The development of an automated microscope image tracking and analysis system.

Microscopy image analysis plays a crucial role in understanding cellular behavior and uncovering important insights in various biological and medical research domains. Tracking cells within the time-lapse microscopy images is a fundamental technique that enables the study of cell dynamics, interactions, and migration. While manual cell tracking is possible, it is time-consuming and prone to subjective biases that impact results. In order to solve this issue, we sought to create an automated software solution, named cell analyzer, which is able to track cells within microscopy images with minimal input required from the user. The program of cell analyzer was written in Python utilizing the open source computer vision (OpenCV) library and featured a graphical user interface that makes it easy for users to access. The functions of all codes were verified through closeness, area, centroid, contrast, variance, and cell tracking test. Cell analyzer primarily utilizes image preprocessing and edge detection techniques to isolate cell boundaries for detection and analysis. It uniquely recorded the area, displacement, speed, size, and direction of detected cell objects and visualized the data collected automatically for fast analysis. Our cell analyzer provides an easy-to-use tool through a graphical user interface for tracking cell motion and analyzing quantitative cell images.

Reference intervals for reticulocyte count and derived reticulocyte parameters in a cohort of healthy adults.

INTRODUCTION: Reticulocyte count and novel derived parameters provide insight into the effectiveness of erythropoiesis and may be useful tools in the classification and diagnosis of anemias. However, there is no standardisation, so we consider it necessary that each laboratory evaluates the parameters according to its own methodology and instrumentation and establishes its own reference ranges. Our aim was to establish the reference intervals (RIs) of reticulocyte profile provided by the Beckman Coulter DxH 900 haematological autoanalyzer in our reference population. METHODS: One hundred and seventy-five healthy adults (18 to 62 years) were included. Subjects were collected from the blood donation centre of the Hospital Clínico San Carlos (Madrid, Spain) upon informed consent. Whole blood was collected and assayed for 14 haematological parameters on the Beckman Coulter DxH 900 analyzer in the haematology laboratory of the Clinical Analysis Department. RIs were established as per Clinical and Laboratory Standards Institute EP28-A3c guidelines using three different statistical approaches. RESULTS: RIs estimated using the non-parametric method and the Harrell-Davis bootstrap method were very similar. RIs estimated by the robust method were narrower. Gender partitioning was required for two haematological parameters (low haemoglobin density (LHD) and microcytic anaemia factor (MAF)). The rest of the parameters did not need to be partitioned according to Lahti's method. CONCLUSION: RIs have been established for 14 hematologic parameters of the reticulocyte profile for the Beckman Coulter DxH 900 haematology analyzer using a healthy cohort of adult subjects.

[Detection and identification of urinary crystals using an automated urine particle analyzer coupled with a digital particle imaging device]. / Détection et identification de cristaux dans les urines à l'aide d'un analyseur de cytologie urinaire couplé à un dispositif d'imagerie numérique.

Polarized light microscopy (POM) remains the gold standard for crystalluria analysis. However, such method is time consuming and requires well-trained staff. Here, to address this issue, we tested the Sysmex UF-4000 analyzer coupled to a UD10 module as an automated flow cytometry-digital particle imaging workflow to assess (i) the ability of the system to detect and identify the crystals species and (ii) the quality of the images provided by the UD-10 module (n = 40) for each urine sample analyzed. First, systematic analysis of 76 samples by POM and the UF-4000/UD-10 analyzer showed that only attentive examination of the 40 photos was able to confidently detect crystalluria-positive samples with no misses and thus serve to discriminate positive-test crystalluria from negative-test crystalluria. These first results were confirmed by sensitivity analysis and the negative predictive value calculated on 200 samples for the results provided by the UF-4000 (39% and 46%) and after examination of the 40 UD-10 photos (100% for the both values). Digital images can therefore serve to screen crystalluria without missing crystals. A part of samples were treated by POM whereas it was not necessary (positive predictive value: 78%). Finally, we compared the crystal identification performances of the Sysmex UF4000/UD10 workflow and the 'gold standard' POM method on 131 urine samples containing crystals. Only calcium oxalate dihydrate crystals were identified by the Sysmex UF-4000. A close examination of the digital photographs enabled exact identification of crystals in 84.7% of the samples, suggesting however that POM is still require as soon as crystals are observed on the photographs. We conclude that a SYSMEX UF-4000 coupled with a UD-10 module can be used in practice with close examination of the photographs to discriminate positive crystalluria from negative crystalluria.

A Clinical Trial to Verify the Efficiency of the LC-1000 Exfoliative Cell Analyzer as a New Method of Cervical Cancer Screening.

OBJECTIVE: The exfoliative cell analyzer, LC-1000 (Sysmex Corporation, Japan), is a medical device that presents the cell proliferation index and 23 research parameters as indicators of cellular proliferative potential. The objective was to evaluate the clinical usability of qualitative assessment by LC-1000 compared with cytology, the human papillomavirus (HPV) test, and histology as gold standard. STUDY DESIGN: Women that visited 3 sites between July 2015 and March 2017 were registered. The primary endpoint in this study was the comparison between LC-1000 measurement and HPV test for sensitivity and specificity for cervical intraepithelial neoplasia 2+ (CIN2+). A tree model algorithm was newly constructed by a statistical method and its relationship with histological results was evaluated. RESULTS: The sensitivity and specificity of LC-1000 were 78.3 and 74.1%, while those of the HPV test were 94.7 and 85.4%, respectively. A tree model comprising five categories was constructed. The proportion of advanced lesions was higher with the change in the rank classification results from 1 to 5. The positive predictive values of CIN2+ in the categories 4 and 5 were high. Despite the small number of subjects, cancer was undetected in categories 1 and 2. In addition, the comparison with follow-up results in 19 women assessed as CIN1 showed that the rate of progression in the categories 3-5 was 50% (7/14); progression in the categories 1 and 2 was 0% (0/5). CONCLUSIONS: LC-1000 may be useful for cervical cancer screening as an index to qualitatively evaluate CIN and cancer based on the changes in characteristics of cells.

Real-time assessment of platinum sensitivity of primary culture from a patient with ovarian cancer with extensive metastasis and the platinum sensitivity enhancing effect by metformin.

The aim of the present study was to perform a rapid evaluation of the efficiency of commonly used platinum-based chemotherapy regimens for patients with ovarian cancer with extensive metastases using an in vitro method combined with culturing primary cells and real-time monitoring, and to further explore the enhanced effect of metformin on susceptibility of ovarian cancer cells to platinum-based chemotherapy. The primary omental metastatic (OM) cells were isolated from the omentum metastasis of a surgical patient with stage IIIc ovarian carcinoma. Drug sensitivity was evaluated using the xCELLigence system, and screening of the most effective platinum chemotherapy was performed through analysis of cell susceptibility to cisplatin, carboplatin, nedaplatin and paclitaxel or docetaxel alone or in combination. At the same time, this system was used to determine whether metformin was able to increase the sensitivity of cancer cells to platinum chemotherapy. The results revealed that nedaplatin exhibited the most marked cytotoxic effect on the OM cells, followed by those of carboplatin and cisplatin. The addition of docetaxel enhanced the cytotoxic effect, and the combination of platinum and paclitaxel also enhanced the effect. Metformin rapidly increased the sensitivity of cells to platinum-based chemotherapy, and this effect was dose-dependent. The sensitivity of OM cells to different platinum-based regimens was varied. The effect of metformin on chemotherapeutic sensitization of cancer cells is clear in vitro, and the real-time cell analyzer assay has the potential to assist in determining individualized drug regimens for patients with metastatic ovarian cancer.

Identification of Positively and Negatively Selected Driver Gene Mutations Associated With Colorectal Cancer With Microsatellite Instability.

Background & Aims: Recent studies have shown that cancers arise as a result of the positive selection of driver somatic events in tumor DNA, with negative selection playing only a minor role, if any. However, these investigations were concerned with alterations at nonrepetitive sequences and did not take into account mutations in repetitive sequences that have very high pathophysiological relevance in the tumors showing microsatellite instability (MSI) resulting from mismatch repair deficiency investigated in the present study. Methods: We performed whole-exome sequencing of 47 MSI colorectal cancers (CRCs) and confirmed results in an independent cohort of 53 MSI CRCs. We used a probabilistic model of mutational events within microsatellites, while adapting pre-existing models to analyze nonrepetitive DNA sequences. Negatively selected coding alterations in MSI CRCs were investigated for their functional and clinical impact in CRC cell lines and in a third cohort of 164 MSI CRC patients. Results: Both positive and negative selection of somatic mutations in DNA repeats was observed, leading us to identify the expected true driver genes associated with the MSI-driven tumorigenic process. Several coding negatively selected MSI-related mutational events (n = 5) were shown to have deleterious effects on tumor cells. In the tumors in which deleterious MSI mutations were observed despite the negative selection, they were associated with worse survival in MSI CRC patients (hazard ratio, 3; 95% CI, 1.1-7.9; P = .03), suggesting their anticancer impact should be offset by other as yet unknown oncogenic processes that contribute to a poor prognosis. Conclusions: The present results identify the positive and negative driver somatic mutations acting in MSI-driven tumorigenesis, suggesting that genomic instability in MSI CRC plays a dual role in achieving tumor cell transformation. Exome sequencing data have been deposited in the European genome-phenome archive (accession: EGAS00001002477).

Rapid diagnosis of mixed phenotype acute leukemia after identifying a blood histogram abnormality.

A 38-year-old woman was suffering from back, right arm, and ankle joint pain, and visited our emergency department. Upon admission, the white blood cell (WBC) count was high (11,700/µL), and low numbers of red blood cells (2.21 × 106/µL) and platelets (PLTs) (42,000/µL) were observed. A PLT histogram showed an abnormally shaped peak at around 20-30 fL, suggesting the presence of giant PLTs or PLT aggregation. The WBC histogram showed abnormal elevation at 35 fL and around 100 fL, suggesting abnormal cells including nucleated red blood cells. A peripheral blood smear was prepared, and morphology was examined. As a result, blasts (4%) including many orthochromatic erythroblasts (48/100 WBCs) were observed. Acute leukemia was suspected, and the patient was transferred the next day to a hospital with a hematology department. Bone marrow aspiration revealed that 99% of cells were blasts positive for B lymphoid lineage markers and myeloperoxidase. The patient was diagnosed with mixed phenotype lineage acute leukemia, treated immediately, and achieved remission. Thus, careful observation of histogram abnormalities of an automatic blood cell analyzer is important for rapid diagnosis of acute leukemia.

Performance evaluation of an automated image-based fluorescence CD4+ cell analyzer.

BACKGROUND: Although AIDS-related mortality has declined since the introduction of antiretroviral therapy (ART), HIV/ AIDS patients are predominantly present in developing countries that lack high-cost diagnostic devices and human expertise. OBJECTIVE: New methods for counting CD4+ cells cost-effectively are needed to replace conventional flow cytometry-based diagnosis. METHODS: We developed a CD4+ cell analyzer, ADAMII, which is a benchtop fluorescence image-based CD3+/4+ cell counting analyzer. It bears a three-channel light source and performs CD3+/4+ counting assays. The automatic 3D stage captures a maximum of 136 images that are subsequently processed and analyzed using a software integrated into the system. RESULTS: Results obtained using ADAMII were compared with data obtained by conventional methods using a FACSCalibur flow cytometer and the point-of-care PIMA CD4 analyzer. Both comparisons between ADAMII vs. FACS and ADAMII vs. PIMA data yielded a strong correlation with an R2 value of 0.98, which ensures the feasibility of CD4 test by ADAMII. CONCLUSIONS: The proposed method using ADAMII can be easily employed in resource-limited areas to replace conventional flow cytometers, which are expensive and require highly trained staff.

Task-shifting of CD4 T cell count monitoring by the touchscreen-based Muse™ Auto CD4/CD4% single-platform system for CD4 T cell numeration: Implication for decentralization in resource-constrained settings.

BACKGROUND: The accuracy of CD4 T cell monitoring by the recently developed flow cytometry-based CD4 T cell counting Muse™ Auto CD4/CD4% Assay analyzer (EMD Millipore Corporation, Merck Life Sciences, KGaA, Darmstadt, Germany) was evaluated in trained lay providers against laboratory technicians. METHODS: After 2 days of training on the Muse™ Auto CD4/CD4% analyzer, EDTA-blood samples from 6 HIV-positive and 4 HIV-negative individuals were used for CD4 T cell counting in triplicate in parallel by 12 trained lay providers as compared to 10 lab technicians. RESULTS: Mean number of CD4 T cells in absolute number was 829 ±â€¯380 cells/µl by lay providers and 794 ±â€¯409 cells/µl by technicians (P > 0.05); and in percentage 36.2 ±â€¯14.8%CD4 by lay providers and 36.1 ±â€¯15.0%CD4 by laboratory technician (P > 0.05). The unweighted linear regression and Passing-Bablok regression analyses on CD4 T cell results expressed in absolute count revealed moderate correlation between CD4 T cell counts obtained by lay providers and lab technicians. The mean absolute bias measured by Bland-Altman analysis between CD4 T cell/µl obtained by lay providers and lab technicians was -3.41 cells/µl. Intra-assay coefficient of variance (CV) of Muse™ Auto CD4/CD4% in absolute number was 10.1% by lay providers and 8.5% by lab technicians (P > 0.05), and in percentage 5.5% by lay providers and 4.4% by lab technicians (P > 0.05). The inter-assay CV of Muse™ Auto CD4/CD4% in absolute number was 13.4% by lay providers and 10.3% by lab technicians (P > 0.05), and in percentage 7.8% by lay providers and 6.9% by lab technicians (P > 0.05). CONCLUSIONS: The study demonstrates the feasibility of CD4 T cell counting using the alternative flow cytometer Muse™ Auto CD4/CD4% analyzer by trained lay providers and therefore the practical possibility of decentralization CD4 T cell counting to health community centers.

A Field-Portable Cell Analyzer without a Microscope and Reagents.

This paper demonstrates a commercial-level field-portable lens-free cell analyzer called the NaviCell (No-stain and Automated Versatile Innovative cell analyzer) capable of automatically analyzing cell count and viability without employing an optical microscope and reagents. Based on the lens-free shadow imaging technique, the NaviCell (162 × 135 × 138 mm³ and 1.02 kg) has the advantage of providing analysis results with improved standard deviation between measurement results, owing to its large field of view. Importantly, the cell counting and viability testing can be analyzed without the use of any reagent, thereby simplifying the measurement procedure and reducing potential errors during sample preparation. In this study, the performance of the NaviCell for cell counting and viability testing was demonstrated using 13 and six cell lines, respectively. Based on the results of the hemocytometer (de facto standard), the error rate (ER) and coefficient of variation (CV) of the NaviCell are approximately 3.27 and 2.16 times better than the commercial cell counter, respectively. The cell viability testing of the NaviCell also showed an ER and CV performance improvement of 5.09 and 1.8 times, respectively, demonstrating sufficient potential in the field of cell analysis.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.

Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin® ) and pertuzumab (Perjeta® ). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells. © 2017 International Society for Advancement of Cytometry.

Image-Based High Content Screening: Automating the Quantification Process for DNA Damage-Induced Foci.

Visual inspection of cellular activities based on conventional fluorescence microscope is a fundamental tool to study the role of DNA damage response (DDR). In the context of drug discovery where the capture of thousands of images is required across parallel experiments, this presents a challenge to data collection and analysis. Manual scoring is laborious and often reliant on trained personnel to intuit biological meaning through visual reasoning. On the other hand, high content screening combines the automation of microscopy image acquisition and analysis in a single platform to quantify cellular events of interests. The data generated is rapid and accurate, lessening the bias of human interpretation. Herein, this chapter will describe an image-based high content screen approach and the data analysis of Ataxia-Telangiectasia Mutated (ATM) DNA damage-induced foci.

In silico cytotoxicity assessment on cultured rat intestinal cells deduced from cellular impedance measurements.

Early and reliable identification of chemical toxicity is of utmost importance. At the same time, reduction of animal testing is paramount. Therefore, methods that improve the interpretability and usability of in vitro assays are essential. xCELLigence's real-time cell analyzer (RTCA) provides a novel, fast and cost effective in vitro method to probe compound toxicity. We developed a simple mathematical framework for the qualitative and quantitative assessment of toxicity for RTCA measurements. Compound toxicity, in terms of its 50% inhibitory concentration IC50 on cell growth, and parameters related to cell turnover were estimated on cultured IEC-6 cells exposed to 10 chemicals at varying concentrations. Our method estimated IC50 values of 113.05, 7.16, 28.69 and 725.15 µM for the apparently toxic compounds 2-acetylamino-fluorene, aflatoxin B1, benzo-[a]-pyrene and chloramphenicol in the tested cell line, in agreement with literature knowledge. IC50 values of all apparent in vivo non-toxic compounds were estimated to be non-toxic by our method. Corresponding estimates from RTCA's in-built model gave false positive (toxicity) predictions in 5/10 cases. Taken together, our proposed method reduces false positive predictions and reliably identifies chemical toxicity based on impedance measurements. The source code for the developed method including instructions is available at https://git.zib.de/bzfgupta/toxfit/tree/master.

Using Red Cell Indices and Reticulocyte Parameters for Carrier Screening of Various Thalassemia Syndromes.

Primary screening for thalassemia carriers usually involves an accurate blood count using an automated blood cell analyzer. We analyzed the red cell and reticulocyte parameters from 200 samples of various types of thalassemias and identified the discrimination criteria for differential diagnosis. These were separated into four groups based on genotypes. These groups included α-thalassemia, ß-thalassemia, ß-thalassemia with Hb E and Hb E trait, which are the important target of thalassemia screening. To compare the effectiveness of the screening strategies, seven selected screening tools were compared, including MCV alone (cutoff <80 fL); MRV alone (cutoff <100 fL); SD-C-NR alone (cutoff >28.5); a combined MCV and MRV; a combined MCV and SD-C-NR; a combined MRV and SD-C-NR; and a combined MCV, MRV and SD-C-NR. The combination of MCV, MRV and SD-C-NR has highest sensitivity for discrimination in all thalassemia, ß-thalassemia, α-thalassemia, Homozygous Hb E and Hb E trait groups as 99.5, 100, 98, 100 and 100 %, respectively. The effectiveness of the combination of MCV, MRV and SD-C-NR parameters in the present study was determined as the faster and higher sensitivity screening tool than the other methods, including simple, reliable, cost-effective, and using only one automated hematology analyzer, saving labor costs.

A general method to regenerate arrayed gold microelectrodes for label-free cell assay.

We developed a method to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes efficient treatment of the gold surface with trypsin (0.25%, v/v) digestion, rinsing with ethanol and deionized water and spinning steps. The proposed method ensured complete regeneration and repeated usage of gold microchips up to 4 times for the real-time electric impedance measurement of anti-cancer drug cytotoxicity.

Performance evaluation of the touchscreen-based Muse™ Auto CD4/CD4% single-platform system for CD4 T cell numeration in absolute number and in percentage using blood samples from children and adult patients living in the Central African Republic.

BACKGROUND: The new microcapillary and fluorescence-based EC IVD-qualified Muse™ Auto CD4/CD4% single-platform assay (EMD Millipore Corporation, Merck Life Sciences, KGaA, Darmstadt, Germany) for CD4 T cell numeration in absolute number and in percentage was evaluated using Central African patients' samples compared against the reference EC IVD-qualified BD FACSCount (Becton-Dickinson, USA) flow cytometer. METHODS: EDTA-blood samples from 124 adults, 10 adolescents, 13 children and 3 infants were tested in parallel at 2 reference laboratories in Bangui. RESULTS: The Muse™ technique was highly reproducible, with low intra- and inter-run variabilities less than 15%. CD4 T cell counts of Muse™ and BD FACSCount in absolute number and percentage were highly correlated (r2 = 0.99 and 0.98, respectively). The mean absolute bias between Muse™ and BD FACSCount cells in absolute number and percentage were -5.91 cells/µl (95% CI -20.90 to 9.08) with limits of agreement from -77.50 to 202.40 cells/µl, and +1.69 %CD4 (95% CI ±1.29 to +2.09), respectively. The percentages of outliers outside the limits of agreement were nearly similar in absolute number (8%) and percentage (10%). CD4 T cell counting by Muse™ allowed identifying the majority of individuals with CD4 T cell <200, <350 or <750 cells/µl corresponding to the relevant thresholds of therapeutic care, with sensitivities of 95.5-100% and specificities of 83.9-100%. CONCLUSIONS: The Muse™ Auto CD4/CD4% Assay analyzer is a reliable alternative flow cytometer for CD4 T lymphocyte enumeration to be used in routine immunological monitoring according to World Health Organization recommendations in HIV-infected adults as well as children living in resource-constrained settings.

Establishment of Age- and Gender-Specific Reference Ranges for 36 Routine and 57 Cell Population Data Items in a New Automated Blood Cell Analyzer, Sysmex XN-2000.

We established age- and gender-specific reference ranges for the 36 routine complete blood cell (CBC) and 57 cell population data (CPD) items in the Sysmex XN-2000 (Sysmex, Japan). In total, 280 peripheral blood samples were obtained from an equal number of healthy adults. Values for 36 routine items and 57 CPD items were obtained for each sample, and the results were categorized into six subgroups (N>39 in each subgroup) according to patient age (20-40, 41-60, and >60 yr) and gender (male and female), and compared with respect to age and gender differences. The majority of data items (22 of 36 routine CBC items and 44 of 57 CPD items) exhibited significant differences (P≤0.05) in their results with respect to age or gender, and several red cell-, lymphocyte-, and platelet-related data tended to decrease in women or older adults. These results provide a basis for establishing age- and gender-specific reference ranges for routine and CPD items in Sysmex XN-2000. Furthermore, these reference ranges could be used to determine clinical significance for new items of Sysmex XN-2000 in further studies.

Ocular safety comparison of non-steroidal anti-inflammatory eye drops used in pseudophakic cystoid macular edema prevention.

Non-steroidal anti-inflammatory drug (NSAID) eye drops are widely used to treat ocular inflammatory conditions related to ophthalmic surgical procedures, such as pseudophakic cystoid macular edema, and they have been used for off-label treatments. The most commonly used NSAIDs are diclofenac and ketorolac and the new molecules bromfenac and nepafenac have also been used. We used primary human keratocytes in cell culture in combination with a novel technology that evaluates dynamic real-time cytotoxicity through impedance analysis. This study also included classic cell viability tests (WST-1(®) and AlamarBlue(®)), wound healing assay, Hen's Egg Test and an ex vivo histopathological assay. NSAIDs were shown to have important cytotoxicities and to retard the healing response. Furthermore, the new eye drops containing bromfenac and nepafenac were more cytotoxic than the more classical eye drops. Nevertheless, no immuno-histochemical changes or acute irritation processes were observed after the administration of any eye drops tested. Due to cytotoxicity and the total absence of discomfort and observable injuries after the administration of these drugs, significant corneal alterations, such as corneal melts, can develop without any previous warning signs of toxicity.

Pathways commonly dysregulated in mouse and human obese adipose tissue: FAT/CD36 modulates differentiation and lipogenesis.

Obesity is linked to adipose tissue hypertrophy (increased adipocyte cell size) and hyperplasia (increased cell number). Comparative analyses of gene datasets allowed us to identify 1426 genes which may represent common adipose phenotype in humans and mice. Among them we identified several adipocyte-specific genes dysregulated in obese adipose tissue, involved in either fatty acid storage (acyl CoA synthase ACSL1, hormone-sensitive lipase LIPE, aquaporin 7 AQP7, perilipin PLIN) or cell adhesion (fibronectin FN1, collagens COL1A1, COL1A3, metalloprotein MMP9, or both (scavenger receptor FAT/CD36). Using real-time analysis of cell surface occupancy on xCELLigence system we developed a new method to study lipid uptake and differentiation of mouse 3T3L1 fibroblasts and human adipose stem cells. Both processes are regulated by insulin and fatty acids such as oleic acid. We showed that fatty acid addition to culture media increased the differentiation rate and was required for full differentiation into unilocular adipocytes. Significant activation of lipogenesis, i.e. lipid accumulation, by either insulin or oleic acid was monitored in times ranging from 1 to 24 h, depending on differentiation state, whereas significant effects on adipogenesis, i.e., surperimposed lipid accumulation and gene transcriptional regulations were measured after 3 to 4 d. Combination of selected times for analysis of lipid contents, cell counts, size fractionations, and gene transcriptional regulations showed that FAT/CD36 specific inhibitor AP5258 significantly increased cell survival of oleic acid-treated mouse and human adipocytes, and partially restored the transcriptional response to oleic acid in the presence of insulin through JNK pathway. Taken together, these data open new perspectives to study the molecular mechanisms commonly dysregulated in mouse and human obesity at the level of lipogenesis linked to hypertrophy and adipogenesis linked to hyperplasia.

In vitro and in vivo ocular safety and eye surface permanence determination by direct and Magnetic Resonance Imaging of ion-sensitive hydrogels based on gellan gum and kappa-carrageenan.

Gellan gum, kappa-carrageenan and alginates are natural polysaccharides able to interact with different cations that can be used to elaborate ion-activated in situ gelling systems for different uses. The interaction between fluid solutions of these polysaccharides and cations presents into the tear made these biopolymers very interesting to elaborate ophthalmic drug delivery systems. The main purpose of this study is to evaluate the ability of mixtures of these polymers to obtain ion-activated ophthalmic in situ gelling systems with optimal properties for ocular use. To achieve this purpose different proportion of the biopolymers were analyzed using a mixture experimental design evaluating their transparency, mechanical properties and bioadhesion in the absence and presence of simulated tear fluid. Tear induces a rapid sol-to-gel phase transition in the mixtures forming a consistent hydrogel. The solution composed by 80% of gellan gum and 20% kappa-carrageenan showed the best mechanical and mucoadhesive properties. This mixture was evaluated for rheological behavior, microstructure, cytotoxicity, acute corneal irritancy, ex-vivo and in vivo ocular toxicity and in vivo corneal contact time using Magnetic Resonance Images (MRI) techniques. Result indicates that the system is safe at ophthalmic level and produces an extensive ocular permanence higher than 6h.