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ABSTRACT We investigated whether the difference of antigen tube 2 (TB2) minus antigen tube 1 (TB1) (TB2−TB1) of the QuantiFERON-TB gold plus test, which has been postulated as a surrogate for the CD8+ T-cell response, could be useful in identifying recent tuberculosis (TB) exposure. We looked at the interferon gamma (IFN-γ) responses and differences in TB2 and TB1 tubes for 686 adults with QFT-plus positive test results. These results were compared among groups with high (368 TB contacts), low (229 patients with immune-mediated inflammatory diseases [IMID]), and indeterminate (89 asylum seekers or people from abroad [ASPFA]) risks of recent TB exposure. A TB2−TB1 value >0.6 IU·ml−1 was deemed to indicate a true difference between tubes. In the whole cohort, 13.6%, 10.9%, and 11.2% of cases had a TB2>TB1 result in the contact, IMID, and ASPFA groups, respectively (P = 0.591). The adjusted odds ratios (aORs) for an association between a TB2−TB1 result of >0.6 IU·ml−1 and risk of recent exposure versus contacts were 0.71 (95% confidence interval [CI], 0.31 to 1.61) for the IMID group and 0.86 (95% CI, 0.49 to 1.52) for the ASPFA group. In TB contact subgroups, 11.4%, 15.4%, and 17.7% with close, frequent, and sporadic contact had a TB2>TB1 result (P = 0.362). The aORs versus the close subgroup were 1.29 (95% CI, 0.63 to 2.62) for the frequent subgroup and 1.55 (95% CI, 0.67 to 3.60) for the sporadic subgroup. A TB2−TB1 difference of >0.6 IU·ml−1 was not associated with increased risk of recent TB exposure, which puts into question the clinical potential as a proxy marker for recently acquired TB infection. IMPORTANCE Contact tuberculosis tracing is essential to identify recently infected people, who therefore merit preventive treatment. However, there are no diagnostic tests that can determine whether the infection is a result of a recent exposure or not. It has been suggested that by using the QuantiFERON-TB gold plus, an interferon gamma (IFN-γ) release assay, a difference in IFN-γ production between the two antigen tubes (TB2 minus TB1) of >0.6 IU·ml−1 could serve as a proxy marker for recent infection. In this large multinational study, infected individuals could not be classified according to the risk of recent exposure based on differences in IFN-γ in TB1 and TB2 tubes that were higher than 0.6 IU·ml−1. QuantiFERON-TB gold plus is not able to distinguish between recent and remotely acquired tuberculosis infection, and it should not be used for that purpose in contact tuberculosis tracing.

A nosocomial case of Legionella pneumophila pneumonia likely caused by a serogroup 3 strain was detected by a urinary antigen test in Spain in 2018. Although Legionella bacteria could not be isolated from respiratory samples, molecular methods implicated the sink faucet of the patient’s room as the probable infection source.

The heterogeneity of Internet of Things (IoT) systems has so far prevented the definition of adequate standards, hence making it difficult to compare meaningfully the security degree of diverse architectural choices. This task can be nonetheless achieved with formal methodologies. However, the dedicated IoT literature shows no evidence of a universal model allowing the security evaluation of any arbitrary system. Based on these considerations, we propose a new model that aims at being global and all-encompassing. Our model can be used to fairly analyse the security level of different IoT systems and compare them in a significant way. It is designed to be adaptive with realistic definitions of the adversary’s (1) actions of interacting with IoT systems; (2) capabilities of accessing the data generated by and exchanged in IoT systems with established rules; and (3) objectives of attacking IoT systems according to the four recognised security properties of confidentiality, integrity, availability and soundness. Such a design enables the straightforward characterization of new adversaries. It further helps in providing a fine-grained security evaluation of IoT systems by either accurately describing attacks against the analysed systems or formally proving their guaranteed level of security.

The rise of wireless applications based on RFID has brought up major concerns on privacy. Indeed nowadays, when such an application is deployed, informed customers yearn for guarantees that their privacy will not be threatened. One formal way to perform this task is to assess the privacy level of the RFID application with a model. However, if the chosen model does not reflect the assumptions and requirements of the analyzed application, it may misevaluate its privacy level. Therefore, selecting the most appropriate model among all the existing ones is not an easy task. This paper investigates the eight most well-known RFID privacy models and thoroughly examines their advantages and drawbacks in three steps. Firstly, five RFID authentication protocols are analyzed with these models. This discloses a main worry: although these protocols intuitively ensure different privacy levels, no model is able to accurately distinguish them. Secondly, these models are grouped according to their features (e.g., tag corruption ability). This classification reveals the most appropriate candidate model(s) to be used for a privacy analysis when one of these features is especially required. Furthermore, it points out that none of the models are comprehensive. Hence, some combinations of features may not match any model. Finally, the privacy properties of the eight models are compared in order to provide an overall view of their relations. This part highlights that no model globally outclasses the other ones. Considering the required properties of an application, the thorough study provided in this paper aims to assist system designers to choose the best suited model.

The genus Acanthamoeba comprises facultative pathogens, causing Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). In both diseases, treatment options are limited, and drug development is challenging. This study aimed to investigate the role of the large thioredoxin reductase selenoprotein of Acanthamoeba (AcTrxR-L) as a potential drug target assessing the effects of the thioredoxin reductase inhibitors auranofin, TRi-1, and TRi-2 on AcTrxR-L activity and on the viability of Acanthamoeba trophozoites. Recombinant expression and purification of AcTrxR-L as a selenoprotein allowed assessments of its enzymatic activity, with reduction of various substrates, including different thioredoxin isoforms. Auranofin demonstrated potent inhibition towards AcTrxR-L, followed by TRi-1, and TRi-2 exhibiting lower effectiveness. The inhibitors showed variable activity against trophozoites in culture, with TRi-1 and TRi-2 resulting in strongly impaired trophozoite viability. Cytotoxicity tests with human corneal epithelial cells revealed lower susceptibility to all compounds compared to Acanthamoeba trophozoites, underscoring their potential as future amoebicidal agents. Altogether, this study highlights the druggability of AcTrxR-L and suggests it to be a promising drug target for the treatment of Acanthamoeba infections. Further research is warranted to elucidate the role of AcTrxR-L in Acanthamoeba pathogenesis and to develop effective therapeutic strategies targeting this redox enzyme.

Quais as principais ações estratégicas do atual governo do estado de São Paulo para a educação básica? Partindo desta questão, o objetivo do presente artigo é apresentar uma avaliação do primeiro ano do governo de Tarcísio de Freitas e das principais tendências para a educação básica verificadas nos discursos e ações efetuadas pelo executivo, em especial no que diz respeito ao avanço da racionalidade neoliberal na educação, às mudanças realizadas no ensino médio, às tecnologias na escola, ao financiamento e à gestão da educação. O tema foi abordado considerando um breve panorama do pensamento neoliberal e do seu projeto político educacional e as correspondentes ações de gestão escolar do governo estadual paulista. Os procedimentos de investigação foram a pesquisa bibliográfica e a documental, com análise do plano de governo para o Estado de São Paulo, do Mapa Estratégico da Secretaria de Estado da Educação e de portarias emitidas. A principal conclusão é a de que se encontra em curso um generalizado processo de consolidação da educação ligada aos princípios e valores privados e gerenciais no âmbito da educação pública no estado de São Paulo.

Abstract Background Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). Methods We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. Results We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. Conclusions This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis. Graphical Abstract

Abstract Background Homecare client services are often distributed across several interdependent healthcare providers, making proper care coordination essential. However, as studies exploring care coordination in the homecare setting are scarce, serious knowledge gaps exist regarding how various factors influence coordination in this care sector. To fill such gaps, this study’s central aim was to explore how external factors (i.e., financial and regulatory mechanisms) and homecare agency characteristics (i.e., work environment, workforce, and client characteristics) are related to care coordination in homecare. Methods This analysis was part of a national multicentre, cross-sectional study in the Swiss homecare setting that included a stratified random sample of 88 Swiss homecare agencies. Data were collected between January and September 2021 through agency and employee questionnaires. Using our newly developed care coordination framework, COORA, we modelled our variables to assess the relevant components of care coordination on the structural, process, and outcome levels. We conducted both descriptive and multilevel regression analyses—with the latter adjusting for dependencies within agencies—to explore which key factors are associated with coordination. Results The final sample size consisted of 1450 employees of 71 homecare agencies. We found that one explicit coordination mechanism (“communication and information exchange” (beta = 0.10, p

Casa Fantikorena en la localidad de Arteta (Navarra) acoge el Museo Etnográfico del Reino de Pamplona, una instalación artística por sí misma, que destaca por su gran colección de objetos históricos. Su objetivo es conservar la memoria del pasado a partir de los relatos de cada uno de los objetos recopilados por Joxe Ulibarrena Arellano a lo largo de toda una vida. Este patrimonio objetual, y esta forma de entender la cultura material, son el eje vertebrador de la propuesta pedagógica Museo Itinerante: 100 objetos para hacer Historia, una actividad que viaja a la escuela y que fomenta la interrogación y análisis de objetos cotidianos para reconstruir de forma significativa la historia reciente y conectarla a la sociedad a través de la Red.

Summary: Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.

Abstract Background Lung metastasis is the most adverse clinical factor and remains the leading cause of osteosarcoma-related death. Deciphering the mechanisms driving metastatic spread is crucial for finding open therapeutic windows for successful organ-specific interventions that may halt or prevent lung metastasis. Methods We employed a mouse premetastatic lung-based multi-omics integrative approach combined with clinical features to uncover the specific changes that precede lung metastasis formation and identify novel molecular targets and biomarker of clinical utility that enable the design of novel therapeutic strategies. Results We found that osteosarcoma-bearing mice or those preconditioned with the osteosarcoma cell secretome harbour profound lung structural alterations with airway damage, inflammation, neutrophil infiltration, and extracellular matrix remodelling with increased deposition of fibronectin and collagens by resident stromal activated fibroblasts, favouring the adhesion of disseminated tumour cells. Systemic-induced microenvironmental changes, supported by transcriptomic and histological data, promoted and accelerated lung metastasis formation. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins involved in extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, an extracellular matrix glycoprotein exclusively secreted by metastatic cells, in the plasma of mice bearing a primary tumour and in biopsy specimens from osteosarcoma patients with poorer overall survival. Depletion of EFEMP1 from the secretome prevents the formation of lung metastasis. Conclusions Integration of our data uncovers neutrophil infiltration and the functional contribution of stromal-activated fibroblasts in ECM remodelling for tumour cell attachment as early pro-metastatic events, which may hold therapeutic potential in preventing or slowing the metastatic spread. Moreover, we identified EFEMP1, a secreted glycoprotein, as a metastatic driver and a potential candidate prognostic biomarker for lung metastasis in osteosarcoma patients. Graphical abstract Osteosarcoma-derived secreted factors systemically reprogrammed the lung microenvironment and fostered a growth-permissive niche for incoming disseminated cells to survive and outgrow into overt metastasis. Daily administration of osteosarcoma cell secretome mimics the systemic release of tumour-secreted factors of a growing tumour in mice during PMN formation; Transcriptomic and histological analysis of premetastatic lungs revealed inflammatory-induced stromal fibroblast activation, neutrophil infiltration, and ECM remodelling as early onset pro-metastatic events; Proteome profiling identified EFEMP1, an extracellular secreted glycoprotein, as a potential predictive biomarker for lung metastasis and poor prognosis in osteosarcoma patients. Osteosarcoma patients with EFEMP1 expressing biopsies have a poorer overall survival.

During the harvesting of the broccoli plant, the leaves are discarded, being considered a by-product that may be up to 47% of total broccoli biomass, representing a large amount of wasted material. The use of broccoli leaves is of great interest in the sense that this wasted material is rich in health promoter compounds, such as isothiocyanates. In this study, C57BL/6J mice ingested 790 mg/kg broccoli leaf flour, and the presence of glucosinolates and isothiocyanates in the plasma, liver, kidney, adipose tissue, faeces and urine was analysed at 1, 2, 4, 8, 12 and 24 h post-ingestion. In plasma, only glucoerucin (GE), glucobrassicin (GB), sulforaphane (SFN) and indol-3-carbinol (I3C) were detected, and all four compounds peaked between 4 and 8 h after ingestion. The compounds SFN, SFN-glutathione (SFN-GSH), SFN–cysteine (SFN-CYS) and SFN-N-acetyl-cysteine (SFN-NAC) were excreted in faeces at high levels, while glucoraphanin (GR), the precursor of SFN, was not detected in any biological samples other than urine. In the liver, the compounds GE, SFN-CYS, SFN-NAC and I3C were detected, while in the kidney, only GE, GB and SFN-GSH were present. None of the glucosinolates and isothiocyanates analysed were detected in fat tissue. These results demonstrate that glucosinolates and their derivatives were absorbed into the bloodstream and were bioavailable after ingestion of powdered broccoli leaves.

The global rise in obesity and its co-morbidities raises worldwide health, social and economic concerns, especially in developed countries. Compounds derived from natural sources are now in the focus of pharmacological therapies. In recent years, sulforaphane (SFN) has been the subject of studies due to its anti-cancer, anti-inflammatory, antioxidant and potential anti-obesity effects. Lately, some studies have also reported the anti-obesogenic potential of chlorophyll. In this study, we evaluated the anti-obesity effects of SFN and chlorophyll a (Chlo.a) in C57BL/6J mice fed with a Western diet, rich in sugar and fat. The study lasted 14 weeks, and for the last 4 weeks SFN (0.25 or 0.5 mg/kg/day) or Chlo.a (0.2 or 0.5 mg/kg/day) was administered orally. The results showed that supplementation with SFN or Chlo.a resulted in an increase in body temperature and a reduction in the size of adipocytes. However, the administration of SFN or Chlo.a for 4 weeks did not decrease the body weight gain or hepatic steatosis, and increased hepatic ROS counterbalancing with an increase in SOD activity. In conclusion, in the animal model used, treatment with SFN or Chlo.a did not show strong anti-obesity effects; however, slight improvements were observed with the supplementation of these compounds.

Abstract Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease, but its fatality rate is high (96%) mainly because of delay in initiation of treatment due to misdiagnosis and lack of a specific treatment. The analysis of gene expression by quantitative real-time PCR in N. gruberi could be a highly efficient means to understand the pathogenicity of N. fowleri and also to find drug targets. Accurate RT-qPCR analysis requires correct normalization of gene expression data using reference genes (RG), whose expression should be constant under different experimental conditions. In this study, six genes, representing the most frequently used housekeeping genes, were selected for evaluation as reference genes in N. gruberi. The expression and stability of these genes was evaluated employing four algorithms (geNorm, NormFinder, BestKeeper and RefFinder). This work shows significant variations of the stability of RGs depending on the algorithms employed and on the experimental conditions (i.e. logarithmic, stationary, heat-shock and oxidative stress). The geNorm, NormFinder and RefFinder analysis of all the experimental conditions in combination revealed that ACT and G6PD were the most stable RGs. While BestKeeper analysis showed that 18S and TBP were the most stable RGs. Moreover, normalization of HSP90 gene expression with the most stable RGs resulted in an upregulation whereas when the normalization was done with the unstable RGs, the gene expression was not reliable. Hence, the implications of this study are relevant to gene expression studies in N. gruberi.

Macrofungi have been previously studied for their nutritional value and medicinal properties. However, despite wild mushrooms being a great source of beneficial bioactive compounds, the literature exploring their biotechnological application as nutraceuticals, cosmeceuticals and nutricosmetics is scarce. In this work, the species Butyriboletus regius, Ganoderma lucidum, Inonotus hispidus, Lanmaoa fragrans, Pisolithus tinctorius, Suillellus luridus, Suillellus mendax and Xerocomus subtomentosus were characterized according to their phenolic composition, antioxidant capacity, antimicrobial activity and cosmeceutical potential. For that purpose, dried and ground mushrooms were extracted with ethanol (40% v/v) using ultrasound-assisted extraction. Of the eight mushrooms analyzed, I. hispidus and P. tinctorius stood out for their high content of phenolic compounds, high antioxidant capacity and anti-hyaluronidase activity. Regarding antimicrobial activity, both mushrooms showed good inhibition of bacterial growth and bactericidal activity, especially on Gram-positive bacteria; however, L. fragans obtained the best results. Cream formulations with I. hispidus and P. tinctorius extracts in their composition improved their antioxidant activity. These results indicate that I. hispidus and P. tinctorius can be proposed as a new potential source of natural compounds with application in the cosmetic industry.

Exocrine pancreatic insufficiency (EPI) is caused by insufficient secretion or limited activity of pancreatic enzymes, especially lipase. Chronic pancreatitis (CP) is the most common EPI disease. CP patients suffer from gastrointestinal symptoms such as diarrhea, abdominal pain, and decreased uptake of nutrients as a consequence of inadequate food digestion. To improve their nutritional status, pancreatic enzyme supplements can be orally administered, known as pancreatic enzyme replacement therapy (PERT). PERT is commonly based on oral porcine preparations, despite their pH sensitivity which results in a limited timeframe for optimal activity. Instead, fungal enzymes could be used to prolong enzyme activity duration, since they are active over a wide pH range. The performance of fungal enzymes was compared to porcine enzymes by measuring total fatty acids under severe pancreatic insufficiency conditions in the tiny-TIMsg dynamic in vitro gastrointestinal model. Administration of 14.000 fungal lipase units (Nortase) led to similar lipid digestion compared to 20.000 porcine lipase units (Kreon and Pangrol). Additionally, opening the Nortase capsules had no negative effect on fungal lipase activity. This work demonstrates that fungal lipases, administered as capsules or powder, can be used as an alternative treatment for EPI patients.

Classically associated with gap junction-mediated intercellular communication, connexin43 (Cx43) is increasingly recognized to possess non-canonical biological functions, including gene expression regulation. However, the mechanisms governing the localization and role played by Cx43 in the nucleus, namely in transcription modulation, remain unknown. Using comprehensive and complementary approaches encompassing biochemical assays, super-resolution and immunogold transmission electron microscopy, we demonstrate that Cx43 localizes to the nuclear envelope of different cell types and in cardiac tissue. We show that translocation of Cx43 to the nucleus relies on Importin-β, and that Cx43 significantly impacts the cellular transcriptome, likely by interacting with transcriptional regulators. In vitro patch-clamp recordings from HEK293 and adult primary cardiomyocytes demonstrate that Cx43 forms active channels at the nuclear envelope, providing evidence that Cx43 can participate in nucleocytoplasmic shuttling of small molecules. The accumulation of nuclear Cx43 during myogenic differentiation of cardiomyoblasts is suggested to modulate expression of genes implicated in this process. Altogether, our study provides new evidence for further defining the biological roles of nuclear Cx43, namely in cardiac pathophysiology.

Herein, we report the preparation of a panel of Schiff bases analogues as antiprotozoal agents by modification of the stereoelectronic effects of the substituents on N-1 and N-4 and the nature of the chalcogen atom (S, Se). These compounds were evaluated towards Trypanosoma cruzi and Trichomonas vaginalis. Thiosemicarbazide 31 showed the best trypanocidal profile (epimastigotes), similar to benznidazole (BZ): IC50 (31)=28.72 μM (CL-B5 strain) and 33.65 μM (Y strain), IC50 (BZ)=25.31 μM (CL-B5) and 22.73 μM (Y); it lacked toxicity over mammalian cells (CC50 > 256 µM). Thiosemicarbazones 49, 51 and 63 showed remarkable trichomonacidal effects (IC50 =16.39, 14.84 and 14.89 µM) and no unspecific cytotoxicity towards Vero cells (CC50 ≥ 275 µM). Selenoisosters 74 and 75 presented a slightly enhanced activity (IC50=11.10 and 11.02 µM, respectively). Hydrogenosome membrane potential and structural changes were analysed to get more insight into the trichomonacidal mechanism.

Obesity is a disease whose incidence has increased over the last few decades. Despite being a multifactorial disease, obesity results essentially from excessive intake of high-calorie foods associated with low physical activity. The demand for a pharmacological therapy using natural compounds as an alternative to synthetic drugs has increased. Natural compounds may have few adverse effects and high economic impact, as most of them can be extracted from underexploited plant species and food by-products. To test the potential anti-obesogenic effects of new natural substances, the use of preclinical animal models of obesity has been an important tool, among which rat and mouse models are the most used. Some animal models are monogenic, such as the db/db mice, ob/ob mice, Zucker fatty rat and Otsuka Long-Evans Tokushima fatty rat. There are also available chemical models using the neurotoxin monosodium glutamate that induces lesions in the ventromedial hypothalamus nucleus, resulting in the development of obesity. However, the most widely used are the obesity models induced by high-fat diets. The aim of this review was to compile detail studies on the anti-obesity effects of natural compounds or their derivatives on rodent models of obesity as well as a critical analysis of the data.

Obesity, classified as an epidemic by the WHO, is a disease that continues to grow worldwide. Obesity results from abnormal or excessive accumulation of fat and usually leads to the development of other associated diseases, such as type 2 diabetes, hypertension, cancer, cardiovascular diseases, among others. In vitro and in vivo models have been crucial for studying the underlying mechanisms of obesity, discovering new therapeutic targets, and developing and validating new pharmacological therapies against obesity. Preclinical animal models of obesity comprise a variety of species: invertebrates, fishes, and mammals. However, small rodents are the most widely used due to their cost-effectiveness, physiology, and easy genetic manipulation. The induction of obesity in rats or mice can be achieved by the occurrence of spontaneous single-gene mutations or polygenic mutations, by genetic modifications, by surgical or chemical induction, and by ingestion of hypercaloric diets. In this review, we describe some of the most commonly used murine models in obesity research.

BackgroundIn vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity.MethodsThe viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy.ResultsIt was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity.DiscussionOur findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.