[Supporting the fragile equilibrium of the family relationship]. / Soutenir le fragile équilibre du lien familial.

Constructing bonds within the family sphere helps to establish a stable relational equilibrium. When a member of the family suffers from psychological disorders, maintaining this bond is akin to walking a tightrope. It must therefore be reinforced by the professionalism of the caregiving teams, attentive to the patient and his or her family. The place and identification of peers and family and friends is, likewise, essential in order to be able to continue walking along this tightrope.

Intravenous Injection of Clinical Grade Human MSCs After Experimental Stroke: Functional Benefit and Microvascular Effect.

Stroke is the leading cause of disability in adults. Many current clinical trials use intravenous (IV) administration of human bone marrow-derived mesenchymal stem cells (BM-MSCs). This autologous graft requires a delay for ex vivo expansion of cells. We followed microvascular effects and mechanisms of action involved after an IV injection of human BM-MSCs (hBM-MSCs) at a subacute phase of stroke. Rats underwent a transient middle cerebral artery occlusion (MCAo) or a surgery without occlusion (sham) at day 0 (D0). At D8, rats received an IV injection of 3 million hBM-MSCs or PBS-glutamine. In a longitudinal behavioral follow-up, we showed delayed somatosensory and cognitive benefits 4 to 7 weeks after hBM-MSC injection. In a separate longitudinal in vivo magnetic resonance imaging (MRI) study, we observed an enhanced vascular density in the ischemic area 2 and 3 weeks after hBM-MSC injection. Histology and quantitative polymerase chain reaction (qPCR) revealed an overexpression of angiogenic factors such as Ang1 and transforming growth factor-1 (TGF-1) at D16 in hBM-MSC-treated MCAo rats compared to PBS-treated MCAo rats. Altogether, delayed IV injection of hBM-MSCs provides functional benefits and increases cerebral angiogenesis in the stroke lesion via a release of endogenous angiogenic factors enhancing the stabilization of newborn vessels. Enhanced angiogenesis could therefore be a means of improving functional recovery after stroke.

Respective effects of oxygen and energy substrate deprivation on beta cell viability.

Deficit in oxygen and energetic substrates delivery is a key factor in islet loss during islet transplantation. Permeability transition pore (PTP) is a mitochondrial channel involved in cell death. We have studied the respective effects of oxygen and energy substrate deprivation on beta cell viability as well as the involvement of oxidative stress and PTP opening. Energy substrate deprivation for 1h followed by incubation in normal conditions led to a cyclosporin A (CsA)-sensitive-PTP-opening in INS-1 cells and human islets. Such a procedure dramatically decreased INS-1 cells viability except when transient removal of energy substrates was performed in anoxia, in the presence of antioxidant N-acetylcysteine (NAC) or when CsA or metformin inhibited PTP opening. Superoxide production increased during removal of energy substrates and increased again when normal energy substrates were restored. NAC, anoxia or metformin prevented the two phases of oxidative stress while CsA prevented the second one only. Hypoxia or anoxia alone did not induce oxidative stress, PTP opening or cell death. In conclusion, energy substrate deprivation leads to an oxidative stress followed by PTP opening, triggering beta cell death. Pharmacological prevention of PTP opening during islet transplantation may be a suitable option to improve islet survival and graft success.

Microvascular plasticity after experimental stroke: a molecular and MRI study.

BACKGROUND: Microvasculature plays a key role in stroke pathophysiology both during initial damage and extended neural repair. Moreover, angiogenesis processes seem to be a promising target for future neurorestorative therapies. However, dynamic changes of microvessels after stroke still remain unclear, and MRI follow-up could be interesting as an in vivo biomarker of these. METHODS: The aim of this study is to characterize the microvascular plasticity 25 days after ischemic stroke using both in vivo microvascular 7T-MRI (vascular permeability, cerebral blood volume (CBV), vessel size index (VSI), vascular density) and quantification of angiogenic factor expressions by RT-qPCR in a transient middle cerebral artery occlusion rat model. CBV and VSI (perfused vessel caliber) imaging was performed using a steady-state approach with a multi gradient-echo spin-echo sequence before and 2 min after intravenous (IV) injection of ultrasmall superparamagnetic iron particles. Vascular density (per mm2) was derived from the ratio [ΔR2/(ΔR2*)²/³]. Blood brain barrier leakage was assessed using T1W images before and after IV injection of Gd-DOTA. Additionally, microvessel immunohistology was done. RESULTS: 3 successive stages were observed: 1) 'Acute stage' from day 1 to day 3 post-stroke (D1-D3) characterized by high levels of angiopoietin-2 (Ang2), vascular endothelial growth factor receptor-2 (VEGFR-2) and endothelial NO synthase (eNOS) that may be associated with deleterious vascular permeability and vasodilation; 2) 'Transition stage' (D3-D7) that involves transforming the growth factors ß1 (TGFß1), Ang1, and tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains 1 (Tie1), stromal-derived factor-1 (SDF-1), chemokine receptor type 4 (CXCR-4); and 3) 'Subacute stage' (D7-D25) with high levels of Ang1, Ang2, VEGF, VEGFR-1 and TGFß1 leading to favorable stabilization and maturation of microvessels. In vivo MRI appeared in line with the angiogenic factors changes with a delay of at least 1 day. All MRI parameters varied over time, revealing the different aspects of the post-stroke microvascular plasticity. At D25, despite a normal CBV, MRI revealed a limited microvessel density, which is insufficient to support a good neural repair. CONCLUSIONS: Microvasculature MRI can provide imaging of different states of functional (perfused) microvessels after stroke. These results highlight that multiparametric MRI is useful to assess post-stroke angiogenesis, and could be used as a biomarker notably for neurorestorative therapy studies. Additionally, we identified that endogenous vessel maturation and stabilization occur during the 'subacute stage'. Thus, pro-angiogenic treatments, such as cell-based therapy, would be relevant during this subacute phase of stroke.

Identification of a novel complex BRAF mutation associated with major clinical response to vemurafenib in a patient with metastatic melanoma.

IMPORTANCE: There is an increasing interest in BRAF V600 mutations in melanomas and their associated sensitivity to vemurafenib, a BRAF inhibitor. However, physicians cannot find information in the literature about vemurafenib response for rare and/or atypical BRAF mutations. OBSERVATIONS: We describe the identification of a novel complex BRAF mutation associated with major clinical response to vemurafenib in a patient with metastatic melanoma. Using a pyrosequencing method, we determined that the tumor positive for mutated BRAF, uncovering a novel c.1799_1803delinsAT; p.V600-K601>D variant. We uncovered this atypical BRAF mutation with 2 different sequencing methods, both in the primary lesion and in 1 metastasis. The patient was immediately treated with vemurafenib as monotherapy and achieved a prolonged (5.5-month) positive response. CONCLUSIONS AND RELEVANCE: We analyzed the consequences of the BRAF V600-K601>D mutation in terms of amino acids. We referred to the published data and databases to screen chemical properties of well-known BRAF V600 mutations and other complex BRAF mutations to find common features of activated BRAF mutations. Importantly, we highlighted that both the site of the mutation and the involved amino acids are important to predict vemurafenib response. Our conclusion is that complex BRAF mutation surrounding codon 600 could also be sensitive to BRAF inhibitors.

The dual effect of mesenchymal stem cells on tumour growth and tumour angiogenesis.

INTRODUCTION: Understanding the multiple biological functions played by human mesenchymal stem cells (hMSCs) as well as their development as therapeutics in regenerative medicine or in cancer treatment are major fields of research. Indeed, it has been established that hMSCs play a central role in the pathogenesis and progression of tumours, but their impact on tumour growth remains controversial. METHODS: In this study, we investigated the influence of hMSCs on the growth of pre-established tumours. We engrafted nude mice with luciferase-positive mouse adenocarcinoma cells (TSA-Luc+) to obtain subcutaneous or lung tumours. When tumour presence was confirmed by non-invasive bioluminescence imaging, hMSCs were injected into the periphery of the SC tumours or delivered by systemic intravenous injection in mice bearing either SC tumours or lung metastasis. RESULTS: Regardless of the tumour model and mode of hMSC injection, hMSC administration was always associated with decreased tumour growth due to an inhibition of tumour cell proliferation, likely resulting from deep modifications of the tumour angiogenesis. Indeed, we established that although hMSCs can induce the formation of new blood vessels in a non-tumoural cellulose sponge model in mice, they do not modify the overall amount of haemoglobin delivered into the SC tumours or lung metastasis. We observed that these tumour vessels were reduced in number but were longer. CONCLUSIONS: Our results suggest that hMSCs injection decreased solid tumour growth in mice and modified tumour vasculature, which confirms hMSCs could be interesting to use for the treatment of pre-established tumours.

Difference in mobilization of progenitor cells after myocardial infarction in smoking versus non-smoking patients: insights from the BONAMI trial.

INTRODUCTION: Although autologous bone marrow cell (BMC) therapy has emerged as a promising treatment for acute myocardial infarction (AMI), trials reported mixed results. In the BONAMI trial, active smoking reduced cardiac function recovery after reperfused AMI. Therefore, we hypothesized that variability in the functionality of BMCs retrieved from patients with cardiovascular risk factors may partly explain these mixed results. We investigated the characteristics of progenitor cells in active smokers and non-smokers with AMI and their potential impact on BMC therapy efficacy. METHODS: Bone marrow and blood samples from 54 smoking and 47 non-smoking patients enrolled in the BONAMI cell therapy trial were analyzed. RESULTS: The white BMC and CD45dimCD34+ cell numbers were higher in active smokers (P = 0.001, P = 0.03, respectively). In marked contrast, either bone marrow or blood endothelial progenitor CD45dimCD34 + KDR + cells (EPCs) were decreased in active smokers (P = 0.005, P = 0.04, respectively). Importantly, a multivariate analysis including cardiovascular risk factors confirmed the association between active smoking and lower EPC number in bone marrow (P = 0.04) and blood (P = 0.04). Furthermore, baseline circulating EPC count predicted infarct size decrease at three months post-AMI in non-smokers (P = 0.01) but not in active smokers. Interestingly, baseline circulating EPCs were no longer predictive of cardiac function improvement in the BMC therapy group. CONCLUSIONS: These data suggest that circulating EPCs play an important role in cardiac repair post-AMI only in non-smokers and that active smoking-associated EPC alterations may participate in the impairment of cardiac function recovery observed in smokers after AMI, an effect that was overridden by BMC therapy.

Magnetic resonance imaging and fluorescence labeling of clinical-grade mesenchymal stem cells without impacting their phenotype: study in a rat model of stroke.

Human mesenchymal stem cells (hMSCs) have strong potential for cell therapy after stroke. Tracking stem cells in vivo following a graft can provide insight into many issues regarding optimal route and/or dosing. hMSCs were labeled for magnetic resonance imaging (MRI) and histology with micrometer-sized superparamagnetic iron oxides (M-SPIOs) that contained a fluorophore. We assessed whether M-SPIO labeling obtained without the use of a transfection agent induced any cell damage in clinical-grade hMSCs and whether it may be useful for in vivo MRI studies after stroke. M-SPIOs provided efficient intracellular hMSC labeling and did not modify cell viability, phenotype, or in vitro differentiation capacity. Following grafting in a rat model of stroke, labeled hMSCs could be detected using both in vivo MRI and fluorescent microscopy until 4 weeks following transplantation. However, whereas good label stability and unaffected hMSC viability were observed in vitro, grafted hMSCs may die and release iron particles in vivo.

Plasmacytoid dendritic cells induce efficient stimulation of antiviral immunity in the context of chronic hepatitis B virus infection.

UNLABELLED: The immune control of hepatitis B virus (HBV) infection is essential for viral clearance. Therefore, restoring functional anti-HBV immunity is a promising immunotherapeutic approach to treatment of chronic infection. Plasmacytoid dendritic cells (pDCs) play a crucial role in triggering antiviral immunity through their ability to capture and process viral antigens and subsequently induce adaptive immune responses. We investigated the potential of pDCs to trigger antiviral cellular immunity against HBV. We used a human leukocyte antigen A (HLA-A)*0201(+) pDC line loaded with HLA-A*0201-restricted peptides derived from hepatitis B core/hepatitis B surface (HBc/HBs) antigens to amplify specific CD8 T cells ex vivo from chronic HBV patients and established a Hepato-HuPBL mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMCs or liver-infiltrating lymphocytes from HLA-A*0201(+) chronic HBV patients by HBc peptide-loaded pDCs elicited up to 23.1% and 76.1% HBV-specific CD8 T cells in 45.8% of cases. The specific T cells from the "responder" group secreted interferon-γ, expressed CD107 upon restimulation, and efficiently lysed HBV antigen-expressing hepatocytes. Circulating hepatitis B e antigen (HBeAg) was found to distinguish the group of patients not responding to the pDC stimulation. The therapeutic efficacy of the pDC vaccine was evaluated in immunodeficient NOD-SCID ß(2) m(-/-) mice reconstituted with HBV patients' PBMCs and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of Hepato-HuPBL mice with the HBc/HBs peptide-loaded pDCs elicited HBV-specific T cells able to specifically lyse the transfected hepatocytes and reduce the systemic viral load. CONCLUSION: pDCs loaded with HBV-derived peptides can elicit functional virus-specific T cells. HBeAg appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. A pDC-based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients.

Intracerebral injection of human mesenchymal stem cells impacts cerebral microvasculature after experimental stroke: MRI study.

Stroke, the leading cause of disability, lacks treatment beyond thrombolysis. The acute injection of human mesenchymal stem cells (hMSCs) provides a benefit which could be mediated by an enhancement of angiogenesis. A clinical autologous graft requires an hMSC culture delay incompatible with an acute administration. This study evaluates the cerebral microvascular changes after a delayed injection of hMSCs. At day 8 after middle cerebral artery occlusion (MCAo), two groups of rats received an intracerebral injection in the damaged brain of either 10 µL of cell suspension medium (MCAo-PBS, n = 4) or 4 × 105 hMSCs (MCAo-hMSC, n = 5). Two control groups of healthy rats underwent the same injection procedures in the right hemisphere (control-PBS, n = 6; control-hMSC, n = 5). The effect of hMSCs on the microvasculature was assessed by MRI using three parameters: apparent diffusion coefficient (ADC), cerebral blood volume (CBV) and vessel size index (VSI). At day 9, eight additional rats were euthanised for a histological study of the microvascular parameters (CBV, VSI and vascular fraction). No ADC difference was observed between MCAo groups. One day after intracerebral injection, hMSCs abolished the CBV increase observed in the lesion (MCAo-hMSC: 1.7 ± 0.1% versus MCAo-PBS: 2.2 ± 0.2%) and delayed the VSI increase (vasodilation) secondary to cerebral ischaemia. Histological analysis at day 9 confirmed that hMSCs modified the microvascular parameters (CBV, VSI and vascular fraction) in the lesion. No ADC, CBV or VSI differences were observed between control groups. At the stroke post-acute phase, hMSC intracerebral injection rapidly and transiently modifies the cerebral microvasculature. This microvascular effect can be monitored in vivo by MRI.

Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC.

BACKGROUND: Epidermal Growth Factor Receptor (EGFR) mutations, especially in-frame deletions in exon 19 (ΔLRE) and a point mutation in exon 21 (L858R) predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells. METHODS: We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue. RESULTS: This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%). In the prospective analysis of 213 samples, 7 (3.3%) samples were not analyzed and EGFR mutations were detected in 18 (8.7%) patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells. CONCLUSIONS: pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.

Intracoronary autologous mononucleated bone marrow cell infusion for acute myocardial infarction: results of the randomized multicenter BONAMI trial.

AIMS: Intracoronary administration of autologous bone marrow cells (BMCs) leads to a modest improvement in cardiac function, but the effect on myocardial viability is unknown. The aim of this randomized multicentre study was to evaluate the effect of BMC therapy on myocardial viability in patients with decreased left ventricular ejection fraction (LVEF) after acute myocardial infarction (AMI) and to identify predictive factors for improvement of myocardial viability. METHODS AND RESULTS: One hundred and one patients with AMI and successful reperfusion, LVEF ≤45%, and decreased myocardial viability (resting Tl201-SPECT) were randomized to either a control group (n = 49) or a BMC group (n = 52). Primary endpoint was improvement of myocardial viability 3 months after AMI. Baseline mean LVEF measured by radionuclide angiography was 36.3 ± 6.9%. Bone marrow cell infusion was performed 9.3 ± 1.7 days after AMI. Myocardial viability improved in 16/47 (34%) patients in the BMC group compared with 7/43 (16%) in the control group (P = 0.06). The number of non-viable segments becoming viable was 0.8 ± 1.1 in the control group and 1.2 ± 1.5 in the BMC group (P = 0.13). Multivariate analysis including major post-AMI prognostic factors showed a significant improvement of myocardial viability in BMC vs. control group (P = 0.03). Moreover, a significant adverse role for active smoking (P = 0.04) and a positive trend for microvascular obstruction (P = 0.07) were observed. CONCLUSION: Intracoronary autologous BMC administration to patients with decreased LVEF after AMI was associated with improvement of myocardial viability in multivariate-but not in univariate-analysis. A large multicentre international trial is warranted to further document the efficacy of cardiac cell therapy and better define a group of patients that will benefit from this therapy. CLINICAL TRIAL REGISTRATION INFORMATION: URL: http://www.clinicaltrials.gov. Unique identifier NCT00200707.

A novel cancer vaccine strategy based on HLA-A*0201 matched allogeneic plasmacytoid dendritic cells.

BACKGROUND: The development of effective cancer vaccines still remains a challenge. Despite the crucial role of plasmacytoid dendritic cells (pDCs) in anti-tumor responses, their therapeutic potential has not yet been worked out. We explored the relevance of HLA-A*0201 matched allogeneic pDCs as vectors for immunotherapy. METHODS AND FINDINGS: Stimulation of PBMC from HLA-A*0201(+) donors by HLA-A*0201 matched allogeneic pDCs pulsed with tumor-derived peptides triggered high levels of antigen-specific and functional cytotoxic T cell responses (up to 98% tetramer(+) CD8 T cells). The pDC vaccine demonstrated strong anti-tumor therapeutic in vivo efficacy as shown by the inhibition of tumor growth in a humanized mouse model. It also elicited highly functional tumor-specific T cells ex-vivo from PBMC and TIL of stage I-IV melanoma patients. Responses against MelA, GP100, tyrosinase and MAGE-3 antigens reached tetramer levels up to 62%, 24%, 85% and 4.3% respectively. pDC vaccine-primed T cells specifically killed patients' own autologous melanoma tumor cells. This semi-allogeneic pDC vaccine was more effective than conventional myeloid DC-based vaccines. Furthermore, the pDC vaccine design endows it with a strong potential for clinical application in cancer treatment. CONCLUSIONS: These findings highlight HLA-A*0201 matched allogeneic pDCs as potent inducers of tumor immunity and provide a promising immunotherapeutic strategy to fight cancer.

Plasmacytoid dendritic cells and dermatological disorders: focus on their role in autoimmunity and cancer.

Dendritic cells (DC), considered as immunological sentinels of the organism since they are antigen presenting cells, create the link between innate and adaptive immunity. DC include myeloid dendritic cells (MDC) and plasmacytoid dendritic cells (PDC). The presence of PDC, cells capable of producing large quantities of interferon alpha (IFN-alpha) in response to pathogenic agents or danger signals, seems to be closely related to pathological conditions. PDC have been observed in inflammatory immunoallergic dermatological disorders, in malignant cutaneous tumours and in cutaneous lesions of infectious origin. They seem to play a crucial role in the initiation of the pathological processes of autoimmune diseases such as lupus or psoriasis. Their function within a tumour context is not as well known and is controversial. They could have a tolerogenic role towards tumour cells in the absence of an activator but they also have the capacity to become activated in response to Toll-like receptor (TLR) ligands and could therefore be useful for therapeutic purposes.

Intravenous administration of 99mTc-HMPAO-labeled human mesenchymal stem cells after stroke: in vivo imaging and biodistribution.

Human mesenchymal stem cells (hMSC) are a promising source for cell therapy after stroke. To deliver these cells, an IV injection appears safer than a local graft. We aimed to assess the whole-body biodistribution of IV-injected (99m)Tc-HMPAO-labeled hMSC in normal rats (n = 9) and following a right middle cerebral artery occlusion (MCAo, n = 9). Whole-body nuclear imaging, isolated organ counting (at 2 and 20 h after injection) and histology were performed. A higher activity was observed in the right damaged hemisphere of the MCAo group [6.5 +/- 0.9 x 10(-3) % of injected dose (ID)/g] than in the control group (3.6 +/- 1.2 x 10(-3) %ID/g), 20 h after injection. In MCAo rats, right hemisphere activity was higher than that observed in the contralateral hemisphere at 2 h after injection (11.6 +/- 2.8 vs. 9.8 +/- 1.7 x 10(-3) %ID/g). Following an initial hMSC lung accumulation, there was a decrease in pulmonary activity from 2 to 20 h after injection in both groups. The spleen was the only organ in which activity increased between 2 and 20 h. The presence of hMSC was documented in the spleen, liver, lung, and brain following histology. IV-injected hMSC are transiently trapped in the lungs, can be sequestered in the spleen, and are predominantly eliminated by kidneys. After 20 h, more hMSC are found in the ischemic lesion than into the undamaged cerebral tissue. IV delivery of hMSC could be the initial route for a clinical trial of tolerance.

Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues.

KRAS mutation status has been reported to be a predictive marker of tumor response to epidermal growth factor receptor (EGFR) inhibitors. We have designed a pyrosequencing assay based on nested polymerase chain reaction (PCR) to characterize KRAS mutation status using formalin-fixed and paraffin-embedded tumor tissues. Mutant and wild-type KRAS cell lines were used to determine the specificity and sensitivity (detection limit approximately 5% mutant alleles) of the method. The results obtained for tumor samples were 95% comparable to those obtained by dideoxy sequencing. Analysis of KRAS mutation using nested PCR and pyrosequencing is a simple, robust, fast, and sensitive method that can be used with formalin-fixed and paraffin-embedded tissues.

Autologous platelet concentrates for bone graft enhancement in sinus lift procedure.

BACKGROUND: Autologous platelet (PLT)-rich plasma has been reported in some studies to promote osteogenesis. The goal of this study was to demonstrate that osteogenesis gained by mixing autologous PLT concentrates (APCs) with a small quantity of autologous bone graft could give a sufficient quality to lead to dental implant placement. The second goal was to compare this osteogenesis with that obtained by a traditional method (iliac bone graft), through clinical, radiologic, and histologic methods. STUDY DESIGN AND METHODS: Eighteen patients needing bilateral sinus floor augmentation were enrolled. One sinus was grafted with iliac crest bone alone, and the other sinus with a small quantity of bone and APC. Panoramic view, computed tomography scan, and biopsies were performed 6 months after the initial surgery to compare ossification. RESULTS: The adjunction of APCs permitted a 60 percent reduction of bone graft required for sinus floor elevation. The bone obtained with APCs had the same histologic and mechanical characteristics as the bone obtained by traditional graft. CONCLUSION: Topical use of APCs might be helpful in bone reconstruction. No clinical, radiologic, or histologic osteogenesis inhibition of high PLT concentration was observed. The resulting osteogenesis was adapted to dental implant placements.

Oct-4, Rex-1, and Gata-4 expression in human MSC increase the differentiation efficiency but not hTERT expression.

Micro-environment seems to exert an important influence on human mesenchymal stem cell (MSC) differentiation and proliferative capacity in bone marrow as well as in culture ex vivo. Oct-4, Rex-1, and TERT genes are well-known for the maintenance of pluripotentiality differentiation and the proliferative capacity of embryonic stem cells. Some previous data report expression of these embryonic factors in selected clones from bone marrow adult stem cells. Our goal was to study expression of Oct-4, Rex-1, and TERT in primary cultured human MSC according to the serum concentration. In addition, we have studied the expression of Gata-4 since this factor plays a key role in organogenesis. We hypothesized that low serum concentration with appropriate growth factors may induce an undifferentiated status with a re-expression of embryonic factors and extend differentiation capacity. Thus, using a defined culture medium, we report on the increased expression of Oct-4, Rex-1, and Gata-4 in human MSC. We have correlated this expression to an increase in differentiation efficiency towards osteogenic and adipogenic phenotypes. Our data suggest that the culture medium used permits the emergence of adult stem cells with a high differentiation capacity and expression of embryonic factors. These cells may have important implications for cell therapy.

Plasma glutathione peroxidase activity as a potential indicator of hypoxic stress in breath-hold diving.

INTRODUCTION: Diving mammals can cope with oxidants which are produced in excess during the reoxygenation of hypoxic tissues. This study addresses the question of whether antioxidants can adapt and whether it allows humans to tolerate the hypoxic stress induced by a single breath-holding in the course of a dynamic diving exercise and protect them from oxidative insult. METHODS: There were 20 male subjects who performed submaximal apnea dynamic diving (ADD). Nine control subjects stayed out of the water and breathed normally. Venous blood samples were collected 1 h before and immediatly after ADD. RESULTS: ADD induced a significant increase in plasma glutathione peroxidase (GPx-3) activity (from 397.5 +/- 44.4 to 410 +/- 43 U x L(-1)), blood reduced glutathione (GSH) (from 1060 +/- 302 to 1292 +/- 213 micromol x L(-1)), and in plasma creatine kinase activity (from 215 +/- 137 to 235 +/- 152 U x L(-1)). The activity of the erythrocyte superoxide dismutase and glutathione peroxidase, as well as the blood oxidized glutathione and the plasma thiobarbituric acid reactive substances concentrations, were maintained at their basal level. The level of training, characterized by the duration and distance of the dive, had no effect on the markers used. CONCLUSION: GPx-3 and GSH could constitute the most readily mobilizable antioxidants that would then contribute to the buffering against a sudden increase in the generation of radical oxygen species. These biomarkers could be used as tools for establishing oxidative stress during hypoxia. The response of GPx-3 to hypoxia could be of physiological relevance.