microscopy, photosensitizer uptake studies, lipid peroxidation level, and actin fibers organization. The optimization of photosensitizer uptake as a function of temperature was also performed. Our results indicated efficient Ce6 delivery into Me45 cells and good photodynamic efficiency enhanced by the electroporation of cancer cells.