Utilization of a mouse/human chimeric model for long term metabolic testing of human skin.

Until now, ex vivo human skin explant utilization in tissue culture has consisted of limited short-term studies (less than a week). This short timeframe does not allow for the investigation of metabolic responses of complex tissues to specific molecules or compounds. Here, we aim to develop an improved mouse transplantation model that maintains the viability, structure and functionality of the human skin explants for prolonged periods of time. Healthy human skin explants derived from biopsies were grafted onto nude mice and used to perform a toxicological study of the reactivity and functionality of grafted skin explants after one month. Histological observations suggest that the tissue properties and phenotype of the human skin graft are conserved as a result of re-vascularization upon tissue integration. The toxicological test performed shows that the human skin graft reacts to systemic exposure of a xenobiotic metabolic inducer when applied to this mouse model. This mouse/human chimeric model can be effective for the long-term study of human skin reactivity to chemicals as well to study in vivo responses to complex co-exposures.

Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by immobilized enzymatically tailored pectins.

The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-alpha) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24h on MHR-alpha-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-alpha secretion were studied. The results indicate that MHR-alpha coating inhibits the LPS-induced activation of macrophages.

Fibronectin modulates osteoblast behavior on Nitinol.

We have previously demonstrated that primary rat osteoclasts behave differently when cultured on austenite and martensite Nitinol. In this study, we coated the two phases of Nitinol with plasma fibronectin and studied if this modifies the proliferation and cell cycle of MC3T3-E1 osteoblasts. The influence of the crystalline structure of Nitinol on the remodeling and conformation of fibronectin was also studied. The results on austenite demonstrated that fibronectin was more strongly remodeled and the cells spread better compared with the martensite phase. Interestingly, the conformation of the protein showed no differences between austenite and martensite. In addition, fibronectin improved cell proliferation in both phases, but the effect of fibronectin coating was stronger on the austenite surface. In addition, in both Nitinol phases, the proportion of cells in the G(1) phase was observed to grow in the presence of fibronectin. This could indicate cell differentiation on Nitinol.

Neonatal screening of cystic fibrosis: diagnostic problems with CFTR mild mutations.

Newborn screening (NBS) of cystic fibrosis (CF) was implemented throughout the whole of France in 2002, but it had been established earlier in three western French regions. It can reveal atypical CF with one or two known CFTR mild mutations, with an uncertain evolution. The sweat test can be normal or borderline. In Brittany, from 1989 to 2004, 196 CF cases were diagnosed (1/2885 births). The incidence of atypical CF diagnosed by NBS is 9.7% (19 from 196). The outcome of 17 (2 lost of view) has been studied, with 9 other atypical CF cases diagnosed by NBS in two other regions. The follow-up period extends from 0.25 to 19.8 years (NBS implemented in Normandy in 1980) with mean age 4.6 years. The most frequent mild mutation is R117H ISV8-7T (50%). At the time of the last visit, nutritional status is normal. All these CF patients are pancreatic sufficient. Only one patient exhibits respiratory infections, whereas 7 others have them intermittently. Two of them had intermittent Pseudomonas aeruginosa colonization at 2.8 and 6.5 years. Mean Shwachman score is 96.7, mean Brasfield score is 22.8. Eight children have had lung function tests (mean follow-up of 10 years): mean FVC was 99% of predicted, mean FEV1 101%, but one of them has FEV1 of 48%. Predicting the phenotype of these atypical CF patients remains difficult, thus complicating any genetic counselling. A regular clinical evaluation is necessary, if possible by a CF unit, because CF symptoms may appear later.

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Gene flow estimation with microsatellites in a Malagasy seed orchard of Eucalyptus grandis.

Eucalyptus grandis has a mixed-mating reproductive system. Malagasy Eucalyptus seed orchards were established 15 years ago with two aims both based on panmixia: open-pollinated seed production and genetic improvement. The panmixia hypothesis has never been confirmed in the seed orchard. From a seedling seed-orchard stand comprising 349 trees and using data obtained with six selected microsatellite markers, paternity analysis was performed for 724 offspring collected on 30 adult trees. Paternity assignment, based on exclusion procedures and likelihood-ratio method, was achieved with high accuracy; the exclusion probability value was 0.997. The outcrossing rate was very high (96.7%). More than 50% of potential male trees (199 out of 349) in the seed orchard contributed to pollination for 440 offspring of 30 progenies (8.6% of the basic population). The pollination rate from outside the seed orchard was high (39.2%), but might be due to the small size of this seed orchard. This study showed that "panmixia-like pollination" can be assumed.

[Herpetic meningitis in the child]. / Méningite herpétique chez l'enfant.

UNLABELLED: In children, viral meningitis is usually caused by Enteroviruses. Herpes simplex viruses (HSV) are known to be a cause of meningo-encephalitis. HSV-2 has been reported to cause recurrent meningitis (Mollaret's meningitis) in adults. CASE REPORT: We report the case of a three-year-old girl with HSV-1 meningitis, whose evolution with treatment by aciclovir was good. CONCLUSION: HSV-1 has rarely been reported as a cause of isolated aseptic meningitis in children. Primary phase of herpes simplex virus infection is not usually associated with neurologic complications.

Effects of nucleus on leukocyte recovery.

The rheological properties of a leukocyte significantly affect its biological and mechanical characteristics. To date, existing physical models of leukocyte are not capable of quantitatively explaining the wide range of deformation and recovery behaviors observed in experiment. However, a compound drop model has gained some success. In the present work, we investigate the effect of nucleus size and position, and the relative rheological properties of cytoplasm and nucleus, on cell recovery dynamics. Two nucleus sizes corresponding to that of neutrophil and lymphocyte are considered. Direct comparison between numerical simulations and experimental observation is made. Results indicate that the time scale ratio between the nucleus and cytoplasm plays an important role in cell recovery characteristics. Comparable time scales between the two cell components yield favorable agreement in recovery rates between numerical and experimental observations; disparate time scales, on the other hand, result in recovery behavior and cell shapes inconsistent with experiments. Furthermore, it is found that the nucleus eccentricity exhibits minimum influence on all major aspects of the cell recovery characteristics. The present work offers additional evidence in support of the compound cell model for predicting the rheological behavior of leukocytes.

[Myocardiopathy and isolated glucocorticoid deficit with ACTH resistance: a fortuitous association?]. / Myocardiopathie et déficit isolé en glucocorticoïdes par résistance à l'ACTH: une association fortuite?

UNLABELLED: Hereditary syndrome of unresponsiveness to ACTH is a rare autosomal recessive disorder characterized by an isolated glucocorticoid deficiency which is exceptionally associated to regressive cardiomyopathy. CASE REPORT: A male newborn had iterative episodes of hypoglycemia since the first hours of life. Acute bronchiolitis at the age of 14 days was associated with transitory dilated cardiomyopathy. Hypoglycemia was due to glucocorticoid deficiency secondary to ACTH insensitivity. Molecular biology showed a composite heterozygotism for the ACTH receptor gene. CONCLUSION: Any congenital glucocorticoid deficiency should lead to search for cardiomyopathy.

Expression of specific white adipose tissue genes in denervation-induced skeletal muscle fatty degeneration.

Denervation of skeletal muscle results in rapid atrophy with loss of contractile mass and/or progressive degeneration of muscle fibers which are replaced to a greater or lesser degree by connective and fatty tissues. In this study, we show that denervated rabbit muscles are transformed into a white adipose tissue, depending on their fiber types. This tissue does express LPL, G3PDH and particularly the ob gene, a white adipose tissue-specific marker, and does not express the brown adipose tissue molecular marker UCP1 mRNA.

Expression of lactate dehydrogenase, myosin heavy chain and myogenic regulatory factor genes in rabbit embryonic muscle cell cultures.

The expression of myogenic regulatory factors (MRFs), lactate dehydrogenase (LDH) and myosin heavy chains (MyHC), as markers of myogenesis, metabolism and contractility respectively, were investigated during differentiation of rabbit embryonic muscle cells in primary culture. Myf5, MyoD and myogenin mRNAs were abundantly expressed at day 1 of culture. The expression of Myf5 and MyoD mRNA transcripts decreased sharply as myoblasts fused and differentiated into myotubes, whilst myogenin mRNA was maintained throughout the duration of the culture. In contrast, MRF4 mRNA was weakly expressed on day 1 of culture, its expression increased slightly as myoblasts fused and reached a maximum level in 7-day-old cultures containing striated myofibres. The specific activity of LDH increased linearly during myoblast proliferation and fusion. In 7-day-old cultures, LDH-M mRNA (dominant in glycolytic muscles) and LDH-H mRNA (predominant in perinatal and oxidative muscles) represented 38% and 62% of total LDH mRNA respectively. At this stage, immunocytochemical staining with perinatal and adult-type MyHC antibodies showed that embryonic and perinatal MyHC isoforms were expressed in all myotubes, while few of them were stained by type I MyHC antibody. However, none of them expressed adult type II MyHC. The latter results were further supported by RT-PCR analysis of adult-type MyHC mRNA which showed that only the type I MyHC mRNA transcript was expressed. These data were in agreement with those reported in vivo on perinatal rabbit muscles. They differed from those obtained on cultured satellite cells isolated from adult rabbit fast-twitch or slow-twitch muscles which did not express embryonic MyHC, and instead expressed fast- or slow-type MyHC according to their muscle origin. Taken together, these results further suggest that myogenic mononucleated cells express different properties in vitro according to their developmental origin as well as properties related to those of the muscles from which they were isolated.

Transformation of slow- or fast-twitch rabbit muscles after cross-reinnervation or low frequency stimulation does not alter the in vitro properties of their satellite cells.

We previously showed that satellite cells isolated from rabbit fast-twitch and slow-twitch muscles presented different behaviours in culture; cells from slow muscle differentiated more quickly and fused into more numerous myotubes than those from fast muscle. Moreover, only slow-muscle derived satellite cells expressed in vitro the slow type I myosin heavy chain isoform (MyHC). We wanted to investigate whether the properties of satellite cells originating from different muscles were under the influence of the adult fibre type on which they were located. For this purpose, we transformed the properties of the adult rabbit fast-twitch semimembranosus accessorius (SMa; approximately 100% type II fibres) and the slow-twitch semimembranosus proprius (SMp; 100% type I fibre) muscles by (1) cross-reinnervating the SMp with the main branch of the fast SMa nerve; or (2) electrical stimulation at 10 Hz of the SMa muscle. We studied their satellite cells in vitro. Five-month cross-reinnervation of the SMp induced a large shift of its MyHC type characteristics towards those of a fast muscle, and three-month electrical stimulation at low frequency transformed the fast-twitch SMa into a slow-twitch muscle, as shown by SDS-PAGE of MyHC. In spite of the transformation of their muscle characteristics, satellite cells in culture kept their original properties. Indeed, as shown by MyoD and myogenin gene expression as markers of fusion, satellite cells isolated from cross-reinnervated and from control SMp began to fuse by eight days of culture, and expressed MyoD and myogenin at that stage. Later they differentiated into numerous myotubes. Satellite cells isolated from electrically stimulated and control SMa presented a similar behaviour in culture: they did not express MyoD and myogenin at eight days, and fused by ten days into only a few myotubes. Moreover, MyHC gene expression showed that, in contrast with slow-muscle derived satellite cells, the type I MyHC gene was not expressed by satellite cells isolated from the stimulated SMa in spite of its homogeneous type I fibre composition. Taken together, these data support the idea that once constituted, muscle fibre types per se do not influence the properties of their associated satellite cells.

Expression of myosin isoforms in denervated, cross-reinnervated, and electrically stimulated rabbit muscles.

The expression of myosin heavy (MyHC) and light (MyLC) chain isoforms was analyzed after denervation and cross-reinnervation by a fast nerve of the slow-twitch Semimembranosus proprius (SMp) muscle, and after denervation and electrical stimulation at low frequency of the fast-twitch Semimembranous accessorius (SMa) muscle of the rabbit. The control SMp (100% type I fibers) expressed 100% type I MyHC and 100% slow-type (1S', 1S and 2S) MyLC isoforms. Five month denervation did not alter significantly the MyHC expression of the muscle, but induced the expression of a new type 1 MyLC corresponding most probably to an embryonic MyLC. Five-month cross-reinnervation of the SMp by the fast SMa nerve induced a large change of its fiber type properties. As shown by immunocytochemistry, almost all fibers were stained by fast myosin antibody, but a high proportion of them co-expressed slow myosin. This result was in agreement with biochemical data showing that fast MyHC and MyLC isoforms became predominant. The control SMa (nearly 100% type II fibers) expressed almost 100% type II MyHC (70% type IIb and 22% IIx/d) and 100% fast-type (1F, 2F and 3F) MyLC isoforms. Five month denervation of the SMa induced a shift in its MyHC, with 98% type IIx/d and 2% type IIb isoforms, and no change in the proportions of its MyLC. Three month electrical stimulation at 10 Hz of the SMa transformed its fiber type composition. All fibers reacted with the slow myosin antibody and a minor proportion of them were stained by the fast myosin antibody. These observations were in agreement with the biochemical analysis showing a large predominance of the slow-type MyHC and MyLC isoforms. Taken together, these results obtained from rabbit muscles which are normally homogeneous in either fast-twitch or slow-twitch fiber types, further support the idea that the different myosin isoforms, particularly the MyHC, are differentially regulated by motor innervation. Type I MyHC is maintained in denervated SMp muscle, but is not expressed in denervated SMa. Type IIb isoform is the most sensitive to neural influence, as it disappears rapidly in denervated and electrically stimulated fast-twitch SMa muscle, and is barely expressed in cross-reinnervated slow-twitch SMp muscle. In contrast, type IIa and type IIx/d are less dependent upon motor innervation. In addition to the previous studies of d'Albis et al. analysis of these results leads us to conclude that, in the rabbit, sensitivity to motor innervation increases from the glycolytic to the oxydative types of fibers, in the order IIB > IIX/IID > IIA > I.

Localization of acetylcholinesterase activity and mRNA in the rabbit embryo between 10 and 15 days.

Histoenzymatic methods and in situ hybridization were used to follow AChE expression in rabbit embryos from 10 to 15 days. Transcripts of AChE are detected at the same developmental stages in all structures where enzymatic activity is found, except in neuronal extension and the ventral part of mesonephros. AChE and BChE expression were compared. BChE transcripts are detected before BChE activity can be revealed in blood cells and mesonephros.

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles.

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.

Effects of thyroid state alterations in ovo on the plasma levels of thyroid hormones and on the populations of fibers in the plantaris muscle of male and female chickens.

Propylthiouracil (PTU), thyroxine (T4) or thyreoliberin (TRH) were injected in ovo to modify the thyroid state of chicken embryos. Significant sexual differences were observed in the effects of these treatments on the plasma concentrations of thyroid hormones and on plantaris muscle characteristics (DNA, RNA, populations of muscle fibers) in 3- and 35-day old male and female chickens. The T4 plasma concentration is lower in control males; it is decreased in PTU treated females and in the T4 treated females at 35 days. The T3 plasma concentration is lowered at 3 days in all treated chickens and also at 35 days in the TRH treated animals. The slow (STnO) and the fast (FTOG) fibers of the plantaris are always more numerous in males. In controls, the number of FTOG fibers remains steady between 3 and 35 days; at the same time, the number of STnO fibers rises in males only. Both PTU and T4 treatments increase the number of the FTOG and the STnO fibers respectively before and after the 3rd day. TRH treatment increases the number of STnO fibers at 3 and 35 days in males, but reduces it at 3 days in females. Thus changes in the number of FTOG fibers can be induced during in ovo myogenesis, whereas the number of STnO fibers may increase after hatching.

[Properties of skeletal muscle fibers. II. Hormonal influences]. / Propriétés des fibres musculaires squelettiques. II. Influences hormonales.

The skeletal muscle contains fibers with various contractile and metabolic properties. These populations of muscle fibers differ in their sensitivity and their response to circulating hormones which also affect the muscular differentiation (multiplication and fusion of myoblasts into myotubes). This review deals with the regulations of energy metabolism and of protein synthesis in muscles by several hormones acting either directly, or in association with other hormones, or by induction of growth factors. In most cases, hormonal effects seem to depend on the type and level of activity of the constitutive muscle fibers. The muscle fiber types involved in the anabolic properties of estrogens have not yet been clearly described. In the case of growth hormone and insulin, the slow fiber type is mainly affected; their effects are partially mediated through an increased secretion of somatomedins (IGFs) or by interaction on IGF receptors. The other reported hormones or factors induce a shift toward a more potent fast contracting activity, ultimately increasing the percentage of fast glycolytic fibers. Androgens, catecholamines and beta-agonists are anabolic and produce an enlargement of these fibers, whereas thyroid hormones or glucocorticoids in excess increase their catabolism.