Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.

The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.

Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system.

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required. Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2. Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34bright/CD38dim cells was obtained in serum-containing culture. When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction. The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture. Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold increase in CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim cells was amplified by 25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB.

Effect of peripheral blood progenitor cell dose on hematopoietic recovery: identification of minimal progenitor cell requirements for rapid engraftment.

Repeated high-dose chemotherapy (HDC) with stem cell support is advocated for curative treatment of epithelial ovarian cancer patients, requiring large quantities of progenitor cell harvest. Although the switchover to peripheral blood stem cell transplantation has generally made possible the harvest of large quantities of progenitor cells, the minimum threshold is still pertinent for planning the safe conduct of HDC. However, as the minimum threshold for safe peripheral blood stem cell transplantation (PBSCT) is not yet established, this study was designed to clarify the minimum amount of progenitor cells required for prompt recovery of hematopoietic. Retrospective analysis was performed on 52 HDCs administered in 37 ovarian cancer patients. After autologous bone marrow aspiration (10 patients) or peripheral blood stem cell harvest (27 patients), colony-forming unit granulocyte macrophage (CFU-GM) were enumerated prior to cryopreservation. Numbers of CFU-GM were again calculated before reinfusion and the patients were divided into eight groups: 0.13-<0.4, 0.4-<0.7, 0.7-<1.0, 1.0-<3.5, 3.5-<5.0, 5.0-<10.0, 10.0-<20.0 and >20.0 (x 10(5)/kg). The minimum CFU-GM threshold (x 10(5)/kg) was found to be 1.0-<3.5 for platelets and 3.5-<5.0 for white blood cells. Higher infusion doses did not lead to significant benefits in hematopoietic reconstruction. These results indicate that preservation of a minimum of 7-10 x 10(5)/kg CFU-GM is recommended for the safe conduct of tandem HDCs.

Peripheral and central structures involved in insect gustation.

Studies in insect gustation have a long history in general physiology, particularly with work on fly labellar and tarsal sensilla and in the general field of insect-plant interactions, where work on immature Lepidoptera and chrysomelid beetles has been prominent. Much more emphasis has been placed on the physiological characteristics of the sensory cells than on the central cellular mechanisms of taste processing. This is due to the fairly direct access for physiological experimentation presented by many taste sensilla and to the obvious importance of tastants in insect feeding and oviposition behaviour. In some of the insect models used for gustatory studies, advances have been made in understanding the basic morphology of the central neuropils involved in the first stages of taste processing. There is much less known about the physiology of interneurons involved. In this review, we concentrate on four insect models (Manduca sexta, Drosophila melanogaster, Neobellieria bullata (and other large flies), and Apis mellifera) to summarize morphological knowledge of peripheral and central aspects of insect gustation. Our views of current interpretations of available data are discussed and some important areas for future research are highlighted.

Transient brain activity used in magnetic resonance imaging to detect functional areas.

Functional areas were detected with short stimuli eliciting transient brain activity using the method of 'transient' regions of interest (ROIs) and functional magnetic resonance imaging (fMRI). This method was validated by comparing the results with sustainedly activated areas identified conventionally. Eighty-eight and 89% of the total areas of transient ROIs derived from 0.2 and 2 s stimulation, respectively, were identified at 5-7 s and 5-9 s, respectively, after stimulus onset. Eighty-eight and 76%, respectively, of these areas overlapped 'conventional' ROIs derived from 20 s stimulation. These results suggest that the delineation of transient ROIs, by targeting a period approximately 7 s after transient neural activity, can be useful for fMRI studies of cognitive functions.

Higher-order brain function analysis by trans-cranial dynamic near-infrared spectroscopy imaging.

Near-infrared spectroscopy is discussed from the viewpoint of human higher-order brain function analysis. Pioneering work in this field is reviewed; then we describe our concept of noninvasive trans-cranial dynamic optical topography and its instrumentation. Also, the validity of its functional images is assessed from both physical and physiological viewpoints. After confirming the validity of this method, we have applied it to a wide variety of fields such as clinical medicine, cognitive science, and linguistics in collaboration with researchers at several other institutes. Further application possibilities and the future of trans-cranial dynamic optical topography are also discussed. © 1999 Society of Photo-Optical Instrumentation Engineers.

Improvements of nuclear magnetic resonance image quality using iterations of adaptive nonlinear filtering.

To improve nuclear magnetic resonance (NMR) image quality, a method using iterations of adaptive nonlinear filtering is presented. The method utilizes the properties of an NMR image's noises which are spike-like in the local region. An NMR image is iteratively filtered by a mode-change-type nonlinear filter which has two modes. The first mode reduces small amplitude noises in the NMR image, so that the S/N ratio of the image is improved, and then the residual noises become more spike-like. The second mode mainly reduces large amplitude spike-like noises of the image improved by the first mode, which cannot reduce spike-like noises. Using the proposed iterative filter, it is found by simulations that the S/N ratio of the NMR image is improved by as much as a factor of 2.7. A significant improvement in the image quality when this filter is used is demonstrated by applying it to an NMR image of a human head obtained from an actual NMR imaging apparatus.

The olfactory responses of the antenna and maxillary palp of the fleshfly, Neobellieria bullata (Diptera: Sarcophagidae), and their sensitivity to blockage of nitric oxide synthase.

The relative sensitivities of the olfactory receptors in the antenna and maxillary palp of the fleshfly, Neobellieria bullata, were assessed using simultaneous electroantennograms (EAGs) and electropalpograms (EPGs). In general, the antennae and maxillary palps were more sensitive to odors related to animals (blood extract and saturated carboxylic acid) than to odors that were plant-derived (citral, hexenol, hexenal). In addition, the maxillary palps were relatively less sensitive to plant-derived odorants than the antennae, perhaps related to their anatomical position. Scanning electron microscopy was also used to assess the types of sensilla found on the two organs. In addition, NADPH-diaphorase histochemistry was used in an attempt to localize the enzyme nitric oxide synthase (NOS) in the antenna and the maxillary palps. We found evidence of NADPH-diaphorase staining in both organs, with localized staining in the antennal cells and more general staining in the maxillary palps. When NOS was selectively blocked using the antagonist L-NAME, the amplitude of the EAGs and EPGs to odorants fell by 30-50%. In contrast, application of the inactive enantiomer, D-NAME, did not change the amplitude of the EAGs or the EPGs. Our results indicate that NOS is involved in the function of olfactory receptor cells in the fleshfly.

Further evaluation of the quantitative chorioallantoic membrane test using trypan blue stain to predict the eye irritancy of chemicals.

The feasibility of using the chorioallantoic membrane (CAM) test using fertile hens' eggs as an alternative method to predict eye irritancy was examined for 12 test chemicals, comprising eight liquids, three powders and one emulsion. The judgement of the injurious effects of chemicals was carried out by a trypan blue staining method that was developed to overcome disadvantages arising from the lack of objectivity and quantitativeness in the original CAM test (Luepke's method). The amounts of pigment adsorbed by the CAM showed a good correlation with the scores obtained in the Draize eye irritation test. The results indicate that the trypan blue staining assay using CAM-TBS assay may be useful as an alternative to the in vivo eye irritation test. The next step will be to validate this method on an interlaboratory basis.

Validation study on five different cytotoxicity assays in Japan-an intermediate report.

In October 1992, the Japanese Society of Alternatives to Animal Experiments (JSAAE) organized a first-step inter-laboratory validation study on five Cytotoxicity assays: crystal-violet staining assay, neutral red uptake, MTT assay, colony formation, and lactate dehydrogenase release assay. This study is to clarify problems in organizing system of validation, protocols and intra- and inter-laboratory variation of ED(50) values on six representative chemicals determined with five cell lines in 45 laboratories, and thereby to evaluate practicability of the Cytotoxicity assays for further validation targetting to the Draize test. Two samples of one of the six chemicals were used to test intra-laboratory variation, so seven chemical preparations were sent to each laboratory after double-blind coding. More than 2300 data files were submitted to the organizing committee in 1993 and now they are under data cleaning and comprehensive analysis. Preliminary results calculated from hand-plotted figures were presented on a typical severe irritant, cetylpyridium chloride monohydrate.

An in vitro alternative to the Draize eye-irritation test: Evaluation of the crystal violet staining method.

To evaluate a simple in vitro method as an alternative to the Draize eye-irritation test, 12 surfactants were tested by both of the above methods, and the correlation between the results was examined. The in vitro method used was the crystal violet staining method. HeLa cells or SIRC cells from rabbit cornea were inoculated into each well of 96-well microplates which contained serially diluted test chemicals. They were cultured for 72 hr, and then fixed and stained with crystal violet. After spectrophotometric measurement, the concentrations of test materials that inhibited the absorbance to 50% of the control level (IC(50)) were determined. The in vivo data used for comparison were the maximum corneal score and the maximum total score obtained by the eye test when the substances were tested at a concentration of 10%. The correlation coefficients between the maximum corneal scores of the 12 surfactants and their IC(50)s in HeLa cells and in SIRC cells were -0.821 and -0.816, respectively, while those between the maximum total scores and IC(50)s in both cell lines were -0.855 and -0.863, respectively. All of these values are statistically highly significant, indicating that the crystal violet staining method may have value in predicting the eye irritancy of surfactants. Further interlaboratory validation will, however, be needed.

Quantitative evaluation to predict the eye irritancy of chemicals: Modification of chorioallantoic membrane test by using trypan blue.

The chorioallantoic membrane (CAM) test using fertile hen's eggs has been reported as a promising alternative method to predict the eye irritancy of chemicals. However, the scoring system of the original CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Furthermore, the test requires skill when the effects of test substances are to be graded. To overcome these disadvantages, a more objective means of judging the injurious effects of chemicals was examined as follows: after treatment with the test chemical, the CAM was stained with trypan blue and the amount of pigment adsorbed was measured spectrophotometrically. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the in vivo eye irritation test scores (r = 0.89). The findings suggest that the trypan blue staining method is very simple and reproducible, and thus would be a promising alternative method for predicting the eye irritancy of chemicals. However, further studies will be required to validate this test method with a wide variety of chemicals and formulations.

Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers.

The aim of this study was to explore the usefulness of a human monocyte cell line in the development of in vitro models for predictive testing of contact sensitizers. Several studies have shown that contact sensitizers induce CD86 expression and enhanced internalization of MHC class II molecules in dendritic cells (DCs). We used THP-1, a human monocyte cell line, as a replacement for DCs for evaluation of these phenotypical alterations as predictive endpoints for contact sensitizers. Known sensitizers and irritants were evaluated. After 24-h exposure to samples, the expression of CD86 on THP-1 cells was measured by flow cytometry. Sensitizers such as dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), eugenol, p-phenylenediamine (PPDA) and ammonium tetrachloroplatinate (Pt) enhanced CD86 expression on THP-1 cells, while nickel sulfate, cobalt sulfate and irritants such as methylsalicylate (MS), sodium dodecyl sulfate (SDS) and dimethyl sulfoxide (DMSO) did not augment CD86 expression. A synergistic effect was observed when DNCB and IFN-alpha were added simultaneously to a culture of THP-1 cells. Furthermore, internalization of MHC class II molecules was observed when the cells were treated with some of sensitizers for 2 h. The inducing effects of chemicals on the two phenotypical alterations were the same. These results suggest that these test systems can be used to predict contact-sensitizing ability of chemicals as an in vitro sensitization assay.

Multivariate factorial analysis of data obtained in seven in vitro test systems for predicting eye irritancy.

Seven in vitro test systems used to predict eye irritancy (EYTEX, SIRC cytotoxicity, HeLa cytotoxicity, chorioallantoic membrane (CAM), liposome, red blood cell and haemoglobin denaturation test system) were applied to 12 surfactants, and the results were subjected to multivariate analysis to evaluate the relative contributions of five factors. These factors were: (1) cellular plasma membrane destruction factor, (2) haemoglobin type protein denaturation factor, (3) EYTEX-type protein denaturation factor, (4) cytotoxicity factor and (5) an unknown (unidentified) factor. The results clarified the basis on which the findings of each test system were related to the Draize results. According to the analysis, the Draize eye irritation test could be explained by the contribution of the protein denaturation factor and cellular plasma membrane destruction factor. This provides support for a previous hypothesis that the major mechanisms of eye irritation are cellular plasma membrane destruction and protein denaturation. The CAM test value showed a higher correlation coefficient (0.906) with the Draize score than did the results of any of the other test systems; this was due to the fact that these two tests showed similar patterns of dependence on the five factors as indicated by the factorial analysis. The haemoglobin denaturation test system had the next highest correlation coefficient at about 0.75. Furthermore, by using further tests to make up the deficiency of the other necessary factors, a desirable battery system could be predicted; for example, the combination of the haemoglobin-denaturation and rat red blood cell tests would provide a result similar to that of the Draize test. This study should contribute to the development of a rational basis for prediction of eye irritancy of chemicals using in vitro test systems.

Liposomes as an in vitro model for predicting the eye irritancy of chemicals.

Liposomes containing 4-methylumbelliferyl phosphate (Um-P) were prepared using the lipid extracts from bovine eyes and were incubated with seven surface-active agents. The Um-P released from the liposomes by each test agent was hydrolysed with alkaline phosphatase and the resultant 4-methylumbelliferone was assayed spectrofluorometrically. The values for Um-P(50) (the concentration of test material at which 50% of Um-P is released) showed a good inverse correlation with the irritation scores obtained by the Draize eye test. The results suggest that the liposomal assay reported here may be useful as an in vitro model for predicting the eye irritancy of chemicals.

Evaluation of stinging-inducing chemicals using cultured neuronal cells: an electrophysiological approach.

The effects of various cosmetic ingredients, including preservatives, humectants, ultraviolet absorbents and solvents, on the electrical properties of neuronal cells were investigated using rat phaeochromocytoma PC12 cells and cultured rat dorsal root ganglion (DRG) neurons. When membrane current was measured under whole-cell voltage-clamp, all nine test compounds inhibited voltage-activated K(+) current in PC12 cells. With the five compounds selected for further experiments with DRG neurons, two types of current responses were observed, namely inhibition of K(+) current by methyl or butyl p-hydroxybenzoate (MPHB or BPHB) and resorcinol, and induction of a cationic inward current by MPHB, BPHB, ethanol and dipropylene glycol. The order of potency of these chemicals to inhibit the K(+) current in PC12 cells was similar to that of their cytotoxicity, which was determined by MTT assay. Capsaicin, BPHB, MPHB and resorcinol, however, inhibited the K(+) current at concentrations lower than those required for cytotoxicity. These results suggest that these cosmetic ingredients exert significant effects on the electrical properties of cultured neuronal cells. This electrophysiological method may be useful in prescreening test systems to detect stinging, and also to clarify mechanisms underlying the irritation induced by a variety of compounds.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test.

The haemolysis test using sheep red blood cells (RBC) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the test according to the standard operating procedure (SOP). Thirty-eight cosmetic ingredients and isotonic sodium chloride solution were used as test substances in this validation study. The concentrations of the test substances that induced 50% haemolysis (HC(50) value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC(50) values were not obtained for coloured or water-insoluble (turbid) substances. Three acids caused denaturation of haemoglobin leaked from RBC and consequently interfered with the determination of the HC(50) value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation coefficient and Spearman's rank correlation between the HC(50) value and maximum average Draize total score (MAS) were -0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 15 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS50) could be correctly classified by this method. These results suggest that the haemolysis test might be applied to cosmetic ingredients as a screening method to distinguish strong irritants that directly affect the cell membrane permeability and do not disturb spectrophotometrical determination of haemoglobin. In order to evaluate the potential for eye irritation of cosmetic ingredients, a combination of haemolysis with other methods based on different mechanism should be employed to improve the predictability.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (8) Evaluation of cytotoxicity tests on SIRC cells.

Two common assays, the neutral red uptake assay (SIRC-NRU) and the crystal violet staining assay (SIRC-CVS), were evaluated as alternatives to the Draize eye irritation test (Draize test).The cytotoxicity of thirty-eight cosmetic ingredients as well as a physiological saline solution was determined on SIRC cells at five to seven laboratories. SIRC-NRU and SIRC-CVS were performed according to the common standard operating procedure (SOP). The 50% effective concentration (EC(50)) was determined for each ingredient. The EC(50) of SIRC-CVS was similar to that of SIRC-NRU, showing a strong correlation (r=0.995). The coefficient of variation (CV) of EC(50) which represents the interlaboratory reproducibility of SIRC-NRU was 32.1%, whereas that of SIRC-CVS was 32.8%. The logarithmically transformed EC(50) values showed a strong correlation with the maximal average Draize total score (MAS) (SIRC-NRU: r=-0.816 (n=30), SIRC-CVS: r=-0.805 (n=29)). Both methods could be applied to water-insoluble substances and dyes. However, strong acids, alkanolamines and alcohols had a tendency to deviate from the linear regression lines which were obtained from the in vivo and in vitro data for both methods in the present study. These results suggest that cytotoxicological testing on SIRC cells may provide an alternative method to the Draize test for cosmetic ingredients.

Induction of tumor necrosis factor alpha and nitric oxide in rabbits inoculated with a cyst extract of Sarcocystis cruzi.

Rabbits develop a toxic reaction similar to endotoxemia following inoculation with a Sarcocystis cruzi cyst extract. To analyze the pathophysiology of the reaction, serum tumor necrosis factor alpha (TNFalpha), nitric oxide (NO) and lipid-lipoprotein profiles were investigated in rabbits given the cyst extract and lipopolysaccharide (LPS) by subcutaneous inoculation. In animals given the cyst extract, overproduction of TNFalpha was detected, together with an increase in NO. The animals developed derangement of lipid metabolism, which was considered to have resulted from TNFalpha induction, consisting of elevated triglyceride and very low density lipoprotein levels and decreased high density lipoprotein (HDL) levels. Serum interleukin-6-like activity also increased transiently in the animals. Capability of the cyst extract to induce TNFalpha, NO and the lipid profile derangement were completely lost by boiling. In animals given LPS, TNFalpha was induced and HDL decreased moderately, without inactivation of those activities of LPS by boiling. These results indicated that S. cruzi cyst extract is a potent, but thermolabile inducer of TNFalpha and NO for rabbits. It is likely that TNFalpha and NO play important roles as mediators in the reaction associated with toxicity of the cyst extract in rabbits.