Human skin in vitro permeation of bentazon and isoproturon formulations with or without protective clothing suit.

Skin exposures to chemicals may lead, through percutaneous permeation, to a significant increase in systemic circulation. Skin is the primary route of entry during some occupational activities, especially in agriculture. To reduce skin exposures, the use of personal protective equipment (PPE) is recommended. PPE efficiency is characterized as the time until products permeate through material (lag time, Tlag). Both skin and PPE permeations are assessed using similar in vitro methods; the diffusion cell system. Flow-through diffusion cells were used in this study to assess the permeation of two herbicides, bentazon and isoproturon, as well as four related commercial formulations (Basagran(®), Basamais(®), Arelon(®) and Matara(®)). Permeation was measured through fresh excised human skin, protective clothing suits (suits) (Microchem(®) 3000, AgriSafe Pro(®), Proshield(®) and Microgard(®) 2000 Plus Green), and a combination of skin and suits. Both herbicides, tested by itself or as an active ingredient in formulations, permeated readily through human skin and tested suits (Tlag < 2 h). High permeation coefficients were obtained regardless of formulations or tested membranes, except for Microchem(®) 3000. Short Tlag, were observed even when skin was covered with suits, except for Microchem(®) 3000. Kp values tended to decrease when suits covered the skin (except when Arelon(®) was applied to skin covered with AgriSafe Pro and Microgard(®) 2000), suggesting that Tlag alone is insufficient in characterizing suits. To better estimate human skin permeations, in vitro experiments should not only use human skin but also consider the intended use of the suit, i.e., the active ingredient concentrations and type of formulations, which significantly affect skin permeation.

A new alternative method for testing skin irritation using a human skin model: a pilot study.

Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (Episkin®, Epiderm®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (Epiderm®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably evaluate reproducibility.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.