Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells.

Engineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 x 12cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

Macrophages promote epithelial repair through hepatocyte growth factor secretion.

Macrophages play a critical role in intestinal wound repair. However, the mechanisms of macrophage-assisted wound repair remain poorly understood. We aimed to characterize more clearly the repair activities of murine and human macrophages. Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of healthy donors (HD) or Crohn's disease (CD) patients or isolated from the intestinal mucosa of HD. In-vitro models were used to study the repair activities of macrophages. We found that murine and human macrophages were both able to promote epithelial repair in vitro. This function was mainly cell contact-independent and relied upon the production of soluble factors such as the hepatocyte growth factor (HGF). Indeed, HGF-silenced macrophages were less capable of promoting epithelial repair than control macrophages. Remarkably, macrophages from CD patients produced less HGF than their HD counterparts (HGF level: 84âeuro0/00±âeuro0/0027âeuro0/00pg/mg of protein and 45âeuro0/00±âeuro0/0034âeuro0/00pg/mg of protein, respectively, for HD and CD macrophages, Pâeuro0/00<âeuro0/000·009) and were deficient in promoting epithelial repair (repairing activity: 90·1âeuro0/00±âeuro0/004·6 and 75·8âeuro0/00±âeuro0/008·3, respectively, for HD and CD macrophages, Pâeuro0/00<âeuro0/000·0005). In conclusion, we provide evidence that macrophages act on wounded epithelial cells to promote epithelial repair through the secretion of HGF. The deficiency of CD macrophages to secrete HGF and to promote epithelial repair might contribute to the impaired intestinal mucosal healing in CD patients.

Study of the mechanisms regulating the proliferative potential of human CD8+T lymphocytes over-expressing telomerase reverse transcriptase (hTERT)

Résumé La plupart des cellules issues du sang ont une durée de vie limitée. Dans les cellules somatiques humaines, y incluant les lymphocytes T, la taille des télomères diminue progressivement à chaque division cellulaire, pouvant aboutir à des instabilités chromosomiques. L'expression ectopique du gène de la transcriptase réverse de la télomérase (hTERT) dans les cellules restaure l'activité de la télomérase, et permet un rallongement de leur vie réplicative. Malgré l'absence de signes caractéristiques de transformation, nous ne savons pas encore si les cellules somatiques qui surexpriment hTERT sont physiologiquement indiscernables des cellules normales. Certaines études récentes proposent que la télomérase joue plusieurs rôles additionnels dans d'autres phénomènes biologiques tels que la réparation de l'ADN, la survie et la croissance des cellules. Dans notre étude, nous avons utilisé des clones issus de lymphocytes T cytotoxiques surexprimant la télomérase afin d'étudier les mécanismes moléculaires qui règlent leur prolifération et leur sénescence. Nous avons montré que les «jeunes » cellules T exprimant ou non hTERT révèlent des taux de croissance identiques suite à des réponses de stimulation induites par des mitogènes. De plus, aucun changement global dans leur expression des gènes n'a pu être mis en évidence. Curieusement, nous avons observé des réponses réduites dans la prolifération des cellules transduites avec la télomérase qui présentaient une élongation des télomères et une durée de vie prolongée. Ces cellules, malgré le maintien d'un niveau élevé de l'expression de gènes impliqués dans la progression du cycle cellulaire, ont également montré une expression accrue de plusieurs gènes trouvés en commun avec nos lymphocytes T vieillissants n'exprimant pas de télomérase. En particulier, les cellules ayant une durée de vie prolongée grâce à l'expression de la télomérase accumulaient également certains inhibiteurs du cycle cellulaire tels que p16Ink4a et p21Cip1, associés à l'arrêt de la croissance cellulaire. En résumé, nos résultats indiquent la présence fonctionnelle de mécanismes alternatifs pouvant contrôler la croissance réplicative de ces cellules; ils sont donc encourageants dans l'optique d'une utilisation à moindre risque de lymphocytes T «immortalisés » à des fins thérapeutiques pour traiter les tumeurs malignes ou les infections. Summary Most mature blood cells have a finite life span. In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division eventually leading to chromosomal instability. Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene in cells restores telomerase activity and results in the extension of their replicative life span. Despite lack of transformation characteristics, it is yet unknown whether somatic cells that over-express telomerase are biologically and physiologically indistinguishable from normal cells. Recent data suggest that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation and senescence. Here we show that early-passage T cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with elongated telomeres and extended life span. These cells, despite maintaining high expression level of genes involved in cell cycle division and progression, also showed increased expression of several genes associated with normal aging T lymphocytes. In particular, late passage T cells over-expressing telomerase accumulated the cyclin-dependent inhibitors p16INK4a and p21CIP1 that have largely been associated with in vitro growth arrest. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p 16 and p21 are still functional in these cells and regulate to some extend their growth potential.

Critical Role of the GM-CSF Signaling Pathway in Macrophage Pro-Repair Activities.

OBJECTIVE: Macrophages play a critical role in intestinal wound repair. However, the molecular pathways that regulate macrophage wound repair activities remain poorly understood. The aim of this study was to evaluate the role of GM-CSF receptor signaling in the wound repair activities of macrophages. METHODS: Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of Crohn's disease (CD) patients. In vitro models were used to study the repair activities of macrophages. RESULTS: We provide evidence that GM-CSF receptor signaling is required for murine macrophages to promote epithelial repair. In addition, we demonstrate that the deficient repair properties of macrophages from CD patients with active disease can be recovered via GM-CSF therapy. CONCLUSION: Our data support a critical role of the GM-CSF signaling pathway in the pro-repair activities of mouse and human macrophages. © 2014 S. Karger AG, Basel.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

ABSTRACT: BACKGROUND: Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. RESULTS: Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000. CONCLUSIONS: A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Laparoscopic inguinal hernia repair with extraperitoneal double-mesh technique.

Totally extraperitoneal laparoscopic hernia repair is an efficient but technically demanding procedure. As mechanisms of hernia recurrence may be related to these technical difficulties, we have modified a previously described double-mesh technique in an effort to simplify the procedure. Extraperitoneal laparoscopic hernia repairs were performed in 82 male and 17 female patients having inguinal, femoral, and recurrent bilateral hernias. A standard propylene mesh measuring 15 x 15 cm was cut into two pieces of 4 x 15 cm and 11 x 15 cm. The smaller mesh was placed over both inguinal rings without splitting. The larger mesh was then inserted over the first mesh and stapled to low-risk zones, reinforcing the large-vessel area and the nerve transition zone. The mean procedure duration was 60 minutes for unilateral and 100 minutes for bilateral hernia repair. Patients were discharged from the hospital within 48 hours. The mean postoperative follow-up was 22 months, with no recurrences, neuralgia, or bleeding complications. Over a 2-year period, this technique was found to be satisfactory without recurrences or significant complications. In our hands, this technique was easier to perform: it allows for a less than perfect positioning of the meshes and avoids most of the stapling to crucial zones.

MACROPHAGES FROM CROHN'S DISEASE PATIENTS EXHIBIT DEFICIENT REPAIR FUNCTIONS

AbstractBackground: Mucosal healing is becoming a major goal in the treatment of Crohn's disease. It has been previously reported that myeloid cells induce mucosal healing in a mouse model of acute colitis. The aim in this study is to investigate the pro-repair function of myeloid cells in healthy donors (HD) and Crohn's disease patients (CD).Methods: Peripheral blood mononuclear cells (PBMC) from HD and CD patients were isolated from blood samples and tested either directly or after differentiation ex-vivo into macrophages (Μφ). Intestinal macrophages (IMACs) were isolated from the bowel mucosa of patients undergoing intestinal surgical resections. Through an in vitro wound healing assay the repairing ability of these various human myeloid cells and the mechanisms responsible of wound healing were evaluated.Results: PBMC and myeloid CD14+ cells from HD and CD were not able to repair at any tested cell concentration. Μφ from HD and ulcerative colitis (UC) patients were able to induce wound healing and this capacity was partially mediated by Hepatocyte Growth Factor (HGF). Remarkably, CD Μφ were unable to promote wound healing and produced lower levels of HGF as compared to Μφ from HD or UC patients. In particular, Μφ from CD in active phase (ACD) exhibited the weakest repair function, but this defect was rescued if rh- GM-CSF was added during the differentiation of PBMCs. Interestingly, IMACs from HD promoted wound healing and produced HGF.Conclusion: We demonstrated that CD Μφ, unlike HD or UC Μφ, were defective in promoting wound healing, in particular if coming from an ACD. This deficient pro-repair function was related to a lower production of HGF. IMACs from HD colonic mucosa induced wound healing, confirming the results obtained with Μφ. Our results are in keeping with the current theory of CD as an innate immunodeficiency. In this context, Μφ may be responsible for the mucosal repair defects observed in CD patients and for the subsequent chronic activation of the adaptive immune response.

Licorice-induced hypertension and common variants of genes regulating renal sodium reabsorption.

AIM: To study if gene alterations affecting renal sodium reabsorption associate with susceptibility to licorice-induced hypertension.METHODS: Finnish subjects (n = 30) with a previously documented incident of licorice-induced hypertension were recruited for the study using a newspaper announcement. Their previous clinical and family histories as well as serum electrolyte levels were examined. DNA samples from all individuals were screened for variants of the genes encoding 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) and alpha-, beta-, and gamma-subunits of the epithelial sodium channel (ENaC).RESULTS: Upon licorice predisposition, the patients had a mean blood pressure of 201/118 mmHg. Circulating potassium, renin, and aldosterone levels were low. No significant DNA variations were identified in the 11betaHSD2 gene. Four subjects were heterozygous for beta- and gammaENaC variants previously shown to be associated with hypertension. Furthermore, a novel G insertion (2004-2005insG) in the SCNN1A gene encoding the alphaENaC was identified in two subjects. The frequency of these ENaC variants was significantly higher in subjects with licorice-induced hypertension (6/30 i.e. 20%) than in blood donors (11/301 i.e. 3.7%, P = 0.002).CONCLUSIONS: Defects of the 11betaHSD2 gene do not constitute a likely cause for licorice-induced hypertension. Variants of the ENaC subunits may render some individuals sensitive to licorice-induced metabolic alterations and hypertension.

Analyses of six homologous proteins of Protochlamydia amoebophila UWE25 encoded by large GC-rich genes (lgr): a model of evolution and concatenation of leucine-rich repeats.

BACKGROUND: Along the chromosome of the obligate intracellular bacteria Protochlamydia amoebophila UWE25, we recently described a genomic island Pam100G. It contains a tra unit likely involved in conjugative DNA transfer and lgrE, a 5.6-kb gene similar to five others of P. amoebophila: lgrA to lgrD, lgrF. We describe here the structure, regulation and evolution of these proteins termed LGRs since encoded by "Large G+C-Rich" genes. RESULTS: No homologs to the whole protein sequence of LGRs were found in other organisms. Phylogenetic analyses suggest that serial duplications producing the six LGRs occurred relatively recently and nucleotide usage analyses show that lgrB, lgrE and lgrF were relocated on the chromosome. The C-terminal part of LGRs is homologous to Leucine-Rich Repeats domains (LRRs). Defined by a cumulative alignment score, the 5 to 18 concatenated octacosapeptidic (28-meric) LRRs of LGRs present all a predicted alpha-helix conformation. Their closest homologs are the 28-residue RI-like LRRs of mammalian NODs and the 24-meres of some Ralstonia and Legionella proteins. Interestingly, lgrE, which is present on Pam100G like the tra operon, exhibits Pfam domains related to DNA metabolism. CONCLUSION: Comparison of the LRRs, enable us to propose a parsimonious evolutionary scenario of these domains driven by adjacent concatenations of LRRs. Our model established on bacterial LRRs can be challenged in eucaryotic proteins carrying less conserved LRRs, such as NOD proteins and Toll-like receptors.

A liver protein fraction regulating hormone-dependent in vitro transcription from the vitellogenin genes induces their expression in Xenopus oocytes.

Xenopus laevis oocytes were used to assay for trans-acting factors shown previously to be involved in the liver-specific regulation of the vitellogenin genes in vitro. To this end, crude liver nuclear extracts obtained from adult estrogen-induced Xenopus females were fractionated by heparin-Sepharose chromatography using successive elutions with 0.1, 0.35, 0.6, and 1.0 M KCl. When these four fractions were injected into oocytes, only the 0.6-M KCl protein fraction significantly stimulated mRNA synthesis from the endogenous B class vitellogenin genes. This same fraction induced estrogen-dependent in vitro transcription from the vitellogenin B1 promoter, suggesting that it contains at least a minimal set of basal transcription factors as well as two positive factors essential for vitellogenin in vitro transcription, i.e. the NF-I-like liver factor B and the estrogen receptor (ER). The presence of these two latter factors was determined by footprinting and gel retardation assays, respectively. In contrast, injection of an expression vector carrying the sequence encoding the ER was unable to activate transcription from the oocyte chromosomal vitellogenin genes. This suggests that the ER alone cannot overcome tissue-specific barriers and that one or several additional liver components participate in mediating tissue-specific expression of the vitellogenin genes. In this respect, we present evidence that the oocyte germinal vesicles contain an NF-I-like activity different from that found in hepatocytes of adult frogs. This observation might explain the lack of vitellogenin gene activation in oocytes injected with the ER cDNA only.

'Good-genes' and 'compatible-genes' effects in an Alpine whitefish and the information content of breeding tubercles over the course of the spawning season.

Some models of sexual selection predict that individuals vary in their genetic quality and reveal some of this variation in their secondary sexual characteristics. Alpine whitefish (Coregonus sp.) develop breeding tubercles shortly before their spawning season. These tubercles are epidermal structures that are distributed regularly along the body sides of both males and females. There is still much unexplained variation in the size of breeding tubercles within both sexes and with much overlap between the sexes. It has been suggested that breeding tubercles function to maintain body contact between the mating partners during spawning, act as weapons for defence of spawning territories, or are sexual signals that reveal aspects of genetic quality. We took two samples of whitefish from their spawning place, one at the beginning and one around the peak of spawning season. We found that females have on average smaller breeding tubercles than males, and that tubercle size partly reveals the stage of gonad maturation. Two independent full-factorial breeding experiments revealed that embryo mortality was significantly influenced by male and female effects. This finding demonstrates that the males differed in their genetic quality (because offspring get nothing but genes from their fathers). Tubercle size was negatively linked to some aspects of embryo mortality in the first breeding experiment but not significantly so in the second. This lack of consistency adds to inconsistent results that were reported before and suggests that (i) some aspects of genetic quality are not revealed in breeding tubercles while others are, or (ii) individuals vary in their signaling strategies and the information content of breeding tubercles is not always reliable. Moreover, the fact that female whitefish have breeding tubercles of significant size while males seem to have few reasons to be choosy suggests that the tubercles might also serve some functions that are not linked to sexual signaling.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Gènes, environnement et neurodéveloppement: te cas de la schizophrénie [Schizophrenia: genes, environment and neurodevelopment].

Psychoses are complex diseases resulting from the interaction between genetic vulnerability factors and various environmental risk factors during the brain development and leading to the emergence of the clinical phenotype at the end of adolescence. Among the mechanisms involved, a redox imbalance plays an important role, inducing oxidative stress damaging to developing neurons. As a consequence, the excitatory/inhibitory balance in cortex and the pathways connecting brain areas are both impaired. Childhood and adolescence appear as critical periods of vulnerability for deleterious environmental insults. Antioxidants, applied during the environmental impacts, should allow preventing these impairments as well as their clinical consequences.