Schistosoma mansoni: Complement and antibody damage,mediated by human eosinophils and neutrophils,in killing schistosomula in vitro

The time and course of damage to Schistosoma mansoni schistosomula mediated by human eosinophils and neutrophils and by antibody (A) and/or complement (C) was studied. The rate of schistosomula death was significantly higher in the complement containing systems (i.e., “A + C” or “C alone”) when compared to A alone. In general, at all the time points studied, the percentage of killing in the three systems was A + C > C alone > A alone irrespective of whether the effector cells were neutrophils or eosinophils. Preferential killing of schistosomula by eosinophils, as compared to neutrophils, was observed with C alone and A + C, but only when eosinophils and neutrophils from the same donor were compared. In contrast, eosinophils and neutrophils appeared to be equally effective in killing antibody-coated schistosomula.A comparison was made of the susceptibility to killing of schistosomula prepared mechanically or by skin penetration. There was no appreciable difference in terms of susceptibility to killing by the various combinations of eosinophils, neutrophils, antibody, and complement between these two forms of schistosomula.The preferential killing of complement-coated, as compared to antibody-coated schistosomula by eosinophils appears to be relatively rapid, an observation which may be of significance both in natural and acquired immunity to migrating larval helminths.     

Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells

Summary The effects of human recombinant interleukin-6 (hrIL-6) on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h⁵¹Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.1A and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100–400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity. hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy.     

OK-432 and IL-2-augmental cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level

Abstract The biology response modifiers OK-432 and interleukin-2 (IL-2) were found to enhance the lytic capacity of cloned CD3⁻ natural killer (NK) cells and CD3⁺ T cells. With respect to NK cells, only those clones with a high proliferative capacity and cultured without phytohaemagglutinin (PHA) responded with enhaced lytic capacity to OK-432. OK-432, but not IL-2, was found to augment the antibody-dependent cellular cytotoxicity of cloned NK cells. With T-cell clones, OK-432 augmented the cellular cytotoxicity of CD3⁺8⁺ but not that of CD3⁺4⁺ cytotoxic T lymphocytes, while IL-2 augmented the cytotoxicity of both types of clone. Taken together, these results indicate that OK-432-augmented lytic capacity is not restricted to NK cells and its pathway of action may be independent of IL-2.     

Comparison of in vitro cytotoxicity of Fusarium mycotoxins,deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines

Three human epithelial cell lines (CaCo-2, HEp-2 and HeLa) implicated as potential targets for three Fusarium toxins were tested for the extent of survival on exposure to increasing toxin concentration and incubation periods. Cytotoxicity assay using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was carried out with deoxynivalenol (DON), T-2 toxins and zearalenone (ZON) on CaCo-2, HEp-2 and HeLa cell lines. Of the three cell lines used, HeLa was the most sensitive, eliciting cell death after 2 days exposure at 100 ng ml^–1with T-2 toxin. HeLa was the only cell line to exhibit cytotoxicity towards ZON showing cell death at 1000 ng ml^–1after 2 days which increased to 4 days, showing substantial cell death at 200 ng ml^–1. HEp-2 was sensitive to DON showing cell death after 2 days (100 ng ml^–1) with complete cell death occurring at 200 ng ml^–1 after 4 days of exposure. Substantial cytoxicity of T-2 towards HEp-2 occurred after 2 days at 1000 ng ml^–1 and complete cell death occurred with 100 ng ml^–1 at day 4. The CaCo-2 cell line was generally resistant to the mycotoxins tested between 100 and 1000 ng ml^–1. This study shows that cytotoxicity of Fusarium toxins to epithelium cell lines is concentration- and time- dependant and results from ZON–HeLa interaction indicate possible cell type-mycotoxin specificity.     

Relative cytotoxicity of psychotropic drugs in cultured rat hepatocytes

The relative cytotoxic effects of ten psychotropic drugs were assessed in rat hepatocyte monolayer cultures. Clear concentration-related toxicity was seen in the narrow range of 10^–5M to S × 10^–5M. The four cytotoxicity endpoints chosen were: release of the cytosolic enzyme, lactate dehydrogenase, and impairment of biosynthesis and secretion of proteins, bile acids and glycerolipids. LDH leakage and inhibition of protein secretion into the culture medium proved to be the parameters which allowed the best differentiation between the test compounds. The inhibition of glycerolipid secretion was the most sensitive test in relation to concentration and time of exposure. Based on the effects of these endpoints, the following ranking of relative in vitro toxicity, using equimolar drug concentrations, could be established: clomipramine > imipramine = thioridazine > chlorpromazine > amitriptyline = fluperlapine > haloperidol > promazine > clozapine sulpiride. This ranking order of in vitro cytotoxicity correlated well with the potential of the drugs to impair liver function in man. Only clozapine had to be classified as a false negative. There was, however, no correlation between the cytotoxicity and the intracellular accumulation of the test drugs. Furthermore, the comparison of the data obtained with psychotropics with the data from five other amphiphilic cationic drugs was consistent with the widely accepted concept of a direct toxic interaction of the drugs with cytomembranes. This nonspecific toxicity of the membrane-active drugs was further corroborated by a positive correlation between their potential to induce LDH leakage in hepatocytes and their ability to induce hemolysis in red cells. In conclusion, the results obtained in our study strongly suggest that it is possible to assess the relative cytotoxicity of psychotropic drugs in rat hepatocyte cultures. It is proposed that this in vitro system provides a useful tool to evaluate new drugs at an early stage of their development, and to identify the most promising candidates within a class of structurally related compounds. In addition, it allows information to be obtained on possible mechanisms of cytotoxicity.Abbreviations AIB aminoisobutyric acid - AMT amitriptyline - BSA bovine serum albumin - CLP clomipramine - CLZ clozapine - CPZ chlorpromazine - FLU fluperlapine - HAL haloperidol - HC₅₀ dose causing 50% hemolysis - IMP imipramine - LDH lactate dehydrogenase - PZ promazine - SUL sulpiride - TCA trichloroacetic acid - TRZ thioridazine     

A light and electron microscopic study of myelopoietic cells in the perihepatic subcapsular region of the liver in the adult aquatic newt,Notophthalmus viridescens

Summary The liver of the newt, Notophthalmus viridescens, consists of several incompletely separated lobes of parenchymal tissue each of which is covered by a perihepatic subcapsular region (PSR) of myeloid tissue. This tissue contains neutrophils and eosinophils in various stages of differentiation. As neutrophils develop from myeloblasts to late neutrophilic myelocytes, two types of granules appear. The primary granules (type of granules formed first) are more electron dense and smaller than the secondary granules (type of granules formed later). The primary granules first appear at the stage designated early neutrophilic myelocyte, and the secondary granules appear at the stage of the maturing neutrophilic myelocyte. The eosinophils present are characterized by much larger granules than those observed in neutrophils. Cells in the PSR which superficially resemble small lymphocytes are primitive stem cells that give rise to neutrophils and eosinophils. The liver PSR is invested by a visceral peritoneum of simple squamous mesothelial cells some of which are ciliated.Supported by ACS IN-105.     

Comparative in vitro cytotoxicity of ethyl acrylate and tripropylene glycol diacrylate to normal human skin and lung cells

Summary The potential for occupational exposure to the esters of acrylic acid (acrylates) is considerable, and, thus, requires a greater understanding of the their toxicity. Confluent (70–90%) cultures of normal human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF), or bronchial epithelium (NHBE) were exposed to the monofunctional ethyl acrylate (EA), the multifunctional tripropylene glycol diacrylate (TPGDA), or TPGDA monomer in a radiation curable lacquer (Lacquer A) at equimolar dosages in order to determine human in vitro cytotoxicity. Viability of the cells after 2–24-h exposure to the representative monofunctional or multifunctional acrylate or solvent control was used to calculate an index of acute cytotoxicity (50% inhibitory dose; ID₅₀) and to determine the shape of the dose-response curves. TPGDA, Lacquer A, and EA were equally cytotoxic (ID₅₀≈0.1 μmol/cm²) to NHEK at equimolar doses. TPGDA or Lacquer A were more cytotoxic (≈100×) to NHDF or NHBE than EA. Sequential exposure of UV_A and TPGDA to NHEK indicate the potential for a synergistic cytotoxic response. These findings are consistent with observed decreases in free sulfhydryl groups (e.g., glutathione or cysteine) that parallel the dose-response-related decreases in viability. Together, these data suggest possible differences in toxicity between the monofunctional EA and multifunctional TPGDA to NHEK, NHDF, or NHBE, possibly due to the difference in the number of functional acrylate groups and/or physicochemical differences (e.g., vapor pressure) between the acrylates investigated.     

Quantification of cytotoxic hemocytes of Mytilus edulis using a cytotoxicity assay in agar

When a thin layer of agar containing a mixture of erythrocytes and Mytilus hemocytes is prepared on slides, the occurrence of plaques of lysed target cells can be observed around a limited number of hemocytes. These hemocytes remain completely intact cells and are viable as evidenced by their ability to phagocytose target cells and/or to form pseudopods. The number of hemocytes releasing cytotoxic molecules has been shown to vary greatly between different animals. The same holds true for the total number of circulating hemocytes, although no correlation exists between the number of hemocytes in the circulation and the percentage of cytotoxic blood cells.     

Synthesis,docking studies,in vitro cytotoxicity evaluation and DNA damage mechanism of new tyrosine-based tripeptides

Peptides are one of the leading groups of compounds that have been the subject of a great deal of biological research and still continue to attract researchers' attention. In this study, a series of tripeptides based on tyrosine amino acids were synthesized by the triazine method. The cytotoxicity properties of all compounds against human cancer cell lines (MCF-7), ovarian (A2780), prostate (PC-3), and colon cancer cell lines (Caco-2) were determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay method, and % cell viability and logIC50 values of the compounds were calculated. Significant decreases in cell viability were observed in all cells (p < 0.05). The comet assay method was used to understand that the compounds that showed a significant decrease in cell viability had this effect through DNA damage. Most of the compounds exhibited cytotoxicity by DNA damage mechanism. Besides, their interactions between investigated molecule groups with PDB ID: 3VHE, 3C0R, 2ZCL, and 2HQ6 target proteins corresponding to cancer cell lines, respectively, were investigated by docking studies. Finally, molecules with high biological activity against biological receptors were determined by ADME analysis.     

Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon γ

 Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line, COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon γ (IFNγ) was also found to enhance the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis, although superoxide anion (O₂ ^–) was observed to be produced. However, the release of tumor necrosis factor α (TNFα) was significantly enhanced during ADMC, while, in addition, apoptosis was significantly inhibited by the addition of anti-TNFα antibody. These findings indicated that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFNγ, while, in addition, TNFα may also be one of the major mediators of apoptosis. Received: 1 August 1996 / Accepted: 27 August 1996     

Glutathione-dependent cytotoxicity of the chloroacetanilide herbicides alachlor, metolachlor, and propachlor in rat and human hepatoma-derived cultured cells

Alachlor, metolachlor, and propachlor are widely used chloroacetanilide herbicides. Their cytotoxicity in rat (Fa32) and human (Hep G2) hepatoma-derived cells was investigated, in connection with their influence on the endogenous glutathione (GSH) content, on the xenobiotic-metabolizing phase I enzymes 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD), and phase II glutathione transferase (GST). The cytotoxicity was measured by the neutral red uptake inhibition assay. The following toxicity range was observed in both cell lines : propachlor>alachlor>metolachlor. When the endogenous GSH content was reduced by pretreatment of the cells with L-buthionine (S,R)-sulfoximine, the cytotoxicity of the herbicides increased strongly in both cell lines. EROD and PROD activities were dose-dependently increased to different degrees in Fa32, as was EROD in Hep G2, but no PROD activity was observed in these cells. The GSH content was not altered after 1 h treatment, and was approximately doubled after 24 h. GST activity was increased in Fa32 cells but not in Hep G2. A comparable cytotoxicity was observed for the investigated chloroacetanilides in both the rat and the human cell lines. Different interactions with xenobiotic-metabolizing phase I and II enzymes were observed, and GSH showed a protective effect against the acetanilides in both cell lines.     

Spermidine cytotoxicity in vitro: Effect of serum and oxygen tension

Summary Plasma amine oxidase activities (benzylamine oxidase and spermine oxidase) were determined in the sera of a number of species of various ages. Benzylamine oxidase (BZO) activity, measured spectrophotometrically, was present in bovine, equine, and ovine species examined. Generally its activity in serum increased with the age of the animal. Spermine oxidase activity (SPO) was estimated by a bioassay of in vitro toxicity and did not necessarily correlate with BZO. Cytotoxicity in the presence of spermidine was found only in the sera of the ruminant species examined. Serum activity tended to rise with animal age; however, great variability was found in perinatal bovine sera. The 50% lethal dose (LD₅₀) of spermidine in the presence of 5% serum and 4×10⁴ NS₁ cells/ml was in the micromolar range. Aminoguanidine, a known inhibitor of SPO, could prevent the cytotoxic effects of exogenously added spermidine in vitro. In contrast, raising the ambient oxygen tension in the incubation environment to 95% lowered the LD₅₀ dose of spermidine required for cytotoxicity. The results suggest that a cell line of hematogenous origin is susceptible to the cytotoxic effects of the products of oxidative deamination of spermidine by SPO, an enzyme present in perinatal bovine sera, and that these cytotoxic effects are potentiated in the presence of an oxygen-enriched environment in vitro. This research was support in part by Grants AM 19899, AM 32237, and HD 00412 from the National Institute of Health, Bethesda, MD.     

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (～300 μg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)‐ or collagen/epinephrine‐stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR‐stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of α_IIbβ₃/FIB and phosphorylation of AKT in THR‐stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

In vitro cytotoxicity testing with fluorescence-based assays in cultured human lung and dermal cells

An in vitro study using human cultured cells was conducted to determine the reliability of fluorescence-based cell viability indicators with traditional in vitro cytotoxicity testing methods. Human lung epithelial carcinoma (A549) cells, and human embryonic skin (WS1) and lung (HFLI) fibroblasts were studied in culture to evaluate their potential to screen for cytotoxicity and to compare to previous protocols conducted in our laboratory. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and fluorescent-labeled probes were used to assess toxicity. Eight chemicals, including mercuric chloride, copper sulfate, sodium fluoride, thioridazine HCl, paraquat, amitriptyline-HCl, verapamil-HCl and chloroquine sulfate, were tested with each cell line using calcein-AM and Sytox. The data suggest that fluorescent probes are sensitive indicators of cytotoxicity and contribute to understanding the mechanisms for each chemical. In combination with previously published reports, the similarity of results among cell lines may be explained by the origin of the cell lines rather than by the diversity of the methods and indicators employed.     

Interaction of diesel exhaust particles with human, rat and mouse erythrocytes in vitro

Inhaled ultrafine (nano) particles can translocate into the bloodstream and interact with circulatory cells causing systemic and cardiovascular events. To gain more insight into this potential mechanism, we studied the interaction of diesel exhaust particles (DEP) with human, rat and mouse erythrocytes in vitro. Incubation of erythrocytes with DEP (1, 10 or 100 μg/ml) for 30 min caused the highest hemolytic effect (up to 38%) in rats, compared to small but significant hemolysis in mice (up to 2.5%) and humans (up to 0.7%). Transmission electron microscopy of erythrocytes revealed the presence of variable degrees of ultrafine (nano)-sized aggregates of DEP either internalized and/or adsorbed onto the erythrocytes in the three species. A significant amount of DEP was found in rat and mouse (but not human) erythrocytes. Lipid erythrocyte susceptibility to in vitro peroxidation measured by malondialdehyde showed a significant and dose-dependent increase in erythrocytes of rats, but not humans or mice. Unlike in human erythrocytes, total antioxidant status (TAS) and superoxide dismutase (SOD) activity in rats were significantly and dose- dependently decreased. In mouse erythrocytes, DEP caused a decreased in SOD (at 10 μg/ml) and TAS (at 100 μg/ml) activities. In conclusion, DEP caused species-dependent erythrocyte hemolysis and oxidative stress, and were either taken up and/or adsorbed onto the red blood cells. Rat (and to a lesser degree mouse) erythrocytes were susceptible to DEP. Human erythrocytes showed the highest resistance to the observed effects. These species difference should be noted when using rats and mice blood as models for humans.     

Reduced amino acid Schiff base containing ruthenium(III) complexes: Synthesis,characterization, DNA interaction,and in vitro cytotoxicity

A number of reduced amino Schiff base ligands and corresponding ruthenium(III) complexes were designed and prepared based on the fact that amino acids not only possess multiple coordinate atoms but also improve the solubility of drugs in the body. The interaction of the complexes with calf thymus DNA was analyzed with spectroscopic methods of ultraviolet‐visible absorption spectra, DNA competitive binding with ethidium bromide, circular dichroism spectra, and DNA melting experiments, and DNA viscosity measurements, indicating that the complexes bind to DNA primarily in the grooving mode. With respect to the ligands, the cytotoxicity in vitro of the complexes against Hela, A549, and MCF‐7 cells was much enhanced, with most of the IC₅₀ values less than 50 μM or even comparable with those of cisplatin.     

Anin vitro model for the biological evaluation of dental materials using human periodontal ligament cells

Anin vitro system is described which uses human diploid cells derived from the periodontal ligament. It simulates the clinical situation and is suitable for rapid cytotoxicity screening of dental materials. It can also be adapted to study various factors influencing biocompatibility.     

Design,synthesis and in vitro cytotoxicity evaluation of 5-(2-carboxyethenyl)isatin derivatives as anticancer agents

Forty four di- or trisubstituted novel isatin derivatives were designed and synthesized in 5–6 steps in 25–45% overall yields. Their structures were confirmed by ¹H NMR and ¹³C NMR as well as LC–MS. The anticancer activity of these new isatin derivatives against three human tumor cell lines, K562, HepG2 and HT-29, were evaluated by MTT assay in vitro. SAR studies suggested that the combination of 1-benzyl and 5-[trans-2-(methoxycarbonyl)ethen-1-yl] substitution greatly enhance their cytotoxic activity, whereas an intact carbonyl functionality on C-3 as present in the parent ring is required to such a potency. This study leads to the identification of two highly active molecules, compounds 2h (IC₅₀ = 3 nM) and 2k (IC₅₀ = 6 nM), against human leukemia K562 cells.