In vitro cytogenetic effects of 2450 MHz waves on human peripheral blood lymphocytes

Cytogenetic analyses were performed on human peripheral blood lymphocytes exposed to 2450 MHz microwaves during 30 and 120 min at a constant temperature of 36.1°C (body temperature). The temperature was kept constant by means of a temperature probe put in the blood sample which gives feedback to a microcomputer that controls the microwave supply. We found a marked increase in the frequency of chromosome aberrations (including dicentric chromosomes and acentric fragments) and micronuclei. On the other hand the microwave exposure did not influence the cell kinetics nor the sister chromatid exchange (SCE) frequency. ©1993 Wiley-Liss, Inc.     

In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells

HLA-A2402-restricted and carcinoembryonic-antigen(CEA)-specific cytotoxic T lymphocytes (CTL) were induced by culturing human peripheral blood mononuclear cells (PBMC) on formalin-fixed autologous adhesive PBMC that had been loaded with CEA-bound latex beads. The CTL killed the CEA-producing HLA-type matched cancer cells, but not the non-producers of CEA, at an effector/target ratio of 10 within 24 h. On the basis of available HLA-A24-binding peptides, we have also attempted to identify the epitope peptide recognized by the CTL. The peptide CEA652(9), TYACFVSNL, stimulated the CTL most strongly when pulsed on HLA-A2402-expressing target cells. The other nine peptides so far tested were also active, but less efficient in their effect on CTL. The CTL failed to kill target cells pulsed with the HLA-A2-binding CEA peptide, CAP-1. The CTL were also generated on the fixed adherent cells previously pulsed with the peptide CEA652(9). Cytotoxic activity of the CTL was inhibited by monoclonal antibodies against CD3, CD8, and MHC class I molecules. These results suggest that human autologous CTL will be inducible on the autologous fixed PBMC without use of the cultured target cancer cells if tumor antigenic protein is available. Received: 31 December 1997 / Accepted: 4 May 1998     

In vitro stimulation of human peripheral blood lymphocytes by Neisseria meningitidis

Abstract Heat-killed Neisseria meningitidis was found to be mitogenic for human peripheral blood lymphocytes (PBL). Separation of lymphocytes by rosetting with sheep erythrocytes indicated that both rosette-forming cells (E⁺, T-enriched) and nonrosetting cells (E⁻, B-enriched) were induced to proliferate by the bacteria. Following meningococcal stimulation, E⁻ cells and PBL displayed proliferative responses of similar magnitude and followed essentially the same kinetics with peak responses occurring after 3–4 days of culture. By comparison, E⁺ lymphocytes gave significantly higher responses and required a longer incubation period (5–7 days) to reach maximum levels of proliferative activity.     

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro trophic effects of synthetic ACTH1-24

In vitro trophic effects of adrenocorticotrophin1-24 (ACTH1-24, Synacthen) on adrenal cells were studied, using an in vitro assay system of guinea-pig adrenal segments kept in organ culture. Two separate methods for detecting growth activity were used, namely the measurement of thymidine kinase and a nucleic acid cytophotometric method. Synthetic ACTH was able to induce growth in the adrenal explants at very low concentrations (10-25 fg ml-1). Biphasic dose-response curves were obtained, comparable to those described for other cytochemical bioassays. The principles of this assay system may allow the development of a new bioassay for the measurement of plasma concentrations of ACTH or antibodies mimicking the growth effect of this trophic hormone.     

In vitro assay for the quantitative measurement of apoptotic lymphocytes phagocytosis by peripheral blood monocytes

Currently used assays for the quantification of apoptotic cells uptake by phagocytes have several methodological problems. Our assay overcomes some of these problems. As a source of apoptotic cells we used peripheral blood lymphocytes obtained from the patients with chronic lymphoblast leukaemia. Apoptosis was induced by incubating cells with cycloheximide for up to 24 h. The assay was performed in suspension of peripheral blood mononuclear cells. For the visualisation of the phagocytes and phagocyted cells and discrimination of phagocyted from bound apoptotic cells we used Acridine orange/Ethidium bromide double staining. Here we offer a simple test which enables reliable measurement and it can show the difference of phagocytic potential between different individuals.     

In vitro palmitoylation of native bovine brain Goα

The native Goα was purified from bovine brain cortex and palmitoylated in vitro. The in vitro palmitoylation site was the same as that in vivo. The internal palmitoylation of purified native Goα was found to be largely maintained. The apparant palmitoylation ratio was significantly increased after the Goa was treated with DTT. The GTPg S binding characteristic of Goα was not influenced by palmitoylation, however, the affinity for LUVs was increased dramatically. The in vitro palmitoylation model of Goα provides a better basis for studying the functional role of G protein palmitoylation in signal transduction.     

Proliferative response of peripheral blood lymphocytes to mitogens in the owl monkey Aotus nancymae

The new world primate Aotus sp. has been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and P. vivax. The present study examined the in vitro proliferative response of peripheral blood mononuclear cells (PBMCs) isolated from Aotus monkeys, utilizing a wide range of mitogens. Results presented herein demonstrate that the in vitro proliferative response of PBMCs from the Aotus sp. is quite variable from monkey to monkey for each of the mitogens assessed. PBMCs from the Aotus monkey exhibited a delayed kinetic proliferative response and, particularly, a different sensitivity to proliferation in response to various concentrations of Phytohemagglutinin-P and favin lectins, the phorbol ester Phorbol myristate acetate and the calcium ionophore ionomycin. Altogether, our findings are consistent with the conclusion that the in vitro proliferative response of PBMCs from the Aotus differ in their activation requirements compared with PBMCs from humans.     

In vitro effects of Celiptium and MR 14504 on mature rat Leydig cell testosterone production

Percoll-purified mature rat Leydig cells have been used to evaluate the testicular toxicity of two highly potent intercalating agents (Celiptium and MR 14505). Testosterone secretion in the absence and in the presence of human chorionic gonadotropin (hCG) was measured to assess Leydig cell function. Celiptium and MR 14504 induce time- and dose-related inhibitory effects on the production of testosterone by Leydig cells, both in the presence and in the absence of hCG, whatever the concentration of hCG used. We have observed that MR 14504 is about 5 times more potent as an inhibitor of rat Leydig cell steroidogenesis than Celiptium without inducing any cell toxicity. The present study indicates that the Leydig cell is an additional potential site for the primary toxic effects of these drugs in the adult rat testis.     

In vitro genotoxicity of polycyclic musk fragrances in the micronucleus test

The synthetic polycyclic musk fragrance compounds galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyrane), tonalide (7-acetyl-1,1,3,4,4,6-hexamerthyltetraline), celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindane), phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane), cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone) and traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) were examined for their genotoxicity in the micronucleus test (MNT) with human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system containing rat liver S9 and the metabolically competent human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses. Galaxolide, tonalide, celestolide, phantolide, cashmeran and traseolide revealed no genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.     

The effects of medium composition and culture conditions on in vitro rooting and ex vitro establishment of jackfruit (Artocarpus heterophyllus Lam.)

A sucrose level of 20 g/l in the culture medium and a 12 h photoperiod were required for optimal in vitro rooting of proliferated jackfruit shoots and these culture conditions also promoted subsequent establishment of plantlets in the glasshouse. High agar levels of 10 and 20 g/l reduced the humidity in culture but also reduced in vitro rooting and had no effect on the establishment of plantlets in the glasshouse. A 12 h photoperiod in ex vitro conditions gave greater growth and development of plantlets than 8 h or 16 h photoperiods.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclearcells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Sister chromatid exchanges of human peripheral blood lymphocytes induced by N,N-diethylaniline in vitro

N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes. To evaluate its genotoxicity, we examined whether it can induce sister chromatid exchanges (SCEs) in human lymphocytes. We found that N,N-diethylaniline significantly increased the frequency of SCEs both in the absence and presence of S-9 mix. The SCEs from cultures treated by N,N-diethylaniline in the presence of S-9 mix displayed a marked increase which was about 5-fold greater than the control. ANOVA analyses indicated that there is a dose–response relationship between doses of N,N-diethylaniline and the frequency of SCEs, especially in the presence of S-9 mix. The results suggested that N,N-diethylaniline has genotoxicity.     

In vitro assessment of the toxicity of metal compounds

A review of the activity of metal compounds in mammalian cell transformation assays has been completed. Results from these assays appear to correlate well with the known carcinogenic activity displayed by specific metal compounds in vivo. Studies of cell transformation in vitro may provide information pertaining to the mechanism of the induction of carcinogenesis by certain metals.     

Functional dissociation of apelin receptor signaling and endocytosis: implications for the effects of apelin on arterial blood pressure

Apelin is a novel neuropeptide involved in the regulation of body fluid homeostasis and cardiovascular functions. It acts through a G protein-coupled receptor, the APJ receptor. We studied the structure-activity relationships of apelin at the rat apelin receptor, tagged at its C-terminal end with enhanced green fluorescent protein and stably expressed in CHO cells. We evaluated the potency of N- and C-terminal deleted fragments of K17F to bind with high affinity to the apelin receptor, and to inhibit cAMP production and to induce apelin receptor internalization. We first characterized the internalization and trafficking of the rat apelin receptor. This receptor was internalized via a clathrin-dependent mechanism and our results suggest that receptor trafficking may follow a recycling pathway. We then tried to identify the amino acids of K17F required for apelin activity. The first five N-terminal and the last two C-terminal amino acids of K17F were not essential for apelin binding or the inhibition of cAMP production. However, the full-length sequence of K17F was the most potent inducer of apelin receptor internalization because successive N-terminal amino-acid deletions progressively reduced internalization and the removal of a single amino acid at the C-terminus abolished this process. Finally, the most novel observation of this work is that hypotensive actions of apelin peptides correlate best with the ability of those ligands to internalize. Thus, apelin receptor signaling and endocytosis are functionally dissociated, possibly reflecting the existence of several conformational states of this receptor, stabilized by the binding of different apelin fragments to the apelin receptor.     

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

In vitro effect of lead acetate on human erythropoietic progenitors

Lead is known to induce hematological disturbances resulting from abnormalities in cell differentiation and hemoglobin synthesis during hematopoiesis. The aim of the present work was to study human erythropoiesisin vitro in the presence of lead. Human erythroblastic progenitors, burst-forming units–erythroid (BFU-E), were exposed to lead acetate at increasing concentrations during 14 days of culture. Hematotoxicity was evaluatedin vitro according to proliferation and differentiation of cell colonies arising from BFU-E development. The ability of cells to synthesize proteins, porphyrins, and hemoglobin was measured by spectrophotometric tests and by high-pressure liquid chromatography (HPLC). Results showed that in the presence of 10^–3 mol/L lead acetate, no hemoglobinized cells were observed in culture and no fluorescent porphyrins were detected in cells. Up to 10^–3 mol/L, lead acetate is not cytotoxic, i.e., it does not induce cell destruction. The present work demonstrates that lead acetate interferes with the porphyrin synthesis of human erythroblastic progenitors in vitro. The decrease of porphyrin content with 10^–5 mol/L lead acetate suggest that δ-aminolevulinic acid dehydratase can be inhibited by lead acetate duringin vitro erythropoiesis. In vivo erythropoiesis occurs in the bone marrow. As about 95% of the body burden of lead in adults is located in the bones with a biological half-life of some years, the concentration of lead acetate found to block porphyrin synthesis in vitro has to be compared with in situ bone marrow lead concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro effects of strontium ranelate on the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaSR) is activated by divalent cations and might mediate some of the effects of strontium ranelate, a new drug for the prevention and treatment of post-menopausal osteoporosis. Here, we showed that the maximal effect of Sr(2+) was comparable to that observed for Ca(2+) for both the cloned rat CaSR expressed in Chinese hamster ovary [CHO(CaSR)] cells and the mouse CaSR constitutively expressed in AtT-20 cells as measured by the accumulation of [(3)H]inositol phosphates (IP) resulting from CaSR activation. Strontium ranelate also displayed comparable agonist activity for the CaSR in both cell lines. Sodium ranelate did not stimulate the IP response in CHO(CaSR) cells. The IP response resulting from activation of other G-protein-coupled receptors was potentiated by Sr(2+), suggesting that entry of Sr(2+) into the cells might influence phospholipase C activity. Modulation of the CaSR activity in bone cells by strontium ranelate may contribute to its reported antiosteoporotic effects.     

Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells

A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1 beta and an increase of interleukin-10.