High Levels Of Cytotoxicity Research Articles

The evaluation of cytotoxicity and cytokine IL-6 production of root canal sealers with and without the incorporation of simvastatin: an invitro study

BackgroundFreshly mixed root canal sealers when proximate the periapical tissues, trigger varying degrees of cytotoxicity/inflammatory reactions. Simvastatin, a class of the drug statin, is a widely used cholesterol-lowering agent with additional anti-inflammatory activities. This study assessed the effects of simvastatin on cytotoxicity and the release of IL-6 (Interleukin-6) production when incorporated in zinc oxide eugenol and methacrylate resin-based sealers.MethodsExperimental groups consisted of conventional zinc oxide eugenol and methacrylate based-EndoREZ sealers (ZE & ER respectively) and 0.5 mg/mL simvastatin incorporated sealers (ZES & ERS). L929 mouse fibroblast cells were exposed to freshly mixed experimental sealers and evaluated for cytotoxicity (MTT assay) and inflammation levels (inflammatory marker IL-6 for ELISA) at various time intervals (0h, 24h and 7th day). The values were compared to the cell control (CC; L929 cells alone) and solvent control (SC; L929 cells + DMSO) groups. All the experiments were conducted in triplicates and subjected to statistical analysis using IBM SPSS Statistics software. Non parametric tests were conducted using Kruskal-Wallis and Friedman tests for inter-group and intra-group comparisons respectively. Pairwise comparison was conducted by post hoc Dunn test followed by Bonferroni correction. P values < 0.05 were considered statistically significant.ResultsAll the experimental groups (ZE, ER, ZES, ERS) exhibited varying degree of cytotoxicity and IL-6 expression compared to the control groups CC and SC. The cell viability for ZE and ER decreased on day 7 as compared to 24 h. ZES and ERS had higher viable cells (75.93% & 79.90%) compared to ZE and ER (54.39% & 57.84%) at all time periods. Increased expression of IL-6 was observed in ZE & ER (25.49 pg/mL & 23.14 pg/mL) when compared to simvastatin incorporated ZE & ER (ZES-12.70 pg/mL & ERS-14.68 pg/mL) at all time periods. Highest level of cytotoxicity and inflammation was observed in ZE compared to all the other groups on day 7.ConclusionsAddition of 0.5 mg/mL of simvastatin to the sealers (ZES and ERS) decreased the cytotoxicity in the freshly mixed state and reduces their inflammatory effect.

ОСОБЛИВОСТІ ПАТОГЕНЕЗУ КОРОНАВІРУСНОЇ ІНФЕКЦІЇ ТА ПРОГНОЗ ЩОДО ПАТОМОРФОЗУ ТУБЕРКУЛЬОЗУ ЛЕГЕНЬ В УМОВАХ ПАНДЕМІЇ СOVID-19

To date, the COVID-19 pandemic has surpassed all other health issues around the world. The interaction between COVID-19 and tuberculosis (TB), which remains the leading cause of death from a single infectious disease in the world, is important, as it can have serious consequences for both confirmed and underdiagnosed TB patients worldwide, especially in low- and middle-income countries, where TB is endemic and health services are poorly equipped. The aim of the study is to assess the prospects of the epidemiology of tuberculosis in the context of the COVID-19 pandemic and the consequences of the interaction between SARS-CoV-2 and Mycobacterium tuberculosis, taking into account the current situation in Ukraine. Materials and methods. Test access to various full-text and abstract databases was used. Results. Pathomorphological changes in COVID-19 patients are due to the direct action of SARS-CoV-2, hyperactivity of the immune system, high levels of cytotoxicity of CD8 + T cells, autoimmune processes. In view of all the above, the governments of TB-affected countries must ensure the continuity and effective provision of TB services during COVID-19. This includes the protection of the most vulnerable groups, including protection from economic hardship, isolation, stigma and discrimination. The global response should identify and mitigate potential risks to the TB-control mission. Conclusions. COVID-19 has pushed back anti-TB efforts for nearly a decade. This failure is likely to affect the long-term increase in TB morbidity and mortality worldwide. With low vaccination rates in TB-endemic countries and the emergence of new genotypes of the virus, this trend is likely to continue. Key words: COVID-19, tuberculosis, pathogenesis, treatment, epidemiology

Enhancement of NK Cell Antitumor Effector Functions Using a Bispecific Single Domain Antibody Targeting CD16 and the Epidermal Growth Factor Receptor.

Simple SummaryStrategies to enhance the preferential accumulation and activation of Natural Killer (NK) cells in the tumor microenvironment can be expected to increase the efficacy of NK cell-based cancer immunotherapy. In this study, we report that a bispecific single domain antibody (VHH) that targets CD16 (FcRγIII) on NK cells and the epidermal growth factor receptor (EGFR) on tumor cells can be used to target and enhance cytolysis of cancer cells. The bispecific VHH enhanced NK cell activation and cytotoxicity in an EGFR- and CD16-dependent and KRAS-independent manner. Moreover, the bispecific VHH induced stronger activity of cancer patient-derived NK cells and resulted in tumor control in a co-culture of metastatic colorectal cancer cells and either autologous peripheral blood mononuclear cells or allogeneic CD16+ NK cells. We believe that this novel approach could represent a valid therapeutic strategy either alone or in combination with other NK cell-based therapies.The ability to kill tumor cells while maintaining an acceptable safety profile makes Natural Killer (NK) cells promising assets for cancer therapy. Strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment can be expected to increase the efficacy of NK cell-based therapies. In this study, we show binding of a novel bispecific single domain antibody (VHH) to both CD16 (FcRγIII) on NK cells and the epidermal growth factor receptor (EGFR) on tumor cells of epithelial origin. The bispecific VHH triggered CD16- and EGFR-dependent activation of NK cells and subsequent lysis of tumor cells, regardless of the KRAS mutational status of the tumor. Enhancement of NK cell activation by the bispecific VHH was also observed when NK cells of colorectal cancer (CRC) patients were co-cultured with EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the bispecific VHH and autologous peripheral blood mononuclear cells or allogeneic CD16 expressing NK cells. The anticancer activity of CD16-EGFR bispecific VHHs reported here merits further exploration to assess its potential therapeutic activity either alone or in combination with adoptive NK cell-based therapeutic approaches.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 134-mG2a-f Exerts Antitumor Activities in Mouse Xenograft Models of Dog Epidermal Growth Factor Receptor-Overexpressed Cells

The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a crucial mediator of cell growth and differentiation and forms homodimers or heterodimers with other HER family members to activate downstream signaling cascades. We previously established an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1), by immunizing mice with the ectodomain of hEGFR. In this study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) and further defucosylated (134-mG2a-f) to facilitate antibody-dependent cellular cytotoxicity (ADCC). Although 134-mG2a-f was developed against hEGFR, it was shown to cross-react with dog EGFR (dEGFR) using flow cytometry. The dissociation constant (KD) of 134-mG2a-f against dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells was determined by flow cytometry to be 3.3 × 10-9 M, indicating that 134-mG2a-f possesses a high binding affinity to dEGFR. Analysis in vitro revealed that 134-mG2a-f contributed to high levels of ADCC and complement-dependent cytotoxicity (CDC) in experiments targeting CHO/dEGFR cells. Furthermore, the in vivo administration of 134-mG2a-f significantly inhibited the development of CHO/dEGFR in comparison with the results observed in response to control mouse IgG. Taken together, the findings of this study demonstrate that 134-mG2a-f could be useful as part of a therapeutic regimen for dEGFR-expressing canine cancers.

Cytotoxic 1,2,3-Triazoles as Potential Leads Targeting the S100A2-p53 Complex: Synthesis and Cytotoxicity.

In silico screening predicted 1 (N-(4-((4-(3-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl) sulfonyl)-phenyl)acetamide) as an inhibitor of the S100A2-p53 protein-protein interaction. S100A2 is a validated pancreatic cancer drug target. In the MiaPaCa-2 pancreatic cell line, 1 was a ∼50 μM growth inhibitor. Synthesis of five focused compound libraries and cytotoxicity screening revealed increased activity from the presence of electron withdrawing moieties on the sulfonamide aromatic ring, with the 3,5-bis-CF3 Library 3 analogues the most active, with GI50 values of 0.91 (3-ClPh; 13 i; BxPC-3, Pancreas) to 9.0 μM (4-CH3 ; 13 d; PANC-1, Pancreas). Activity was retained against an expanded pancreatic cancer cell line panel (MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, PANC-1 and HPAC) and the normal cell line MCF10A (breast). Bulky 4-disposed substituents on the terminal phenyl ring enhanced broad spectrum activity with growth inhibition values spanning 1.1 to 3.1 μM (4-C(CH3 )3 ; 13 e; BxPC-3 and AsPC-1 (pancreas), respectively). Central alkyl spacer contraction from propyl to ethyl proved detrimental to activity with Library 4 and 5.5- to 10-fold less cytotoxic than the propyl linked Library 2 and Library 3. The data herein was consistent with the predicted binding poses of the compounds evaluated. The highest levels of cytotoxicity were observed with those analogues best capable of adopting a near identical pose to the p53-peptide in the S100A2-p53 binding groove.

An in vitro study of dual drug combinations of anti-viral agents, antibiotics, and/or hydroxychloroquine against the SARS-CoV-2 virus isolated from hospitalized patients in Surabaya, Indonesia.

A potent therapy for the infectious coronavirus disease COVID-19 is urgently required with, at the time of writing, research in this area still ongoing. This study aims to evaluate the in vitro anti-viral activities of combinations of certain commercially available drugs that have recently formed part of COVID-19 therapy. Dual combinatory drugs, namely; Lopinavir-Ritonavir (LOPIRITO)-Clarithromycin (CLA), LOPIRITO-Azithromycin (AZI), LOPIRITO-Doxycycline (DOXY), Hydroxychloroquine (HCQ)-AZI, HCQ-DOXY, Favipiravir (FAVI)-AZI, HCQ-FAVI, and HCQ-LOPIRITO, were prepared. These drugs were mixed at specific ratios and evaluated for their safe use based on the cytotoxicity concentration (CC50) values of human umbilical cord mesenchymal stem cells. The anti-viral efficacy of these combinations in relation to Vero cells infected with SARS-CoV-2 virus isolated from a patient in Universitas Airlangga hospital, Surabaya, Indonesia and evaluated for IC50 24, 48, and 72 hours after viral inoculation was subsequently determined. Observation of the viral load in qRT-PCR was undertaken, the results of which indicated the absence of high levels of cytotoxicity in any samples and that dual combinatory drugs produced lower cytotoxicity than single drugs. In addition, these combinations demonstrated considerable effectiveness in reducing the copy number of the virus at 48 and 72 hours, while even at 24 hours, post-drug incubation resulted in low IC50 values. Most combination drugs reduced pro-inflammatory markers, i.e. IL-6 and TNF-α, while increasing the anti-inflammatory response of IL-10. According to these results, the descending order of effective dual combinatory drugs is one of LOPIRITO-AZI>LOPIRITO-DOXY>HCQ-AZI>HCQ-FAVI>LOPIRITO-CLA>HCQ-DOX. It can be suggested that dual combinatory drugs, e.g. LOPIRITO-AZI, can potentially be used in the treatment of COVID-19 infectious diseases.

Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvβ6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvβ6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvβ6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvβ6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvβ6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvβ6 expression and short survival time (p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvβ6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvβ6-positive CCA cells (p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvβ6-positive CCA tumor spheroids (p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvβ6-positive CCA.

The Antiproliferative and Apoptotic Effect of a Novel Synthesized S-Triazine Dipeptide Series, and Toxicity Screening in Zebrafish Embryos.

Several derivatives containing morpholine/piperidine, anilines, and dipeptides as pending moieties were prepared using s-triazine as a scaffold. These compounds were evaluated for their anticancer activity against two human breast cancer cell lines (MCF-7 and MDA-MB-231), a colon cancer cell line (HCT-116), and a non-tumorigenic cell line (HEK 293). Tamoxifen was used as a reference. Animal toxicity tests were carried out in zebrafish embryos. Most of these compounds showed a higher activity against breast cancer than colon cancer. Compound 3a—which contains morpholine, aniline, and glycylglycinate methyl ester—showed a high level of cytotoxicity against MCF-7 cells with IC50 values of less than 1 µM. This compound showed a much lower level of toxicity against the non-tumorigenic HEK-293 cell line, and in the in vivo studies using zebrafish embryos. Furthermore, it induced cell cycle arrest at the G2/M phase, and apoptosis in MCF-7 cells. On the basis of our results, 3a emerges as a potential candidate for further development as a therapeutic drug to treat hormone receptor-positive breast cancer.

Tumor-selective new piperazine-fragmented silicon phthalocyanines initiate cell death in breast cancer cell lines

A new silicon phthalocyanine with piperazine-furan ring and its quaternized form were synthesized. All compounds were analyzed by spectroscopic techniques (FT-IR, 1H-NMR, MS, and UV–vis), and the absorbance characteristics of silicon phthalocyanines were evaluated with the expected strong typical absorption bands in the far-red spectrum. The cytotoxic effects of these phthalocyanines induced by photodynamic therapy (PDT) were determined in a dose-dependent manner. Following cytotoxicity analysis, flow cytometric research of cell death was performed. The formation of reactive oxygen species (ROS) was determined by confocal microscopy. High levels of cytotoxicity and decreased viable cell population have been detected in cancer cells after treatment. In addition, ROS formation was observed in PDT treated cancer cells. However, low levels of cell death and ROS formation were observed in non-tumorigenic cells. According to western blot data, PDT-mediated treatment was found to provide different expression patterns of the cleaved PARP1 protein. The presented study demonstrates that PDT-mediated treatment of newly synthesized phthalocyanines has significant anti-cancer effects on breast cancer cells and may induce different cell death pathways.

5-O-Demethylnobiletin Alleviates CCl4-Induced Acute Liver Injury by Equilibrating ROS-Mediated Apoptosis and Autophagy Induction.

Polymethoxyflavanoids (PMFs) have exhibited a vast array of therapeutic biological properties. 5-O-Demethylnobiletin (5-DN) is one such PMF having anti-inflammatory activity, yet its role in hepatoprotection has not been studied before. Results from in vitro study revealed that 5-DN did not exert a high level of cytotoxicity on HepG2 cells at 40 μM, and it was able to rescue HepG2 cell death induced by carbon tetrachloride (CCl4). Subsequently, we investigated acute liver injury on BALB/c mice induced by CCl4 through the intraperitoneal injection of 1 mL/kg CCl4 and co-administration of 5-DN at (1 and 2 mg/kg) by oral gavage for 15 days. The results illustrated that treatment with 5-DN attenuated CCl4-induced elevated serum aminotransferase (AST)/alanine aminotransferase (ALT) ratio and significantly ameliorated severe hepatic damage such as inflammation and fibrosis evidenced through lesser aberrations in the liver histology of 5-DN dose groups. Additionally, 5-DN efficiently counteracted and equilibrated the production of ROS accelerated by CCl4 and dramatically downregulated the expression of CYP2E1 vitally involved in converting CCl4 to toxic free radicals and also enhanced the antioxidant enzymes. 5-DN treatment also inhibited cell proliferation and inflammatory pathway abnormally regulated by CCl4 treatment. Furthermore, the apoptotic response induced by CCl4 treatment was remarkably reduced by enhanced Bcl-2 expression and noticeable reduction in Bax, Bid, cleaved caspase 3, caspase 9, and apaf-1 expression. 5-DN treatment also induced the conversion of LC3 and promoted the autophagic flux. Conclusively, 5-DN exhibited hepatoprotective effects in vitro and in vivo and prevented liver fibrosis induced by CCl4.

Fecal indicators, pathogens, antibiotic resistance genes, and ecotoxicity in Galveston Bay after Hurricane Harvey

Unprecedented rainfall after Hurricane Harvey caused a catastrophic flood in the southern coast of Texas, and flushed significant floodwater and sediments into Galveston Bay, the largest estuary along the Texas Gulf Coast. This study investigated the immediate and long-term (6 months post-Harvey) fecal indicators, pathogenic bacteria, antibiotic resistance genes (ARGs), and ecotoxicity in the Galveston Bay. Dramatic decrease of salinity profile to zero, increased levels of fecal indicator bacteria and pathogenic bacteria, and detection of various ARGs were observed in the water and sediment samples collected 2 weeks post-Harvey. High levels of BlaTEM and cytotoxicity measured by yeast bioluminescent assay (BLYR) were also observed especially near the river mouths. While Vibrio spp. was dominant in water, much higher abundance of fecal indicator bacteria and pathogen were detected in the sediments. A decreasing trend of BlaTEM and cytotoxicity was observed in March 2018 samples, suggesting the Bay has returned to its pre-hurricane conditions 6 months post-Harvey. Interestingly, the abundance of fecal indicator bacteria and pathogens were shifted dramatically according to high-streamflow and low-streamflow seasons in the Bay. The data are useful to construct the model of risk assessment in coastal estuaries system and predict the effects of extreme flooding events in the future.

Chlorin e6 embedded in phospholipid nanoparticles equipped with specific peptides: Interaction with tumor cells with different aminopeptidase N expression

A promising direction in Biopharmaceuticals is the development of specific peptide-based systems to improve drug delivery. This approach may increase tumor specificity and drug penetration into the target cell. Similar systems have been designed for several antitumor drugs. However, for photodynamic therapy drugs, such studies are not yet enough. Previously, we have developed a method of inclusion of chlorin e6 (Ce6), a photosensitizer used in photodynamic therapy, in phospholipid nanoparticles with a diameter of up to 30 nm, and reported an increase in its effectiveness in the experiments in vivo. In this work, we propose to modify a previously developed delivery system for Ce6 by the addition of cell-penetrating (R7) and/or targeting NGR peptides. The interaction of the compositions developed with HepG2 and MCF-7 tumor cells is shown. The expression of CD13 protein with affinity to NGR on the surface of these cells has been studied using flow cytometry. The expression of this protein on the HepG2 cells and its absence on MCF-7 was demonstrated. After incubation of tumor cells with the resulting Ce6 compositions, we evaluated the cellular accumulation, photoinduced, and dark cytotoxicity of the drugs. After irradiation, the highest level of cytotoxicity was observed when R7 peptide was added to the system, either alone or in combination with NGR. In addition to R7, the NGR-motif peptide increased the internalization of Ce6 in HepG2 cells without affecting its photodynamic activity. In this work we also discuss possible mechanisms of action of the cell-penetrating peptide when attached to phospholipid nanoparticles.

Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

Objective:Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.Materials and Methods:We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors’ and ALL/NHL patients’ peripheral blood mononuclear cells.Results:We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice.Conclusion:This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.

CD56dim CD57- NKG2C+ NK cells retaining proliferative potential are possible precursors of CD57+ NKG2C+ memory-like NK cells.

Formation of the adaptive-like NK cell subset in response to HCMV infection is associated with epigenetic rearrangements, accompanied by multiple changes in the protein expression. This includes a decrease in the expression level of the adapter chain FcεRIγ, NKp30, and NKG2A receptors and an increase in the expression of NKG2C receptor, some KIR family receptors, and co-stimulating molecule CD2. Besides, adaptive-like NK cells are characterized by surface expression of CD57, a marker of highly differentiated cells. Here, it is shown that CD57-negative CD56dim NKG2C+ NK cells may undergo the same changes, as established by the similarity of the phenotypic expression pattern with that of the adaptive-like CD57+ NKG2C+ NK cells. Regardless of their differentiation stage, NKG2C-positive NK cells had increased HLA-DR expression indicating an activated state, both ex vivo and after cultivation in stimulating conditions. Additionally, CD57- NKG2C+ NK cells exhibited better proliferative activity compared to CD57+ NKG2C+ and NKG2C- NK cells, while retaining high level of natural cytotoxicity. Thus, CD57- NKG2C+ NK cells may represent a less differentiated, but readily expanding stage of the adaptive-like CD57+ NKG2C+ NK cells. Moreover, it is shown that NK cells have certain phenotypic plasticity and may both lose NKG2C expression and acquire it de novo during proliferation, induced by IL-2 and K562-mbIL21 feeder cells.

Draft Genome Sequence of CH_48, a Highly Cytotoxic Bacillus thuringiensis Strain Isolated from Rosemary at the Retail Level.

Bacillus thuringiensis CH_48 exhibits extremely high levels of Vero cell cytotoxicity and sphingomyelinase activity.

IMMUNOPHYSIOLOGICAL EFFECTS IN EXPERIMENTAL ANIMALS AFTER ADMINISTRATION OF MYASTHENOGENIC SERUMS WITH VARYING DEGREES OF CYTOTOXICITY

Introduction. The action of foreign antigens on the body leads to its sensitization, reflecting the degree of reactivity of the organism and the degree of immunogenicity of the introduced material. The animal immunization model can be used to elucidate the nature of various antigenic agents, including myasthenogenic factors. It is of interest to assess the nature of the impact of immunogenic myasthenogenic factors on the immune system (phagocytosis, opsonizing complement factors).Aim. To estimate the immunophysiological effects after animals’ immunization with myasthenogenic blood serum with varying cytotoxicity degrees obtained from patients with thymus-independent and thymus-dependent myasthenia.Methods. The animals were immunized 4 times with myasthenogenic serums with different levels of cytotoxicity: M – myasthenia gravis without morphological and functional thymus changes (low cytotoxicity), MH – myasthenia gravis with thymus hyperplasia (medium cytotoxicity), MT – myasthenia gravis with thymoma (high cytotoxicity). The activity of oxygen-independent and oxygen-dependent phagocytosis was determined using light microscopy; the concentration of C3 and C4 complement components was measured by immunoturbidimetric method.Results. As a result of immunization, a change in temperature was detected in animals as a reaction to the administration of immunogenic myasthenogenic serum. In animals immunized with serums with medium and high cytotoxicity, a decrease in the adhesive properties (phagocytic index) of phagocytic neutrophils by 1.7 and 1.5 times, respectively, and a 20 % decrease in the digesting function of neutrophils (index of phagocytosis completion) compared with the control was revealed. In all immunized animals, the stimulation index reflecting the activity of NADPH-N-oxidase neutrophils was reduced. After the action of myasthenogenic serum with low and high levels of cytotoxicity, the concentration of the C3 component of the complement was 15 % lower than the control. The administration of the highly cytotoxic serum led to an increase in the concentration of the C3 complement component against a decrease in temperature. A twofold increase in the concentration of the C4 complement component in animals sensitized with the serum with a low degree of cytotoxicity was accompanied by an increase in temperature.Conclusion. Myasthenogenic serums of varying cytotoxicity degrees sensitized innate immunity factors – phagocytosis and complement proteins, which led to a violation in the processes of opsonization, absorption, processing and presentation of the antigen, and was accompanied by a change in physiological parameters.Keywords: immunization; myasthenogenic serum; cytotoxicity; phagocytosis; complement.

&lt;p&gt;Cordycepin Nanoencapsulated in Poly(Lactic-Co-Glycolic Acid) Exhibits Better Cytotoxicity and Lower Hemotoxicity Than Free Drug&lt;/p&gt;

PurposeCordycepin, a natural product isolated from the fungus Cordyceps militaris, is a potential candidate for breast cancer therapy. However, due to its structural similarity with adenosine, cordycepin is rapidly metabolized into an inactive form in the body, hindering its development as a therapeutic agent. In the present study, we have prepared cordycepin as nanoparticles in poly(lactic-co-glycolic acid) (PLGA) and compared their cellular uptake, cytotoxicity and hemolytic potential with free cordycepin.Materials and MethodsCordycepin-loaded PLGA nanoparticles (CPNPs) were prepared by the double-emulsion solvent evaporation method. Physico-chemical characterization of the nanoparticles was done by zetasizer, transmission electron microscopy (TEM) and reverse-phase high-pressure liquid chromatography (RP-HPLC) analyses. Cellular uptake and cytotoxicity of CPNPs and free drug were tested in human breast cancer cells (MCF7). Hemolytic potential of both of these forms was evaluated in rat red blood cells (RBCs).ResultsPhysico-chemical characterization revealed that CPNPs were spherical in shape, possessed a size range of 179–246 nm, and released the encapsulated drug sustainably over a period of 10 days. CPNPs exhibited a high level of cellular uptake and cytotoxicity than the free drug in MCF-7 cells. While CPNPs were not toxic to rat RBCs even at high concentrations, free cordycepin induced hemolysis of these cells at relatively low concentration.ConclusionOur results reveal that delivery as CPNPs could enhance the clinical efficacy of cordycepin substantially.

9,10-Anthraquinone Dithiocarbamates as Potential Pharmaceutical Substances with Pleiotropic Actions: Computerized Prediction of Biological Activity and Experimental Validation

The pleiotropic pharmacological actions of 9,10-anthraquinone dithiocarbamates were studied. The Way2Drug web portal was used to establish that cytotoxic, antiviral, and anticonvulsant actions were predicted for the study molecules, along with moderate toxicity. In vitro experimental studies showed that all study compounds had cytotoxic effects on tumor cell lines Hep-2, CHO, HeLa, Raji, BL, and B95-8. The high levels of cytotoxicity against cell lines Raji and B95-8 seen with derivatives II, IV, VI, and X was evidence of direct actions on Epstein-Barr virus (EBV). Along with the cytotoxic action, dithiocarbamates V, VIII, and IX showed antiviral effects against herpes virus HSV-2. Compounds VI and X, which had cytotoxic and antiviral actions, produced anticonvulsant effects 3 and 6 h after administration, while dithiocarbamate III, which had cytotoxic activity, retained its antiviral effect 24 h after administration. Dithiocarbamates with pleiotropic actions were moderately toxic (LD50 > 1000 mg/ml, mice, p.o.). Interactions between the structures and the three types of biological activity studied experimentally were assessed.

In-vitro evaluation of the cytotoxicity, mitotoxicity and lipotoxicity potentials of Helichrysum petiolare Hilliard &amp; B.L. Burtt using HepG2 (C3A) Cell-lines

This study evaluated the hepatotoxicity (cytotoxicity, mitotoxicity and lipotoxicity) potentials of the boiled aqueous (BAQ), cold aqueous (CAQ) and ethanol (ETQ) extracts of Helichrysum petiolare using standard procedures that employed the use of C3A cell-lines. The results obtained showed negligible BAQ and CAQ cytotoxicities, which were further, corroborated by stability in the mitochondrial membrane potentials and were also congruent with the CAQ and BAQ results for steatosis and phospholipidosis. The data suggested a favourable CAQ and BAQ toxicity profiles with limited risks for hepatotoxicity. However, ETQ extract showed significantly high levels of cytotoxicity and lipotoxicity, and a low level of mitotoxicity. Our result suggested a potential risk of the ETQ extract for hepatotoxicity but appears partly independent of direct mitochondrial involvement.

Anti-PLA2R1 Antibodies Containing Sera Induce In Vitro Cytotoxicity Mediated by Complement Activation.

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy (MN). However, the pathogenic role of anti-PLA2R1 autoantibodies is unclear. Our aim was to evaluate the in vitro cytotoxicity of anti-PLA2R1 antibodies mediated by complement. Forty-eight patients with PLA2R1-related MN from the prospective cohort SOURIS were included. Anti-PLA2R1 titer, epitope profile, and anti-PLA2R1 IgG subclasses were characterized by ELISA. Cell cytotoxicity was evaluated by immunofluorescence in HEK293 cells overexpressing PLA2R1 incubated with patient or healthy donor sera in the presence or absence of rabbit complement or complement inhibitors. Mean cytotoxicity of anti-PLA2R1 sera for HEK293 cells overexpressing PLA2R1 was 2 ± 2%, which increased to 24 ± 6% after addition of rabbit complement (p < 0.001) (n = 48). GVB-EDTA, which inhibits all complement activation pathways, completely blocked cell cytotoxicity, whereas Mg-EGTA, which only inhibits the classical and lectin pathways, highly decreased suggesting a limited role of the alternative pathway. A higher diversity of IgG subclasses beyond IgG4 and high titer of total IgG anti-PLA2R1 were associated with increased cytotoxicity (p = 0.01 and p = 0.03 respectively). In a cohort of 37 patients treated with rituximab, high level of complement-mediated cytotoxicity was associated with less and delayed remission at month 6 after rituximab therapy (5/12 vs. 20/25 (p = 0.03) in 8.5 months ± 4.4 vs. 4.8 ± 4.0 (p = 0.02)). Kaplan-Meier analysis demonstrated that high level of cytotoxicity (≥40%) (p = 0.005), epitope spreading (defined by immunization beyond the immunodominant CysR domain) (p = 0.002), and high titer of anti-PLA2R1 total IgG (p = 0.01) were factors of poor renal prognosis. Anti-PLA2R1 antibodies containing sera can induce in vitro cytotoxicity mediated by complement activation, and the level of cytotoxicity increases with the diversity and the titer of anti-PLA2R1 IgG subclasses. These patients with high level of complement-mediated cytotoxicity could benefit from adjuvant therapy using complement inhibitor associated with rituximab to induce earlier remission and less podocyte injury.