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Results for “In vitro assay”

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At least 19 recordsLinked to original sources

PubMed1992

The effect of Lasso herbicide on human immune function as measured by in vitro assays.

Using in vitro assays, this study was undertaken to determine whether the components of Lasso herbicide formulation had an effect on the human immune system. Mononuclear cells from human peripheral blood were exposed to analytical alachlor, alachlor conjugated to human serum albumin or Lasso formulation over a concentration range from .01 microM-1.0 microM. The effects of the test materials on the following immunological functions were determined: lymphocyte proliferation induced by mitogen or antigen; antibody synthesis of IgG and IgM isotypes in pokeweed stimulated mononuclear cell cultures; cytotoxic T cell proliferation; lysis of target cells by natural killer cells and lymphokine activated killer cells. The data demonstrated that the test compounds had no significant, dose related effect on the function of immunocompetent cells. Hence, the data suggest that the components of the Lasso formulation have no effect on the human immune system.

Acetamides↗

PubMed1992

Synthetic lipopeptide immunomodulators derived from bacterial lipoprotein: tools for the standardization of in vitro assays.

For the evaluation of immunomodulators by in vitro assays, agents working reproducibly are difficult to obtain; conventional preparations of bacterial immunomodulators tend to vary for different preparations. Here we suggest that synthetic lipopeptide analogues derived from bacterial lipoprotein can be used as standards for various in vitro assays: studying B lymphocyte activation, lipopeptides act as potent mitogens and polyclonal activators inducing immunoglobulin synthesis. In monocytes/macrophages, lipopeptide stimulate the secretion of IL-1, IL-6, tumor necrosis factor (TNF) and nitrogen oxide (NO); they also induce tumor cytotoxicity. Lipopeptides also constitute potent immuno-adjuvants in vitro and in vivo, either in combination with or covalently bound to antigen. These activities are displayed in various species; in mice they are found in LPS non-responder and responder strains. The novel synthetic lipopeptides described here can be synthesized readily in gram amounts with high purity and reproducibility; they are non-toxic and can be stored for a long time even at room temperature. Thus, lipopeptides meet the requirements to serve as effective standards for a multitude of relevant biological assays.

Amino Acid Sequence↗

PubMed1992

Study of dose-response relationship in contact sensitivity using an in vitro assay.

Dose-response relationships in contact sensitivity were evaluated in guinea pigs using an in vitro assay. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with DNCB and 2,4-dinitrobenzene sulfonic salt (DNBS). Lymph node cells from sensitized and control guinea pigs were cultured in the presence of different doses of DNCB and DNBS at 8 x 10(5) cells/well, respectively. The sensitivity was evaluated by the lymphocyte transformation test (LTT), which was assessed by uptake of 3H-thymidine. The results indicated that there were significant correlations between the doses of sensitizers and the values of LTT in both phases of induction and challenge. Thus, the presence of higher numbers of LTT-reactive lymphocytes in the circulation may well correlate with the sensitizing doses. The values examined by in vitro assay correlated well with patch test readings (r = 0.653), indicating that following the increment of degree of patch test reactions, the values of SI were also increased. The in vitro LTT may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. The in vitro assay reproduced the cross-reaction between DNCB and DNBS which was confirmed in vivo.

Animals↗

PubMed1977

Comparison of antiplaque agents using an in vitro assay reflecting oral conditions.

An in vitro assay is described using saliva-treated bovine enamel slabs for determining the potential of chemotherapeutic agents to adsorb to tooth surfaces and act against plaque-forming bacteria. Chlorhexidine was found to inhibit the formation of in vitro plaque by Actinomyces viscosus, A naeslundii, Streptococcus mutans and S sanguis. Actinobolin was found to have marked antibacterial properties but limited adsorptive qualities.

Actinomyces↗

PubMed1979

In vivo and in vitro assays of immunocompetence in bronchogenic carcinoma.

To determine the degree of correlation among the various in vivo and in vitro assays that could be used to assess immunocompetence in bronchogenic carcinoma, the response to common recall skin antigens, primary sensitization to DNCB and lymphocyte function tests based on blastogenic response to mitogens and in mixed lymphocyte culture (MLC) were tested in 48 patients with bronchogenic carcinoma. The values were compared to responses in 94 age-matched healthy control subjects. There were impaired skin tests reactions among squamous cell carcinoma patients, with little impairment of their lymphocyte blastogenesis reactions. Anaplastic carcinoma patients had notable defects in lymphocyte function tests but less impairment of the skin test reactions. These data suggest that the mitogen concanavalin A, phytohemagglutinin, and the MLC are more useful screening assays of in vitro immunocompetence than are the other commonly used mitogens.

Aged↗

PubMed1979

Activation of the bactericidal capacity of polymorphonuclear granulocytes after surgery, measured with a new in vitro assay.

A classic in vitro polymorphonuclear (PMN) granulocyte bactericidal system was used alongside a newly developed modification to see whether the new assay would increase the possibility to detect a stimulation of PMN bactericidal functions. In the new assay each granulocyte was provided with 30--40 bacteria, which is quite close to the maximal killing capacity (usually 60 bacteria per PMN). Granulocytes were obtained from 8 patients the day before, the day after and 2 d after they underwent thoracotomy with cardiopulmonary by-pass (CPS). The granulocytes from all patients showed an increased capacity to kill Staph. aureus in vitro 2 d after the operation, compared to before, when the submaximal bacterial concentration per granulocyte was used, whereas no change was observed with the standard bacterial concentration (3--4 bacteria per granulocyte). Thus, the new assay might make it possible to observe an enhanced PMN bactericidal ability.

Adult↗

PubMed1992

[Cell culture and its application. Application of cultured vascular endothelial cells to the in vitro assay for the invasion of cancer cells through endothelium-matrix membrane system].

Usefulness of cultured human vascular endothelial cells for the in vitro assay of cancer invasion was reviewed. Selective isolation procedures for the vascular endothelial cells from the different types of vascular tubes were described. Human umbilical cord vein is one of the most useful sources for the isolation of fresh normal endothelial cells. The cells are isolated by perfusing trypsin solution through the umbilical cord vein and successively subcultivated up to 80 population doubling levels as in vitro life span. In case of human thoracic aorta, endothelial cells were removed from endothelium layer of aorta ring by a jet flow of collagenase solution through a needle. Purity of these cells as endothelial cells can be determined by indirect staining of the cytoplasma with anti-endothelin antibody. Usefulness of these cultured endothelial cells has been proved in the studies of regulation of endothelin biosynthesis, ageing of vascular cell, isolation of some new growth factors, interaction with other blood cells, etc. In addition to these studies, we consider that interaction of tumor cells with endothelial cells plays an important role in tumor metastasis. So we established in vitro invasion assay system through the interaction with endothelial cells to examine the invasion and adhesion of tumor cells. Human umbilical cord endothelial cells (HUVEC) were cultured on porous membranes coated with laminin (LN). HT1080 fibrosarcoma cells were seeded onto HUVEC, and HT 1080 cells passed through the membrane were counted. HUVEC were easily distinguished from tumor cells by specific staining of endothelial cells with UEA-1 lectin. Using scanning electron microscopy, we confirmed that HT1080 cells invaded between HUVEC. Roles of adhesion molecules induced by some cytokines on invasion of cancer cells through the endothelial cells-matrix membrane system were discussed.

Cell Adhesion↗

PubMed1975

Specific and nonspecific antitumor immunity. I. Description of an in vitro assay based on inhibition of DNA synthesis in tumor cells.

As in vitro assay of cell-mediated antitumor immunity based on the inhibition of tumor cell DNA synthesis (IDS) was devised. It was reasoned that both cytolytic and cytostatic antitumor effects could be measured in a quantitative yet generalized manner with this approach. By the use of microculture techniques and simplified methods for the determination of tritiated-thymidine incorporation by all cells in individual cultures, normal and immune effector (E) cell populations were compared in their ability to inhibit the DNA synthesis of a fixed initial number of various target tumor cells. Doubling dilutions of E cells were used to titrate the antitumor effects of normal and immune cells at many E/T (effector to target) ratios. Under conditions of alloimmunization, significant immunologically specific IDS effects could routinely be detected at an E/T ratio of less than 1 or 0.1:1, and under certain conditions at 1:100 or less. Results were highly reproducible with respect to the individual E cell donors, replicate cultures, and repeat experiments. The effects were proportional to visually determined cell destruction and independent of obvious culture artifacts. The IDS method was compared with the 51Cr release technique under various experimental conditions. The results demonstrated that decreases in E/T ratio and/or the addition of excess nonimmune cells to immune effector populations had a similar effect in both assays, which was to decrease the magnitude of the immune cell activity. Addition of excess normal cells reduced the activity of immune cells to a level below that of an equal number of immune cells tested at the same E/T ratio without added nonimmune cells. Both assays detected primarily a T lymphocyte-mediated lytic event when effectors generated by the described alloimmunizations were used. The IDS assay also detected a weak non-T-cell activity in anti-theta plus complement-treated alloimmune spleen. The possibility that this represented antibody-dependent cell-mediated cytotoxicity was raised by the finding that normal spleen ceels plus antitarget antibody had significant activity in the IDS system. The sensitivities of the two methods were compared and the potential of the IDS method was evaluated.

Animals↗

PubMed1991

A new in vitro assay for carbon dioxide excretion by trout red blood cells: effects of catecholamines.

A new in vitro assay was developed and critically characterized to measure the rate of CO2 excretion by trout red blood cells (RBCs) from HCO3- in their natural plasma under normal in vivo conditions of acid-base status. The assay is based on the addition of [14C]bicarbonate to the whole blood and collection of the resultant 14CO2 in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO2 excretion rates are representative of those occurring in vivo. Rates are linear over the 3 min time course of the assay, related to haematocrit in a non-linear fashion, elevated by the addition of carbonic anhydrase, reduced by blockade with acetazolamide, and sensitive to variations of equilibration PCO2. Large variations in plasma [HCO3-] have only a small effect on CO2 excretion rates when the blood is chronically equilibrated at these levels. Acute elevations in [HCO3-], however, create a non-equilibrium situation, resulting in large increases in CO2 excretion. When the blood is acidified, to duplicate typical post-exercise metabolic acidosis, adrenaline causes a marked inhibition of RBC CO2 excretion. The response is transient, reaching a peak 5-8 min after addition of adrenaline and disappearing by 30-60 min. The magnitude of the adrenergic inhibition is correlated with the magnitude of the RBC pHi regulatory response, expressed as the RBC transmembrane pH difference (pHe-pHi). These results support the 'CO2 retention theory' explaining observed increases in blood PCO2 in vivo after exhaustive exercise and catecholamine infusions in fish.

Animals↗

PubMed1976

Cryopreservation of human lymphocyte function as measured by in vitro assays.

Methods are described by which cryopreserved cells can be utilized in a number of in vitro assays. On a per cell basis, nearly total recovery of function can be demonstrated for lymphocyte transformation (mitogens, antigens and MLC) and rosette-forming cells. Excellent recovery of mononuclear cell production of leukocyte migration inhibitory factor was also observed. Highly reproducible activity was recovered on a per lymphocyte basis in lymphocyte cytotoxicity but with a definite decrement in the percentage recovery. Both for longitudinal studies of immune function and for standardization of these assays in one or more laboratories such cryopreserved cells are ofimmense value and should be widely utilized.

Blood Specimen Collection↗

PubMed1992

In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes.

In vitro assays have been developed for three membrane-bound carotenogenic enzymes, phytoene desaturase, lycopene cyclase and beta-carotene hydroxylase, expressed in Escherichia coli. Transformants of E. coli containing different deletion constructs of the Erwinia herbicola carotenogenic gene cluster were employed, allowing the estimation of enzyme activities without interference from subsequent reactions. New HPLC systems were developed to separate substrates and reaction products enabling the determination of radioactivity on-line. The newly developed assays facilitate the purification of these enzymes which have never been isolated before.

Carotenoids↗

PubMed1979

A micromethod for in vitro assay of lymphocyte proliferation in mouse whole blood.

A microplate culture system to assay the in vitro proliferative responses of mouse peripheral blood lymphocytes to phytohaemagglutinin is described. The method utilises whole blood, a gamma-emitting isotopic label and semi-automated harvesting. Data are presented to show the effects of several variables on the culture system. The technique is suitable for serial study of the in vitro phytohaemagglutinin response in individual animals.

Animals↗

PubMed1978

Immunologic monitoring of transplant rejection: correlation of in vitro assays with morphologic changes on transplant biopsy.

The assays of lymphocyte-mediated cytotoxicity (LMC), antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were correlated with histopathologic criteria of refection in 35 transplant biopsies. A positive LMC was seen with 6/8 biopsies showing moderate to severe cellular rejection and in 8/17 with mild cellular rejection. Positive ADCC and/or CDC assays were associated with 14/14 biopsies containing rejection vasculitis. These results suggest that selected in vitro assays may be useful in monitoring the immunologic events occurring in the rejecting allograft.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1992

Prediction of response to drug therapy of cancer. A review of in vitro assays.

Cancer chemotherapy has witnessed a great deal of progress since the introduction of the nitrogen mustards in the 1940s. Unfortunately, individual patients with apparently identical tumour histologies do not always respond identically to the same drug regimen. Determining the sensitivity and resistance of an organism before treatment has been the standard of care in infectious diseases for many years, while in oncology treatment has been initiated according to tumour histology rather than the tumour's sensitivity to a given agent. Attempts to individualise therapy have been the goal of oncologists since the 1950s. Since that time a number of in vitro assays have been developed to predict therapeutic outcome prior to the start of therapy. In the 1970s, with the introduction of the human tumour stem cell assay, it was generally believed that oncology was on the threshold of entering an era of predictive in vitro chemosensitivity testing. Unfortunately, this assay was shown to have a number of technical drawbacks including the low plating efficiencies of many primary tumour samples which thus limits the percentage which can be evaluated, leaving us still at this threshold today. Several recent developments, such as the Kern assay, which measures inhibition of radioactive precursors into tumour cells in the presence of antineoplastic agents, ATP bioluminescence assays, and the fluorescent cytoprint assay offer the promise of rapid and sensitive results. Other assays, such as the tetrazolium-based MTT and the sulphorhodamine blue assay appear to hold more promise in the screening and evaluation of potential new agents in established tumour cell lines than for evaluating chemosensitivity of clinical specimens. However, before a particular assay can be considered as an in vitro test of chemosensitivity or resistance, controlled prospective studies must be carried out to validate the assay in a number of different tumour types.

Antineoplastic Agents↗

PubMed1992

Comparison of the potency of three Brazilian Bothrops antivenoms using in vivo rodent and in vitro assays. BIASG (Butantan Institute Antivenom Study Group).

Three Brazilian polyspecific Bothrops antivenoms were compared using standard W.H.O. rodent in vivo and in vitro assays of their ability to neutralize the principal venom activities of pooled whole Bothrops jararaca venom. On a volume basis, the antivenoms were equally effective in neutralizing lethal activity in mice, and there were only minor differences in their ability to neutralize venom-induced haemorrhage, necrosis and procoagulant activity. Antivenom efficacy in neutralizing defibrinogenation varied. However, when equal amounts of antivenom IgG were compared, it was found that the FUNED antivenom best neutralized lethality, haemorrhage, necrosis and fibrinogen clotting activity. Vital Brazil and FUNED antivenoms were equally effective in neutralizing plasma coagulant activity but Vital Brazil antivenom was the more effective in neutralizing defibrinogenation.

Animals↗

PubMed1978

Use of lysostaphin to remove cell-adherent staphylococci during in vitro assays of phagocyte function.

Lysostaphin, a bacteriolytic enzyme, has been used to remove cell-adherent and extracellular Staphylococcus aureus from phagocyte-bacterial mixtures in vitro. Lysostaphin kills S. aureus more rapidly than penicillin, is not toxic for phagocytic cells and, when used for short periods at low concentrations, appears to enter neither human nor mouse mononuclear phagocytes. The use of lysostaphin provides the basis of a simple reliable direct in vitro assay for measuring the attachment and ingestion of S. aureus by phagocytic cells.

Bacteriolysis↗

PubMed1976

[In vitro assay for ACTH-releasing activity using ACTH radioimmunoassay: ACTH releasing activities by various drugs (author's transl)].

Several procedures have been reported for the assay of corticotrophine-releasing factor (CRF), each having its advantages and disadvantages. This report deals with an in vitro assay of ACTH releasing activity utilizing pituitary incubation combined with ACTH radioimmunoassay. Rat half pituitary was preincubated in 2 ml Krebs Ringer bicarbonate buffer containing 0.2% glucose and 0.25 % BSA (KRBG-BSA) for 1.5 hr (45 min X 2). The medium was replaced by 1 ml KRBG-BSA and incubated for 30 min. Then the medium was again replaced by 1 ml KRBG-BSA or KRBG-BSA containing test materials and incubated for another 30 min. The amount of ACTH assayed by radioimmunoassay in the 2nd 30 min incubation was compared with in the 1st 30 min incubation and expressed as percentage. In ACTH radioimmunoassay, anti-ACTH serum was diluted to 1 : 1,500-3,000. The 125I-alpha 1-24ACTH-antibody system was not affected by lysine-vasopressin (LVP), arginine-vasopressin (AVP), rat's pituitary LH, GH and prolactin. Human 1-39ACTH was used as ACTH standard, and the dilution curve of incubation medium was paralleled with the standard curve. Repeatability of immunoassayable ACTH within-assay was 174 +/- 5.0 pg/tube (CV = 2.9%). A log dose-relationship was observed between the amounts of stalk median eminence extracts (SME ; NIAMDD) added to the incubation medium and its ACTH releasing activities. The sensitivity of this assay method was at least 0.1 SME or 10 mU of LVP and AVP. Using this method, it found that LVP, AVP, norepinephrine (100 ng/ml200 ng/ml) and 5-hydroxytryptophane (1 mug/ml) had ACTH releasing activities but LH-RH, TRH, glucagon, dopamine, phentolamine, propranolol, haloperidol, prostaglandin E1 and indomethacin did not affect the release of ACTH.

5-Hydroxytryptophan↗

PubMed1979

Characterization of a UGA-suppressing serine tRNA from Schizosaccharomyces pombe with the help of a new in vitro assay system for eukaryotic suppressor tRNAs.

Two different allele-specific suppressor mutants of the fission yeast Schizosaccharomyces pombe produce opal (UGA) suppressor tRNAs. This was shown by the use of a new in vitro assay for eukaryotic nonsense suppression: a wheat germ extract is programmed with rabbit globin mRNAs and the readthrough products are studied. alpha-Globin is elongated upon addition of ochre (UAA) suppressor tRNAs, whereas beta-globin yields a readthrough product with opal suppressor tRNAs. This simple and very sensitive assay allowed the purification of the opal suppressor tRNA from S. pombe strain sup3-e. The pure tRNA can be aminoacylated with serine; thus, we conclude that this suppressor tRNA inserts serine in response to the UGA termination codon of pure rabbit beta-globin mRNA.

Alleles↗