Mechanistic Model with Empirical Pitting Onset Approach for Detailed and Efficient Virtual Analysis of Atmospheric Bimetallic Corrosion.

A mechanistic model of atmospheric bimetallic corrosion with a simplified empirical approach to the onset of localized corrosion attacks is presented. The model was built for a typical bimetallic sample containing aluminum alloy 1050 and stainless steel 316L sheets. A strategy was developed that allowed the model to be calibrated against the measured galvanic current, geometrical corrosion attack properties, and corrosion products. The pitting-onset simplification sets all pits to be formed at a position near the nobler metal and treated all pits as being of the same shape and size. The position was based on the location of the highest pitting events and the pit attributes on an average of the deepest pits. For 5 h exposure at controlled RH (85%, 91%, and 97%) and salt load (86 µg NaCl/cm2), the model was shown to be promising: both for analysis of local bimetallic corrosion chemistry, such as pH and corrosion products, and for efficient assessment of pitting damage by computing a single largest pit depth. Parametric studies indicated that the pitting-onset approximation deviated the most at the beginning of exposure and when RH was below 91%.

Effect of Cathodic Polarisation Switch-Off on the Passivity and Stability to Crevice Corrosion of AISI 304L Stainless Steel.

The effects of cathodic polarisation switch-off on the passivation of AISI 304L stainless steel in air and its crevice corrosion susceptibility in 3.5 wt.% NaCl aqueous electrolyte were investigated. Scanning Kelvin probe (SKP) data showed that the oxide film is significantly destabilised and the rate of steel passivation in air is slowed down. Thermal desorption analysis (TDA) highlighted that hydrogen absorption is proportional to the applied cathodic current density. A special crevice corrosion set-up was designed to realise simultaneous reproducible monitoring of potential and galvanic current to study the impact of prior cathodic polarisation on crevice corrosion onset.

Electroactive Bacteria Associated With Stainless Steel Ennoblement in Seawater.

Microorganisms can increase the open-circuit potential of stainless steel immersed in seawater of several hundred millivolts in a phenomenon called ennoblement. It raises the chance of corrosion as the open-circuit potential may go over the pitting corrosion potential. Despite the large impact of the ennoblement, no unifying mechanisms have been described as responsible for the phenomenon. Here we show that the strict electrotroph bacterium "Candidatus Tenderia electrophaga" is detected as an ennoblement biomarker and is only present at temperatures at which we observe ennoblement. This bacterium was previously enriched in biocathode systems. Our results suggest that "Candidatus Tenderia electrophaga," and its previously described extracellular electron transfer metabolism coupled to oxygen reduction activity, could play a central role in modulating stainless steel open-circuit potential and consequently mediating ennoblement.

In situ monitoring of corrosion mechanisms and phosphate inhibitor surface deposition during corrosion of zinc-magnesium-aluminium (ZMA) alloys using novel time-lapse microscopy.

In situ time-lapse optical microscopy was used to examine the microstructural corrosion mechanisms in three zinc-magnesium-aluminium (ZMA) alloy coated steels immersed in 1% NaCl pH 7. Preferential corrosion of MgZn(2) lamellae within the eutectic phases was observed in all the ZMA alloys followed by subsequent dissolution of Zn rich phases. The total extent and rate of corrosion, measured using time-lapse image analysis and scanning vibrating electrode technique (SVET) estimated mass loss, decreased as Mg and Al alloying additions were increased up to a level of 3 wt% Mg and 3.7 wt% Al. This was probably due to the increased presence of MgO and Al(2)O(3) at the alloy surface retarding the kinetics of cathodic oxygen reduction. The addition of 1 × 10(-2) mol dm(-3) Na(3)PO(4) to 1% NaCl pH 7 had a dramatic influence on the corrosion mechanism for a ZMA with passivation of anodic sites through phosphate precipitation observed using time-lapse image analysis. Intriguing rapid precipitation of filamentous phosphate was also observed and it is postulated that these filaments nucleate and grow due to super saturation effects. Polarisation experiments showed that the addition of 1 × 10(-2) mol dm(-3) Na(3)PO(4) to the 1% NaCl electrolyte promoted an anodic shift of 50 mV in open circuit potential for the ZMA alloy with a reduction in anodic current of 2.5 orders of magnitude suggesting that it was acting primarily as an anodic inhibitor supporting the inferences from the time-lapse investigations. These phosphate additions resulted in a 98% reduction in estimated mass loss as measured by SVET demonstrating the effectiveness of phosphate inhibitors for this alloy system.

Changes in transcriptome after in vivo exposure to ionising radiation reveal a highly specialised liver response.

PURPOSE: To identify transcriptional gene-networks involved in the early in vivo response of liver cells to radiation exposure and improve our understanding of the molecular processes responsible for tissue radiosensitivity. MATERIALS AND METHODS: Transcriptome variations of liver RNA samples were measured 3 hours post-irradiation using microarray technology. The results were confirmed and extended using real-time polymerase-chain-reaction (RT-PCR). RESULTS: We identified quantitative changes in the expression of 126 genes, most of which were observed for the first time. We show that some modifications, such as the upregulation of the cyclin-dependent kinase inhibitor 1A (Cdkn1A) gene, persisted for at least two months after the initial exposure. Other genes regulated by the transformation-related protein 53 (Trp53/p53) such as Bcl2-associated X protein (Bax) or etoposide-induced-2.4 (Ei24/PIG8) were not upregulated. Grouping differentially expressed genes into functional categories revealed that the primary response of liver cells to radiation exposure was the enhancement of oxidoreductase activity and inhibition of cell proliferation, involving cell cycle progression and apoptosis-related genes. CONCLUSIONS: The data provides evidence of gene expression modifications associated with the hepatic response to radiation exposure. One of the main differences observed with radiation-sensitive tissues such as the spleen was cell proliferation. The comparison of our data with transcriptome modifications in different biological models enabled the identification of networks of genes that might be co-regulated. Overall, our expression data revealed genes and cellular pathways that might help to improve our understanding of the molecular basis underlying tissue radiosensitivity and to identify possible targets for novel therapeutic strategies.

Leukemia inhibitory factor: Role in human mesenchymal stem cells mediated immunosuppression.

The interactions between mesenchymal stem cells (MSCs) and immune system are currently being explored. Leukemia inhibitory factor (LIF) is linked to regulatory transplantation tolerance. Our aim was to study the expression of LIF on human MSCs at both gene and protein level in mixed lymphocyte reaction (MSC/MLR), and its implication in MSC immunosuppressive effect. There was a 7-fold increase (611pg/ml) in LIF in MSC/MLR as compared to MSCs alone. Using LIF neutralizing antibody, a significant restoration of up to 91% of CD3+ lymphocyte proliferation in MSC/MLR was observed (p=0.021). LIF was implicated in the generation of regulatory lymphocytes, as demonstrated by decrease of Foxp3+ regulatory cells after using LIF neutralizing antibody in MSC/MLR (p=0.06) by flow cytometry. A positive correlation between LIF and human leukocyte antigen (HLA-G) gene expression by MSCs was found (R(2)=0.74). Our findings provide evidence supporting the immunomodulatory effect of MSCs.

Correlation between plasma Flt3-ligand concentration and hematopoiesis during G-CSF-induced CD34+ cell mobilization.

This study aimed to correlate blood Flt3-ligand (FL) concentration with CD34(+) cell number in blood and bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization. Nonhuman primates were injected with 10 microg/kg of G-CSF (Lenograstim) daily over a period of 5 days. Daily blood sampling and repeated BM sampling showed that FL concentration before mobilization was negatively correlated to the absolute number of BM CD34(+) cells, but also to the number of G-CSF-mobilized CD34(+) cells on days 3-5 of treatment. This showed that FL concentration in the blood reflected BM status before mobilization, and suggested that this parameter could be used as a predictive indicator of G-CSF-induced CD34(+) cell mobilization.

Immunosuppressive effects of mesenchymal stem cells: involvement of HLA-G.

INTRODUCTION: Mesenchymal stem cells (MSCs) possess unique immunomodulatory properties. They are able to suppress allogenic T-cell response and modify maturation of antigen-presenting cells. Their role in the treatment of severe graft versus host disease has been reported. The underlying molecular mechanisms of immunosuppression are currently being investigated. Histocompatibility locus antigen (HLA)-G is a nonclassical major histocompatibility complex class I antigen with strong immune-inhibitory properties. METHODS: We studied the role of HLA-G on MSC-induced immunosuppression. The expression of HLA-G on human MSCs cultured alone and in mixed lymphocytes reaction (MSC/MLR) was analyzed. RESULTS: We found that HLA-G can be detected on MSCs by real-time reverse-phase polymerase chain reaction, immunofluorescence, flow cytometry (52.4+/-3.6%), and enzyme-linked immunosorbent assay in the supernatant (38.7+/-5.2 ng/mL). HLA-G protein expression is constitutive and the level is not modified upon stimulation by allogenic lymphocytes in MSC/MLR. The functional role of HLA-G protein expressed by MSCs was analyzed using the 87G anti-HLA-G blocking antibody in a MSC/MLR. We found that blocking HLA-G molecule significantly raised lymphocyte proliferation in MSC/MLR (35.5%, P=0.01). CONCLUSION: Our findings provide evidences supporting involvement of HLA-G in the immunosuppressive properties of MSCs. These results emphasize the potential application of MSCs as a relevant therapeutic candidate in transplantation.

Identification of IL-10 and TGF-beta transcripts involved in the inhibition of T-lymphocyte proliferation during cell contact with human mesenchymal stem cells.

Mesenchymal stem cells (MSC) inhibit the response of allogeneic T lymphocytes in culture. Because the mechanisms of this effect may differ according to the existence of cell contact, we investigated the differences in gene expression of inhibitory molecules during MSC-T lymphocyte coculture when cell contact does and does not occur. Human MSC and T lymphocytes were cultured together in standard and transwell cultures. MSC gene expression was analyzed by semiquantitative real-time RT-PCR. MSC elicited a high dose-dependent inhibition of T lymphocytes in cultures with cell contact, but inhibition occurred even without cell contact. In both cases, we observed significant upregulation of IDO, LIF, and HLA-G, along with downregulation of HGF and SDF1. In cultures with cell contact, IL-10 and TGF-beta transcripts were expressed in a significantly higher level than in cultures without this contact. Furthermore, in the latter, the increased inhibition of T-cell proliferation was positively correlated with IDO gene expression and negatively correlated with SDF1 gene expression. MSC appear to induce T-cell tolerance by two distinct mechanisms. The first of these, which does not require cell contact, induces expression of the tolerogenic genes IDO, LIF, and HLA-G. The second mechanism, which is contact dependent, modulates IL-10 and TGF-beta gene expression. These two mechanisms probably play separate roles in MSC-induced tolerance in allogeneic hematopoietic stem cell transplantation.

Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model.

It has been suggested that human mesenchymal stem cells (hMSC) could be used to repair numerous injured tissues. We have studied the potential use of hMSC to limit radiation-induced skin lesions. Immunodeficient NOD/SCID mice were locally irradiated to the leg (30 Gy, dose rate 2.7 Gy/min) using a (60)Co source to induce a severe skin lesion. Cultured bone marrow hMSC were delivered intravenously to the mice. The irradiated skin samples were studied for the presence of the human cells, the severity of the lesions and the healing process. Macroscopic analysis and histology results showed that the lesions were evolving to a less severe degree of radiation dermatitis after hMSC transplant when compared to irradiated non-transplanted controls. Clinical scores for the studied skin parameters of treated mice were significantly improved. A faster healing was observed when compared to untreated mouse. Immunohistology and polymerase chain reaction analysis provided evidence that the human cells were found in the irradiated area. These results suggest a possible use of hMSC for the treatment of the early phase of the cutaneous radiation syndrome. A successful transplant of stem cells and subsequent reduction in radiation-induced complication may open the road to completely new strategies in cutaneous radiation syndrome therapy.

Mesenchymal stem cells increase self-renewal of small intestinal epithelium and accelerate structural recovery after radiation injury.

Patients who undergo pelvic or abdominal radiotherapy may develop side effects that can be life threatening. Tissue complications caused by radiation-induced stem cell depletion may result in structural and functional alterations of the gastrointestinal (GI) tract. Stem cell therapy using mesenchymal stem cells (MSC) is a promising approach for replenishment of the depleted stem cell compartment during radiotherapy. There is little information on the therapeutic potential of MSC in injured-GI tract following radiation exposure. In this study, we addressed the ability of MSC to support the structural regeneration of the small intestine after abdominal irradiation. We isolated MSC from human bone marrow and human mesenchymal stem cells (hMSC) were transplanted into immunotolerent NOD/SCID mice with a dose of 5.10(6) cells via the systemic route. Using a model of radiation-induced intestinal injury, we studied the link between damage, hMSC engraftment and the capacity of hMSC to sustain structural recovery. Tissue injury was assessed by histological analysis. hMSC engraftment in tissues was quantified by PCR assay. Following abdominal irradiation, the histological analysis of small intestinal structure confirms the presence of partial and transient (three days) mucosal atrophy. PCR analysis evidences a low but significant hMSC implantation in small intestine (0.17%) but also at all the sites of local irradiation (kidney, stomach and spleen). Finally, in presence of hMSC, the small intestinal structure is already recovered at three days after abdominal radiation exposure. We show a structural recovery accompanied by an increase of small intestinal villus height, three and fifteen days following abdominal radiation exposure. In this study, we show that radiation-induced small intestinal injury may play a role in the recruitment of MSC for the improvement of tissue recovery. This work supports, the use of MSC infusion to repair damaged GI tract in patients subjected to radiotherapy. MSC therapy to avoid extended intestinal crypt sterilization is a promising approach to diminish healthy tissue alterations during the course of pelvic radiotherapy.

Use of flt3 ligand to evaluate residual hematopoiesis after heterogeneous irradiation in mice.

We evaluated the possibility of using plasma Flt3 ligand (FL) concentration as a biological indicator of bone marrow function after heterogeneous irradiation. Mice were irradiated with 4, 7.5 or 11 Gy with 25, 50, 75 or 100% of the bone marrow in the field of irradiation. This model of irradiation resulted in graded and controlled damage to the bone marrow. Mice exhibited a pancytopenia correlated with both the radiation dose and the percentage of bone marrow irradiated. The FL concentration in the blood increased with the severity of bone marrow aplasia. Nonlinear regression analysis showed that the FL concentration was strongly correlated with the total number of residual colony-forming cells 3 days after irradiation, allowing a precise estimate of residual hematopoiesis. Moreover, the FL concentration on day 3 postirradiation was correlated with the duration and severity of subsequent pancytopenia, suggesting that variations in FL concentrations might be used as a predictive indicator of bone marrow aplasia, especially by the use of linear regression equations describing these correlations. Our results provide a rationale for the use of FL concentration as a biological indicator of residual hematopoiesis after heterogeneous irradiation.

Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs: a study of their quantitative distribution after irradiation damage.

Mesenchymal stem cells (MSCs) have been shown to migrate to various tissues. There is little information on the fate and potential therapeutic efficacy of the reinfusion of MSCs following total body irradiation (TBI). We addressed this question using human MSC (hMSCs) infused to nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice submitted to TBI. Further, we tested the impact of additional local irradiation (ALI) superimposed to TBI, as a model of accidental irradiation. NOD/SCID mice were transplanted with hM-SCs. Group 1 was not irradiated before receiving hMSC infusion. Group 2 received only TBI at a dose of 3.5 Gy, group 3 received local irradiation to the abdomen at a dose of 4.5 Gy in addition to TBI, and group 4 received local irradiation to the leg at 26.5 Gy in addition to TBI. Fifteen days after irradiation, quantitative and spatial distribution of the hMSCs were studied. Histological analysis of mouse tissues confirmed the presence of radio-induced lesions in the irradiated fields. Following their infusion into nonirradiated animals, hMSCs homed at a very low level to various tissues (lung, bone marrow, and muscles) and no significant engraftment was found in other organs. TBI induced an increase of engraftment levels of hMSCs in the brain, heart, bone marrow, and muscles. Abdominal irradiation (AI) as compared with leg irradiation (LI) increased hMSC engraftment in the exposed area (the gut, liver, and spleen). Hind LI as compared with AI increased hMSC engraftment in the exposed area (skin, quadriceps, and muscles). An increase of hMSC engraftment in organs outside the fields of the ALI was also observed. Conversely, following LI, hMSC engraftment was increased in the brain as compared with AI. This study shows that engraftment of hMSCs in NOD/ SCID mice with significantly increased in response to tissue injuries following TBI with or without ALI. ALI induced an increase of the level of engraftment at sites outside the local irradiation field, thus suggesting a distant (abscopal) effect of radiation damage. This work supports the use of MSCs to repair damaged normal tissues following accidental irradiation and possibly in patients submitted to radiotherapy.

Kinetics of plasma FLT3 ligand concentration in hematopoietic stem cell transplanted patients.

The present study aimed to follow-up variations in plasma Flt3 ligand (FL) concentration after hematopoietic stem cell transplantation and to compare the influence of conditioning regimens on variations in FL concentration. Ten patients undergoing a conditioning regimen, including BEAM, cyclophosphamide (Cy) + total body irradiation or Cy + anti-thymocyte globulins (ATG), which was then followed by hematopoietic stem cell transplantation, were studied. Plasma FL concentrations, white blood cell (WBC) expression of both FL mRNA and the membrane-bound form of FL were carried out at different times post-treatment. The results indicated that plasma FL concentration increased rapidly after the conditioning regimen in all patients, in correlation with the decrease in number of WBCs. The area under the curve of FL according to time was directly correlated with the duration of pancytopenia, except when ATG was included in the conditioning regimen. Although the number of patients was limited in this study, the comparison of ATG-treated patients and other patients suggests that plasma FL concentration is regulated by a complex mechanism partly involving circulating blood cells.

[Mesenchymal stem cells from human proximal femurs possess immunosuppressive activity].

OBJECTIVE: To evaluate whether mesenchymal stem cells (MSCs) obtained from human proximal femurs possess immunosuppressive effect so as to look for ideal bank of MSCs for clinical prophylaxis and treatment of graft versus host disease (GVHD). METHODS: Human marrows were collected from the proximal femurs of patients undergoing hip replacement to isolate MSCs. The puncture materials obtained from the iliac bone marrow of 12 healthy donors were used as controls. Peripheral blood lymphocytes (PBLs) were obtained from the peripheral blood of healthy persons. 1 x 10(5) PBLs were mixed with allogeneic PBLs radiated by 60 cobalt and put into the wells of a 96-well plate. MSCs of the concentrations of 1 x 10(5), 3 x 10(4), 1 x 10(4), and 3 x 10(3), were added into the culture fluid of the mixed PBLs to be co-cultivated for 5 days. 1 microCi/well [(3)H] TdR was added in the last 18 hours. The cells were collected and the counts per minute (cpm) was detected. 3 x 10(4) and 1 x 10(4) MSCs were put into the wells. When the MSCs adhered to the wall, a membrane with micropores was inserted value of 1 x 10(5) PBLs and radiated allo-PBLs were put onto the top of which. Five days after cultivation 1 microCi/well [(3)H] TdR was added and the cpm was tested. MSCs were cultured in RPMI-1640 culture fluid and then contacted MLR constructed by 1 x 10(5) PBLs and 1 x 10(5) allo-PBL directly or via Transwell membrane with micropores. Five days after the supernatant was collected. ELISA was used to detect the content of TGF-beta(1). 0.1 microg/ml, 1 microg/ml, or 10 microg/ml anti-human TGF-beta1 antibody was added to the co-cultivation system. Five days after [(3)H] TdR was added so as to test the value cpm. RESULTS: 3 x 10(3) - 1 x 10(5) MSCs from proximal femurs inhibited the PBLs proliferation to 62 +/- 18% - 28 +/- 12% of maximal response, however, not significantly different from that observed in MSCs collected from bone marrow of healthy donors via iliac crest aspiration (58 +/- 12% - 27 +/- 6%, P > 0.05). When these cells were separated physically from MLR system via a membrane with micropores 0.2 microm in diameter, 3 x 10(4) and 1 x 10(4) cells still markedly suppressed the PBLs proliferation to 36 +/- 8% and 53 +/- 13% of maximal response. ELISA showed that TGF-beta1 was measured in the supernatants of MSCs, MSCs + MLR and MSCs + MLR in Transwell system, without significant differences among these experimental conditions. Furthermore, the presence of increasing amounts of neutralizing anti- TGF-beta1 antibody did not reverse the inhibitory effect. CONCLUSION: MSCs obtained from the proximal femurs possess immunosuppressive activity. A soluble factor/factors was/were involved in such effect, however, TGF-beta1 was not a candidate.

Comparison of autologous cell therapy and granulocyte-colony stimulating factor (G-CSF) injection vs. G-CSF injection alone for the treatment of acute radiation syndrome in a non-human primate model.

PURPOSE: To compare the efficacy of autologous cell therapy after irradiation combined with granulocyte-colony stimulating factor (G-CSF) injections with G-CSF treatment alone in a heterogeneous model of irradiation representative of an accidental situation. MATERIAL AND METHODS: Non-human primates were irradiated at 8.7 Gy whole-body dose with the right arm shielded to receive 4.8 Gy. The first group of animals received G-CSF (lenograstim) injections starting 6 h after irradiation, and a second group received a combination of G-CSF (lenograstim) injections and autologous expanded hematopoietic cells. Animals were followed up for blood cell counts, circulating progenitors, and bone marrow cellularity. RESULTS: No significant differences were seen between the two treatment groups, whatever the parameter observed: time to leukocyte or platelet recovery and duration and severity of aplasia. CONCLUSION: Our results indicated that identical recovery kinetic was observed when irradiated animals are treated with G-CSF independently of the reinjection of ex vivo expanded autologous hematopoietic cells. Thus G-CSF injections might be chosen as a first-line therapeutic strategy in the treatment of accidental acute radiation victims.

Radiation-induced increase in plasma Flt3 ligand concentration in mice: evidence for the implication of several cell types.

Circulating T lymphocytes were proposed as the main producer of Flt3 ligand. However, during aplasia, there is a drastic reduction in the number of T lymphocytes, while plasma Flt3 ligand concentration is increased. This contradiction prompted us to compare variations in plasma Flt3 ligand during radiation-induced aplasia in BALB/c mice and in T-lymphocyte-deficient NOD-SCID mice to delineate the role of T lymphocytes in the increase in Flt3 ligand concentration. The results showed that plasma Flt3 ligand concentration was increased similarly in the two strains of mice, and that Flt3 ligand concentration was negatively correlated to the number of residual hematopoietic progenitors. Moreover, the Flt3 ligand mRNA expression and Flt3 ligand protein concentration were similar in the two strains of mice in all organs tested, i.e. thymus, spleen, bone marrow, liver, brain and blood cells. These results confirm that Flt3 ligand concentration in the blood is a reflection of bone marrow function and that T lymphocytes are not the main regulator of Flt3 ligand variations during aplasia.

Application of autologous hematopoietic cell therapy to a nonhuman primate model of heterogeneous high-dose irradiation.

We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.

[Therapeutic effect of human mesenchymal stem cells in skin after radiation damage]. / Potentiel thérapeutique des cellules souches mésenchymateuses humaines dans les lésions cutanées radioinduites.

Over 50% of all cancer patients presently receive radiotherapy at one stage in their treatment course. Inevitably skin is one of the most frequently damaged tissue due to its localization and constant turn-over. Our present goal is to reduce radiation-induced complications in human skin through stem cell therapy, particulary in human epidermis. Mesenchymal Stem Cells (MSCs) have been shown to be multipotent cells able to engraft in many tissues after injury. Herein, we isolated human MSCs and tested their capability to improve skin wound healing after irradiation. This potential was assessed in NOD/SCID mice which received 30 Gy locally on the thigh. This dose caused within 3 weeks local epidermis necrosis which was repaired within 13 weeks. MSCs were intravenously injected in irradiated mice 24 hours after exposure. Clinical scoring throughout 6 weeks gave indications that human MSCs reduced the extent of damage and accelerated the wound healing process. We show by quantitative qPCR and histological studies the presence of human MSCs derived cells into the scar. Human MSCs homed to the damaged skin and participated to the wound healing process. These results open prospects for cellular therapy by MSCs in irradiated epithelial tissues and could be extended to the whole general field of cutaneous cicatrization, particularly after burns.

In vivo gene targeting of IL-3 into immature hematopoietic cells through CD117 receptor mediated antibody gene delivery.

BACKGROUND: Targeted gene transfection remains a crucial issue to permit the real development of genetic therapy. As such, in vivo targeted transfection of specific subsets of hematopoietic stem cells might help to sustain hematopoietic recovery from bone marrow aplasia by providing local production of growth factors. METHODS: Balb/C mice were injected intravenously, with an anti-mouse c-kit (CD117) monoclonal antibody chemically coupled to a human IL-3 gene-containing plasmid DNA. Mice were sacrificed for tissue analyses at various days after injection of the conjugates. RESULTS: By ELISA, the production of human IL-3 was evidenced in the sera of animals 5 days after treatment. Cytofluorometric analysis after in vivo transfection of a reporter gene eGFP demonstrated transfection of CD117+/Sca1+ hematopoietic immature cells. By PCR analysis of genomic DNA and RNA using primer specific pIL3 sequences, presence and expression of the human IL-3-transgene were detected in the bone marrow up to 10 days in transfected mice but not in control animals. CONCLUSIONS: These data clearly indicate that antibody-mediated endocytosis gene transfer allows the expression of the IL-3 transgene into hematopoietic immature cells, in vivo. While availability of marketed recombinant growth factors is restricted, this targeting strategy should permit delivery of therapeutic genes to tissues of interest through systemic delivery. In particular, the ability to specifically target growth factor expression into repopulating hematopoietic stem cells may create new opportunities for the treatment of primary or radiation-induced marrow failures.