Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector

Induced pluripotent stem (iPS) cells have generated keen interestdue to their potential use in regenerative medicine. They havebeen obtained from various cell types of both mice and humans byexogenous delivery of different combinations of Oct4, Sox2, Klf4,c-Myc, Nanog, and Lin28. The delivery of these transcription factorshas mostly entailed the use of integrating viral vectors (retrovirusesor lentiviruses), carrying the risk of both insertional mutagenesisand oncogenesis due to misexpression of these exogenousfactors. Therefore, obtaining iPS cells that do not carry integratedtransgene sequences is an important prerequisite for their eventualtherapeutic use. Here we report the generation of iPS cell linesfrom mouse embryonic fibroblasts with no evidence of integrationof the reprogramming vector in their genome, achieved by nucleofectionof a polycistronic construct coexpressing Oct4, Sox2, Klf4,and c-Myc

A protocol describing the genetic correction of somatic human cells and subsequent generation of IPS cell

The generation of patient-specific induced pluripotent stem cells (iPSCPSCPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSCPSCPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCPSCPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCPSCPSCs can be obtained in 4–5 months.

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

Background: The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.Results: The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.Conclusion: Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.

Population pharmacokinetics of imatinib and the role of alpha-acid glycoprotein

AIMS: The aims of this observational study were to assess the variability in imatinib pharmacokinetics and to explore the relationship between its disposition and various biological covariates, especially plasma alpha1-acid glycoprotein concentrations. METHODS: A population pharmacokinetic analysis was performed using NONMEM based on 321 plasma samples from 59 patients with either chronic myeloid leukaemia or gastrointestinal stromal tumours. The influence of covariates on oral clearance and volume of distribution was examined. Furthermore, the in vivo intracellular pharmacokinetics of imatinib was explored in five patients. RESULTS: A one-compartment model with first-order absorption appropriately described the data, giving a mean (+/-SEM) oral clearance of 14.3 l h-1 (+/-1.0) and a volume of distribution of 347 l (+/-62). Oral clearance was influenced by body weight, age, sex and disease diagnosis. A large proportion of the interindividual variability (36% of clearance and 63% of volume of distribution) remained unexplained by these demographic covariates. Plasma alpha1-acid glycoprotein concentrations had a marked influence on total imatinib concentrations. Moreover, we observed an intra/extracellular ratio of 8, suggesting substantial uptake of the drug into the target cells. CONCLUSION: Because of the high pharmacokinetic variability of imatinib and the reported relationships between its plasma concentration and efficacy and toxicity, the usefulness of therapeutic drug monitoring as an aid to optimizing therapy should be further investigated. Ideally, such an approach should take account of either circulating alpha1-acid glycoprotein concentrations or free imatinib concentrations.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

Assessing ageing of individual T lymphocytes: Mission impossible?

Effector T lymphocytes are the progeny of a limited number of antigen- specific precursor cells and it has been estimated that clonotypic human T cells may expand million fold on their way reaching high cell numbers that are sufficient for immune protection. Moreover, memory T cell responses are characterized by repetitive expansion of antigen-specific T cell clonotypes, and limitations in the proliferative capacity could lead to immune senescence. Because telomeres progressively shorten as a function of cell division, telomere length is a powerful indicator of the replicative in vivo history of human T lymphocytes. In this review, we summarize observations made over the last decade on telomere length dynamics of well-defined T cell populations derived from healthy donors and patients with infectious disease or cancer. We focus on T cell differentiation, T cell ageing, and natural and vaccine induced immune responses. We also discuss the scientific evidence for in vivo replicative senescence of antigen-specific T cells, and evaluate the available methods for measuring telomere lengths and telomerase activity, and their potential and limitations to increase our understanding of T cell physiology. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Diffusion-weighted MRI in metastatic gastrointestinal tumours (GIST): a pilot study on the assessment of treatment response in comparison with 18 F-FDG PET/CT

Purpose: To evaluate the clinical potential of diffusion-weighted MR imaging with apparent diffusion coefficient (ADC) mapping for the assessment of gastrointestinal stromal tumour (GIST) response to targeted therapy in comparison with 18F-FDG PET/CT Methods and Materials: Five patients (3 W/2M, aged 56±13 y) with metastatic GIST underwent both a 18F-FDG PET/CT (Discovery LS, GE Healthcare) and a MRI (VIBE T1 Gd, DWI [b = 50,300,600] and ADC mapping) before and after change in therapy. Exams were first analysed blindly and then PET/CT images were coregistered to T1 Gd MR images for lesion detection. SUVmax and ADC were measured for the six largest lesions on MRI. The relationship between SUVmax and ADC was analysed using Spearman's correlation. Results: Altogether, 24 lesions (15 hepatic and 9 non-hepatic) were analysed on both modalities. Three PET/CT lesions (12.5%) were initially not considered on ADC and 4 lesions on the second PET/CT were excluded because of hepatic vascular activity spillover. SUVmax decreased from 7.2±7.7 g/mL to 5.9±5.9 g/mL (P = 0.53) and ADC increased from 1.2x10-3 mm2/s ± 0.4 to 1.4x10-3 mm2/s ± 0.4 (P = 0.07). There was a significant association between SUVmax decrease and ADC increase (rho= -0.64, P = 0.004). Conclusion: Changes in ADC from diffusion-weighted MRI reflect response of 18F-FDG-avid GIST to therapy. The exact diagnostic value of DWI needs to be investigated further, as well as the effect of lesion size and time under therapy before imaging. Furthermore, the proven association between SUVmax and ADC may be useful for the assessment of treatment response in 18F-FDG non-avid GIST.

Chitosan-based nanogels for selective delivery of photosensitizers to macrophages and improved retention in and therapy of articular joints.

Macrophages play key roles in inflammatory disorders. Therefore, they are targets of treatments aiming at their local destruction in inflammation sites. However, injection of low molecular mass therapeutics, including photosensitizers, in inflamed joints results in their rapid efflux out of the joints, and poor therapeutic index. To improve selective uptake and increase retention of therapeutics in inflamed tissues, hydrophilic nanogels based on chitosan, of which surface was decorated with hyaluronate and which were loaded with one of three different anionic photosensitizers were developed. Optimal uptake of these functionalized nanogels by murine RAW 264.7 or human THP-1 macrophages as models was achieved after <4h incubation, whereas only negligible uptake by murine fibroblasts used as control cells was observed. The uptake by cells and the intracellular localization of the photosensitizers, of the fluorescein-tagged chitosan and of the rhodamine-tagged hyaluronate were confirmed by fluorescence microscopy. Photodynamic experiments revealed good cell photocytotoxicity of the photosensitizers entrapped in the nanogels. In a mouse model of rheumatoid arthritis, injection of free photosensitizers resulted in their rapid clearance from the joints, while nanogel-encapsulated photosensitizers were retained in the inflamed joints over a longer period of time. The photodynamic treatment of the inflamed joints resulted in a reduction of inflammation comparable to a standard corticoid treatment. Thus, hyaluronate-chitosan nanogels encapsulating therapeutic agents are promising materials for the targeted delivery to macrophages and long-term retention of therapeutics in leaky inflamed articular joints.

LDLs induce fibroblast spreading independently of the LDL receptor via activation of the p38 MAPK pathway.

Because adventitial fibroblasts play an important role in the repair of blood vessels, we assessed whether elevation in LDL concentrations would affect fibroblast function and whether this depended on activation of intracellular signaling pathways. We show here that in primary human fibroblasts, LDLs induced transient activation of the p38 mitogen-activated protein kinase (MAPK) pathway, but not the c-Jun N-terminal kinase MAPK pathway. This activation did not require the recruitment of the LDL receptor (LDLR), because LDLs efficiently stimulated the p38 MAPK pathway in human and mouse fibroblasts lacking functional LDLR, and because receptor-associated protein, an LDLR family antagonist, did not block the LDL-induced p38 activation. LDL particles also induced lamellipodia formation and cell spreading. These effects were blocked by SB203580, a specific p38 inhibitor. Our data demonstrate that LDLs can regulate the shape of fibroblasts in a p38 MAPK-dependent manner, a mechanism that may participate in wound healing or vessel remodeling as in atherosclerosis.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Comparison of [3H]tamsulosin and [3H]prazosin binding to wild-type and constitutively active alpha1B-adrenoceptors.

We have tested the hypothesis that smaller alpha1B-adrenoceptor labeling by [3H]tamsulosin compared to [3H]prazosin is related to differential recognition of agonist low affinity states. Paired saturation binding experiments with [3H]prazosin and [3H]tamsulosin were performed in membrane preparations from rat liver and Rat- fibroblasts stably transfected with wild-type hamster alpha1B-adrenoceptors or a constitutively active mutant thereof. In all three settings [3H]tamsulosin labeled significantly fewer alpha1B-adrenoceptors than [3H]prazosin. In noradrenaline competition binding experiments, the percentage of agonist low affinity sites was smallest for the constitutively active alpha1B-adrenoceptor but the percentage of agonist low affinity sites recognized by [3H]tamsulosin and [3H]prazosin did not differ significantly. We conclude that [3H]tamsulosin labels fewer alpha1B-adrenoceptors than [3H]prazosin but this is not fully explained by a poorer labeling of agonist low affinity sites.

Clinical consequences of imatinib plasma concentrations variability in hemato-oncologic patients

Background: Imatinib has revolutionized the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Considering the large inter-individual differences in the function of the systems involved in its disposition, exposure to imatinib can be expected to vary widely among patients. This observational study aimed at describing imatinib pharmacokinetic variability and its relationship with various biological covariates, especially plasma alpha1-acid glycoprotein (AGP), and at exploring the concentration-response relationship in patients. Methods: A population pharmacokinetic model (NONMEM) including 321 plasma samples from 59 patients was built up and used to derive individual post-hoc Bayesian estimates of drug exposure (AUC; area under curve). Associations between AUC and therapeutic response or tolerability were explored by ordered logistic regression. Influence of the target genotype (i.e. KIT mutation profile) on response was also assessed in GIST patients. Results: A one-compartment model with first-order absorption appropriately described the data, with an average oral clearance of 14.3 L/h (CL) and volume of distribution of 347 L (Vd). A large inter-individual variability remained unexplained, both on CL (36%) and Vd (63%), but AGP levels proved to have a marked impact on total imatinib disposition. Moreover, both total and free AUC correlated with the occurrence and number of side effects (e.g. OR 2.9±0.6 for a 2-fold free AUC increase; p<0.001). Furthermore, in GIST patients, higher free AUC predicted a higher probability of therapeutic response (OR 1.9±0.5; p<0.05), notably in patients with tumor harboring an exon 9 mutation or wild-type KIT, known to decrease tumor sensitivity towards imatinib. Conclusion: The large pharmacokinetic variability, associated to the pharmacokinetic-pharmacodynamic relationship uncovered are arguments to further investigate the usefulness of individualizing imatinib prescription based on TDM. For this type of drug, it should ideally take into consideration either circulating AGP concentrations or free drug levels, as well as KIT genotype for GIST.

The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome.

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.

Multi-layer amniotic membrane (MLAM) transplantation for non traumatic corneal perforations or descemetoceles

Purpose:To describe the indications, the surgical procedure and the clinical outcome of MLAM in the treatment of non traumatic corneal perforations and descemetoceles . Methods:A prospective, non comparative, interventional case series of eight consecutive patients (mean age 59 years old, 6 men and 2 women) with non traumatic corneal perforations or descemetoceles.The surgery consisted in a MLAM transplantation of a cryopreservated human amniotic membrane. The series included: three active herpetic keratitis, one rosacea, one perforation of an hydrops, one cicatricial pemphigoid, one perforation after an abcess in a corneal graft and one perforation after protonbeamtherapy. The clinical outcome included: the follow-up, the integrity of the eye, corneal epithelialization, inflammation and neovascularization, and the integration of the MLAM. Stromal thickness was followed precisely with the slit lamp. A corneal graft was performed at one patient after the MLAM, allowing microscopic investigation of the removed MLAM integrated in the cornea. Results:The mean follow-up was 8.78 months (range 3.57 to 30.17). Amniotic membrane transplantation was successful and reduced inflammation in 7 patients out of 8 ,after one procedure.One patient who presented a large herpetic keratitis epithelial defect with corneal anaesthesia had his MLAM dissolved after two weeks with an aqueous leakage. Epithelium healed within 3 weeks above 7 MLAM and remained stable at 3 months in 7 out of 8 patients. MLAM opacification gradually disappeared over a few months, however, stromal layers filling in the corneal perforations or above the descemetoceles remained stable. Conclusions:MLAM transplantation is a safe, effective and useful technique to cure non traumatic corneal perforations and descemetoceles. It can be performed in emergency despite the presence of an active inflammation or infection. By facilitating epithelialization, reducing inflammation and neovascularization, it allows corneal surface reconstruction in patients with persistent epithelial defects and corneal melting that usually ends in a perforation. For full visual rehabilitation, a delayed penetrating keratoplasty is required.

Impaired glutathione synthesis in schizophrenia: convergent genetic and functional evidence

Schizophrenia is a complex multifactorial brain disorder with a genetic component. Convergent evidence has implicated oxidative stress and glutathione (GSH) deficits in the pathogenesis of this disease. The aim of the present study was to test whether schizophrenia is associated with a deficit of GSH synthesis. Cultured skin fibroblasts from schizophrenia patients and control subjects were challenged with oxidative stress, and parameters of the rate-limiting enzyme for the GSH synthesis, the glutamate cysteine ligase (GCL), were measured. Stressed cells of patients had a 26% (P = 0.002) decreased GCL activity as compared with controls. This reduction correlated with a 29% (P < 0.001) decreased protein expression of the catalytic GCL subunit (GCLC). Genetic analysis of a trinucleotide repeat (TNR) polymorphism in the GCLC gene showed a significant association with schizophrenia in two independent case-control studies. The most common TNR genotype 7/7 was more frequent in controls [odds ratio (OR) = 0.6, P = 0.003], whereas the rarest TNR genotype 8/8 was three times more frequent in patients (OR = 3.0, P = 0.007). Moreover, subjects with disease-associated genotypes had lower GCLC protein expression (P = 0.017), GCL activity (P = 0.037), and GSH contents (P = 0.004) than subjects with genotypes that were more frequent in controls. Taken together, the study provides genetic and functional evidence that an impaired capacity to synthesize GSH under conditions of oxidative stress is a vulnerability factor for schizophrenia.