RESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , SARS-CoV-2/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Humanos , Mutación , Pruebas de Neutralización , Conformación de Ácido Nucleico , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/fisiología , Técnica SELEX de Producción de Aptámeros , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Retroviruses have been invading mammalian germlines for millions of years, accumulating in the form of endogenous retroviruses (ERVs) that account for nearly one-tenth of the mouse and human genomes. ERVs are epigenetically silenced during development, yet the cellular factors recognizing ERVs in a sequence-specific manner remain elusive. Here we demonstrate that ZFP809, a member of the Krüppel-associated box zinc finger protein (KRAB-ZFP) family, initiates the silencing of ERVs in a sequence-specific manner via recruitment of heterochromatin-inducing complexes. ZFP809 knockout mice display highly elevated levels of ZFP809-targeted ERVs in somatic tissues. ERV reactivation is accompanied by an epigenetic shift from repressive to active histone modifications but only slight destabilization of DNA methylation. Importantly, using conditional alleles and rescue experiments, we demonstrate that ZFP809 is required to initiate ERV silencing during embryonic development but becomes largely dispensable in somatic tissues. Finally, we show that the DNA-binding specificity of ZFP809 is evolutionarily conserved in the Muroidea superfamily of rodents and predates the endogenization of retroviruses presently targeted by ZFP809 in Mus musculus. In sum, these data provide compelling evidence that ZFP809 evolved to recognize foreign DNA and establish histone modification-based epigenetic silencing of ERVs.
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Proteínas de Unión al ADN/metabolismo , Retrovirus Endógenos/genética , Epigénesis Genética , Silenciador del Gen , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Retrovirus Endógenos/fisiología , Genoma , Histonas/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Activación Viral/genética , Integración Viral/genéticaRESUMEN
BACKGROUND: Short dimeric or mulitmeric peptides derived from a highly conserved stretch of amino acids from gammaretroviral envelope proteins has been found to have immunosuppressive properties in vitro. Here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders. RESULTS: The anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of 17 amino acids was tested in two murine skin inflammation models, a TPA-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. Overall, mice (n = 24) treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of 28.8% in ear thickness (range 20.1-42.5), whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect (p = 0.028). Furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mRNA of the pro-inflammatory cytokines TNF-α, IL-17C, and IL-6 as well as the chemokine CXCL2 compared to mice treated with control peptides. CONCLUSION: Using two murine skin inflammation models, we show that an immunosuppressive retroviral peptide is capable of reducing inflammatory disorders. The results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation.
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Dermatitis por Contacto/inmunología , Dermatitis Irritante/inmunología , Inmunosupresores/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Quimiocina CXCL2/genética , Dermatitis por Contacto/etiología , Dermatitis por Contacto/prevención & control , Dermatitis Irritante/etiología , Dermatitis Irritante/prevención & control , Dimerización , Modelos Animales de Enfermedad , Expresión Génica/inmunología , Humanos , Inmunosupresores/farmacología , Interleucina-17/genética , Interleucina-6/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxazolona/inmunología , Oxazolona/toxicidad , Péptidos/química , Péptidos/farmacología , Retroviridae/química , Retroviridae/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/inmunología , Acetato de Tetradecanoilforbol/toxicidad , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The possibility that retroviruses play a role in multiple sclerosis (MS) has long been considered; accumulating findings suggest this to be most likely in the form of human endogenous retroviruses (HERVs). A genetic test series of fifty endogenous retroviral loci for association with MS in Danes showed SNP markers near a specific endogenous retroviral locus, HERV-Fc1 located on the X-chromosome, to be positive. Bout Onset MS was associated with the HERV-Fc1 locus, while a rarer form, Primary Progressive MS, was not. Moreover, HERV-Fc1 Gag RNA in plasma was increased 4-fold in patients with recent history of attacks, relative to patients in a stable state and to healthy controls.Finally, genetic variations in restriction genes for retroviruses influence the risk of MS, providing further support for a role of retroviral elements in disease.We speculate that endogenous retroviruses may activate the innate immune system in a variety of ways, involving the host proteins, TRIMs, TLRs, TREXs and STING. Observations in HIV-positive patients suggest that antiretroviral drugs can curb MS. Thus, these new findings regarding the etiology and pathogenesis of MS, suggest alternative ways to challenge autoimmune diseases.
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Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidad , Esclerosis Múltiple/genética , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Marcadores Genéticos , Humanos , Esclerosis Múltiple/virología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Human endogenous retroviruses (HERVs) are suspected to play a role in the development of multiple sclerosis (MS). This suspicion has in part been based on increased expression of viral RNA or proteins or antibodies targeting retroviral products in MS patients. Recently, our group provided genetic evidence for association between the endogenous retrovirus HERV-Fc1 and MS, suggesting that HERV-Fc1 plays a role in this multifactorial disease. We have found increased expression of HERV-Fc1 in MS patients suffering from recent attack, but the underlying mechanism for association is still unknown. FINDINGS: Evidence from animal models indicates that ERV implication in the pathogenesis of diseases can be a result of extra copies of the virus in the germ line. Therefore, we investigated the genome of 81 individuals, 74 patients with MS and 7 healthy controls, by means of Southern blotting, for presence of extra HERV-Fc1 copies. The known insertion at the Xq21.33 position was readily detectable, but no additional insertions in other genomic contexts could be identified in any studied individuals. This substantiates our previous copy-number PCR findings of a 2:1 ratio of HERV-Fc1 DNA between women and men, as expected from the X-chromosome location; there was no difference between patient and control individuals. CONCLUSIONS: No additional germ line copies of HERV-Fc1 could be identified, precluding such copies to underlie the association between this provirus and multiples sclerosis.
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Variaciones en el Número de Copia de ADN , Retrovirus Endógenos/genética , Células Germinativas/patología , Células Germinativas/virología , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Southern Blotting , Femenino , Humanos , MasculinoRESUMEN
The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni. Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni-specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.
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Células Estrelladas Hepáticas , Macrófagos del Hígado , Animales , Células Estrelladas Hepáticas/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C3H , Vitamina A/metabolismoRESUMEN
Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.
Asunto(s)
Septinas/metabolismo , Animales , Western Blotting , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Septinas/genéticaRESUMEN
This study evaluates the immunogenicity of the HIV envelope protein (env) in mice presented either attached to γ-retroviral virus-like-particles (VLPs), associated with cell-derived microsomes or as solubilized recombinant protein (gp160). The magnitude and polyfunctionality of the cellular immune response was enhanced when delivering HIV env in the VLP or microsome form compared to recombinant gp160. Humoral responses measured by antibody titres were comparable across the groups and low levels of antibody neutralization were observed. Lastly, we identified stronger IgG2a class switching in the two particle-delivered antigen vaccinations modalities compared to recombinant gp160.
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Anticuerpos Anti-VIH/sangre , Proteínas gp160 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Inmunidad Celular , Animales , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Virosomas/inmunologíaRESUMEN
The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.
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Elementos Transponibles de ADN/genética , Vectores Genéticos/genética , Teratocarcinoma/patología , Transposasas/genética , Línea Celular Tumoral , Humanos , Transcripción Genética , Transposasas/metabolismoRESUMEN
A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2h. Following adsorption of albumin onto Ta and PS(ox) there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PS(ox) substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.
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Materiales Biocompatibles Revestidos/química , Cicloheximida/farmacología , Matriz Extracelular/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Cuarzo , Adsorción , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/química , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Oxidación-Reducción , Poliestirenos/química , Suero/química , Albúmina Sérica Bovina/química , Ovinos , Especificidad por Sustrato , Tantalio/química , Factores de TiempoRESUMEN
Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.
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Resorción Ósea , Diferenciación Celular , Osteoclastos/fisiología , Septinas/metabolismo , Animales , Células Cultivadas , Fémur/crecimiento & desarrollo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Septinas/deficienciaRESUMEN
The quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden) has been established as a useful tool for evaluating interactions between various biological and non-biological systems, and there has been increasing interest in using the QCM-D technique for cell monitoring applications. This study investigated the potential of the QCM-D to characterise the initial adhesion and spreading of cells in contact with protein precoated biocompatible surfaces. The QCM-D technique is attractive for monitoring cell adhesion and spreading as it allows in situ real-time measurements. The adhesion of NIH3T3 (EGFP) fibroblasts to tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces precoated with serum proteins was examined using the QCM-D for a period of either 2 or 4 h. Time-lapse photography was performed at 30 min intervals to visually examine cell adhesion and spreading in order to relate cell morphology to the QCM-D response. Following adsorption of albumin, fibronectin or newborn calf serum onto the surfaces, QCM-D measurements showed that cells adhered and spread on the fibronectin and serum coated surfaces, while few cells adhered to the albumin coated surfaces. Cells adhered to albumin coated surfaces had a rounded morphology. The responses to fibronectin and serum precoated surfaces were quite different for each of the underlying substrates indicating that the process of cell adhesion and spreading elicits different responses depending on both the protein coating composition and the influence of the underlying substrate. The different response may be due to extracellular matrix remodelling as well as cytoskeletal changes. Frequency (f) and dissipation (D) changes associated with cell adhesion were less than would be expected from the Sauerbrey relation due to the viscoelastic properties of the cells.
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Adhesión Celular , Materiales Biocompatibles Revestidos/metabolismo , Ensayo de Materiales/métodos , Poliestirenos/metabolismo , Cuarzo , Tantalio/metabolismo , Absorción , Albúminas/química , Animales , Bovinos , Materiales Biocompatibles Revestidos/química , Fibronectinas/química , Ratones , Células 3T3 NIH , Oxidación-Reducción , Poliestirenos/química , Suero/química , Propiedades de SuperficieRESUMEN
The quartz crystal microbalance with dissipation (QCM-D) technique was employed to characterize initial cell adhesion in terms of attachment and spreading of pre-osteoblastic MC3T3-E1 cells on Ta and Cr surfaces. Evaluation of initial cell adhesion established a correlation between input cell number and the shifts in frequency (f) and dissipation (D). The f-shift was found to be much larger in serum-free medium as compared to a medium including serum; hence, initial cell adhesion was subsequently evaluated in serum-free medium. During the first hour of adhesion, we found a positive correlation between the QCM-D f-shift and the average area of the spread cells, as measured by cryo-scanning electron microscopy (cryo-SEM). Finally, the QCM-D technique was used to study cell adhesion on different metal oxide surfaces. Initial cell adhesion on Ta was found to induce a larger f-shift as compared to Cr, indicating larger spreading of cells on Ta. Cryo-SEM data confirmed that spreading of cells on Cr was on average only two-thirds the spreading on Ta. Our results demonstrate that the QCM-D technique is a versatile technique to quickly distinguish initial cell-surface interactions on different biomaterials.
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Movimiento Celular/fisiología , Cromo , Osteoblastos/citología , Osteoblastos/fisiología , Cuarzo , Células Madre/citología , Células Madre/fisiología , Tantalio , Animales , Materiales Biocompatibles , Técnicas Biosensibles , Adhesión Celular/fisiología , Línea Celular , Microscopía por Crioelectrón , Cristalización , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica de RastreoRESUMEN
INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno-associated virus (rAAV)-mediated and retroviral gene delivery. Each method for gene delivery was optimized. Gene transfer efficiency was compared on the basis of eGFP expression as assessed by fluorescence microscopy and fluorescence-activated flow cytometry. BMP-2 gene expression and osteogenic differentiation were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were transiently transduced. The efficiency of rAAV-mediated gene delivery was proportional to the rAAV vector titer applied. Retroviral gene delivery resulted in long-term transgene expression of porcine MSCs. A 26-fold increase in percentage of eGFP expressing cells (1.7%+/-0.2% versus 44.1% +/-5.0%, mean +/-SD) and a 68-fold increase in mean fluorescence intensity (327.4+/-56.6 versus 4.8+/-1.3) was observed by centrifugation of retroviral particles onto the target cell layer. Porcine MSCs that were BMP-2 transduced by optimized retroviral gene delivery demonstrated a significant increase in BMP-2 gene expression and showed increased osteogenic differentiation. Retrovirally transduced porcine MSCs were furthermore tested free of replication-competent viruses. DISCUSSION: The non-viral gene transfer methods applied were significantly less efficient compared to the viral methods tested. However, due to advantages with respect to safety issues and ease of handling, improvement of non-viral gene delivery to primary MSCs deserves further attention. The high efficiency of rAAV-mediated gene delivery observed at high titers can be explained by the ability of rAAV vector to transduce nondividing cells and by its tropism towards porcine MSCs. rAAV-mediated gene delivery resulted in transient transgene expression due to lack of stable AAV genome integration. MLV-mediated retroviral gene delivery can be considered a safe method for long-term transgene expression by porcine MSCs, and is therefore particularly attractive for advanced tissue engineering strategies requiring extended transgene expression.
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Técnicas de Transferencia de Gen , Células Madre Mesenquimatosas/citología , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Virus de la Leucemia Murina/metabolismo , Ratones , Células 3T3 NIH , Retroviridae/metabolismo , Porcinos , Tibia/patología , Ingeniería de Tejidos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , TransgenesRESUMEN
Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously. The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.
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Remodelación Ósea , Callo Óseo/fisiología , Fracturas Óseas/terapia , Terapia Genética , Glicoproteínas/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/genética , Tibia/lesiones , Animales , Fenómenos Biomecánicos , Peso Corporal , Callo Óseo/citología , Callo Óseo/diagnóstico por imagen , Femenino , Fracturas Óseas/patología , Humanos , Osteoprotegerina , Radiografía , Ratas , Ratas Wistar , Tibia/citología , Tibia/diagnóstico por imagenRESUMEN
We recently described that the autoimmune, central nervous system disease, multiple sclerosis (MS), is genetically associated with the human endogenous retroviral locus, HERV-Fc1, in Scandinavians. A number of dominant human genes encoding factors that restrict retrovirus replication have been known for a long time. Today human restriction genes for retroviruses include amongst others TRIMs, APOBEC3s, BST2 and TREXs. We have therefore looked for a role of these retroviral restriction genes in MS using genetic epidemiology. We here report that markers in two TRIMs, TRIM5 and TRIM22 and a marker in BST2, associated statistically with the risk of getting MS, while markers in or near APOBEC3s and TREXs showed little or no effect. This indicates that the two TRIMs and BST2 influence the risk of disease and thus supports the hypothesis of a viral involvement.
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Esclerosis Múltiple/genética , Retroviridae/genética , Desaminasas APOBEC , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Citidina Desaminasa , Citosina Desaminasa/genética , Retrovirus Endógenos/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Antígenos de Histocompatibilidad Menor , Proteínas Represoras/genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasAsunto(s)
Farmacorresistencia Viral/genética , Proteína gp41 de Envoltorio del VIH/genética , Inhibidores de Fusión de VIH/farmacología , VIH-1/genética , Fragmentos de Péptidos/farmacología , Enfuvirtida , Genotipo , Proteína gp41 de Envoltorio del VIH/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Mutación , Polimorfismo GenéticoRESUMEN
BACKGROUND: Concominant with the widespread use of combined immunotherapy in the management of Crohn's disease (CD), the incidence of hepato-splenic gamma-delta (γδ)-T cell lymphoma has increased sharply in CD patients. Malignant transformation of lymphocytes is believed to be a multistep process resulting in the selection of malignant γδ-T cell clones. We hypothesised that repeated infusion of anti-TNF-α agents may induce clonal selection and that concurrent treatment with immunomodulators further predisposes patients to γδ-T cell expansion. METHODOLOGY/PRINCIPAL FINDINGS: We investigated dynamic changes in the γδ-T cells of patient with CD following treatment with infliximab (Remicade®; n=20) or adalimumab (Humira®; n=26) using flow cytometry. In patients with a high γδ-T cell level, the γδ-T cells were assessed for clonality. Of these 46 CD patients, 35 had a γδ-T cells level (mean 1.6%) comparable to healthy individuals (mean 2.2%), and 11 CD patients (24%) exhibited an increased level of γδ-T cells (5-15%). In the 18 patients also receiving thiopurines or methotrexate, the average baseline γδ-T cell level was 4.4%. In three male CD patients with a high baseline value, the γδ-T cell population increased dramatically following infliximab therapy. A fourth male patient also on infliximab monotherapy presented with 20% γδ-T cells, which increased to 25% shortly after treatment and was 36% between infusions. Clonality studies revealed an oligoclonal γδ-T cell pattern with dominant γδ-T cell clones. In support of our clinical findings, in vitro experiments showed a dose-dependent proliferative effect of anti-TNF-α agents on γδ-T cells. CONCLUSION/SIGNIFICANCE: CD patients treated with immunomodulators had constitutively high levels of γδ-T cells. Infliximab exacerbated clonal γδ-T cell expansion in vivo and induced γδ-T cell proliferation in vitro. Overall, young, male CD patients with high baseline γδ-T cell levels may be at an increased risk of developing malignant γδ-T cell lymphomas following treatment with anti-TNF-α agents.
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Antiinflamatorios/efectos adversos , Anticuerpos Monoclonales/efectos adversos , Enfermedad de Crohn/tratamiento farmacológico , Linfoma/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Adulto , Anciano , Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedad de Crohn/inmunología , Femenino , Citometría de Flujo , Humanos , Infliximab , Linfoma/etiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Adulto JovenRESUMEN
We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.
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Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiología , Esclerosis Múltiple/etiología , Alelos , Estudios de Casos y Controles , Estudios de Cohortes , ADN Viral/análisis , ADN Viral/fisiología , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Desequilibrio de Ligamiento , Masculino , Esclerosis Múltiple/genética , Esclerosis Múltiple/virología , Oportunidad Relativa , Polimorfismo de Nucleótido Simple/fisiologíaRESUMEN
The aim of the study was to investigate the expression of angio- and lymphangiogenic molecules (vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4) in non-Hodgkin lymphomas (NHL) treated in the pre-rituximab era. Pre-therapeutic lymph-node biopsies from 155 patients with NHL (64 follicular lymphomas (FLs), 47 de novo diffuse large B-cell lymphomas (DLBCL) and 44 peripheral T-cell lymphomas (PTCL)) were stained by in situ hybridization and immunohistochemistry. Tumor cell expression of VEGF, VEGF-C and their receptors was detected in most of the analyzed biopsies. In FL, diffuse intratumoral VEGF staining correlated with shorter overall survival (OS) (p = 0.008) and diffuse KDR staining was associated with a higher risk of histologic transformation (p = 0.05). In DLBCL, high KDR expression predicted poor treatment response (p = 0.03) and had a significant adverse impact on OS (p < 0.001). In PTCL, diffuse tissue distribution of VEGF mRNA correlated with an unfavorable 5-year OS (p = 0.004).