High Degree Of Cytotoxicity Research Articles

Toxicological properties of mixtures of binary silver-copper, silver-zinc, and copper nanoparticles on cell culture model and laboratory animals

The development, testing, and implementation of antimicrobial agents necessitates the determination of their toxicological characteristics. The objective of our research was to ascertain the direction and magnitude of the impact of experimental samples of disinfectants based on binary silver-copper, silver-zinc, and copper nanoparticles on biological entities of disparate levels of organization (cell culture, laboratory animals). The culture of calf coronary vessel cells (CVCs), 220 mature white laboratory rats, and 45 Chinchilla rabbits were used for the study. Mixtures of binary nanoparticles D1: MeNPs content – 5.4 mmol/L; D2: MeNPs content – 4.9 mmol/L were used as antimicrobial compounds. Toxic effects on cell culture were determined by the percentage of monolayer integrity, and biological effects in animals were determined by determining acute and subacute toxicity by clinical and biochemical parameters. The results of the culture studies demonstrated that the CC50 value of D2 was 6.2 times lower than that of D1, indicating a higher degree of cytotoxicity. No animal deaths were observed in the acute toxicity test (single intragastric administration to white rats at a dose of 30000 mg/kg body weight), which permitted the classification of the test samples as Class VI toxicity (relatively harmless) and Class IV hazardous (low-hazardous). When applied topically to rabbits' skin and mucous membranes, the experimental samples did not exhibit a pronounced irritant effect. A 30-day dermal application of the drugs to rats at doses of 0.5 and 5.0 mL/kg was conducted to determine the impact on hematological and biochemical parameters of a tenfold dose. Following the cessation of the administration of the aforementioned experimental disinfectant samples, the levels of all indicators were observed to return to the control levels within 14 days. The higher toxicity of the D2 drug for biological systems of different levels of organization, compared to the D1 drug, may be attributed to two factors: the higher concentration of AgNPs (2.4 times) and the potentiation of the toxic effect of two binary compounds (Ag-Zn and Ag-Cu) in its composition. Further research is needed to determine the biological impact of experimental samples of disinfectants based on silver, copper, and zinc metal nanoparticles on other functional systems of laboratory animals and the clinical and biochemical status of productive agricultural animals in production conditions.

Molecular Docking and Dynamics Study of Acetogenins Targeting Cyclin-dependent Kinase and In vitro Evaluation of Annona Muricata Fruit Extracts using MDA-MB-231 Breast Cancer Cell Line

Background: Overexpressed CDK1 and CDK2 are targeted as potential sites for cancer treatment. Annona muricata fruit has been reported to have more than 100 acetogenins showing cytotoxic activities against cancer cell lines. Hence the study aims to demonstrate the cytotoxicity of ethyl acetate fruit extract, its role in cell cycle progression, and apoptosis using the MDMBA-231 breast cancer cell line. Docking, dynamics, and ADME studies were also demonstrated to generate lead molecules of AM fruit responsible for cancer treatment. Method: Cell viability was quantified by the MTT assay. Cell cycle arrest and apoptotic cells were determined by flow cytometry and PI annexin V-FITC staining by flow cytometry, respectively. Molecular docking, molecular dynamics, and ADME properties of 11 acetogenins were studied using the schrödinger maestro suite 2018-1. Results: The MTT assay revealed IC50 232.9μg/ml with a high degree of cytotoxicity. The extract effectively caused cell cycle arrest at the G2M and S phases; early and late apoptosis was induced at 160 μg/ml and 320 μg/ml. Docking scores of muricin L, J, and annomuricin A complexed with CDK2 and muricin J, K, and L with CDK1 binding energy ranging is mentioned as a molecular dynamic study envisaged muricin j against CDK2 stable hydrogen and hydrophobic interactions with critical residues like ASP-86, GLN-131, HIS-84, LYS-89, PHE80, PHE82, and PHE83 throughout 200 ns (hinge region). ADMET profiling also confirmed that all 11 ligands passed the rule of 5 and 3. The in vitro and in silico studies revealed that these acetogenins could be CDK1 and CDK2 inhibitors for cancer treatment. Conclusion: The in vitro studies presume that the ethyl acetate fruit extract of AM is an excellent cytotoxic agent. In silico studies demonstrated that muricin j could lead molecules to target kinase proteins responsible for cell proliferation. ADME study enlightened us to take 11 acetogenins for the drug discovery process in managing cancer treatment.

RESEARCH ON VARIOUS MODELS OF THE BIOLOGICAL EFFECTS OF COMPONENTS OF A COMPLEX EFFECT (PHOTOIRRADIATION; EXOSOMES OF MESENCHYMAL STEM CELLS AND NANOPARTICLES) FOR THE CORRECTION OF THE INFLAMMATORY PROCESS

RESEARCH ON VARIOUS MODELS OF THE BIOLOGICAL EFFECTS OF COMPONENTS OF A COMPLEX EFFECT (PHOTOIRRADIATION; EXOSOMES OF MESENCHYMAL STEM CELLS AND NANOPARTICLES) FOR THE CORRECTION OF THE INFLAMMATORY PROCESS

Outer-Membrane Vesicles of Fusobacterium necrophorum: A Proteomic, Lipidomic, and Functional Characterization.

Outer-membrane vesicles (OMVs) are extruded nanostructures shed by Gram-negative bacteria, containing periplasmic contents, and often including virulence factors with immunogenic properties. To assess their potential for use in vaccine development, we purified OMVs from the Fusobacterium necrophorum subspecies necrophorum, an opportunistic necrotic infection-causing pathogen, and characterized these structures using proteomics, lipid-profiling analyses, and cytotoxicity assays. A proteomic analysis of density-gradient-purified F. necrophorum OMVs identified 342 proteins, a large proportion of which were outer-membrane proteins (OMPs), followed by cytoplasmic proteins, based on a subcellular-localization-prediction analysis. The OMPs and toxins were among the proteins with the highest intensity identified, including the 43-kDa-OMP-, OmpA-, and OmpH-family proteins, the cell-surface protein, the FadA adhesin protein, the leukotoxin-LktA-family filamentous adhesin, the N-terminal domain of hemagglutinin, and the OMP transport protein and assembly factor. A Western blot analysis confirmed the presence of several OMPs and toxins in the F. necrophorum OMVs. The lipid-profiling analysis revealed phospholipids, sphingolipids, and acetylcarnitine as the main lipid contents of OMVs. The lactate-dehydrogenase-cytotoxicity assays showed that the OMVs had a high degree of cytotoxicity against a bovine B-lymphocyte cell line (BL-3 cells). Thus, our data suggest the need for further studies to evaluate the ability of OMVs to induce immune responses and assess their vaccine potential in vivo.

Nano zinc oxide induced lipid peroxidation, oxidative stress, genotoxicity, phagocytic alteration, and detoxification response in the coelomocytes of an anecic earthworm of India

Zinc oxide nanoparticle (ZnO NP) is an emerging soil pollutant of industrial origin. The unique physicochemical properties of ZnO NP viz. the bioavailability and subsequent ecotoxicological effects on organisms have made it an ecological concern. Lampito mauritii (Kinberg, 1866) is a common anecic earthworm and is considered as a keystone species of soil ecosystems in India. They are continuously exposed to various environmental toxins during their feeding and other activities. The current study aimed to estimate the immunotoxicological effects of ZnO NP in the coelomocytes, the chief immunoeffector cells of L. mauritii. The results of the study indicated decreased total count, phagocytic activity, antioxidative, and detoxifying potential of the coelomocytes in L. mauritii at 7 and 14 days of exposure. ZnO NP exposure resulted in the generation of oxidative molecules, lipid peroxidation, and DNA damage within coelomocytes. The higher degree of cytotoxicity on ZnO NP exposure was suggestive of immunocompromisation in L. mauritii. An integrated biomarker response (IBR) guided methodology was also utilized in estimating the comprehensive effects of ZnO NP on the health of L. mauritii. The present study would provide useful information on the ZnO NP induced chemical stress in L. mauritii, with the selected endpoints having the potential to function as markers for ZnO NP toxicity.

Torpor/hibernation cycle may enhance the risk of insecticides for bats: an in vitro study

Exposure to pollutants is considered one of the potential reasons of population declines in bats. In the context of previous studies, we managed to create and keep a wide collection of cell lines from European bat species. Liver cells were chosen for testing, as they represent the preferred model for toxicological studies. Bats are protected, cell lines replacing experimental animals thus represent a unique opportunity to examine effects of pollutants which animals are exposed to in their environments. Moreover, cell incubation temperature variation may simulate physiological states of heterothermic bats. Liver cell lines were cultivated to the required cell number. Exposure to five different concentrations of permethrin (PM) and imidacloprid (IMI) were used to determine cytotoxic effects of these pesticides on Nyctalus noctula-derived liver cells cultivated at 37 °C and 8 °C for 24 h. An assay based on the measurement of activity of lactate dehydrogenase released from damaged cells was used for quantitating cytotoxicity. Cytotoxicity of IMI ranged from 0% to 47% and from 56% to 67% at 37 °C and 8 °C, respectively. Cytotoxicity of PM ranged from 36% to 56% and from 43% to 88% at 37 °C and 8 °C, respectively. Permethrin was tested on the cells at an order of magnitude lower concentrations; even so, higher degree of cytotoxicity was recorded. Imidacloprid was more toxic to bat liver cells at a hibernation temperature than at body temperature of 37 °C.

Synthesis and characterization of lanthanide complexes as potential therapeutic agents

Herein, two different biologically active moieties; 2-carbaldehyde thiophene and salicylic acid hydrazide were simply combined to produce ((E)-2-hydroxy-N/-((thiophen-2-yl)methylene)benzohydrazide) (H2L). This hybrid ligand was used for synthesis of four lanthanide complexes (Ln3+ = Sm3+, Eu3+, Dy3+ and La3+) as novel potential biological agents. Sequentially, the coordination behavior of lanthanide ions with a dianionic tetradentate hydrazone ligand (OONS donation sites) was examined. The lanthanide complexes were characterized on the basis of FT-IR, 1H-NMR and mass spectrometry as well as elemental and thermal analyses and molar conductivity. The complexes were screened for in vitro antibacterial and antifungal activity against the multidrug resistant pathogens, such as Klebsiella pneumonia, Escherichia coli, Staphylococcus aureus and Candida albicans. The antimicrobial results revealed that La3+ complex has a good potency against different gram positive and gram negative bacteria in comparision with different series of standard drugs. On other hand, Dy3+ complex demonstrate a noticeable antifungal activity against C. albican. All lanthanide complexes showed a moderate antioxidant activity with IC50 (0.5 mM) except La3+ complex. Also, in vitro cytotoxic and growth inhibitory activities against Erlish Acities Carcinoma for all produced complexes were determined. The highest degree of cytotoxicity (IC50 = 50 μM) was obtained in case of Sm3+ complex.

A multiparametric study of gold nanoparticles cytotoxicity, internalization and permeability using an in vitro model of blood–brain barrier. Influence of size, shape and capping agent

Gold nanoparticles (AuNPs) have biomedical application on imaging and due to increased optical performance, star-shaped AuNPs are of special interest. Because shape, size and capping greatly influence their toxicokinetics and toxicodynamics, a systematic multiparametric comparative study of the influence of these parameters on the cytotoxicity, internalization, and in vitro permeability was conducted in human Cerebral Microvascular Endothelial Cell line (hCMEC/D3), an in vitro model of the human blood-brain barrier (BBB). AuNPs of different size (14 nm and ∼50 nm), shape (spheres and stars), and coating (11-mercaptoundecanoic acid or MUA and sodium citrate) were synthesized and fully characterized. The time- and concentration-dependent cytotoxic profile of the tested AuNPs differed for the different AuNPs. Generally, toxicity was greater for stars relative to sphere-shaped AuNPs, and citrate coating was more toxic than MUA. Regarding the influence of size, smaller-sized AuNPs were more cytotoxic when compared at the same Au concentration. However, when the concentration of AuNPs was expressed as the number of AuNPs/mL, a higher degree of cytotoxicity was noted for the larger ̴50 nm AuNPs. To understand the influence of size, shape and capping, a systematic study design, in which only one of the variables changes, is determinant for correct data interpretation. Considering the results herein presented, for the sake of comparison of differently-sized AuNPs, it is preferable to design the study based upon the number of nanoparticles, since at a fixed Au concentration the number of particles available to promote effect is higher for smaller-sized AuNPs. Cellular internalization also differed among the tested AuNPs; although all were unable to cross the in vitro BBB, the intracellularly accumulated AuNPs can induce cell damage and later compromise BBB integrity and permeability.

Music therapy improves the mood of patients undergoing hematopoietic stem cells transplantation (controlled randomized study).

The allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a therapeutic medical treatment for various neoplastic hematologic, congenital, genetic, or acquired disorders. In this procedure which combines high-dose chemotherapy and/or radiotherapy and has a high degree of cytotoxicity, the patient experiences solitary confinement, which causes psychological distress, pain, anxiety, mood disorders and can lead him/her to depression. Music therapy was applied with the purpose of decreasing this social confinement. This is a randomized controlled trial. Patients (n=100) were selected randomly. Patients (n=50) were selected for the Experimental Music Therapy Group (EMG) and n=50 for the control group (CG) who received the standard treatment. The intervention of live music was applied using music therapy techniques. Assessment and quantification were made using the visual analog scale (VAS). The dependent variables were pain, anxiety, and mood of patients. The Mann-Whitney test (p<0.05) applied was considered statistically significant when comparing the groups, improving mood significantly (EMG). Music therapy proved to be a strong ally in the treatment of patients undergoing allo-HSCT, providing bio-psychosocial welfare.

In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

Cancer is commonly diagnosed in dogs over the age of 10 and is a leading cause of death due to the lack of effective drugs. Flavonoids possess antioxidant, anti-inflammatory and anticarcinogenic properties and have been studied as chemopreventive agents in human cancer therapy. However, the literature on dogs is sparse. In this study, we analyzed the effect of nine flavonoids on cell viability, DNA damage and topoisomerase IIa/IIb gene expression in a canine tumor cell line (DH82). Apigenin, luteolin, trans-chalcone and 4-methoxychalcone showed the highest degree of cytotoxicity in the absence of considerable DNA damage, whereas genistein exhibited low cytotoxicity but induced a high level of DNA damage. These five flavonoids inhibited topoisomerase IIa and IIb gene expression to variable extents and with variable specificity. Genistein exerted a lower inhibitory effect on the two topoisomerases than luteolin and apigenin. trans-Chalcone and 4-methoxychalcone exerted greater inhibition of topoisomerase IIa expression than topoisomerase IIb. The differences in the effects between genistein and luteolin and apigenin might be explained by the position of ring B, whereas the more specific effect of chalcones on topoisomerase IIa might be due to their open chain structure.

Thiopurine Methyltransferase Predicts the Extent of Cytotoxicty and DNA Damage in Astroglial Cells after Thioguanine Exposure

Thiopurine methyltransferase (Tpmt) is the primary enzyme responsible for deactivating thiopurine drugs. Thiopurine drugs (i.e., thioguanine [TG], mercaptopurine, azathioprine) are commonly used for the treatment of cancer, organ transplant, and autoimmune disorders. Chronic thiopurine therapy has been linked to the development of brain cancer (most commonly astrocytomas), and Tpmt status has been associated with this risk. Therefore, we investigated whether the level of Tpmt protein activity could predict TG-associated cytotoxicity and DNA damage in astrocytic cells. We found that TG induced cytotoxicity in a dose-dependent manner in Tpmt+/+, Tpmt+/− and Tpmt−/− primary mouse astrocytes and that a low Tpmt phenotype predicted significantly higher sensitivity to TG than did a high Tpmt phenotype. We also found that TG exposure induced significantly more DNA damage in the form of single strand breaks (SSBs) and double strand breaks (DSBs) in primary astrocytes with low Tpmt versus high Tpmt. More interestingly, we found that Tpmt+/− astrocytes had the highest degree of cytotoxicity and genotoxicity (i.e., IC50, SSBs and DSBs) after TG exposure. We then used human glioma cell lines as model astroglial cells to represent high (T98) and low (A172) Tpmt expressers and found that A172 had the highest degree of cytoxicity and SSBs after TG exposure. When we over-expressed Tpmt in the A172 cell line, we found that TG IC50 was significantly higher and SSB's were significantly lower as compared to mock transfected cells. This study shows that low Tpmt can lead to greater sensitivity to thiopurine therapy in astroglial cells. When Tpmt deactivation at the germ-line is considered, this study also suggests that heterozygosity may be subject to the greatest genotoxic effects of thiopurine therapy.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

The Effects of Hyaluronic Acid, Calcium Hydroxide, and Dentin Adhesive on Rat Odontoblasts and Fibroblasts

The aim of this study was to investigate the effects and efficiency of pulp capping preparations based on hyaluronic acid, calcium hydroxide, and dentin adhesive on the pulp tissue of Sprague-Dawley rats. The rats were killed and extracted teeth sectioned transversely through the pulp. The slices were placed in a RPMI 1640 cell culture medium supplemented with 10 % foetal calf serum. During 14 days of cultivation cultures were treated with preparations that contained hyaluronic acid (Gengigel Prof®), and calcium hydroxide (ApexCal®), or with dentin adhesive (Excite®). Cellularity and viability of fibroblasts and odontoblasts was analysed using a haemocytometer. Hyaluronic acid proved most efficient and the least toxic for direct pulp capping. Even though calcium hydroxide and dentin adhesive demonstrated a higher degree of cytotoxicity, their effects were still acceptable in terms of biocompatibility.

Coding Polypurine Hairpins Cause Target-Induced Cell Death in Breast Cancer Cells

Polypurine reverse-Hoogsteen hairpins (PPRHs) are double-stranded DNA molecules formed by two polypurine stretches linked by a pentathymidine loop, with intramolecular reverse-Hoogsteen bonds that allow a hairpin structure. PPRHs bind to polypyrimidine targets by Watson-Crick bonds maintaining simultaneously a hairpin structure due to intramolecular Hoogsteen bonds. Previously, we described the ability of Template-PPRHs to decrease mRNA levels because these PPRHs target the template DNA strand interfering with the transcription process. Now, we designed Coding-PPRHs, a new type of PPRHs that directly target the pre-mRNA. The dihydrofolate reductase (dhfr) gene was selected as a target in breast cancer therapy. These PPRHs caused a high degree of cytotoxicity and a decrease in DHFR mRNA and protein levels, but by a different mechanism of action than Template-PPRHs. Coding-PPRHs interfere with the splicing process by competing with U2 auxiliary factor 65 for binding to the polypyrimidine target sequence, leading to a lower amount of mature mRNA. These new PPRHs showed high specificity as no off-target effects were found. The application of these molecules as therapeutic tools was tested in breast cancer cells resistant to methotrexate, obtaining a noticeable cytotoxicity even though the dhfr locus was amplified. Coding-PPRHs can be considered as new molecules to decrease gene expression at the mRNA level and an alternative to other antisense molecules.

Synthesis of 2,6-dioxatricyclo[3.3.1.03,7]nonanes by intramolecular haloetherification and/or transannular hydroxycyclization of alkenes in [4+3]-cycloadducts

The synthesis of new difunctionalized 2,6-dioxatricyclo[3.3.1.03,7]nonanes is described. This type of structure is an interesting synthetic building block for potential bioactive molecules and it was prepared from 8-oxabicyclo[3.2.1]oct-6-en-3-one having a NHBoc function on C-1. This precursor was obtained by a [4+3] cycloaddition reaction of 2-tert-butoxycarbonylaminofuran and the oxyallyl cation generated in situ from 2,4-dibromo-3-pentanone. Reduction of the carbonyl group at C-3 was accomplished in high yield and stereoselective manner to afford the corresponding axial alcohol at C-3 as a major product. Further intramolecular haloetherification of this type of alcohols with NBS and I(py)2BF4 led to the corresponding bromo and iodo-derivatives at C-8 of the 2,6-dioxatricyclo[3.3.1.03,7]nonane framework, in high yield. Epoxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-ol followed by treatment with NaCN, NaN3, and/or NaOH in MeOH afforded 8-hydroxy-2,6-dioxatricyclo[3.3.1.03,7]nonanes in high yield via a transannular hydroxycyclization mediated by a base and through an alkoxide intermediate. The new 2,6-dioxatricyclo[3.3.1.03,7]nonanes were tested for biological activity against HIV-1 virus and MT-4 lymphoid cell line, showing a low anti-HIV activity and a high degree of cytotoxicity.

Characterization of Bizzy Nut extracts in estrogen-responsive MCF-7 breast cancer cells

Kola acuminate, also known as Bizzy Nut or Kola Nut, is a natural product that contains bioactive chemicals that possess hormonal properties. The purpose of this study was to characterize the putative phytoestrogenic compounds present in Bizzy Nut for estrogenic-like activity. As an initial step, five extracts (E1 - hexane, E2 - ether, E3 - acetone, E4 - methanol and E5 - water) were sequentially generated using solid-liquid phase extraction and their bioactivity was examined in MCF-7, MDA-MB-468 and LNCaP cancer cell models. MTT cell viability, dye exclusion, caspase activity and microscopic assessment of apoptotic cells demonstrated that extracts of Bizzy were cytotoxic to MCF-7, MDA-MB 468 and LNCaP cells. In MCF-7 cells, the acetone extract (E3) at 100 ppm elicited a potent cytotoxic response with a growth-inhibitory concentration (GI(50)) of 67 ppm. In contrast, E3 stimulated growth in LNCaP cells. The ether extract (E2) showed a dose-dependent cytotoxic response with a GI(50) of 13 ppm in the LNCaP cell line. Examination of the apoptotic response induced by E2 and E3 paralleled the level of cell cytotoxicity observed in both cell lines. The methanol extract (E4) was the only extract that showed a time-, dose-, and estrogen-receptor-dependent stimulation of pS2 gene expression. On the other hand, the acetone extract (E3), which showed the highest degree of cytotoxicity, showed no transcription stimulation of pS2 in MCF-7 cells. Altogether, these data indicate that Bizzy contains unique active hormonal compounds that have specific biological properties that are cell line-dependent.

Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells

Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin's lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k "antiapoptotic" gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells.

Effect of cytokines on the toxicity of cytostatic drugs to normal bone marrow cells in vitro.

We evaluated in vitro how growth factors influenced the effect of cytostatic drugs on normal hematopoietic progenitor cells. Bone marrow was obtained from 15 donors for bone marrow transplantation. After separation the mononuclear fraction was incubated with granulocyte colony-stimulating factor (G-CSF) at 5 and 50 ng/ml, with granulocyte/macrophage colony-stimulating factor (GM-CSF) at 1 and 10 ng/ml, and with interleukin 3 (IL-3) at 0.5 and 5 ng/ml for 24 h prior to incubation with cytostatic drugs. These incubations were performed with 0.05 microM mitoxantrone and 0.2 microM daunorubicin for 1 h, and cells were thereafter cultured for colony-forming units--granulocyte/macrophage (CFU-GM) in soft agar for 10-12 days. Incubation with 0.05 microM cytosine arabinoside was performed continuously throughout the culture period. The proliferation of normal hematopoietic progenitor cells stimulated with GM-CSF at 10 ng/ml and with IL-3 at 5 ng/ml was significantly increased to 218% and 215% colonies, respectively, as compared with the control stimulated with conditioned medium only. Stimulation with G-CSF, on the other hand, did not induce any significantly enhanced proliferation relative to the control. Daunorubicin applied in combination with G-CSF at 5 ng/ml or with IL-3 at 0.5 ng/ml exerted a significantly higher degree of cytotoxicity on normal hematopoietic progenitor cells, resulting in 21% and 30% surviving colonies as compared with the 38% recorded for daunorubicin alone (P < 0.05). Neither G-CSF nor IL-3 at a higher concentration nor GM-CSF exerted a significantly altered degree of toxicity relative to cells incubated with daunorubicin alone. The cytotoxic effect exerted on normal hematopoietic cells by mitoxantrone or ara-C was unchanged or significantly decreased after stimulation with growth factors as compared with the effect on cells incubated with cytostatic drugs alone. We conclude that G-CSF and IL-3 augment the effect of daunorubicin on normal hematopoietic progenitor cells.

Characterization of a Renal Cell Carcinoma Cell Line Suitable as a Target for Immunological Studies in Vitro and in the NU/NU Mouse

A continuous human renal carcinoma cell line designated RPMI-SE was established from a patient with a poorly to moderately differentiated renal cell carcinoma. The cells are anchorage dependent, have a well defined globular shape with pseudopod-like structures, a doubling time in vitro of 24 hr. and are able to grow subcutaneously in female ICR Swiss nu/nu mice. RPMI-SE cells obtained from tissue culture and the nude mouse had the chromosome number of neartetraploidy. Common morphologic rearrangements were present in both the cell line and nu/nu mouse tumor. RPMI-SE was evaluated as a target cell line in an in vitro Indium-111 release assay. Three patterns of cytotoxicity were observed at an effector to target cell ratio of 100:L The highest degree of cytotoxicity (75 per cent) was obtained with autologous lymphocytes. An intermediate level of cytotoxicity (50 per cent) was obtained with allogeneic lymphocytes. The lowest level of cytotoxicity (27 per cent) was obtained with normal lymphocytes and was comparable to the level of cytotoxicity observed with the NK sensitive K562 target cell line. Morphologic data and chromosomal analysis indicate that the RPMI-SE cell line has maintained characteristics of the original tumor. This cell line will be useful for immunological studies as a target cell line in vitro as well as in vivo in the nude mouse.

Suppressive effect by cooperation of splenic and peritoneal adherent cells on generation of cytotoxic T lymphocytes.

When spleen cells primed in vivo against allogeneic lymphoid cells were used as responder cells in secondary mixed lymphocyte cultures, a high degree of cytotoxicity was generated even in the absence of splenic adherent cells. However, removal of adherent cells from such primed responder spleen cells reduced the cytotoxicity to some extent. On the other hand, when these responder cells were transferred into the peritoneal cavity of irradiated syngeneic mice together with antigenic cells, unseparated responder cells generated a lower degree of cytotoxicity than did adherent cell-depleted responder cells. In an in vitro system, peritoneal adherent cells also suppressed the generation of cytotoxic T lymphocytes by unseparated responders; however they augmented the cytotoxic T lymphocyte generation by adherent cell-depleted responders. These adherent cell populations with augmenting activity became inhibitory when they coexisted. The mechanism of this inhibitory action remains unclear.