Extensive natural variation for cellular hydrogen peroxide release is genetically controlled.

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H(2)O(2) release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H(2)O(2) release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10-8) and 54 suggestive associations (p<1.00×10-5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H(2)O(2) release was observed in Down Syndrome (DS) individuals (p<2.88×10-12). Taken together, our results show strong evidence of genetic control of H(2)O(2) in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.

Bone tissue engineering using foetal cell therapy.

Different cell sources for bone tissue engineering are reviewed. In particular, adult cell source strategies have been based on the implantation of unfractionated fresh bone marrow; purified, culture expanded mesenchymal stem cells, differentiated osteoblasts, or cells that have been modified genetically to express rhBMP. Several limiting factors are mentioned for these strategies such as low number of available cells or possible immunological reaction of the host. Foetal bone cells are presented as an alternative solution and review of actual treatments using these cells is presented. Finally, foetal cells used specifically for bone tissue engineering are characterised and potentially interesting therapeutic options are proposed.

Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects.

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response.

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4+ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4+ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35-/- and IL-12p40-/-) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4+ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in purified CD4+ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-gamma and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-gamma mRNA in CD4+ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-gamma in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.

Correlation between vasoconstrictor roles and mRNA expression of alpha1-adrenoceptor subtypes in blood vessels of genetically engineered mice.

We examined the contribution of each alpha(1)-adrenoceptor (AR) subtype in noradrenaline (NAd)-evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration-response curves (CRCs) for NAd in the thoracic aortas of wild-type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD(2) value in rank order is as follows: WT mice (8.21) > alpha(1B)-adrenoceptor knockout (alpha(1B)-KO) (7.77) > alpha(1D)-AR knockout (alpha(1D)-KO) (6.44) > alpha(1B)- and alpha(1D)-AR double knockout (alpha(1BD)-KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and alpha(1B)-KO mice (7.12) or alpha(1D)-KO (6.19) and alpha(1BD)-KO (6.29) mice. However, the CRC maximum responses to NAd in alpha(1D)- and alpha(1BD)-KO mice were significantly lower than those in WT and alpha(1B)-KO mice. Except in the thoracic aortas of alpha(1BD)-KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the alpha(1D)-AR gene. In the thoracic aorta, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.25 and 8.46, respectively, and in alpha(1B)-KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.34 and 7.47, respectively, and in alpha(1B)-KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and alpha(1B)-KO mice, which showed low affinities to BMY7378. We performed a quantitative analysis of the mRNA expression of each alpha(1)-AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each alpha(1)-AR subtype in mediating vascular contraction. The rank order of each alpha(1)-AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, alpha(1D)-AR > alpha(1B)-AR > alpha(1A)-AR in the thoracic aorta and alpha(1D)-AR > alpha(1A)-AR > alpha(1B)-AR in the mesenteric artery. No dramatic compensatory change of alpha(1)-AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.

Modified Corneal Collagen Crosslinking (CXL) Reduces Corneal Edema and Diurnal Visual Fluctuations in Fuchs' Dystrophy

Purpose: Crosslinking of corneal collagen with riboflavin and ultraviolet-A irradiation (CXL) induces crosslinks within and between collagen fibers. CXL increases corneal biomechanical and biochemical stability and is currently used clinically to treat keratectasia. CXL also significantly reduces the stromal swelling capacity. We investigated whether a modified CXL treatment protocol would be beneficial in early Fuchs' dystrophy with various degrees of corneal edema and diurnal variations in visual acuity. Methods: CXL was performed as published previously with the following modification: in cases where the stroma was thicker than 450 µm after abrasion and 30 minutes of instillation of isoosmolar riboflavin solution, glycerol 70% solution was applied every 5 seconds for two minutes, and central corneal thickness (CCT) was measured using ultrasound pachymetry. Glycerol 70% solution was administered repeatedly until the target corneal thickness of 370-430 µm was reached. During irradiation, CCT was monitored by ultrasound pachymetry every five minutes and glycerol 70% solution was applied, if necessary. Results: Three eyes in two patients were treated using the modified CXL protocol. Representative case: a 50-year-old woman with Fuchs' dystrophy and a history of 3 years of diurnal visual fluctuations was referred to us in March 2008. Preoperative best spectacle-corrected visual acuity (BSCVA) was 20/50. We performed modified CXL in the left eye. At one month after CXL, Scheimpflug analysis of CCT showed a reduction of more than 100 µm, and the Corneal Thickness Spatial Profile (CTSP) and Percentage of Increase in Thickness (PIT) showed a regularization of the "flattening" typical for Fuchs' dystrophy. Accordingly, diurnal analysis of corneal thickness showed a distinct postoperative reduction in CCT at all time points measured. At one month after CXL, the patient reported a reduction of diurnal visual fluctuations and we measured an increase in BSCVA to 20/32. The patient showed stable topographical and visual acuity at the three months follow-up. Conclusions: We saw a distinct reduction in CCT, an improvement of the corneal thickness spatial profile (CTSP) and an increase in BSCVA at one month after treatment, which remained stable at the three months follow-up. Patients with early Fuchs' dystrophy and disturbing diurnal visual fluctuations represent a novel application for CXL. Although CXL may not prevent the outcome of the dystrophy, it may increase the patients' visual comfort until keratoplasty becomes necessary.

Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells.

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.

Evaluation of a modified Broström-Gould procedure for treatment of chronic lateral ankle instability: A retrospective study with critical analysis of outcome scoring.

BACKGROUND: Chronic lateral ankle instability accounts for 20% of the ankle injuries. This study evaluates functional outcome of the modified Broström-Gould technique using suture anchors, with 4 different clinical scores. METHODS: A consecutive series of 41 patients were included with a minimum follow-up of one year. The function was assessed using 4 clinical scores including: the AOFAS for hind foot; the FAAM; the CAIT and the CAIS. RESULTS: Out of 41 patients; 27 patients were very satisfied, 11 satisfied and 3 were not satisfied. Ankle mobility returned to normal in 93% of patients. At follow-up the AOFAS was 89/100 (37-100), the FAAM 85/100% (35-100%), the CAIT 20/30 (5-30), and the CAIS 74/100% (27-100%). CONCLUSION: Outcome of modified Broström-Gould procedure is good with high satisfaction rate in terms of ankle mobility. The disparity in outcome of scores, signals towards the need of a standard evaluation system.

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production.

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor beta (IL-2R beta; CD122) and the polymorphic antigen NK1.1. These "NK-like" T cells are distinguished by their highly skewed V alpha and V beta repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1+ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2R beta+ T cells with a skewed V beta repertoire characteristic of "NK-like" T cells. Furthermore lack of NK1.1 expression on IL-2R beta+ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2R beta+ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that "NK-like" T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.

Advantages of a modified gastroscope for video-assisted internal mammary artery harvesting.

Instead of standard rigid thoracoscopes, we used a modified gastroscope for video assistance during 12 minimally invasive left internal mammary harvesting. Flexibility and remote control of its last centimeters give to the gastroscope a total freedom of movements, and perfect positioning in every direction. The scope is equipped with cold light, a suction canal and an irrigation canal, which allow for in situ washing without needing to remove it from the thoracic cavity. Thanks to these advantages, vision and lighting are always perfect.

Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1.

Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by cell density via diffusible signalling molecules ('autoinducers') of the N-acylhomoserine lactone (AHL) type. Two Bacillus sp. isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria. From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain. Expression of the aiiA homologue from isolate A24 in P. aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, N-butyryl-L-homoserine lactone. This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming. However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface. In conclusion, introduction of an AHL degradation gene into P. aeruginosa could block cell-cell communication and exoproduct formation, but failed to interfere with surface colonization.

Innate immune response to poxvirus vectors

Summary : A large body of evidence indicates that the innate immune system plays a key role in host response to viral infection. Recently, Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptor receptors (NLRs) have emerged as key innate immune sensors of microbial products, eliciting intracellular signaling and leading to the production of chemokines, cytokines and interferons (IFNs) that shape innate immune responses and coordinate the development of adaptive immunity. Poxviruses are currently developed as vaccines vectors for infectious diseases such as HIV, tuberculosis and malaria. Modified vaccinia virus Ankara (MVA) and New York vaccinia virus (NWAC) are attenuated, replication deficient strains of poxvirus. The mechanisms underlying innate immune responses to MVA and NYVAC are poorly characterized. Thus, the objectives of the project were to determine the innate immune profile stimulated by poxviruses in innate immune cells and to evaluate the impact of modifications in the viral genome on MVA and NYVAC immunogenicity. MVA stimulated the production of abundant amounts of chemokines and IFNß but low levels of cytokines by human macrophages. In contrast, NYVAC weakly stimulated the production of all mediators. Interestingly, MVA and NYVAC strongly stimulated innate immune responses in vivo and in human whole blood, suggesting that a soluble factors}, possibly a complement component, was required for optimal activation of innate immune cells by poxviruses. Modified MVA and NYVAC produced by single or multiple deletions of viral genes targeting crucial pathways of host innate immunity, and mutant poxviruses with limited replication capacity, increased the production of pro-inflammatory molecules by human whole blood. Gene expression profiling in human macrophages confirmed the increased immunologic stimulatory capacity of modified poxviruses. The pathways activated by MVA and NYVAC in innate immune cells were described by analysing the response of knockdown or shRNA transduced macrophages with impaired expression of TLRs and their adaptors (MyD8$ and TRIF), RLRs (RIG-I, MDA-5 and the adaptor IPS-1) and the NALP3 inflammasome composed óf the NLR NALP3, caspase-1 and ASC. These experiments revealed a critical role for TLR2-TLR6-MyD88 in the production of tFNß-independent chemokines and of MDA-5-IPS-1 in the production of IFNß and IFNßdependent chemokines. The transcription of the iL1b gene encoding for the IL-1ß cytokine was initiated through TLR2-MyD88, whereas the maturation and the secretion of IL-1ß were controlled by the NALP3 inflammasome. Finally, we analyzed the role of macrophage migration inhibitory factor (MIF), a mediator of inflammation and innate immune responses, in MVA infection. We observed that MVA infection increased MIF production by innate immune cells and that MIF deficiency impaired macrophage and dendritic cell responses (ie migration, maturation, cytokine and IFN production) to MVA infection in vitro and in vivo. Moreover, MIF-deficiency resulted in delayed anti-MVA specific antibody production in mice immunized with the virus. In conclusion, we demonstrate. that poxviruses can be modified genetically to improve their immunogenicity. We also report the first comprehensive analysis of poxvirus sensing by innate immune cells, showing that the TLR, RLR and NLR pathways play specific and coordinated roles in regulating cytokine, chemokine and IFN response to poxvirus infection. Finally, we show that MIF is an integral host component involved in innate and adaptive immune responses to MVA infection. The present findings provide important information relevant to the study of the pathogenesis of poxvirus infections and allow a better understanding of the immunogenic potential of vaccine vectors, which is required for the development of optimized modìfied pox-vaccine vectors.

Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

Phacomatoses et tumeurs génétiquement déterminées: la transition enfant-adulte [Phacomatosis and genetically determined tumors: the transition from childhood to adulthood]

Les phacomatoses regroupent des maladies du développement du neurectoderme, engendrant des manifestations cutanées ou du système nerveux central. Les symptômes de ces maladies peuvent affecter les individus atteints à différents moments de leur vie. Il s'agit de maladies, héréditaires ou congénitales, qui sont transmises de façon variable. Effectivement, certaines, telles que la neurofibromatose, la sclérose tubéreuse ou la maladie de von Hippel-Lindau sont autosomiques dominantes, alors que d'autres, telles que la maladie de Sturge-Weber sont sporadiques. Des transmissions autosomiques récessives liées à X ou des formes mosaïques existent également. Une revue de la littérature, comprenant les cinq phacomatoses les plus fréquemment vues par un neurochirurgien (neurofibromatose de type I et II, sclérose tubéreuse de Bourneville, maladie de Sturge-Weber-Krabbe, maladie de von Hippel-Lindau) a été effectuée en se centrant sur le diagnostic, la variabilité de la symptomatologie selon l'âge du patient et son traitement. Les cas de patients adultes et pédiatriques vus aux consultations de neurologie et neurochirurgie de l'hôpital de Lille (France) et Lausanne (Suisse), de 1961 à nos jours, ont été revus pour illustrer les différentes pathologies rencontrées, selon l'âge des patients atteints. Le phénotype de ces maladies se modifie avec l'âge, car les gènes incriminés sont des gènes impliqués dans la différentiation tissulaire et sont activés à des âges différents suivant les tissus. Le rôle du neurochirurgien sera variable selon l'âge et le syndrome du patient. Il importe de connaître les variations du phénotype de ces maladies avec l'âge ainsi que les conséquences à long terme des traitements pour proposer au patient un suivi neurochirurgical personnalisé. Phacomatoses, or neurocutaneous disorders, are a group of congenital and hereditary diseases characterized by developmental lesions of the neuroectoderm, leading to pathologies affecting the skin and the central nervous system. There is a wide range of pathologies affecting individuals at different moments of life. The genetics is variable: while neurofibromatosis 1 and 2, tuberous sclerosis and von Hippel-Lindau disease are all inherited as autosomal dominant traits, Sturge-Weber syndrome is sporadic. Other neurocutaneous disorders can be inherited as autosomal recessive traits (i.e., ataxia-telangiectasia), X-linked (i.e., incontinentia pigmenti) or explained by mosaicism (i.e., hypomelanosis of Ito, McCune-Albright syndrome). In this review, we discuss the major types of neurocutaneous disorders most frequently encountered by the neurosurgeon and followed beyond childhood. They include neurofibromatosis types 1 and 2, tuberous sclerosis, Sturge-Weber syndrome and von Hippel-Lindau disease. In each case, a review of the literature, including diagnosis, genetics and treatment will be presented. The lifespan of the disease with the implications for neurosurgeons will be emphasized. A review of cases, including both pediatric and adult patients, seen in neurosurgical practices in the Lille, France and Lausanne, Switzerland hospitals between 1961 and 2007 is presented to illustrate the pathologies seen in different age-groups. Because the genes mutated in most phacomatoses are involved in development and are activated following a timed schedule, the phenotype of these diseases evolves with age. The implication of the neurosurgeon varies depending on the patient's age and pathology. While neurosurgeons tend to see pediatric patients affected with neurofibromatosis type 1, tuberous sclerosis and Sturge-Weber syndrome, there will be a majority of adult patients with von Hippel-Lindau disease or neurofibromatosis type 2