Addition Of Fetal Calf Serum Research Articles

Effect of delayed addition of fetal calf serum to the culture medium on in vitro embryo production from creole heifers raised in a high altitude in Ecuador

El estudio tuvo como objetivo evaluar el efecto del suero fetal bovino (FCS) agregado al medio de cultivo los días 5 a 7 después de la fertilización in vitro sobre la producción y posterior criotolerancia de blastocistos de vaquillonas criollas ecuatorianas. Se recolectaron complejos cúmulo-ovocitos (COCs) inmaduros mediante aspiración intravaginal guiada por ultrasonido (OPU) de 10 vaquillonas criollas y de ovarios de matadero en ocho sesiones. Los COCs se sometieron a maduración in vitro (MIV), fecundación (FIV) y cultivo (IVC). El día 1 después de la FIV, los presuntos embriones de cada fuente de ovocitos (OPU [O] y Matadero [A]) se asignaron aleatoriamente en dos grupos según si se añadió FCS al 2.5% (v/v) al medio de cultivo el día 5 después de la FIV: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+ y 4) A-FCS-. El día 7 después de la FIV se vitrificaron embriones de alta calidad. La criotolerancia de los embriones vitrificados-calentados se evaluó según la reexpansión del blastocele a las 2 h y la posterior reexpansión y eclosión a las 24 y 48 h de incubación. La tasa de blastocistos el día 7 no difirió entre la fuente de ovocitos y el grupo FCS. Después del proceso de vitrificación/calentamiento, la adición de FCS solo afectó la tasa de reexpansión a las 2 h, independientemente de la fuente de ovocitos (p<0.05). Asimismo, la tasa de eclosión de los blastocistos a las 48 h de incubación se vio drásticamente afectada únicamente en los ovocitos derivados de OPU (p<0.01). En conclusión, la suplementación de FCS el día 5 después de la FIV no mejoró la producción de blastocistos y afectó negativamente la criotolerancia de embriones bovinos in vitro derivados de vaquillonas criollas criadas en los Andes ecuatorianos.

P381 Kinetics of Titan cells generation and transcriptome modifications comparing three in vitro protocols

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesTo face and escape his environment and the host immune response, Candida neoformans is able to change his morphology (Titan cells) and his metabolism (dormancy, quiescence). Titan cells (TC) have been observed in lungs and brains in experimental mouse models of cryptococcosis and in patients. In 2018, three independent teams published three protocols allowing the reproducible generation of TC in vitro [Hommel B et al., (AA); Trevijano-Contador N, et al., (OZ), and Dambuza IM et al., (EB), in PLoS Pathogens 2018]. TC generated in these protocols exhibited the same features as the TC described in vivo. Our objective was to compare and describe the three protocols in parallel to highlight common and different features that can impact further study using those specific protocols.MethodsA total of 22 h before starting the kinetics of the TC generation, the three protocols requested a pre-culture at 30°C in three different liquid media. The medium for TC production for the three labs was also different but a common factor is the addition of fetal calf serum (FBS) for OZ and EB. This kinetics was evaluated for size and quantity (%) of TC produced over a 72 h period (H0, H18, H24, H48, H72) at 30°C under shaking for AA while OZ and EB protocols incubate the cells at 37°C and 5% of CO2, while the whole transcriptome was analyzed at H0, H3, H7, and H18 in triplicates.ResultsOZ generated the highest percentage of TC, 63.1% and 58.2% at H18 and H24, and decreased drastically down to 6.7% at H48. EB reached a high percentage of TC at H24 for 46.7% and dropdown <10% until the end of the kinetics. AA did not reach a quantity of TC as high as the two other protocols but it remained constant over a period of H72 (Table 1). RNA sequencing preliminary analysis showed some differences in genes expressed at the different time points analyzed. The PCA analysis revealed that the triplicates of each protocol for the 4-time points analyzed are closed to each other, related to the good quality of our experiments. The differential gene expression (DGE) showed significant (P <.01 and Log2 fold change >1) differences at H0 which highlights the impact of the preculture on the TC process. The highest numbers of DGE are observed between H0 and H7 for the three protocols, where about two 450 DGE, two 000 DGE, and two 300 AA, OZ, and EB, respectively. After analysis of the PCA plot during the kinetics, EB and OZ are grouped while AA is not. That could be explained by the presence of FBS in OZ and EB protocols.ConclusionBy running the three protocols in parallel, we showed here that the kinetics of TC generation differed between each other with a significant variation of the transcriptome. This is an important finding that paves the way to compare more deeply the transcriptome of C. neoformans during TC generation with the final goal is to identify the genes associated with TC generation.

CD200 Is a Potent New Growth Factor in Multiple Myeloma through IGF-1R Interaction

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the accumulation of clonal plasma cells within the bone marrow.We previously identified that CD200 is expressed in Multiple Myeloma Cells (MMCs) of 78% of newly MMdiagnosed patients and this expression is associated with a bad prognosis. CD200 is a type I immunoglobulin superfamily transmembrane glycoprotein that is expressed by various cell types including B cells and T cells. In tumors cells, interaction of CD200 with its known receptor CD200R, inhibits T-cell mediated immune response and induces immunosuppressive responses. However, the role of CD200 in tumorigenesis is poorly understood. Recently, a study shown that CD200 can induce neuritogenesis and promote neuronal survival of primary neurons through interaction and activation of the fibroblast growth factor receptor.Here, we investigated for the first time the role of CD200 in MM cell biology. First, we identified that CD200 protein expression is present only in 3 out of 9 Human Myeloma Cell lines (HMCLs). Then, we confirmed that CD200 protein is strongly expressed in MMCs of 73% of the 35 previously untreated MM patients and that CD200 is never expressed in the normal plasma cell counterparts.CD200 overexpression using lentiviral vector significantly increased HMCLs cell growth in a serum free culture medium (SYN-H) compared to GFP overexpression (fold increasing: 1.6 and 1.4 at day 4; 2.5 and 2.1 at day 7 for LP1 and XG1 respectively). Furthermore, CD200 overexpression results in significant increasing of the number of colonies for HMCLs CD200positive compared to HMCLs CD200negative in clonogenic assays (4.3 fold and 13.1 fold for LP1 and XG1 respectively, p<0.001). We confirmed also the growth advantage linked to CD200 overexpression in coculture model. These effects are significantly inhibited by a monoclonal antibody (MoAb) against CD200 compared to treatment with a no-relevant anti-IgG1. We also confirmed the significant inhibition of proliferation with the anti-CD200 MoAb using clonogenic assays. The anti-CD200 MoAb has no effect on the GFP transduced HMCLs.To investigate the implication of CD200R in CD200-induced MM cell growth, inducible CD200R depletion was analyzed in CD200positive and CD200negative, using lentiviral vectors. After doxycycline addition, the shCD200R reduced CD200R expression by 95-100% but had no significant effects on the proliferation advantage mediated by CD200 demonstrating that the growth advantage supported by CD200 overexpression is independent of CD200R.Thus, to investigate the possibility of another receptor activated by CD200 interaction, we added different essential MM growth factors to the serum free medium. Addition of fetal calf serum, IGF1 or insulin in SYN-H medium abrogated the growth advantage provided by CD200 overexpression in HMCLs. These data suggest that CD200 could act as a growth factor through interaction and activation of IGF1R. In addition, overexpression of CD200 in a IGF-1R negative XG12 HMCL did not stimulate its cell growth or clonogenic activity. Furthermore, anti-IGF-1R monoclonal antibody completely abrogated the CD200-dependent growth activity at day 4 and at day 7 in LP1CD200positive cell line compared to non-relevant antibody. The anti-IGF-1R MoAb has no effect on control LP1GFPpositive cell line cultured in serum free culture medium.In order to identify a direct interaction between CD200 and IGF-1R in serum free culture conditions, IGF-1R immunoprecipitation in denaturing conditions was performed at day 4 using LP1GFP and LP1CD200 cell lines. CD200 and IGF-1R interaction was identified by mass spectrometry and western-blotting.According to these data, MM patients with CD200high and IGF1Rhigh gene expression have a poor prognosis (OS) compared to patient with only CD200high or IGF1Rhigh expression (P=0.03, 52.7 vs 61.9 months in HM series; and P=0.02, 58.9 vs 68.1 months in TT2 series) and compared to patients with CD200low and IGF1Rlow gene expression (P=0.002, 52.7 vs 71.6 months in HM series; and P=0.01, 58.9 vs 70.2 months in TT2 series). Same results are observed for EFS.Altogether, these data demonstrated a new role of CD200 as a MM growth factor through interaction with IGF1R. Our results suggest that CD200 and IGF1R inhibition by monoclonal antibodies could represent a promising strategy in MM. DisclosuresCartron:Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Celgene: Consultancy, Employment; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:Sanofi: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; EngMab: Research Funding.

Abstract B01: Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors

Abstract Over the last decades, it has become increasingly clear that tumors are characterized by inter-individual heterogeneity, which can account as one of the prominent reasons for treatment failure. To address this problem, personalized precision medicine approaches need to be applied such as comprehensive genomic profiling to identify actionable driver mutations in individual tumors. Unfortunately, genomics alone is not sufficient in tumors that are driven by mutated but otherwise undruggable targets and a typically low mutational burden. This is a characteristic of many pediatric malignancies including rhabdomyosarcoma (RMS), the most common childhood soft-tissue sarcoma. Hence, we aim to develop an in vitro drug-profiling platform to identify and prioritize treatment strategies for RMS patients. To this end, we generated a panel of patient-derived xenografts (PDXs) including some diagnostic and relapse samples. We then screened 18 different culture conditions to identify suitable parameters to establish in vitro primary cultures of PDX tumors. Interestingly, addition of fetal calf serum to cell culture media has a detrimental effect on viability of most primary RMS cell cultures (PRCCs). In contrast, defined serum-free conditions allow to grow primary cultures for several passages that closely preserve the clonal composition and phenotypic characteristics of the parental tumor, as assessed by genomic and copy number analysis. Pharmacologic profiles of PRCCs using a targeted drug library of more than 200 compounds revealed patient-specific vulnerabilities, among them an unexpected sensitivity to AKT inhibitors in some fusion-positive RMS. Interestingly, hierarchical clustering of drug sensitivities clustered PAX3-FOXO1 fusion-positive tumors together and separated them from fusion-negative RMS. Moreover, a screen to establish effective drug combinations in a highly resistant high-risk relapse sample using standard chemotherapeutics (doxorubicin, etoposide, vincristine) together with our targeted compound library revealed the BH3-only mimetic ABT-263 as the top-scoring drug capable of resensitizing recurrent PRCCs to first-line treatment. Resensitizing with ABT-263 was not a patient-specific vulnerability as it was observed in several additional PRCCs. Mechanistically, genetic loss-of-function validation experiments revealed that this occurs via blockade of the BCL-XL-MCL-1 axis and is dependent on upregulation of the BH3-only protein NOXA. Taken together, our study provides an in vitro tool kit to prioritize actionable drug targets or combinatorial options for RMS patients for whom conventional therapies are failing. Citation Format: Beat W. Schaefer, Gabriele Manzella, Michaela Roemmele, Luduo Zhang, Joëlle Tchinda, Felix Niggli, Marco Wachtel. Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B01.

54 Effect of Different Cryopreservation Protocols for Sheep Embryonic Stem Cells

Particular attention is required to improve cryopreservation of embryonic stem cells (ESC) and study their characteristics. Stem cells were obtained from the inner cell mass of Day 5-6 blastocysts. The ESC were then cultured on mTeSR™1 medium (Stemcell Technologies, Cambridge, MA, USA). We studied the survival of ESC after slow freezing and vitrification. Slow freezing was carried out using a Planer Kryo 360-3.3 freezer (Planer plc, Sunbury-on-Thames, United Kingdom), using various cryoprotectants: 1.5 M dimethyl sulfoxide (Me2SO), 1.5 M ethylene glycol (EG), or 1.5 M propylene glycol (PG). Six vitrification solutions (VS) were used to vitrify ESC: VS1 = 20% Me2SO + 20% EG + 0.5 M sucrose; VS2 = 20% Me2SO + 20% PROH + 0.5 M sucrose; VS3 = 20% EG + 20% PG + 0.5 M sucrose; VS4 = 20% Me2SO + 20% EG + 0.5 M sucrose + 10% FCS; VS5 = 20% Me2SO + 20% PROH + 0.5 M sucrose + 10% FCS; and VS6 = 20% EG + 20% PG + 0.5 M sucrose + 10% FCS. For the dehydration of cells and the addition of vitrification solutions, a 3-step equilibration was used. The proliferative properties of the cells were determined using an Apel PD-303S spectrophotometer (Apel Co. Ltd., Kawaguchi, Japan), using an MTT test (staining with methylthiazolyl-diphenyl tetrazolium). After slow freezing, the highest percentage of frozen–thawed cells proliferating was observed when using 1.5 M EG (P &gt; 0.05). At the same time, the highest cell doubling after thawing was observed when using 1.5 M EG, and 1.5 M Me2SO. After vitrification, the highest percentage of proliferation was observed in the VS2 and VS4 groups (49.7 ± 3.2% and 53.2 ± 3.8%, respectively). It should be noted that the addition of fetal calf serum to the vitrification solution also increased the proliferation of ESC after vitrification and thawing.

Exploring influences on the cellular uptake of medium-sized silver nanoparticles into THP-1 cells

The evaluation of the uptake of nanomaterials by cells in vitro tests is of great relevance to understand potential toxicity mechanisms of nanomaterials. As an example, the uptake of medium-sized nanosilver (size range of 50 and 75nm) was studied closely for a relevant human lung cell line (THP-1). Time dependent uptake was studied in relation to different cell culture media with or without the addition of fetal calf serum (FCS). After cell isolation, washing, acid digestion and quantification of silver (Ag) by inductively coupled plasma mass spectrometry (ICPMS) were applied to study the general uptake. It is demonstrated that the uptake of Ag (from 75nm Ag nanoparticles) is a factor of 5 higher in a medium without FCS in comparison to the medium with FCS.In addition, the stability and the abundance of Ag nanoparticles in the supernatant after cell exposure were studied in relation to different timings. By means of asymmetric flow field flow fractionation (AF4) for the size dependent particle separation and an on-line hyphenation to the sensitive elemental detection by ICPMS, different Ag particles were separated and further identified. By combining results from the total uptake of Ag by cells with results obtained from the analysis of the supernatant, total recoveries of 98 to 104% were determined in relation to the exposed concentration of Ag.Finally, the influence of the medium composition (with or without FCS) on the stability of 50nm Ag nanoparticles was studied directly after spiking. A method by AF4-ICPMS was applied and the obtained fractograms confirm a clear influence of the different composed media composition and particle size on cellular uptake.The developed and applied ICPMS methods were found to be suitable approaches to evaluate the potential uptake of inorganic nanoparticles by cells.

Correlation of Changes of Cho?K1 Cells Metabolism to Changes in Protein Expression in Camp Differentiation

A correlated investigation of cell metabolism and protein expression of Chinese Hamster Ovary cells (CHO-K1) under different growth conditions was performed by 1H nuclear magnetic resonance (1H-NMR), mass spectrometry (MS) and biochemical assays. CHO fibroblasts have shown different metabolic products when grown in different media and when differentiation is stimulated by adenosine 3’, 5’ cyclic monophosphate (cAMP). In particular, while addition of Fetal Calf Serum causes an increase in the glucose metabolism correlated to changes in lipid composition of the membrane observed by 1 H-NMR, differentiation induced by cAMP causes biochemical differences in glucose and lipidic metabolism uniquely correlated both to the specific changes in the composition of nuclear proteins, revealed by mass spectrometry, and to the differences in metabolism, determined by NMR.

Hyperplastic obesity and liver steatosis as long-term consequences of suboptimal in vitro culture of mouse embryos.

In the present study, we identify and describe an obese phenotype in mice as a long-term consequence of a suboptimal in vitro culture that resulted from the addition of fetal calf serum (FCS) into the culture medium. Mice produced with FCS displayed a high mortality rate (approximately 55% versus 15% in control mice within 20 mo) and increased sensitivity to the development of obesity in adulthood when fed either a standard or a high-fat diet. These mice developed hyperplastic obesity that was characterized by a significant expansion of the fat pads (approximately 25% and 32% higher body weight in male and female mice over controls, respectively) with unchanged adipocyte size. We observed a sexual dimorphism in the development of obesity in the mice produced with FCS. Whereas the female mice displayed hypertension, hyperleptinemia, and fatty liver, the male mice only displayed glucose intolerance. The mRNA expression of metabolically relevant genes in the adipose tissue was also affected. The males produced with FCS expressed higher mRNA levels of the genes that activate fatty acid oxidation (peroxisome proliferator-activated receptor alpha [Ppara, PPARalpha] and acyl-CoA oxidase 1 [Acox1, ACOX1]) and thermogenesis (uncoupling protein 1 [Ucp1, UCP1]), which may counteract the metabolic phenotype. Conversely, the females produced with FCS generally expressed lower levels of these metabolic genes. In the females, the obese phenotype was associated with inhibition of the lipogenic pathway (peroxisome proliferator-activated receptor gamma [Pparg, PPARgamma] and fatty acid synthase [Fasn, FAS]), indicating a saturation of the storage capacity of the adipose tissue. Overall, our data indicate that the exposure to suboptimal in vitro culture conditions can lead to the sexually dimorphic development of obesity in adulthood.

Forskolin effect on the cryosurvival ofin vitro-produced bovine embryos in the presence or absence of fetal calf serum

The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Investigation of the cytotoxicity of nanozeolites A and Y

Nanosized zeolite particles are important materials for many applications in the field of nanotechnology. The possible adverse effects of these nanomaterials on human health have been scarcely investigated and remain largely unknown. This study reports the synthesis of nanozeolites Y and A with particle sizes of 25–100 nm and adequate colloidal stability for in vitro cytotoxicity experiments. The cytotoxic response of macrophages, epithelial and endothelial cells to these nanocrystals was assessed by determining mitochondrial activity (MTT assay) and cell membrane integrity (LDH leakage assay). After 24 h of exposure, no significant cytotoxic activity was detected for nanozeolite doses up to 500 μg/ml. The addition of fetal calf serum to the cell culture medium during exposure did not significantly change this low response. The nanozeolites showed low toxicity compared with monodisperse amorphous silica nanoparticles of similar size (60 nm). These results may contribute to the application of safe nanozeolites for purposes such as medical imaging, sensing materials, low-k films and molecular separation processes.

Meiosis initiation in the human ovary requires intrinsic retinoic acid synthesis

The initiation of meiosis is crucial to fertility. While extensive studies in rodents have enhanced our understanding of this process, studies in human fetal ovary are lacking. We used RT-PCR and immunohistochemistry to investigate expression of meiotic factors in human fetal ovaries from 6 to 15 weeks post fertilization (wpf) and developed an organ culture model to study the initiation of human meiosis. We observed the first meiotic cells at 11 wpf, when STRA8, SPO11 and DMC1 are first expressed. In culture, meiosis initiation is observed in 10 and 11 wpf ovaries and meiosis is maintained by addition of fetal calf serum. Meiosis is stimulated, compared with control, by retinoic acid (RA) (P < 0.05). No major change occurred in mRNA for CYP26B1, the RA-degrading enzyme proposed to control the timing of meiosis in mice. We did, however, observe increased mRNA levels for ALDH1A1 in human ovary when meiosis began, and evidence for a requirement to synthesize RA and thus sustain meiosis. Indeed, ALDH inhibition by citral prevented the appearance of meiotic cells. Finally, 8 wpf ovaries (and earlier stages) were unable to initiate meiosis whatever the length of culture, even in the presence of RA and serum. However, when human germ cells from 8 wpf ovaries were placed in a mouse ovarian environment, some did initiate meiosis. Our data indicate that meiosis initiation in the human ovary relies partially on RA, but that the progression and regulation of this process appears to differ in many aspects from that described in mice.

The Hexosamine Biosynthesis Pathway Is Essential for Pancreatic Beta Cell Development

Pancreatic exocrine and endocrine cells develop during embryonic life from endodermal progenitors. This process depends on activation of a hierarchy of transcription factors. Although information is available regarding the mesodermal signals controlling pancreas development, little is known about the role of environmental factors such as nutrients, including glucose, that also may impact development. Previously, we showed that glucose plays an important and specific role in beta cell development by activating the transition of Neurogenin3-positive endocrine progenitors into beta cells. Here, we examined the implication of glucose metabolism and more precisely the role of the hexosamine biosynthesis pathway (HBP) to understand the mechanisms by which glucose regulates beta cell development. We have established an in vitro model of endocrine and exocrine cells development from embryonic day 13.5 rat pancreases in a manner that replicates in vivo pancreas development perfectly. Using this model, we tested the effect of selective inhibitors and activators of the HBP and found that the HBP has a modest effect on cell proliferation and exocrine cell differentiation. On the other hand, beta cell development is tightly controlled by the HBP. Specifically, HBP activators increase beta cell development, whereas inhibitors repress such development. Importantly, both the HBP and glucose control the same steps in beta cell development.

Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage

To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm(2) lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm(2). Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm(2) and 2478/mm(2) centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm(2). The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue.

Carbon black and titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells: Need for multiparametric evaluation due to adsorption artifacts

The initiation of an inflammatory process is the main adverse effect observed following the exposure of the airway epithelium to nanoparticles (NPs). This study was designed to explore the pro-inflammatory potential of two different NPs of similar size but of different compositions (CB 13 nm and TiO2 15 nm) on a human bronchial epithelial cell line (16HBE14o-). The expression of granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL-6), and tumor necrosis factor alpha (TNFα) was evaluated in terms of mRNA, intracellular proteins, and released cytokines. Exposure to NPs induced a dose-dependent expression of all these cytokines, depending upon the chemical composition of NPs. The released cytokines appeared to be an inaccurate methodology to evaluate the pro-inflammatory response. Indeed, NPs adsorbed cytokines, and the binding was dependent on the nature of both the cytokine and NPs. Furthermore, addition of fetal calf serum or bovine serum albumin improved the detection of cytokines but also reduced cellular responses. Use of different detergents (Tween, Triton, and NP40) demonstrated limited efficiency to desorb cytokines from NPs. Thus, this study demonstrated the pro-inflammatory potential for CB and TiO2 NP but underlines the methodological artifacts faced during the in vitro evaluation of cytokine release that necessitates a multiparametric evaluation.

Controlling α-SMA expression in adult human pancreatic stem cells by soluble factors

In the application of adult stem cells in regenerative medicine, it is indispensable to control stem cell behaviour in vitro. Since stem cells spontaneously differentiate into several cell types, it is mandatory to identify methods to enrich the desired cell types and concurrently block other differentiation pathways. More precisely, generation of a defined cell population is a key prerequisite for a therapeutic application of stem cells. Here we have demonstrated that it is possible to influence the differentiation of human pancreatic stem cells (hPSCs). During activation of mesodermal differentiation, the cytoskeletal protein alpha-smooth muscle actin (alpha-SMA) seems to play an important role in different cell systems and can usually be detected in hPSCs during in vitro cultivation. We cultured stem cells under different conditions and analyzed the impact on alpha-SMA expression. On the one hand, supplements like retinoic acid (RA) and dimethyl sulfoxide (DMSO) were added to the cultivation medium; on the other hand, different media with or without the addition of fetal calf serum (FCS) were used. Expression of alpha-SMA was determined by immunocytochemistry, Western blot analysis and quantitative RT-PCR. After the treatment of hPSCs with RA, a strong induction of alpha-SMA protein expression was observed when 2mM RA was added to the medium. DMSO in turn induced a marked reduction in alpha-SMA-positive cells. This could also be observed using a keratinocyte serum-free medium (KSFM). Furthermore, the general addition of FCS to the medium had a blocking effect on alpha-SMA expression and decreased the number of alpha-SMA-positive cells to a minimum. The controlled modulation of hPSCs by soluble factors is a first success on the way to a promising application for transplantation medicine and cell therapy of degenerative diseases.

Photoreactivity of indirubin derivatives

Twenty-nine analogs of indirubin, an isomer of indigo, have been synthesized to optimize its promising kinase inhibitory scaffold. These compounds being also pigmented, have been tested for their photoreactivity. Absorption maxima were between 485 nm and 560 nm. Addition of fetal calf serum induced fluorescence and time dependent absorption modifications. Appropriate illumination induced Reactive Oxygen Species (ROS) production for nineteen compounds out of twenty-nine. The relationship between fluorescence and ROS production is discussed. Six compounds showed an important toxicity on F98 cells, a murine glioma cell line. Three of these were found to be also phototoxic, as four other non-toxic compounds. All but one phototoxic compounds were detected as ROS producers by in vitro tests. Photoreactivity assessment is important to anticipate adverse reactions for compounds that might be clinically developed. The experimental assay was found to be the only way to evaluate the photoreactivity of this family of compounds since no predictive criteria on structures could be found. Combining the vascular tumor growth inhibition induced by kinase inhibitors with the massive local blood flow arrest following photodynamic treatment may be an efficient anti-cancer strategy. These data could orientate further syntheses of either non-photoreactive compounds or compounds displaying both kinase inhibitory activity and strong phototoxicity.

Isolation and cytotoxicity of low-molecular-weight metabolites of Candida albicans

In this study, the low molecular weight lypophilic metabolites of C. albicans and C. dubliniensis strains produced in a synthetic medium with the addition of fetal calf serum were identified using LC/MS and MS/MS technique and quantified. All strains investigated produce a metabolite with a UV spectra maximum at 224 and 279 nm and minimum at 243 nm. Following comparison with ESI, MS/MS spectral data of a reference compound, the metabolite was identified as 3-indoleethanol (tryptophol). The concentration of extracellular tryptophol in the biosynthesis of C. albicans and C. dubliniensis ranged from 2.45 microg/mL to 191 microg/mL, respectively. Contrary to previously published data, gliotoxin or gliotoxin-like compounds were not detected, and all investigated C. albicans and C. dubliniensis strains have the same metabolite profile. Cytotoxic effects of tryptophol and 3-indolelactic acid (precursor of tryptophol biosynthesis) were cell-line-dependent. The EC50 of tryptophol ranged between 2 and 7 mM, with the EC50 of 3-indolelactic acid approximately double (between 4 and 8 mM). Tryptophol exhibited cell-type dependent cytotoxicity in relatively high concentrations, with domination of apoptosis.

A modified coproantigen test used for surveillance of Echinococcus spp . in Tibetan dogs

Recently an immunological test for Echinococcus spp. antigens in dog faeces has been developed. The antigens appear to be carbohydrates, which survive proteolytic digestion and environmental degradation. For ELISA a capture antibody is used to capture the antigens, followed by a detection antibody. This paper describes a modification of the test whereby capture and detection antibodies are generated exclusively to the carbohydrate portion of the parasite tegument. Faecal extracts were heated to 70 °C overnight and the addition of foetal calf serum to the extracts was not necessary. The use of this modification as a surveillance tool in an extensive field trial of hydatid control in Western Sichuan is described. From 2003 onwards all dogs received a treatment with praziquantel pills in the spring and the autumn of each year. On each of six occasions 580 faecal samples were collected from 29 villages and analysed. Prevalence of Echinococcus spp. coproantigen-positive samples was 50% in year 2000, and decreased from 35% to 17% through 2003–2005. This coproantigen technique is now being used as part of the Chinese National Hydatid Disease Control Program, initially in 10 counties in Sichuan Province.

Identification of a Signal Peptide for Unconventional Secretion

Homeoproteins are a class of transcription factors defined by the structure of their DNA-binding domain, the homeodomain. In addition to their nuclear cell-autonomous activities, homeoproteins transfer between cells, thanks to two separate steps of secretion and internalization, which both rely on unconventional mechanisms. Internalization is driven by the third helix of the homeodomain (Penetratin) through a non-vesicular and endocytosis-independent mechanism. In contrast, homeoprotein secretion involves vesicular compartments and requires the presence of a sequence of 11 amino acids (Sec sequence) spanning between the second and third helix of the homeodomain. In this study, we report that the SecPen polypeptide, which combines the two identified domains, Penetratin and Sec, bears all of the necessary information to go in and out of cells. We have analyzed key mechanisms and demonstrated that this peptide can efficiently cross a tight junction epithelium.

Hematopoietic development from human embryonic stem cell lines

The most common human cell-based therapy applied today is hematopoietic stem cell (HSC) transplantation. Currently, human bone marrow, mobilized peripheral blood, and umbilical cord blood represent the major sources of transplantable HSCs, but their availability for use is limited by both compatibility between donor and recipient and required quantity. Although increasing evidence suggests that somatic HSCs can be expanded to meet current needs, their in vivo potential is concomitantly compromised after ex vivo culture. In contrast, human embryonic stem cells (hESC) possess indefinite proliferative capacity in vitro and have been shown to differentiate into the hematopoietic cell fate, giving rise to erythroid, myeloid, and lymphoid lineages using a variety of differentiation procedures. Human ESC-derived hematopoietic cells emerge from a subset of embryonic endothelium expressing PECAM-1, Flk-1, and VE-Cadherin, but lacking CD45 (CD45negPFV). These CD45negPFV precursors are exclusively responsible for hematopoietic potential of differentiated hESCs. hESC-derived hematopoietic cells show similar clonogenic capacity and primitive phenotype to somatic sources of hematopoietic progenitors and possess limited in vivo repopulating capacity in immunodeficient mice, suggestive of HSC function. Here, we will review current progress in studies of hESC-derived hematopoietic cells and discuss the potential precincts and applications.