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IMPORTANCE: ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. OBJECTIVE: To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. DESIGN, SETTING, AND PARTICIPANTS: This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. INTERVENTIONS: In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. MAIN OUTCOMES AND MEASURES: The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). RESULTS: In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n = 36); ApTOLL, 0.2 mg/kg (n = 36), or placebo (n = 47) in a 1:1:√2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, −45%; 95% CI, −67% to −10%), smaller final infarct volume (mean difference log-transformed vs placebo, −42%; 95% CI, −66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). CONCLUSIONS AND RELEVANCE: In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04734548

An in silicomethod has been developed that permits the binary differentiation between pure liquids causing serious eye damage or eye irritation, and pure liquids with no need for such classification, according to the UN GHS system. The method is based on the finding that the Hansen Solubility Parameters (HSP) of a liquid are collectively important predictors for eye irritation. Thus, by applying a two-tier approach in which in silico-predicted pKa values (firstly) and a trained model based solely on in silico-predicted HSP data (secondly) were used, we have developed, and validated, a fully in silicoapproach for predicting the outcome of a Draize test (in terms of UN GHS Cat. 1/Cat. 2A/Cat. 2B or UN GHS No Cat.) with high validation set performance (sensitivity = 0.846, specificity = 0.818, balanced accuracy = 0.832) using SMILES only. The method is applicable to pure non-ionic liquids with molecular weight below 500 g/mol, fewer than six hydrogen bond donors (e.g. nitrogen–hydrogen or oxygen–hydrogen bonds) and fewer than eleven hydrogen bond acceptors (e.g. nitrogen or oxygen atoms). Due to its fully in silicocharacteristics, this method can be applied to pure liquids that are still at the desktop design stage and not yet in production.

This review article delves into the details of the 3R-Refinement principles as a vital framework for ethically sound rodent research laboratory. It highlights the core objective of the refinement protocol, namely, to enhance the well-being of laboratory animals while simultaneously improving the scientific validity of research outcomes. Through an exploration of key components of the refinement principles, the article outlines how these ethics should be implemented at various stages of animal experiments. It emphasizes the significance of enriched housing environments that reduce stress and encourage natural behaviors, non-restraint methods in handling and training, refined dosing and sampling techniques that prioritize animal comfort, the critical role of optimal pain management and the importance of regular animal welfare assessment in maintaining the rodents well-being. Additionally, the advantages of collaboration with animal care and ethics committees are also mentioned. The other half of the article explains the extensive benefits of the 3R-Refinement protocol such as heightened animal welfare, enhanced research quality, reduced variability, and positive feedback from researchers and animal care staff. Furthermore, it addresses avenues for promoting the adoption of the protocol, such as disseminating best practices, conducting training programs, and engaging with regulatory bodies. Overall, this article highlights the significance of 3R-Refinement protocol in aligning scientific advancement with ethical considerations along with shaping a more compassionate and responsible future for animal research.

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Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, non-tumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols — for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.

Contractions of guinea‐pig tracheal preparations to cysteinyl‐leukotrienes (LTC4, LTD4and LTE4) were characterized in organ baths, and cysteinyl‐leukotriene metabolism was studied using radiolabelled agonists and RP‐HPLC separation.In the presence of S‐hexyl GSH (100 μM) the metabolism of [3H]‐LTC4into [3H]‐LTD4was inhibited and the LTC4‐induced contractions were resistant to CysLT1receptor antagonism but inhibited by the dual CysLT1/CysLT2receptor antagonist BAY u9773 (0.3–3 μM) with a pA2‐value of 6.8±0.2.In the presence of L‐cysteine (5 mM), the metabolism of [3H]‐LTD4into [3H]‐LTE4was inhibited and the LTD4‐induced contractions were inhibited by the CysLT1receptor antagonist ICI 198,615 (1–10 nM) with a pA2‐value of 9.3±0.2. However, at higher concentrations of ICI 198,615 (30–300 nM) a residual contraction to LTD4was unmasked, and this response was inhibited by BAY u9773 (1–3 μM).In the presence of the combination of S‐hexyl GSH with L‐cysteine, the LTD4‐induced contractions displayed the characteristics of the LTC4contractile responses, i.e. resistant to CysLT1receptor antagonism, increased maximal contractions and slower time‐course. This qualitative change of the LTD4‐induced contraction was also observed in the presence of S‐decyl GSH (100 μM), GSH (10 mM) and GSSG (10 mM).S‐hexyl GSH, S‐decyl GSH, GSH and GSSG all stimulated a formation of [3H]‐LTC4from [3H]‐LTD4.In conclusion, GSH and GSH‐related compounds changed the pharmacology of the LTD4‐induced contractions by stimulating the conversion of LTD4into LTC4. Moreover, the results indicate that, in addition to the metabolism of LTC4into LTD4and LTE4, also the formation of LTC4from LTD4may regulate cysteinyl‐leukotriene function.

Using a novel dual-channel FACS methodology, the organotin compound TBT (2 μM) was shown to induce rapid (maximal by 3 min) and sustained elevations in intracellular calcium levels [Ca2+]i in Jurkat T cells. This was preceded by mitochondrial hyperpolarization (maximal at 1 min), with subsequent loss of membrane potential, (Δψm) over the next 15 min and was associated with the release of mitochondrial cytochrome c and the activation of type II caspases. The activation of the caspases was blocked by calcium chelation with EGTA and/or BAPTA. Interestingly, changes in Δψm caused by TBT were not affected by chelation of intra- and extracellular calcium or by performing the experiments in a Ca2+-free medium. TBT also caused rapid elevation of [Ca2+]i in cells lacking glycolytic ATP production. Despite this, the loss of Δψm and the activation of type II caspases were delayed (maximal by 2 h) in these cells. Further, there was a failure to activate type II caspases in cells treated with TBT in a Ca2+-free medium, despite rapid release of mitochondrial cytochrome c. Consequently, these cells evaded the induction of apoptosis and were diverted to delayed necrotic deletion. Taken together, these data strongly suggest that the rapid rise in [Ca2+]i caused by TBT in Jurkat T cells is not directly coupled to the induction of mitochondrial permeability transition, which rather results from a direct interaction of TBT with mitochondrial component(s) controlling pore transition. However, the rise in [Ca2+]i is a prerequisite for postmitochondrial events involved in caspase activation prior to the induction of apoptosis.

Diaryl tellurides carrying electron-donating substituents in the para positions were found to efficiently inhibit peroxidation of rat hepatocytes, rat liver microsomes and a chlorobenzene solution of phosphatidylcholine. The most active compound in the microsomal assay, bis(4-dimethylaminophenyl) tellurite, showed an IC50-value of 30 nM. This compound also caused a dose-dependent delay of the onset of the linear phase of microsomal peroxidation stimulated by iron/ADP/ascorbate. The peak oxidation potentials of the diaryl tellurides (0.50-1.14 V in MeCN) correlated linearly with the IC50-values in this assay, with a point of inflection around 0.85 V. In the hepatocyte system, all compounds showed similar protective activity. It is proposed that diaryl tellurides exert an antioxidative effect by deactivating both peroxides and peroxyl radicals under the formation of telluroxides. These oxides may regenerate the active divalent organotellurides upon exposure to a suitable reducing agent.

Smooth muscle cells have been isolated from human umbilical cord veins, characterized and cultured for the development of an endothelialsmooth muscle cell co-culture system. After harvesting endothelial cells, the umbilical cords were trimmed of amnion, connective tissue and arteries, split into pieces, cut open longitudinally and placed with the luminal surface of the explant down onto a culture plate, without the use of proteolytic enzymes. Adherent primary cells were sequentially passaged and various cytological/biochemical characterizations were performed between passages 2 and 10. Cells stained positive for antibodies against a smooth muscle actin, negative for antibodies against factor VIII and displayed typical ‘hill and valley’ morphology when confluent. Cell proliferation was stimulated and supported in a concentration-dependent manner by both human serum and fetal bovine serum over the range 1%–20%. The use of human serum at concentrations >10% decreased the population doubling time during exponential growth by circa 50%. The cells were also characterized by high seeding efficiencies (>70%) and retained their diploid karyotype for up to 3 months in culture. Endothelial cells and smooth muscle cells prepared from umbilical veins were then seeded at varying densities onto either side of porous tissue culture inserts coated with fibronectin. Utilising the measurement of electrical resistance, the optimal seeding density of 5×104 cells/cm2 for each cell type gave maximal resistance across the cell bi-layer already after 24 hours, which remaining essentially unaltered for up to 4 days of culture and which was always substantially higher than the resistance of filters seeded only with endothelial cells on one side. This was not substantially affected either by increasing passage of the HUVSM cells cultured with a fixed passage of endothelial cells, or by varying the donor origin of the endothelial cells. In terms of functionality of the selective permeability of the model, the calcium ionophore ionomycin (25 µM), added to the endothelial side of the bi-layer, caused a 30% reduction in the electrical resistance across the co-culture within 60 minutes, with control resistance being re-established within 1 hour of removal of the ionophore by washing. These results clearly indicate that smooth muscle cells and endothelial cells prepared from the same human blood vessel can be reconstituted into a functional vascular model suitable for the study of biochemical, physiological and toxicological phenomena in the human vascular wall.

Exposure of human umbilical vein endothelial cells to oxidants such as hydrogen peroxide, tert-butyl hydroperoxide and diamide has been shown to induce oxidant-specific S-thiolation of cellular proteins with glutathione. In this study we show that the diamide-induced S-thiolation of a 42 KDa protein is accompanied by cell shrinkage and the loss of cell to cell contact due to a structural reorganization of the cytoskeleton, as assessed by cytofluorometric visualization by confocal microscopy. Both events are simultaneously and rapidly reversed upon the removal of the stimulus. The 42 KDa protein was identified as actin by sequence analysis of the excised and electro-blotted protein band from SDS-PAGE gels, by immunochemical detection and by binding to immobilized deoxyribonuclease I. Additionally, we have shown that diamide exposure of the cells resulted in a S-thiolation of both polymerized F-actin and monomeric G-actin.

In this report the protein human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been examined to clarify the roles of (a) direct oxidation and (b) thiol-disulphide exchange (with glutathione disulphide) on the modification of its catalytic activity. Anin vitrosystem using purified human GAPDH and [35S]-GSSG (glutathione disulphide), has permitted clarification of these possibilities by showing that S-glutathionylation of GAPDH does not result in an inactivation of the enzyme. Rather, the direct oxidation of GAPDH with hydrogen peroxide is responsible for inhibition of the catalytic activity of the protein. Furthermore, pre-treatment of the enzyme with hydrogen peroxide enhances the formation of glutathione-GAPDH mixed disulphides in the presence of glutathione disulphide. This may serve as a molecular “switch” directing the protein to other reported functions in the cell. It is also shown that the efficiency of S-glutathionylation of either native or oxidised GAPDH is enhanced by the presence of recombinant glutaredoxin (thiol transferase) of either bacterial or human origin. Under the conditions of analysis the glutaredoxin itself is also shown to readily undergo S-glutathionylation external to its active site. Taken together, the data indicate the complexity of mechanisms likely to be involved in regulating cellular proteins during oxidative stress and implicate controlled enzyme-catalysed S-glutathionylation as a potential selectivity factor in the redox modification of protein function by glutathione.

Human alveolar macrophages (AMs) obtained from smokers and non-smokers by bronchoalveolar lavage (BAL) were subjected to various concentrations of NO2in an inverted monolayer exposure model. Culture super natants were collected 4 h after the exposure and assayed for secreted TNF-α, IL-1β, IL-8 and MIP-1α. The steady state levels of the mRNAs for these cytokines were also analysed in the cells. The adherence of BAL cells to plastic prior to exposure to the gas elevated the steady state mRNA levels of all four cytokines tested in smoker's cells and that of TNF-α and IL-1β, but not IL-8 (MIP-1α not tested), in non-smoker's cells. Interestingly, adherent cells from non-smokers released circa 15-, 3-,1.5- and 3-fold the amounts of IL-1β, IL-8, TNF-α and MIP-1α, respectively, than smoker's cells during control incubation or exposure to air. A 20 min exposure to NO2(5 or 20 p.p.m.) did not increase the secretion of any of the cytokines from either cell type. In contrast, NO2caused a concentration- dependent inhibition of the secretion of all cytokines except IL-1β from smoker's cells. Additionally, NO2greatly diminished the release of all cytokines in response to further treatment with lipopolysaccharide (LPS). In contrast, only the secretion of TNF-α from non-smoker's cells was inhibited by the gas in a concentration- dependent manner, whilst LPS-induced secretion of the cytokines was not affected by the gas. The steady state levels of the respective mRNAs for each of the cytokines were not significantly affected in smoker's cells by exposure to NO2, except for a negative, dose-dependent trend in the case of TNF-α. Nitrogen dioxide also failed to elevate the levels of the mRNAs in non-smoker's cells but, again, tended to diminish the levels, particularly of IL-1β mRNA. However, exposure to the gas inhibited LPS- induced accumulation of cytokine mRNAs in smoker's cells only. The data suggest that macrophage-derived cytokine mediators of the sepsis response may not play a role in the generation of NO2-induced inflammation in the human lung. Conversely, the gas seems to non-specifically inhibit the release and/or production of cytokines, particularly from smoker's cells, at the post-transcrip tional level, and impairs the ability of the cells to increase the transcription and release of the cytokines in response to bacterial LPS. The fact that NO2seriously impaired the already diminished capacity of smoker's cells to release several important pro-inflammatory cytokines, both under control conditions and in response to LPS, strongly suggest that the inhalation of NO2in cigarette smoke may contribute to impairing host defence against infection in the lung.

Fluorescence microscopy of A549 cells stained with a glutathione (L‐γ‐glutamyl‐Lcysteinylglycine, GSH)‐specific polyclonal antibody displayed uniform staining of the perinuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol‐reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)‐dependent inhibition of GSH synthesis. However, confocal visualization of antibody‐stained A549 cells in the z‐plane revealed the majority of the perinuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z‐plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z‐section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near‐complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z‐plane. The antibody also provided initial visualization of the compartmentalization of protein‐GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti‐GSH antibody‐stained Jurkat T lymhocytes, we also demonstrated population variations in the cellular compliment of GSH and protein‐GSH mixed disulfides, formed in response to diamide. In addition, we showed cell‐cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.

Muscle atrophy and cachexia are associated with many human diseases. These catabolic states are often associated with the loss of glutathione (GSH), which is thought to contribute to the induction of oxidative stress within the muscle. Glutathione synthesis and secretary characteristics were studied in human skeletal muscle myoblasts and myotube‐like cells derived from the myoblasts by growth factor restriction. Differentiation was associated with a shift in the sulfur amino acid precursor specificity for synthesis of GSH from cystine to cysteine, as well as loss in ability to use extracellular glutathione and activation of methionine use. The thiol drug N‐acetylcysteine was also shown to be an effective precursor irrespective of the state of differentiation. Additionally, myoblasts and myotube cultures were shown to secrete GSH continually, but only the differentiated cells responded to stress hormones such as glucagon, vasopressin, and phenylephrine, by increased secretion of the tripeptide. The data suggest that the skeletal muscle cells may provide an important hormonally regulated extra‐hepatic source of systemic GSH and also shed light on the mechanisms of accelerated turnover of GSH operating during strenuous muscle activity and trauma. The data may also provide biochemical rationales for the nutritional and/or pharmacological manipulation of GSH with sulfur amino acid precursors during the treatment of muscle‐specific oxidative stress and atrophy.

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