The Human Induced Pluripotent Stem Cell Test as an Alternative Method for Embryotoxicity Testing.

The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity rely mainly on animal studies, which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Here, human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized a fetal bovine serum (FBS)-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as beating frequency and area of beating cardiomyocytes by video analyses are measured. The embryotoxic positive and non-embryotoxic negative controls, 5-Fluorouracil (5-FU) and Penicillin G (PenG), respectively, were correctly assessed in the hiPS Test. More compounds need to be screened in the future for defining the assay's applicability domain, which will inform us of the suitability of the hiPS Test for detecting adverse effects of substances on embryonic development.

Comparison of intermittent and consecutive balneological outpatient treatment (hydrotherapy and peloidotherapy) in fibromyalgia syndrome: a randomized, single-blind, pilot study.

To compare the efficacy of intermittent and consecutive balneological outpatient treatment (hydrotherapy and peloidotherapy) in fibromyalgia syndrome (FMS). A parallel 1:1, single-blind, pilot study was performed. Patients were recruited from musculoskeletal disorders outpatient clinic. Eligible participants were patients aged 18-60, diagnosed as FMS according to ACR 2010 criteria. They were randomly assigned to either consecutive or intermittent treatment groups. Both groups received 20 min of full body immersion in a tap water pool at 38-39 °C and 30 min of mud pack application on the back region at 45 °C. Delivery of the treatment was five times weekly during 2 weeks in consecutive group and two times weekly during 5 weeks in intermittent group. The primary outcomes were pain intensity and the number of patients achieving a minimal clinically important difference (MCID) on Fibromyalgia Impact Questionnaire (FIQ) at the 1st month after the completion of the treatment. Statistical analyses were based on intention to treat method. The assessing physician was blinded. Pain intensity significantly decreased in all post-treatment evaluations of both groups (except after treatment in the intermittent group). There was no significant difference between the groups. MCID for FIQ was achieved in 6 (24%) patients in the consecutive group and 12 (48%) in the intermittent group at the 1st month. There was no statistical difference in the secondary judgment criteria. The consecutive and intermittent deliveries of balneological outpatient treatment (hydrotherapy and peloidotherapy) seem to have similar effects on the clinical status of patients with FMS.

Does addition of 'mud-pack and hot pool treatment' to patient education make a difference in fibromyalgia patients? A randomized controlled single blind study.

The aim of this randomized controlled single-blind study is to explore whether addition of mud-pack and hot pool treatments to patient education make a significant difference in short and mild term outcomes of the patients with fibromyalgia. Seventy women with fibromyalgia syndrome were randomly assigned to either balneotherapy with mud-pack and hot pool treatments (35) or control (35) groups. After randomization, five patients from balneotherapy group and five patients from control group were dropped out from the study with different excuses. All patients had 6-h patient education programme about fibromyalgia syndrome and were given a home exercise programme. The patients in balneotherapy group had heated pool treatment at 38 °C for 20 min a day, and mud-pack treatment afterwards on back region at 45 °C. Balneotherapy was applied on weekdays for 2 weeks. All patients continued to take their medical treatment. An investigator who was blinded to the intervention assessed all the patients before and after the treatment, at the first and the third months of follow-up. Outcome measures were FIQ, BDI and both patient's and physician's global assessments. Balneotherapy group was significantly better than control group at after the treatment and at the end of the first month follow-up assessments in terms of patient's and physician's global assessment, total FIQ score, and pain intensity, fatigue, non-refreshed awaking, stiffness, anxiety and depression subscales of FIQ. No significant difference was found between the groups in terms of BDI scores. It is concluded that patient education combined with 2 weeks balneotherapy application has more beneficial effects in patients with fibromyalgia syndrome as compared to patient education alone.

Characterization of the C17.2 cell line as testing system for endocrine disruption-induced developmental neurotoxicity.

Hormone signaling plays an essential role during fetal life and is vital for brain development. Endocrine-disrupting chemicals can interfere with the hormonal milieu during this critical time-period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the applicability of the neural progenitor C17. 2 cell-line, as an in vitro test system to aid in the detection of endocrine disruption (ED) induced DNT. For this, C17.2 cells were exposed during 10 days of differentiation to agonists and antagonists of the thyroid hormone (Thr), glucocorticoid (Gr), retinoic acid (Rar), retinoic x (Rxr), oxysterols (Lxr), estrogen (Er), androgen (Ar), and peroxisome proliferator activated delta (Pparß/δ) receptors, as well as to the agonist of the vitamin D (Vdr) receptor. Upon exposure and differentiation, neuronal morphology (neurite outgrowth and branching), and the percentage of neurons in culture were assessed by immunofluorescence. For this, the cells were incubated with Hoechst (nuclear staining) and stained for ßIII-tubulin (neuronal marker). The C17.2 cells were responsive to the Rar, Rxr and Pparß/δ agonists which decreased neurite outgrowth and branching. Additionally, exposure to the Gr agonist increased the number of cells differentiating into neurons, while exposure to the Rxr agonist had the opposite effect. With this approach, we have identified that the C17.2 cells are responsive to Gr, Rar, Rxr, and Pparß/δ agonists, hence contributing to the development of test systems for hazard assessment of ED-induced DNT.

Endocrine disrupting chemicals (EDCs) interfere with hormonal signaling. As hormones play a vital role for an organism's development, EDC exposure is of high concern. In European regulations, the use of a chemical can be restricted if its toxicity is mediated by hormonal interference. A number of EDCs affect brain development. However, in animal tests, it is impossible to prove that a chemical induces developmental neurotoxicity (DNT) via endocrine disruption (ED). Furthermore, the regulatory DNT tests require large amounts of animals. Thus, there is an urgent need for in vitro test systems to identify ED-induced DNT. Herein we present the development of such a method based on the murine neural progenitor cell-line C17.2 with which neuronal differentiation processes can be mimicked. We show that differentiation of C17.2 cells are sensitive to retinoid, glucocorticoid, and peroxisome proliferator activated receptor signaling disruption, thus providing an alternative method for identifying ED-induced DNT.

Molecular and Functional Characterization of Different BrainSphere Models for Use in Neurotoxicity Testing on Microelectrode Arrays.

The currently accepted methods for neurotoxicity (NT) testing rely on animal studies. However, high costs and low testing throughput hinder their application for large numbers of chemicals. To overcome these limitations, in vitro methods are currently being developed based on human-induced pluripotent stem cells (hiPSC) that allow higher testing throughput at lower costs. We applied six different protocols to generate 3D BrainSphere models for acute NT evaluation. These include three different media for 2D neural induction and two media for subsequent 3D differentiation resulting in self-organized, organotypic neuron/astrocyte microtissues. All induction protocols yielded nearly 100% NESTIN-positive hiPSC-derived neural progenitor cells (hiNPCs), though with different gene expression profiles concerning regional patterning. Moreover, gene expression and immunocytochemistry analyses revealed that the choice of media determines neural differentiation patterns. On the functional level, BrainSpheres exhibited different levels of electrical activity on microelectrode arrays (MEA). Spike sorting allowed BrainSphere functional characterization with the mixed cultures consisting of GABAergic, glutamatergic, dopaminergic, serotonergic, and cholinergic neurons. A test method for acute NT testing, the human multi-neurotransmitter receptor (hMNR) assay, was proposed to apply such MEA-based spike sorting. These models are promising tools not only in toxicology but also for drug development and disease modeling.

A human iPSC-based in vitro neural network formation assay to investigate neurodevelopmental toxicity of pesticides.

Proper brain development is based on the orchestration of key neurodevelopmental processes (KNDP), including the for­mation and function of neural networks. If at least one KNDP is affected by a chemical, an adverse outcome is expected. To enable a higher testing throughput than the guideline animal experiments, a developmental neurotoxicity (DNT) in vitro testing battery (DNT IVB) comprising a variety of assays that model several KNDPs was set up. Gap analysis revealed the need for a human-based assay to assess neural network formation and function (NNF). Therefore, we established the human NNF (hNNF) assay. A co-culture comprised of human induced pluripotent stem cell (hiPSC)-derived excitatory and inhibitory neurons as well as primary human astroglia was differentiated for 35 days on microelectrode arrays (MEA), and spontaneous electrical activity, together with cytotoxicity, was assessed on a weekly basis after washout of the compounds 24 h prior to measurements. In addition to the characterization of the test system, the assay was challenged with 28 com­pounds, mainly pesticides, identifying their DNT potential by evaluating specific spike-, burst-, and network parameters. This approach confirmed the suitability of the assay for screening environmental chemicals. Comparison of benchmark con­centrations (BMC) with an NNF in vitro assay (rNNF) based on primary rat cortical cells revealed differences in sensitivity. Together with the successful implementation of hNNF data into a postulated stressor-specific adverse outcome pathway (AOP) network associated with a plausible molecular initiating event for deltamethrin, this study suggests the hNNF assay as a useful complement to the DNT IVB.

The impact of biostatistics on hazard characterization using in vitro developmental neurotoxicity assays.

In chemical safety assessment, benchmark concentrations (BMC) and their associated uncertainty are needed for the toxicological evaluation of in vitro data sets. A BMC estimation is derived from concentration-response modelling and results from various statistical decisions, which depend on factors such as experimental design and assay endpoint features. In current data practice, the experimenter is often responsible for the data analysis and therefore relies on statistical software, often without being aware of the software default settings and how they can impact the outputs of data analysis. To provide more insight into how statistical decision-making can influence the outcomes of data analysis and interpretation, we have developed an automated platform that includes statistical methods for BMC estimation, a novel endpoint-specific hazard classification system, and routines that flag data sets that are outside the applicability domain for an automatic data evaluation. We used case studies on a large dataset produced by a developmental neurotoxicity (DNT) in vitro battery (DNT IVB). Here we focused on the BMC and its confidence interval (CI) estimation as well as on final hazard classification. We identified five crucial statistical decisions the experimenter must make during data analysis: choice of replicate averaging, response data normalization, regression modelling, BMC and CI estimation, and choice of benchmark response levels. The insights gained are intended to raise more awareness among experimenters on the importance of statistical decisions and methods but also to demonstrate how important fit-for-purpose, internationally harmonized and accepted data evaluation and analysis procedures are for objective hazard classification.

Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals.

Developmental neurotoxicity (DNT) is a major safety concern for all chemicals of the human exposome. However, DNT data from animal studies are available for only a small percentage of manufactured compounds. Test methods with a higher throughput than current regulatory guideline methods, and with improved human relevance are urgently needed. We therefore explored the feasibility of DNT hazard assessment based on new approach methods (NAMs). An in vitro battery (IVB) was assembled from ten individual NAMs that had been developed during the past years to probe effects of chemicals on various fundamental neurodevelopmental processes. All assays used human neural cells at different developmental stages. This allowed us to assess disturbances of: (i) proliferation of neural progenitor cells (NPC); (ii) migration of neural crest cells, radial glia cells, neurons and oligodendrocytes; (iii) differentiation of NPC into neurons and oligodendrocytes; and (iv) neurite outgrowth of peripheral and central neurons. In parallel, cytotoxicity measures were obtained. The feasibility of concentration-dependent screening and of a reliable biostatistical processing of the complex multi-dimensional data was explored with a set of 120 test compounds, containing subsets of pre-defined positive and negative DNT compounds. The battery provided alerts (hit or borderline) for 24 of 28 known toxicants (82% sensitivity), and for none of the 17 negative controls. Based on the results from this screen project, strategies were developed on how IVB data may be used in the context of risk assessment scenarios employing integrated approaches for testing and assessment (IATA).

Balneotherapy in fibromyalgia: a single blind randomized controlled clinical study.

We aimed to evaluate the effectiveness of balneotherapy in fibromyalgia management. Fifty women with fibromyalgia under pharmacological treatment were randomly assigned to either the balneotherapy (25) or the control (25) group. Four patients from the balneotherapy group and one patient from the control group left the study after randomization. The patients in the balneotherapy group (21) had 2 thermomineral water baths daily for 2 weeks in Tuzla Spa Center. The patients in the control group (24) continued to have their medical treatment and routine daily life. An investigator who was blinded to the study arms assessed the patients. All patients were assessed four times; at the beginning of the study, at the end of the 2nd week, the 1st month, and the 3rd month after balneotherapy. Outcome measures of the study were pain intensity, Fibromyalgia Impact Questionnaire (FIQ), Beck Depression Inventory (BDI), patient's global assessment, investigator's global assessment, SF-36 scores, and tender point count. Balneotherapy was found to be superior at the end of the cure period in terms of pain intensity, FIQ, Beck Depression Inventory, patient's global assessment, investigator's global assessment scores, and tender point count as compared to the control group. The superiority of balneotherapy lasted up to the end of the 3rd month, except for the Beck Depression Inventory score and the investigator's global assessment score. Significant improvements were observed in PF, GH, and MH subscales of SF-36 during the study period in the balneotherapy group; however, no such improvement was observed in the control group. Balneotherapy was superior only in VT subscale at the end of therapy and at the end of the third month after the therapy as compared to the controls. It was concluded that balneotherapy provides beneficial effects in patients with fibromyalgia.

Effect of balneotherapy on temporospatial gait characteristics of patients with osteoarthritis of the knee.

Effects of balneotherapy on gait properties of patients with osteoarthritis of the knee were investigated prospectively. A total of 30 patients with knee osteoarthritis received balneotherapy consisting of two daily thermomineral water baths for 2 weeks. Patients were evaluated using gait analysis and clinical scores, both within 2 weeks, before and after spa treatment. Patients were walking faster in their control analyses (0.81 +/- 0.21 to 0.89 +/- 0.19 m/s; P = 0.017), with a shorter mean stance time (63.0 +/- 3.3 to 61.8 +/- 2.5% stride; P = 0.007), an increased cadence (96 +/- 13.1 to 100 +/- 11.9 steps/min; P = 0.094) and stride length (996 +/- 174 to 1,058 +/- 142 mm; P = 0.017). Balneotherapy also resulted in a significant decrease in Lequesne knee osteoarthritis index (12.1 +/- 3.7 to 10.0 +/- 3.3 points; P = 0.003), VAS for pain (58 +/- 25 to 33 +/- 15; P = 0.0001), VAS for patients' (56 +/- 24 to 29 +/- 19; P < 0.001) and investigator's global assessment (55 +/- 20 to 26 +/- 15; P < 0.0001) and WOMAC score (2.1 +/- 0.7 to 1.6 +/- 0.8; P = 0.0004). Balneotherapy has positive effects on gait properties and clinical health quality parameters of patients with knee osteoarthritis in short-term evaluations.

A 10-day course of SPA therapy is beneficial for people with severe knee osteoarthritis. A 24-week randomised, controlled pilot study.

The objective of this study was to test if spa therapy can play a role in the management of severe knee osteoarthritis (OA). Twenty patients with radiologically and clinically severe knee OA were randomly assigned into spa and drug therapy groups. Spa group (n = 10) traveled to a spa town and stayed at a hotel for a 10-day spa therapy course. They followed a balneotherapy regimen including thermal pool baths at 37 degrees C for 20 min two times daily. Drug therapy group (n = 10) stayed at home and followed their individually prescribed drug therapy (NSAIDs and paracetamol). Patients were assessed at baseline (week 0), after spa therapy at 2 weeks (week 2) and during follow-up period at 12 (week 12) and 24 (week 24) weeks by a blinded investigator. Patients assessed with Lequesne algofunctional index (LAFI), pain (visual analogue scale, VAS), patient's and investigator's global evaluation (VAS), ten-stairs stepping up and down time, 15 m walking time and three times squatting up and down time. Significant improvement in pain and LAFI scores were found at week 2, week 12 and week 24 in the spa therapy group compared to baseline. Comparing the two group differences, spa therapy was superior to drug therapy in pain reduction and in physician's global assessment at all time points. This superiority was also found in LAFI scores and patients' global assessments at week 12 and week 24. A 10-day course of spa therapy may be beneficial in short- and medium-term up to 24 weeks by reducing pain and improving functional status and overall well-being in patients with severe knee OA and may be considered as an effective therapeutic tool for such patients in countries like Turkey where it is widely available and (at least partly) reimbursed.

SPA therapy in fibromyalgia: a randomised controlled clinic study.

OBJECTIVE: The aim of the present study is to evaluate the effectiveness of spa therapy in the management of fibromyalgia. METHODS: Thirty women with fibromyalgia were randomly assigned to either a spa therapy group or a control group. The spa therapy group (n = 16) had spa treatment for 2 weeks in addition to their medical treatment. The control group (n = 14) continued to have their medical treatment and/or daily exercises. An investigator who was blinded for the intervention assessed all the patients for 9 months. Improvements in Fibromyalgia Impact Questionnaire (FIQ), pain and number of tender points were primary outcomes. Secondary outcome measures were improvement in sleep disturbance, fatigue, gastrointestinal symptoms, anxiety, Beck Depression Inventory and patient's global evaluation. RESULTS: the spa group was found to be superior to the control group at the end of intervention in terms of FIQ, pain, tender point count, fatigue and patients' global assessment. This superiority remained for 6 months in FIQ, 1 month in pain and tender point count. CONCLUSION: It was concluded that the addition of spa therapy to medical therapy has both short- and long-term beneficial effects in female patients with fibromyalgia.