Membrane Topology of Pestiviral Non-Structural Protein 2 and determination of the minimal autoprotease domain.

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae A distinctive feature of the Flaviviridae is the importance of non-structural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes.In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the Substituted Cysteine Accessibility Method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the ER lumen and is followed by three transmembrane segments (TM) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM segment was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to the one of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2.ImportancePestiviral NS2 is central to the regulation of RNA replication and virion morphogenesis via its autoprotease activity. This activity is temporally regulated by the cellular DNAJC14 as a cofactor: while free NS3 is required for RNA replication as a component of the viral replicase, only uncleaved NS2-3 supports virion morphogenesis. For a better understanding of the underlying molecular interactions, topological and structural data are required. The topology-based determination of the minimal NS2-protease domain in the present study will facilitate future attempts to determine the structure of this unusual protease cofactor complex. In the hepatitis C virus system, NS2 functions as a hub in virion morphogenesis by interacting with structural as well as non-structural proteins. Our knowledge of the membrane topology will significantly support future detailed interaction studies for pestiviral NS2.

Knock-down of oncohistone H3F3A-G34W counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells.

Giant cell tumors of bone (GCTB) are semi-malignant tumors associated with extensive osteolytic defects and massive bone destructions. They display a locally aggressive behavior and a very high recurrence rate. Recently, a single mutation has been identified in GCTB affecting the H3F3A gene coding for the histone variant H3.3 (H3.3-G34W). The aim of this study was to investigate whether H3.3-G34W is sufficient to drive tumorigenesis in GCTB. Initially, we confirmed the high frequency of this mutation in 94% of 84 analyzed tissue samples. Using a siRNA based approach we could selectively knockdown H3.3-G34W in primary neoplastic stromal cells isolated from tumor tissue (GCTSC). H3.3-G34W knockdown caused a significant inhibition of cell proliferation, migration and colony formation capacity in vitro. Xenotransplantation of GCTSCs onto the chorioallantoic membrane of fertilized chicken eggs further demonstrated a significant impact of H3.3-G34W knockdown on tumor engraftment and growth in vivo. Our data indicate that H3.3-G34W is sufficient to drive tumorigenesis in GCTB. Apart from the application of H3.3-G34W screening as diagnostic tool, our data suggest that H3.3-G4W represents a promising target for the development of new GCTB therapies.

Consequences of telomerase inhibition by BIBR1532 on proliferation and chemosensitivity of chondrosarcoma cell lines.

PURPOSE: Human chondrosarcomas are generally resistant to conventional treatments like chemotherapy and radiotherapy. We investigated the effects of BIBR1532, an inhibitor of telomerase on chondrosarcoma cells in vitro. METHODS: Telomerase activity, telomere lengths, growth kinetics and chemosensitivity were analyzed in chondrosarcoma cell lines treated with BIBR1532. RESULTS: BIBR1532 treatment resulted in telomerase inhibition, decrease of telomere length and reduction of growth capacity of telomerase positive chondrosarcoma cells. Although resistant to cisplatin, telomerase positive cells were sensitive to paclitaxel, which rapidly induced telomere erosion. CONCLUSION: Targeting of telomeres might represent a valid strategy for the (re-)sensitization of chemoresistant chondrosarcomas.

Survival of human mesenchymal stromal cells from bone marrow and adipose tissue after xenogenic transplantation in immunocompetent mice.

INTRODUCTION: Mesenchymal stromal cells (MSC) represent an attractive cell population for tissue engineering purposes. As MSC are described as immunoprivileged, non-autologous applications seem possible. A basic requirement is the survival of MSC after transplantation in the host. The purpose of the current paper was to evaluate the survival of undifferentiated and osteogenically induced human MSC from different origins after transplantation in immunocompetent mice. METHODS: Human MSC were isolated from bone marrow (BMSC) and adipose tissue (ASC). After cultivation on mineralized collagen, MSC were transplanted subcutaneously into immunocompetent mice (n=12). Undifferentiated MSC (group A) were compared with osteogenic-induced MSC (group B). Human-specific in situ hybridization and anti-vimentin staining was used to follow MSC after transplantation. Quantitative evaluation of lymphocytes and macrophages was performed as a measure of immunologic rejection. Unloaded scaffolds served as controls (group C). Specimens were harvested at 4 and 8 weeks. RESULTS: Undifferentiated BMSC and ASC were detected in the majority of cases after xenogenic transplantation (group A, a total of 22 out of 24 cases), while osteogenic-induced MSC (group B) could be detected in only three of 24 cases. Quantification of lymphocytes and macrophages revealed significantly higher cell numbers in group B compared with group A (P<0.05). DISCUSSION: Our results suggest that undifferentiated MSC are candidates for non-autologous cell transplantation, while osteogenic-induced MSC seem to be eliminated by the host's immune system. This observation seems independent of the origin of MSC and applies to BMSC and ASC.

Screening, identification, and functional analysis of three novel missense mutations in the TRADD gene in children with ALL and ALPS.

BACKGROUND: Apoptosis is known to be a crucial process involved in embryogenesis, development and homeostasis of the immune system. Impaired apoptosis causes dysfunction of lymphocyte homeostasis, growth advantage of tumor cells as well as resistance to current treatment protocols. To investigate the role of the apoptosis adaptor molecules TRADD and FADD in the development of hematological diseases, patient samples were screened for mutations in these genes. PROCEDURE: Genomic DNA from 51 children suffering from B-lineage-ALL (n = 17), T-lineage-ALL (n = 24), ALPS Type Ia (n = 3) and ALPS Type III (n = 7) were analyzed. Genomic DNA from 50 unrelated donors without hematological diseases served as controls. Identified mutations were cloned and their influence on cell viability and NFkappaB activation was analyzed by flow cytometry and luciferase assay, respectively. RESULTS: In the FADD gene no genetic alteration could be detected. However, three novel missense mutations in the TRADD gene could be detected. They are located within a region of TRADD known to exert mainly anti-apoptotic effects for example through the activation of the NFkappaB pathway. Functional analysis of cells overexpressing mutant TRADD cDNA demonstrated a reduced NFkappaB activity and consequently increased cell death compared to wild-type TRADD. CONCLUSION: Mutations in the TRADD gene may contribute to the development of different hematological diseases. The identified mutations demonstrate a putative impact on TRADD signaling and cell survival but may not mainly explain the pathology of the diseases investigated.

A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy.

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumour's course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.

A phase I study of a 4-week course of SDZ-RAD (RAD) quiescent cyclosporine-prednisone-treated renal transplant recipients.

BACKGROUND AND METHODS: This phase I, randomized, blinded, placebo-controlled study assessed the safety profile and pharmacokinetics of a 4-week course of once-daily, sequential ascending doses (0.75, 2.5, or 7.5 mg/day) of SDZ-RAD (RAD) capsules in renal transplant recipients on a stable regimen of cyclosporine (CsA; Neoral) and prednisone. RESULTS: RAD displayed a similar spectrum of side effects as observed with rapamycin, namely, an increased incidence of infection associated with the augmented immunosuppression and a dose-related occurrence of thrombocytopenia, hypercholesterolemia, and hypertriglyceridemia, particularly at the 7.5-mg dose. The pharmacokinetic parameters of RAD showed dose proportionality, a good correlation between trough and area under the curve (AUC) concentrations, and a moderate accumulation of 2.5-fold. The drug was absorbed within 2 hr and displayed a 16-19-hr half-life, which is shorter than that of rapamycin. RAD reached steady state at 4 days. Preliminary kinetic-dynamic correlations indicate a correlation between thrombocytopenia (but not hyperlipidemia) and AUC, as well as maximum drug concentrations, and weight-adjusted dose. At the end of a 4-week course of simultaneous dosing, there was no evidence of a pharmacokinetic interaction between CsA and RAD. CONCLUSION: This study suggests that the shorter half-life of RAD compared to rapamycin may confer the benefits of rapid attainment of steady state and dissipation of effects upon drug cessation. Controlled, multicenter trials are being planned to assess the impact of these features on clinical outcomes.

Drug-induced apoptosis in osteosarcoma cell lines is mediated by caspase activation independent of CD95-receptor/ligand interaction.

Osteosarcoma is one of the most common primary malignant tumors of bone. Treatment of this tumor with systemic chemotherapy dramatically improves the prognosis, although the molecular mechanisms involved in the drug action are poorly understood. In chemosensitive leukaemic T cells and certain solid tumors, cytotoxic drugs mediate the induction of apoptosis by activation of the CD95/APO-1/Fas system. Triggering of the corresponding signaling pathway may involve CD95-receptor/ligand interaction, activation of caspases, or alterations in mitochondrial function. The purpose of our study was to determine if similar mechanisms are involved in the chemosensitivity of osteosarcomas. We found that cytotoxic drugs induce characteristic biochemical and morphological alterations related to apoptosis in osteosarcoma cell lines, including activation of caspases and disturbance of mitochondrial function. However, drug treatment did not result in activation of CD95-receptor or CD95-ligand mRNA. In addition, drug-induced apoptosis was blocked by caspase inhibitors but not by inhibition of CD95-ligand action, indicating a CD95-receptor/ligand-independent mechanism in osteosarcoma cell lines.

Enrichment of c-Met+ tumorigenic stromal cells of giant cell tumor of bone and targeting by cabozantinib.

Giant cell tumor of bone (GCTB) is a very rare tumor entity, which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis, emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells, stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation, differentiation, migration, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, immunohistochemistry, antibody protein array, Alu in situ hybridization, FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies, differentiated to osteoblasts, migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4, indicating self-renewal, invasion and differentiation potential. Compared with adherent-growing cells, markers for pluripotency, stemness and cancer progression, including the CSC surface marker c-Met, were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib, an inhibitor of c-Met in phase II trials, eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met(+) tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB.

Transplantation of mesenchymal stromal cells on mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation.

BACKGROUND: Tissue engineering using mesenchymal stromal cells (MSC) represents a promising approach for bone regeneration. Nevertheless, the optimal constructs have yet to be determined. It still remains unclear if there is a benefit of in vitro differentiation of MSC prior to transplantation or if undifferentiated MSC hold the optimal potential concerning new tissue formation. METHODS: After isolation and in vitro expansion, MSC were seeded on mineralized collagen sponges and transplanted in a heterotopic SCID mice model (n=12). While group A contained undifferentiated MSC, in group B cells were cultivated for 14 days in vitro under osteogenic conditions prior to implantation. Results were compared with non-loaded scaffolds (group C). Animals were killed for investigation at 4 and at 8 weeks. RESULTS: In situ hybridization demonstrated integration of MSC for up to 8 weeks in groups A and B. Histology revealed significantly more extracellular matrix synthesis in MSC-seeded scaffolds containing calcium phosphate and collagen type I at 4 and 8 weeks after transplantation compared with unloaded controls. At a biochemical level, higher levels of specific alkaline phosphatase expression were detected in MSC-loaded scaffolds (P<0.05). Scaffolds containing undifferentiated and differentiated MSC did not appear to differ in terms of matrix synthesis and protein expression, while the number of avital cells was significant higher in those probes loaded with differentiated MSC (P<0.01). DISCUSSION: The integration of transplanted cells and MSC-associated matrix synthesis encourages the use of MSC-loaded mineralized collagen for tissue engineering of bone. Furthermore, our data suggest that in vitro differentiation of MSC does not have a positive influence in terms of improved matrix synthesis.

[Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen]. / Mesenchymale Stammzellen zum Tissue Engineering von Knochen: Dreidimensionale osteogene Differenzierung auf mineralisiertem Kollagen.

AIM: Due to their plasticity and high proliferation capacity in vitro, human mesenchymal stem cells (MSC) are promising candidates for substitution of mesenchymal tissues, such as bone. According to the tissue engineering concept, combinations of cells and three dimensional scaffolds are used to replace damaged tissue. Although various attempts have been made, the optimal combination of cells and artificial scaffold has not been found so far. METHODS: In this work, human MSC were isolated from bone marrow aspirates according to standard protocols and cultivated on mineralized collagen. Osteogenic differentiation was induced by medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. Cell proliferation on the scaffold (WST-1 vitality assay, total protein measurement) and osteogenic differentiation (quantitative Real-Time-RT-PCR) were monitored for 24 days. RESULTS: Viable cells were found within the matrix throughout the cultivation period using histological and histochemical methods. Effective osteogenic differentiation could be demonstrated by the increase of expression of osteogenic marker genes (such as alkaline phosphatase) on a molecular level. CONCLUSION: Our results make the cell/matrix construct investigated in this work a promising candidate for tissue engineering of bone using mesenchymal stem cells. This has to be tested further by in vivo analysis.

Modulation of resistance to anti-APO-1-induced apoptosis in osteosarcoma cells by cytokines.

The CD95/APO-1 Fas receptor/ligand system plays a crucial role in growth control by mediating apoptosis in lymphoid and non-lymphoid cells. To investigate the role of CD95-mediated apoptosis in osteosarcoma, we studied 3 human osteosarcoma cell lines (HOS/TE 85, MG 63 and Saos-2) and osteoblasts derived from bone biopsies. In contrast to osteoblast-like cells, all cell lines were resistant to anti-APO-1-induced apoptosis despite constitutive CD95 expression at intermediate levels. Blocking of macromolecular synthesis by cycloheximide or actinomycin D or modulation of CD95 expression by cytokines (TNF-alpha and/or gamma-interferon) restored sensitivity to anti-APO-1-induced cell death. PCR analysis of the CD95 transcripts revealed the production of a truncated splice variant that codes for a soluble form of the CD95 receptor. Synthesis and secretion of soluble CD95 protein into the culture supernatant was demonstrated by Western blot analysis. Treatment with sensitizing cytokines led to up-regulation of full-length CD95 transcripts and the encoded membrane-bound CD95 protein but not the truncated mRNA splice variant and the corresponding soluble receptor, as shown by PCR and Western blot analysis. The biological activity of soluble CD95 secreted by osteosarcoma cells was demonstrated by the ability of osteosarcoma supernatants to protect the sensitive T-cell line Jurkat from anti-APO-1-mediated apoptosis. Our results suggest that the production of soluble CD95 by osteosarcoma cell lines that may block physiological death signals and the production of membrane-bound CD95 are differently regulated by cytokines via modulation of RNA splicing.

[Clinical and molecular genetic analysis of 4 Swiss families with the pure form of hereditary spastic spinal paralysis]. / Klinische und molekulargenetische Analyse bei vier schweizerischen Familien mit der reinen Form der hereditären spastischen Spinalparalyse.

Hereditary spastic paraplegia (HSP) is a rare neurodegenerative disease of the spinal cord with a progressive gait disorder, associated with other neurological abnormalities in the complicated form. A cluster of families with this disorder in the central part of the country has long been known to Swiss neurologists. In the present report, we describe our clinical and molecular findings in four large families originating from this region and suffering from a pure HSP form. Clinical presentation was similar in the four families. The age of onset varied widely from 2 to 70 years with the appearance of a gait disorder, which slowly progressed to wheelchair confinement after 30-70 years. No other neurological abnormality was found except for impairment of the vibration sense and sphincter abnormalities. In three families an association with markers of the SPG4 locus on chromosome 2 was found. In the fourth, the largest one, no linkage could be found with either SPG4, or with the other two known loci, SPG3 on chromosome 14 and SPG6 on chromosome 15. These data demonstrate the genetic heterogeneity in HSP, even in families from the same region. They also suggest the presence of at least one additional locus for the pure form.

Phenotype of autosomal dominant spastic paraplegia linked to chromosome 2.

We report the clinical features of 12 families with autosomal dominant spastic paraplegia (ADSP) linked to the SPG4 locus on chromosome 2p, the major locus for this disorder that accounts for approximately 40% of the families. Among 93 gene carriers, 32 (34%) were unaware of symptoms but were clinically affected. Haplotype reconstruction showed that 90% of the asymptomatic gene carriers presented increased reflexes and/or extensor plantar responses independent of age at examination. The mean age at onset was 29 years, ranging from 1 to 63 years. Intra- as well as inter-familial variability of age at onset was important, but did not result from anticipation. Phenotype-genotype correlations and comparison with SPG3 and SPG5 families indicated that despite the variability of age at onset, SPG4 is a single genetic entity but no clinical features distinguish individual SPG4 patients from those with SPG3 or SPG5 mutations.