[The Orthopaedic and Traumatology Research Laboratory]. / Le Laboratoire de Recherche en Orthopédie Traumatologie - LROT.

The history of the Orthopaedics and Traumatology Research Laboratory (LROT) is summarized during the last thirty years. The approach, initially focused on bone biomechanics and strength of materials, underwent a rapid diversification as expressed by the list of research topics: monitoring of bone healing; bone strains measurements for different level of activities including microgravity and prevention of disuse osteoporosis; biological effects of electromagnetic fields; evolution of the viscoelastic properties of the callus during bone healing; improvement of the osteoinductive properties of bone substitutes produced by the Tissues Bank of the University Hospital Erasme; Kashin-Beck disease; SICOT telediagnostic, and biomechanics of threaded implants. Those topics, event the most fundamental ones, have immediate significant clinical applications allowing a decrease of the morbidity and an acceleration of the rehabilitation of the patients. The results show the need of multidisciplinary collaborations coordinated around one autonomous laboratory, able to handle specific protocols requiring a dedicate environment.

[The Tissue Bank of the University Hospital Erasme]. / La Banque de Tissus de l'Hôpital Universitaire Erasme.

The evolution of the Tissue Bank belonging to the University Hospital Erasme is summarized during its 13 years of experience. In parallel with this evolution, the important modifications of the legislation, the selection criteria and the bone graft processing are reported. The significant improvement of the safety of the allograft related to the risk of infection is also mentioned. In constant progression, the ongoing research within the BTE studies the osteogenic activity of the graft, mostly of bone demineralized matrix.

Effects of physical environment on the evolution of Kashin-Beck disease in Tibet.

In previous studies we observed a proximo-distal gradient of lesion frequencies along the limb, with the distal joints being the most often affected. This suggests an associated effect of environmental factors on the most exposed joints. On a population of 820 children (mean age 13 years) of endemic areas distributed in groups of healthy and severity stages I to III of KBD (Kashin-Beck disease), the effects of different working activities were studied. Heavy work like that of a ploughman were compared to light physical work, e.g. school children, and exposure to cold and history of frostbite were also considered. The most severe stages, II and III, were present in 72% of the ploughman vs. 29% of the schoolchildren, 70% of the shepherds vs. 30% (p < 0.001) of the schoolchildren, and in 65% of the shepherds working in winter vs. 40% of those working in the other seasons (p < 0.001). In the group with history of frostbite, 58% present the severest stages vs. 40% without (p < 0.001). The results confirm a highly significant relation between microtrauma and cold and the severity of the KBD alterations.

Cellular processes involved in human epidermal cells exposed to extremely low frequency electric fields.

We observed on different tissues and organisms a biological response after exposure to pulsed low frequency and low amplitude electric or electromagnetic fields but the precise mechanism of cell response remains unknown. The aim of this publication is to understand, using bioinformatics, the biological relevance of processes involved in the modification of gene expression. The list of genes analyzed was obtained after microarray protocol realized on cultures of human epidermal explants growing on deepidermized human skin exposed to a pulsed low frequency electric field. The directed acyclic graph on a WebGestalt Gene Ontology module shows six categories under the biological process root: "biological regulation", "cellular process", "cell proliferation", "death", "metabolic process" and "response to stimulus". Enriched derived categories are coherent with the type of in vitro culture, the stimulation protocol or with the previous results showing a decrease of cell proliferation and an increase of differentiation. The Kegg module on WebGestalt has highlighted "cell cycle" and "p53 signaling pathway" as significantly involved. The Kegg website brings out interactions between FoxO, MAPK, JNK, p53, p38, PI3K/Akt, Wnt, mTor or NF-KappaB. Some genes expressed by the stimulation are known to have an exclusive function on these pathways. Analyses performed with Pathway Studio linked cell proliferation, cell differentiation, apoptosis, cell cycle, mitosis, cell death etc. with our microarrays results. Medline citation generated by the software and the fold change variation confirms a diminution of the proliferation, activation of the differentiation and a less well-defined role of apoptosis or wound healing. Wnt and DKK functional classes, DKK1, MACF1, ATF3, MME, TXNRD1, and BMP-2 genes proposed in previous publications after a manual analysis are also highlighted with other genes after Pathway Studio automatic procedure. Finally, an analysis conducted on a list of genes characterized by an accelerated regulation after extremely low frequency pulsed stimulation also confirms their role in the processes of cell proliferation and differentiation. Bioinformatics approach allows in-depth research, without the bias of pre-selection, on cellular processes involved in a huge gene list.

Mice overexpressing the human neurofilament heavy gene as a model of ALS.

We discuss the evidence, based on the analysis of transgenic mice overexpressing the human neurofilament (NF) heavy gene, that abnormal NF accumulations can provoke neurodegeneration of motor neurons. Transgenic mice overexpressing by two-fold the normal levels of human NF-H proteins develop a progressive motor neuron disease with several pathologic features reminiscent of those found in amyotrophic lateral sclerosis (ALS). A plausible mechanism for the selective motor neuron degeneration is that exceeding levels of NF-H cross-linkages impede transport of newly synthesized NF structures. The abnormal NF accumulations in perikarya and proximal axons is accompanied by a disruption in axonal transport of not only NF proteins but also of other components required for maintenance of axons. The relevance of the NF-H transgenics as a model of ALS is discussed in light of our current knowledge of motor neuron disease.

The role of the two E1a mRNA products of subgroup B adenoviruses in the regulation of early promoters of subgroup C adenoviruses.

HeLa cells were co-transfected with recombinant plasmids carrying adenovirus (Ad)2 or Ad3 E1a promoters fused to the chloramphenicol acetyl transferase gene (cat), and a plasmid encoding the Ad3 E1a promoter. Whereas no stimulating effect was observed on the Ad3 E1a promoter, the Ad2 promoter was inhibited. To determine which of the E1a gene products of Ad3 was responsible for the repressive effect, plasmids were constructed in which only the 13S or 12S mRNA product of Ad3 was expressed. Both the 12S and 13S mRNA products of Ad3 E1a were found to depress the transcription from the Ad2 E1a promoter. Each Ad3 E1a gene product was able to stimulate transcription from the Ad5 E2a early promoter in a manner similar to that of the Ad2 E1a gene products. In the case of the Ad5 E3 promoter, neither of the Ad3 E1a gene products stimulated transcription, but an inhibition was observed. These results suggest that both mRNA products of the Ad3 E1a region inhibit transcription at the TATA box transcription complex.

Copy-dependent and correct developmental expression of the human neurofilament heavy gene in transgenic mice.

We recently produced four lines of transgenic mice bearing a 34 kb DNA fragment that includes the human gene coding for the neurofilament heavy (NF-H) chain. Analysis of the NF-H transgenics revealed an increase in human NF-H mRNA and protein that parallels the increase in gene copy number, providing the first example of a transgene with copy-dependent expression in neurons. In addition, expression of the human NF-H transgene is induced post-natally following a developmental pattern similar to the endogenous mouse NF-H gene.

Statistical validation of the acceleration of the differentiation at the expense of the proliferation in human epidermal cells exposed to extremely low frequency electric fields.

An acceleration of differentiation at the expense of proliferation is observed in our previous publications and in the literature after exposure of various biological models to low frequency and low-amplitude electric and electromagnetic fields. This observation is related with a significant modification of genes expression. We observed and compared over time this modification. This study use microarray data obtained on epidermis cultures harvested from human abdominoplasty exposed to ELF electric fields. This protocol is repeated with samples collected on three different healthy patients. The sampling over time allows comparison of the effect of the stimulus at a given time with the evolution of control group. After 4 days, we observed a significant difference of the genes expression between control (D4C) and stimulated (D4S) (p < 0.05). On the control between day 4 and 7, we observed another group of genes with significant difference (p < 0.05) in their expression. We identify the common genes between these two groups and we select from them those expressing no difference between stimulate at 4 days (D4S) and control after 7 days (D7C). The same analysis was performed with D4S-D4C-D12C and D7S-D7C-D12C. The lists of genes which follow this pattern show acceleration in their expressions under stimulation appearing on control at a later time. In this list, genes such as DKK1, SPRR3, NDRG4, and CHEK1 are involved in cell proliferation or differentiation. Numerous other genes are also playing a function in mitosis, cell cycle or in the DNA replication transcription and translation.

Transfection of human lamins A and C into mouse embryonal carcinoma cells possessing only lamin B.

The peripheral lamina of eukaryotic nuclei is composed of polypeptides called lamins that vary in number from one to four according to organism, cell type, and differentiated state of the cells. Early embryonic cells and stem cells of mammals generally possess only lamin B while lamins A and C appear later during differentiation. To study the role of the late appearance of lamins A and C in the differentiated phenotype, we have performed transfection of cDNAs coding for human lamins A or C into mouse embryonal carcinoma (EC) cell lines F9 and P19 lacking these two lamins. Transient transfections have shown that lamins A or C could be expressed, translocated to the peripheral lamina, and distributed into daughter cell nuclei after mitosis. These results demonstrated that EC cells devoid of lamins A and C nevertheless possessed the appropriate mechanisms for the localization and mitotic redistribution of exogenous lamins A and C.

Phorbol esters induce transient changes in the accessibility of the carboxy-terminal domain of nuclear lamin A.

Treatment of human epithelial cells in culture with phorbol esters (TPA) gives rise to a transient and reversible loss of accessibility to antibodies of the nonhelical carboxy-terminal domain of nuclear lamin A that distinguishes it from lamin C. No change in the accessibility of epitopes present in the common domain of lamins A and C was observed. Loss of accessibility of lamin A was not due to proteolytic degradation nor to modification of the isoelectric point of lamin A and did not depend upon protein kinase C activation nor protein synthesis. Perturbation of desmosome organization by growth in low calcium blocked the effect of TPA on lamin A. Prolonged exposure to nocodazole, one of the effects of which is a perinuclear collapse of intermediate filaments, also blocked the effect of TPA on lamin A. These results suggest that the initial target of TPA may be at the level of cell-cell contacts and that the perturbation induced by TPA may be propagated via the structural link formed by intermediate filaments between the cell surface and the nucleus, giving rise to a change in conformation of the carboxy-terminal domain of lamin A or to an interaction of this domain with another nuclear component. These results form the basis for the hypothesis that the interphase nuclear lamina may play an active role in the process of mechanochemical signal transduction.

A simple test to monitor the motor dysfunction in a transgenic mouse model of amyotrophic lateral sclerosis.

We reported recently that transgenic mice overexpressing human neurofilament heavy (NF-H) proteins develop a progressive neurological disorder with pathological features resembling those found in amyotrophic lateral sclerosis (ALS) (Côté et al 1993). A simple behavioral test, the grasping ability, has been used here for evaluating the motor dysfunction during aging of NF-H transgenic mice. Transgenic mice overexpressing NF-H proteins are normal at birth but they progressively fail to uphold their weight when tested for their grasping ability. The deficits in motor function during aging correlate with a progressive disruption of peripheral nerve function as evidenced by the atrophy and degeneration of distal axons.

Differential accessibility of the tail domain of nuclear lamin A in interphase and mitotic cells.

Human autoantibodies reactive against the tail domain exclusive to lamin A and absent from lamin C have been used for immunofluorescence studies on human fibroblast and epithelial cells. These autoantibodies were seen to react on mitotic cells where lamin A is present in a soluble depolymerized form and to react against lamin A in assembled interphase nuclear lamina after in situ extraction of chromatin. Taken together, these results support the suggestion that the tail domain of lamin A may be involved in the putative interaction of lamin A with chromatin.

Redistribution of nuclear lamin A is an early event associated with differentiation of human promyelocytic leukemia HL-60 cells.

The nuclear lamina of mammalian somatic cells is characterized by the constitutive presence of lamin B polypeptides while the appearance of lamins A and C generally occur during establishment of a differentiated phenotype. We have used antibodies specific to the unique carboxy-terminal domain of lamin A, i.e. distinct from the shared domains of lamins A and C, to study the individual behaviour of lamin A during establishment of a macrophage phenotype in human HL-60 cells. Lamin A was present as a nuclear cap in the majority of undifferentiated cells and it was redistributed to a full peripheral nuclear location very early after induction of differentiation by phorbol esters, even in the presence of a protein synthesis inhibitor. Induction of the cells into a reversible precommitment state by bromodeoxyuridine was accompanied by a similar redistribution of lamin A that however reverted to a cap after removal of inducer. No changes were observed in the uniform peripheral nuclear location of lamin C under all of these conditions. These results strongly suggest that lamin A plays a role in the early events of cell differentiation. Taken together with previous results on the interaction of A-type lamins with chromatin, these findings offer experimental evidence consistent with the proposed role of A-type lamins, and particularly lamin A, in the process of chromatin reorganization that accompanies the expression of a differentiated phenotype.

Regulation of the expression of lamins A and C is post-transcriptional in P19 embryonal carcinoma cells.

The polypeptide composition of the nuclear lamina can display important variations: undifferentiated cells express only lamin B and they acquire lamins A and C only after differentiation. We have analyzed the expression of lamins A and C in P19 pluripotent mouse embryonal carcinoma cells. Undifferentiated P19 cells are completely devoid of lamins A and C. We show that undifferentiated P19 cells contain low, but detectable steady-state levels of RNAs for lamins A and C that begin to increase by 24 h of retinoic acid-induced differentiation. However, the rate of transcription of the lamin A and C gene(s), analyzed by run-on transcription assays, remains unchanged during the differentiation process. These results demonstrate that, at least in P19 embryonal carcinoma cells, regulation of the expression of lamins A and C is a post-transcriptional event.

Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease of motor neurons, characterized by depositions of neurofilaments in the perikarya and proximal axons. The pathogenesis of ALS remains poorly understood, but two lines of evidence suggest that neurofilament accumulation may play a causal role. First, transgenic mice that overexpress neurofilament proteins show motor neuron degeneration and, second, variant alleles of the neurofilament heavy-subunit gene (NF-H) have been found in some human ALS patients. To investigate how disorganized neurofilaments might cause neurodegeneration, we examined axonal transport of newly synthesized proteins in mice that overexpress the human NF-H gene. We observed dramatic defects of axonal transport, not only of neurofilament proteins but also of other proteins, including tubulin and actin. Ultrastructural analysis revealed a paucity of cytoskeletal elements, smooth endoplasmic reticulum and especially mitochondria in the degenerating axons. We therefore propose that the neurofilament accumulations observed in these mice cause axonal degeneration by impeding the transport of components required for axonal maintenance, and that a similar mechanism may account for the pathogenesis of ALS in human patients.

Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis.

We generated four transgenic mice with a 34 kb genomic fragment including the complete human neurofilament heavy (NF-H) gene. This human NF-H fragment contained all regulatory elements for tissue-specific expression, and in two transgenic lines, human NF-H proteins were produced at levels up to 2-fold the levels of endogenous mouse NF-H protein. By 3-4 months of age, these NF-H transgenics progressively develop neurological defects and abnormal neurofilamentous swellings that are highly reminiscent of those found in amyotrophic lateral sclerosis (ALS). We propose that a modest up-regulation of NF-H cross-linkers can result in an impairment of neurofilament transport, causing neuronal swellings with ensuing axonopathy and muscle atrophy, a mechanism of pathogenesis pertinent to the possible etiology of ALS.